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Extracellular Vesicles Slow Down Aβ(1–42) Aggregation by Interfering with the Amyloid Fibril Elongation Step
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  • Vesa Halipi
    Vesa Halipi
    Division of Chemical Biology, Department of Life Sciences, Chalmers University of Technology, Kemivägen 10, S-412 96 Gothenburg, Sweden
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  • Nima Sasanian
    Nima Sasanian
    Division of Chemical Biology, Department of Life Sciences, Chalmers University of Technology, Kemivägen 10, S-412 96 Gothenburg, Sweden
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  • Julia Feng
    Julia Feng
    Division of Chemical Biology, Department of Life Sciences, Chalmers University of Technology, Kemivägen 10, S-412 96 Gothenburg, Sweden
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  • Jing Hu
    Jing Hu
    Division of Physical Chemistry, Department of Chemistry, Lund University, SE-22100 Lund, Sweden
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    Orcidhttps://orcid.org/0009-0000-3605-2185
  • Quentin Lubart
    Quentin Lubart
    Division of Chemical Biology, Department of Life Sciences, Chalmers University of Technology, Kemivägen 10, S-412 96 Gothenburg, Sweden
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  • David Bernson
    David Bernson
    Division of Chemical Biology, Department of Life Sciences, Chalmers University of Technology, Kemivägen 10, S-412 96 Gothenburg, Sweden
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  • Daniel van Leeuwen
    Daniel van Leeuwen
    Division of Chemical Biology, Department of Life Sciences, Chalmers University of Technology, Kemivägen 10, S-412 96 Gothenburg, Sweden
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  • Doryaneh Ahmadpour
    Doryaneh Ahmadpour
    Division of Chemical Biology, Department of Life Sciences, Chalmers University of Technology, Kemivägen 10, S-412 96 Gothenburg, Sweden
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  • Emma Sparr
    Emma Sparr
    Division of Physical Chemistry, Department of Chemistry, Lund University, SE-22100 Lund, Sweden
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  • Elin K. Esbjörner*
    Elin K. Esbjörner
    Division of Chemical Biology, Department of Life Sciences, Chalmers University of Technology, Kemivägen 10, S-412 96 Gothenburg, Sweden
    *Email: [email protected]
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    Orcidhttps://orcid.org/0000-0002-1253-6342
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ACS Chemical Neuroscience

Cite this: ACS Chem. Neurosci. 2024, 15, 5, 944–954
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https://doi.org/10.1021/acschemneuro.3c00655
Published February 26, 2024

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Abstract

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Formation of amyloid-β (Aβ) fibrils is a central pathogenic feature of Alzheimer’s disease. Cell-secreted extracellular vesicles (EVs) have been suggested as disease modulators, although their exact roles and relations to Aβ pathology remain unclear. We combined kinetics assays and biophysical analyses to explore how small (<220 nm) EVs from neuronal and non-neuronal human cell lines affected the aggregation of the disease-associated Aβ variant Aβ(1–42) into amyloid fibrils. Using thioflavin-T monitored kinetics and seeding assays, we found that EVs reduced Aβ(1–42) aggregation by inhibiting fibril elongation. Morphological analyses revealed this to result in the formation of short fibril fragments with increased thicknesses and less apparent twists. We suggest that EVs may have protective roles by reducing Aβ(1–42) amyloid loads, but also note that the formation of small amyloid fragments could be problematic from a neurotoxicity perspective. EVs may therefore have double-edged roles in the regulation of Aβ pathology in Alzheimer’s disease.

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Introduction

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Alzheimer’s disease (AD) is a neurodegenerative disorder that is characterized by a progressive loss of neurons in the brain and an associated decline in memory and cognitive functions. (1) One of the major pathological hallmarks of AD is the accumulation of extracellular senile plaques consisting of fibrillar aggregates of amyloid-β (Aβ) peptides. (1,2) The exact causes of the formation of plaques remain unclear, and a better understanding of the molecular and cellular mechanisms that drive the underlying aberrant Aβ self-assembly and aggregation is needed. Much evidence suggests that amyloid proteins, alongside forming fibrils, also populate a variety of small and soluble oligomeric states. These have been identified as the most neurotoxic species in many cases. (3−6) Antibodies targeting such soluble amyloid species have in fact recently reached some success in clinical trials. (7) Furthermore, cell studies suggest that short amyloid fibril fragments, alongside various oligomers, can be neurotoxic. (8) Thus, even though the appearance and abundance of amyloid plaques are typically not well correlated with disease severity in AD, (9) it is still important to understand the Aβ aggregation cascade and how it can be modulated in order to provide a clearer molecular view of the pathology of the disease and thereby facilitate the identification of targets for the future much-needed development of new treatments.
Aβ peptides exist in various isoforms, of which the Aβ(1–40) variant is most abundant. (3) The second most common and two amino acids longer Aβ(1–42) variant has been shown to aggregate more rapidly (10) and to be the major protein component of senile plaques. (3,11) Recent advances in the analysis of amyloid formation kinetics (12,13) have provided detailed mechanistic insights into how the Aβ(1–42) peptide aggregates in vitro. This has pinpointed the importance of secondary nucleation (the nucleation of new aggregates on the surface of existing fibrils) as a rate-limiting step and the major source of toxic oligomeric species. (12) Further, it has opened new avenues to understand, in molecular detail, the effects of intrinsic (mutational) modifiers (14) as well as various external effectors (15) and putative pharmacological agents. (16)
This study focuses on the role of extracellular vesicles (EVs) in Aβ(1–42) amyloid formation. EVs are small membrane vesicles that are secreted from most, if not all, cell types. They can, due to their small size, diffuse through biological fluids to deliver cargos such as lipids, proteins, and nucleic acids to other cells, (17,18) even at distal sites of the body and across the blood–brain barrier. (19) This suggests that they have functional and targeted roles in nonsynaptic intercellular communication. (20) However, early work on EVs suggested that they also function as a means for cells to rid protein waste, (21) which is interesting in relation to the cellular need for clearance of amyloid aggregates formed through protein misfolding and aggregation. EVs have indeed been implicated in the pathologies of many protein misfolding-related neurodegenerative diseases, (22,23) including AD. (24−26) Studies have reported that EVs can, in this context, have both beneficial and detrimental effects, (9) but relatively few studies have, so far, explored their direct crosstalk with amyloid proteins. (22,24,27) One study has shown that EVs from neuroblastoma cells accelerate the aggregation of the Parkinson-related protein α-synuclein, (22) whereas another study showed that EVs from human pancreatic islets suppressed amyloid formation of the diabetes-associated islet amyloid polypeptide (IAPP). (28) In AD, it is notable that a large proportion of the amyloid precursor protein (APP) cleavage events that generate Aβ peptides take place in endolysosomal compartments, (29,30) which are conspicuously also sites for EV biogenesis. This presents an interesting and putatively pathogenic cross-section. Amyloid proteins have also been found in association with EVs, and it has therefore been suggested that EVs may be transporters in the cell–cell propagation of amyloid, which is considered important for disease progression. (31−33) In this context, it has been reported that circulating EVs preferentially interact with prefibrillar Aβ aggregates (34) and that EVs from AD patients contain Aβ oligomers and can transfer these to recipient neurons in cell culture. (32) It has, on the other hand, also been reported that EVs can transport Aβ to microglia for degradation and hence contribute to clearance. (35) These observations suggest that the cross talk between EVs and amyloid in neurodegenerative diseases may be multifaceted and with both pathological and protective outcomes.
This biophysical study focuses on the role of EVs in Aβ(1–42) amyloid fibril formation and explores their effects on the peptide’s aggregation kinetics and self-assembly mechanism, as well as on the effect of EVs on Aβ(1–42) fibril morphologies. Since EVs have been reported to be biophysically and biochemically heterogeneous, (36,37) we compared EVs from two cell lines of different origin using human neuroblastoma SH-SY5Y as a representation of neurons and human embryonic kidney (HEK293-T) as a representation of non-neuronal cells. We used thioflavin-T (ThT) fluorescence assays to probe Aβ(1–42) aggregation kinetics in the absence and presence of the EVs, together with kinetic modeling (38) to identify the inhibitory mechanism. The main finding of this study is that EVs from both of these cell types, potently and with surprisingly similar efficacy, reduce the rate of Aβ(1–42) fibril formation by specifically interfering with the fibril elongation step. We discuss this result in relation to observations of significant morphological changes to the resulting fibrils by atomic force and cryo-electron microscopies. Our study provides important molecular and mechanistic insights into the impact of EVs on extracellular Aβ aggregation and contributes to the understanding of their role(s) in Aβ-mediated neurodegeneration.

Results

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Isolation and Characterization of EVs

EVs from adherent cultures of SH-SY5Y neuroblastoma and HEK293-T embryonic kidney cells that had been kept under serum-free conditions were isolated by centrifugal filtration of the conditioned media, (39) as described in the Methods section. This collects all cell-secreted EVs below the set size cutoff (220 nm, see Methods), including subtypes such as exosomes and small microvesicles. The particle size distributions and concentrations in the EV samples were determined by using nanoparticle tracking analysis (NTA) (Figure 1a, Supporting Information Table S1). The samples contained particles that were within the expected size range for exosomes (∼40 to 160 nm) and microvesicles (∼50 nm to 1 μm). (18) We found that HEK293-T cells released more and larger (∼1.5× larger mean diameter) EVs than the SH-SY5Y cells. The protein content of the EVs was determined to be 1.65 ± 1.30 × 10–9 μg per particle for SH-SY5Y and 3.05 ± 0.54 × 10–9 μg per particle (Supporting Information Figure S1) using a BCA assay. This agrees well with other published data. (28,40) The higher protein content in the HEK 293-T EVs can be explained with its approximately two times larger surface area (as estimated by their mean radii, Supporting Information Table S1). Western blot analysis of the EVs and of the corresponding whole cell lysates confirmed the presence of the EV-enriched protein Flotillin-1 in both EV types, but the amount was lower in SH-SY5Y EVs, despite similar abundances in the cell lysates (Figure 1b). HEK293-T, but not SH-SY5Y, EVs were also positive for EV-associated protein Alix. The lack of Alix in SH-SY5Y EVs can likely be explained by low cellular expression levels even though Alix expression in SH-SY5Y EVs was confirmed in another study. (41) None of the EV samples contained calnexin, confirming that they were free from cellular contaminations.

Figure 1

Figure 1. Size and molecular identity of EVs. (a) Size distributions and particle concentrations of EVs isolated from SH-SY5Y (black) and HEK293-T (blue), as determined by NTA. Mean EV diameters ± standard deviations are given in the legend. (b) Western blots showing the presence of different protein markers in the EV samples and whole cell lysates. Abbreviations: SH = SH-SY5Y and HEK = HEK293-T.

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EVs Slow Down Aβ(1–42) Aggregation into Amyloid Fibrils

ThT fluorescence (42) was used to monitor the kinetics of Aβ(1–42) amyloid fibril formation in the absence and presence of the SH-SY5Y- and HEK293-T-derived EVs. We used size exclusion chromatography-purified monomeric peptide solutions (2 μM) as the starting material to obtain reproducible kinetics. (12,43) Figure 2a,b shows the kinetic curves for Aβ(1–42) amyloid fibril formation across a range of different EV particle concentrations that are in line with reported abundances of EVs in cerebrospinal (44) or interstitial brain fluid. (45) The data show that both EV types slow the aggregation rate of Aβ(1–42) in a concentration-dependent manner. This was accompanied by a decrease in end-point ThT fluorescence (taken as the mean ThT signal over the final 3 h of the plateau phase for each sample, especially for the HEK293-T-derived EVs Figure 2c). SDS-PAGE analysis of the residual monomer content at the aggregation end points showed that the presence of EVs somewhat reduced the monomer conversion into fibrils (Supporting Information Figure S2). This may suggest that the EVs both slow down the aggregation kinetics and shift the monomer–fibril equilibrium or apparent solubility of Aβ(1–42), although it should be noted that we could not observe any clear trend in residual monomer concentration with increasing EV concentration. The cell culture media on its own (without EVs) had no effect on Aβ(1–42) aggregation (Supporting Information Figure S3). The change in Aβ(1–42) aggregation reaction half-times (thalf) and growth-times (tgrowth; defined as the reaction time to increase the fibril content from 10 to 90% of the end-point maximum) as a function of EV concentration was analyzed (Figure 2d,e), which indicated an approximate sixfold reduction in the reaction rate at the highest EV concentration tested. Interestingly, there were very small differences in the aggregation modulatory effect of the SH-SY5Y- and HEK293-T-derived EVs, despite their different cellular origins, significantly different mean diameters, and differences in the abundance of at least some generic surface markers (Figure 1).

Figure 2

Figure 2. Aβ(1–42) aggregation kinetics in the presence of EVs. (a,b) Change in ThT fluorescence as a function of time, representing the aggregation kinetics of 2 μM Aβ(1–42) into amyloid fibrils in the presence of increasing concentrations of EVs purified from (a) SH-SY5Y and (b) HEK293-T cells. The EV concentrations are given in particles/mL, as indicated by the legend in (a). Three replicate kinetic curves are overlaid for each condition. (c) Change in end-point ThT fluorescence (defined as the mean ThT signal over the final 3 h of the plateau phase, which corresponds to 37 data points) as a function of increasing EV concentration. (d) Reaction half-times and (e) reaction growth-times, extracted from the data in (a,b). The error bars represent standard deviation (n = 3).

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EVs Inhibit the Elongation Step in Aβ(1–42) Fibril Formation

Having determined an aggregation-reducing effect of EVs, we next used kinetic analyses and seeded aggregation experiments to pinpoint the inhibitory mechanism, which is important to explain the mode of action of a modulator, its overall efficacy, and putative downstream consequences. (16) To first discriminate between primary nucleation and secondary mechanisms (secondary nucleation and aggregation), we repeated the aggregation kinetics experiments in Figure 2, adding different concentrations of preformed Aβ(1–42) fibrils to seed the reactions (Figure 3a–f). Seeding increased the reaction rates, which is expected as the presence of preformed fibrils bypasses the, typically slow, primary nucleation reaction step. (12,46,47) Importantly, the inhibitory effects of the EVs remained in the presence of the seeds, as further illustrated by the half-time plots in Figure 3g,h. Previous studies have shown this to be an indication of the fact that the modulator acts on secondary reaction steps (secondary nucleation or elongation). (12,16) Since the inhibitory effect of EVs remained even under the highest seeded conditions (25%) where elongation has been shown to dominate the amyloid formation rate, (47) these data qualitatively suggest that EVs inhibit fibril elongation.

Figure 3

Figure 3. Effect of EVs on the seeded aggregation of Aβ(1–42). (a–f) Normalized Aβ(1–42) aggregation kinetic curves showing the effects of EVs in the absence (a,d) and presence (b,c and e,f) of 5 or 25% preformed Aβ(1–42) fibril seeds. Panels (a–c) and (d–f) show data for SH-SY5Y and HEK293-T EVs, respectively. The solid lines were fitted to the data using a multistep secondary nucleation model of amyloid formation setting the rate constant for elongation (k+) as a free parameter as described in the main text. The parameters underlying these fits are given in Tables S4 and S5. (g,h) Reaction half-times as a function of EV and seed concentration, derived from the data in, respectively, (a–c and d–f). The error bars represent the standard deviation (n = 3). (i) Change in the elongation rate constant (k+) as a function of EV concentration, as determined by the fitting of the data in a–f. The elongation rates are reported relative to that of 2 μM Aβ(1–42) aggregating in the absence of EVs.

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These observations were followed up by fitting of the kinetic data using the Amylofit web interface, (38) as described in the Methods. We used a mathematical model that accounts for the fact that Aβ(1–42) intrinsically forms fibrils via a secondary nucleation-dominated reaction mechanism (12) that can become saturated (i.e., lose its monomer concentration dependence) at high monomer concentrations. (48) The model operates with two variable rate constants: the product of fibril elongation and primary nucleation (k+kn) and the product of fibril elongation and secondary nucleation (k+k2). We obtained a good fit to the data in Figure 3a,d with k+k2, but not with k+kn, as a free parameter (Supporting Information Figure S4a,b and d,e) (see Supporting Information Tables S2 and S3 for all fitted parameters and associated mean residual errors). Fitting with both k+kn and k+k2 as free parameters resulted, expectedly, in the best possible fit (Supporting Information Figure S4c,f) due to the highest degree of freedom. However, only the k+k2 rate constant changed in a systematic (decreasing) manner with increasing EV concentration (Supporting Information Figure S4g,h). This supports the conclusion that EVs preferentially interfere with secondary growth processes over the primary nucleation steps.
Finally, to quantitatively determine the rate constants for primary nucleation (kn), elongation (k+), and secondary nucleation (k2) independently, we applied the secondary nucleation-dominated model to the seeded data in Figure 3, as described in ref (12and38) and the Methods section. The best fit to data was obtained with the elongation rate constant (k+) as the free parameter, as shown by the solid lines in Figure 3a–c; the other fits are shown in Supporting Information Figures S5 and S6, and all fitted rate constants are given in Supporting Information Tables S4 and S5. Taken together, all kinetic analyses, including curve shape analysis (Figure 2) and data fitting (Figure 3), support the idea that EVs inhibit the elongation step in Aβ(1–42) fibril formation. The inhibition decreases the Aβ(1–42) fibril elongation rate constants ∼30-fold for SH-SY5Y EVs and 40-fold for HEK293-T EVs at the highest EV concentrations tested (Figure 3i).

Presence of EVs during Aggregation Alters the Size and Morphology of the Aβ(1–42) Fibrils

In addition to kinetic analysis, we examined if the presence of EVs affected the morphologies of the resulting Aβ(1–42) fibrils using atomic force microscopy (AFM) and cryogenic electron microscopy (cryo-TEM). Figure 4a–c and Supporting Information Figure S7 show representative AFM images recorded from dried samples of Aβ(1–42) fibrils taken at the end point of aggregation reactions with or without EVs (at 7.2 × 109 particles/mL). The images confirm that fibrils had formed under all assayed conditions, consistent with the ThT and kinetic data (Figure 2). Analysis of the AFM images to determine fibril lengths (Figure 4d) revealed that the EVs significantly reduced average fibril lengths (from 805 ± 39 nm in the absence of EVs to 290 ± 8 and 311 ± 9 nm for SH-SY5Y and HEK293-T, respectively). The difference in the mean Aβ(1–42) fibril length in the different EV-containing samples was small and not statistically significant (one-way ANOVA, p = 0.37). The fragmentation of amyloid fibrils into shorter fibril length has been associated with higher toxicity. (8,49) We did, however, only observe minor reductions in cell viability under experimental conditions relevant to the present aggregation study and no apparent differences between Aβ(1–42) fibrils formed in the absence or presence of EVs or upon treatment with EVs alone (Supporting Information Figure S8), suggesting that neither the short nor the long fibril fragments had acute cytotoxic effects at the assayed concentration.

Figure 4

Figure 4. Morphological characterization of Aβ(1–42) fibrils formed in the absence and presence of EVs. (a–c) AFM images of Aβ(1–42) fibrils formed (a) in phosphate buffer with DPBS (see Methods) and (b,c) in the presence of SH-SY5Y and HEK293-T EVs. Scale bars = 2 μm. (d,e) AFM-based analysis of the distributions of (d) fibril lengths (e) and cross-sectional heights of the Aβ(1–42) fibrils formed in the absence and presence of EVs (n = 100–120 per condition, *** denotes p < 0.001 by one-way ANOVA). (f–k) Cryo-TEM images of Aβ(1–42) fibrils formed in the absence of EVs (f,g) and in the presence of EVs from, respectively, SH-SY5Y(h,i) and HEK293-T (j,k) cells. The Aβ(1–42) fibrils formed in the presence of EVs contained small dark dots, indicated by the white arrows in (i) and (k), suggestive of the dense association of EV components. Scale bars = 250 nm. All analyses have an EV concentration of 7.2 × 109 particles/mL.

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The average heights of the Aβ(1–42) fibrils were significantly altered when formed in the presence of EVs (Figure 4e; from 4.5 ± 0.2 nm in the absence EVs to 5.6 ± 0.1 and 6.6 ± 0.1 nm for SH-SY5Y and HEK293-T EVs, respectively). This difference between fibrils formed in the presence of SH-SY5Y and HEK293-T EVs is statistically significant (one-way ANOVA, p < 0.001) and may relate to the fact that HEK293-T-derived EVs were larger and hence make more material available for potential co-aggregation (when supplied at the same particle concentration as used here). The Aβ(1–42) fibrils formed in the absence of EVs had an average height of 4.5 ± 0.2 nm, which is consistent with structural models of in vitro-formed Aβ(1–42) fibrils with two intertwined protofilaments obtained by solid-state NMR. (50,51) In the case of HEK293-T-derived EVs, the increase in fibril thickness could potentially be consistent with the formation of a different fibril polymorph with three or four protofilaments, (52) but for SH-SY5Y-derived EVs, the change is likely too small to accommodate such major rearrangement in the fibril structure. Alternatively, the change in height could result from differences in the packing of the two filaments and/or from coaggregation or coating of the Aβ(1–42) fibrils with EV components. (53)
We proceeded with carrying out cryo-TEM imaging to probe for potential fibril–EV interactions. The cryo-TEM images provided more detailed information about the morphology of the Aβ(1–42) fibrils (Figure 4k–f). The fibrils formed without EVs had, expectedly, clear filament twists (Figure 4g, Supporting Information Figure S9a–c), whereas twists were less apparent in the Aβ(1–42) fibrils formed in the presence of the EVs (Figure 4i,k, Supporting Information Figure S9d–i). The Aβ(1–42) fibrils formed in the presence of the EVs also appeared to have some small dark dots, indicating locally increased electron densities that could be related to the dense association of EV components. Notably, colocalization between intact EVs and the Aβ(1–42) fibrils was only rarely observed. An example of the colocalization of an EV to a fibril end is shown Supporting Information Figure S10. This is reasonable given that very low EV concentrations and high Aβ(1–42):EV ratios were used throughout this study due to the potency of the EV-mediated aggregation inhibition.

Discussion

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EVs have been implicated in several aspects of AD pathology, for example, as plausible candidates for systemic spreading of aggregated Aβ(1–42) peptides, as recently reviewed by Picca et al. and Jiang et al. (23,25) Aβ peptide association with EVs has also been reported (26,54) as well as crosstalk between EV (exosome) biogenesis and the regulation of APP processing within multivesicular body organelles. (55) In this study, we explored another aspect of this intersection of EVs and Aβ peptides using a biophysical approach and chemical kinetics to explore a putative role of EVs as regulators in extracellular Aβ(1–42) aggregation.
The main finding of this study is that EVs, of both neuronal and non-neuronal origin, effectively slow down Aβ(1–42) aggregation by interfering with the fibril elongation step (i.e., the addition of monomers to fibril ends). This, furthermore, resulted in the formation of shorter (∼300 nm) and thicker fibrils. We observed a remarkable similarity in the effect of the two EV types, suggesting that their inhibition capacities are related to generic physical and biochemical attributes of EVs rather than to cell-type-specific EV molecular fingerprints. Our results can also be viewed in relation to the relatively scarce literature on EV-mediated effects on the aggregation of other amyloidogenic proteins. EVs have qualitatively similar effects on Aβ(1–42) and IAPP, (28) an equally sized polypeptide but with opposite net charge at neutral pH [+2 compared to −3 for Aβ(1–42)]. The mechanism of inhibition in the IAPP case was not established, making it difficult to directly compare the results, but the opposing charges of the two peptides indicate differences in the electrostatic interactions between the peptides and the EVs. α-Synuclein aggregation has, on the other hand, been reported to accelerate in the presence of EVs. (22) One notable difference between Aβ(1–42) and α-synuclein is the higher propensity of the latter to bind to membranes in monomeric form. (56)
We found that EVs, under the conditions and concentrations used in our work [EV concentrations of ∼109 particles/mL and an initial Aβ(1–42) monomer concentration of 2 μM], caused up to ∼6-fold reductions in the overall Aβ(1–42) fibrillation rate (Figure 2d) and 30- to 40-fold decreases in fibril elongation rates (Supporting Information Tables S4 and S5). To put this in context, one may first consider EVs as pure lipid vesicles, which, according to the steadily growing literature on EV lipid compositions, (57−59) are enriched in cholesterol and phosphatidylcholine or phosphatidylserine, as well as sphingolipids such as sphingomyelin and glycosphingolipids. Thus, even though the exact lipid composition of the SH-SY5Y- and HEK293-T-derived EVs used here have not been assessed, it is possible to make some general comparisons to the published literature. For example, we and others have found that dioleoylphosphatidylcholine (DOPC) and (60) or dimyristoylphosphatidylcholine (DMPC)/cholesterol (61) synthetic lipid vesicles with sizes comparable to those of EVs can catalyze Aβ(1–42) fibrillation. It has also been shown that sphingomyelin promotes Aβ(1–42) oligomerization. (62) This suggests that synthetic lipid membranes that contain several of the most abundant EV lipid components in fact have opposite effects on Aβ(1–42) aggregation as the inhibitory effect we report with the EVs. On the other hand, a study by Sanguanini et al. (63) suggests that phosphatidylserine may inhibit Aβ(1–42) aggregation, and we have found that monosialoganglioside 1 (GM1) also acts inhibitory (Supporting Information Figure S11). This suggests that the fine-tuned combination of lipids present in a biological vesicle may, in fact, be decisively important for their aggregation modulatory effect. However, it should also be noted that the elongation inhibitory mechanism of EVs that we report here has hitherto not been observed with lipid vesicles. Reported effects include only the modulation of primary or secondary nucleation. This may be uniquely tied to the biological complexity of the EV membrane, where variations in lipid distributions are relevant and may be important, as well as the presence of transmembrane and surface-associated EV proteins.
Importantly, there is a rather large difference in the concentrations needed to modulate Aβ(1–42) fibrillation using synthetic lipid vesicles compared to EVs. The modulatory effects observed in the abovementioned references required synthetic lipid vesicle concentrations of typically around 1010–1011 particles/ml (see Supporting Information text for calculation) to achieve smaller effects (typically no more than twofold changes to the Aβ(1–42) aggregation rate) than with EVs. Thus, although it has been recognized that the compositional complexity of lipid membranes (as existing in cell-derived EVs) is important for the outcome of lipid membrane-mediated modulation of Aβ(1–42) aggregation, (63) our work indicates that EVs are much more potent than their synthetic, pure lipid vesicle counterparts. This, in turn, further emphasizes that EV proteins or specific lipids may be important for the inhibitory effect on Aβ(1–42). For example, EVs have been observed to contain chaperones (64) such as Hsp70, (20) which could potentially contribute to the inhibition of Aβ(1–42) aggregation. (65) We report a high degree of similarity in the inhibitory effect of the SH-SY5Y- and HEK-293T-derived EVs, despite their different cellular origins and mean particle size. Ribeiro et al. reported that pancreatic EVs from healthy individuals inhibit IAPP aggregation, whereas EVs from type II diabetes patients had no effect. They speculate that observed differences in protein coverage on the EV surface (or conversely the accessibility to the lipid bilayer of the EVs) could underlie this result. (28) We observe, on the other hand, that the protein content of the SH-SY5Y and HEK-293T differs by a factor of ∼2 (Supporting Information Figure S1), which correlates with their respective surface areas (as estimated by the difference in EV size, Supporting Information Table S1). This suggests that the SH-SY5Y- and HEK-293T-derived EVs have similar protein coverage, and this could, if one reasons along the same lines as Ribeiro et al., contribute to their similar effects on Aβ(1–42).
We concluded, using different kinetic analyses, that the EVs slow down Aβ(1–42) aggregation by primarily inhibiting the fibril elongation step. This conclusion is in agreement with the formation of shorter fibril fragments in EV-containing Aβ(1–42) samples (Figure 4). Notably, inhibition of primary or secondary nucleation should, by contrast, increase fibril lengths in a system with finite amounts of available monomer, simply because the rate of formation of new fibrils in the system is slowed down and elongation of existing fibrils is therefore favored. Fibril elongation inhibition has previously not been reported in relation to lipid-membrane-mediated amyloid modulation. It has rather been associated with end-capping molecules such as certain chaperones (66,67) or divalent metal ions. (43,68) The latter bind to, and induce, N-terminal loops in Aβ monomers, (69) thus impeding their abilities to associate with fibril ends. Even though we found rare examples of intact EVs attaching to fibril ends (Supporting Information Figure S9), this appears as an energetically unfavorable interaction geometry. Moreover, based on simple geometric considerations (see Supporting Information text for calculation), and the reasonable assumption that most Aβ(1–42) monomers converted into fibrils, (70) one can estimate the Aβ(1–42) fibril concentration in the EV-containing samples to be on the order of 1015 fibrils/mL at the aggregation end point. Thus, on a particle basis, the EVs exert their effects under extreme substoichiometric conditions, which appear inconsistent, on a numerical basis, with a prevalent end-capping effect. However, similar extremes in substoichiometric inhibition have been observed with linoleic acid and the amyloid-forming peptide NACore, and the effect is ascribed to the co-assembly of fatty acid aggregates and the amyloid species at the early stages of aggregation. (71) Moreover, whereas the EV concentrations used in this work were within the physiological range, (44,45) total Aβ(1–42) concentrations in brain fluids are generally lower (72) even though significant concentration variation likely exists. (73) This suggests that Aβ(1–42):EV ratios in the brain may be lower than those assessed here and that elongation inhibitory mechanisms could potentially be even more effective in vivo.
We found that the presence of EVs during the aggregation reaction increased the thickness of the Aβ(1–42) fibrils from ∼4.5 to ∼6 nm (Figure 4e) and that the thicker fibrils appeared to have a smoother surface (less apparent twist, Figure 4g,i). This could result from differences in the lateral assembly of protofilaments alone. Similar alterations to Aβ fibril morphologies have also been observed in the presence of lipid vesicles composed of POPC, GM1, and cholesterol. (74) Furthermore, aggregation of the Parkinson’s related protein α-synuclein in the presence of phospholipid vesicles leads to the formation of lipid–protein co-aggregates with distinct morphologies compared to assemblies formed by the protein alone. (53) It is therefore possible that Aβ(1–42), through fibril growth, can sequester lipids from the EVs. However, the larger inhibitory effect of EVs compared to that of synthetic lipid vesicles suggests that EV proteins also play an important role. It is possible that co-aggregation and coating of the fibril surface impede elongation even though ends are not specifically capped. Co-aggregated proteins and lipids may alter electrostatic and hydrophobic properties of the fibrils or induce a different fibril fold. This is important since fibril elongation mechanistically may initiate by monomer adsorption to the fibril surface and subsequent sliding of the monomer toward the fibril end, (75) a process that could be significantly impeded by the presence of co-aggregated molecules that alter the fibril surface. Inhibition of Aβ(1–42) elongation could also, at least in part, occur indirectly due to monomer sequestration onto EVs. However, this would likely have manifested as effects on primary nucleation, as well.
A second consequence of the EV-mediated inhibition of Aβ(1–42) fibril elongation is the formation of short fibrils (∼300 nm), resulting from the skewed balance between nucleation events (formation of new aggregates) and elongation (extension of existing ones) in the reaction. This will lead to accumulation, over time, of smaller protein aggregates. Even though we could not confirm a clear difference in the toxicity of fibrils formed in the presence or absence of EVs under the conditions and low concentrations used in this study, elongation inhibition per se is associated with both amyloid toxicity (16) and the poor efficacy of some anti-Aβ antibodies. (76) We (8) and others (49) have, furthermore, shown a direct inverse correlation between the cytotoxicity of amyloid fibrils and their length, an effect that we ascribe to an increased propensity for cellular uptake, (8) which in turn can enable higher mobility of the fibrils and promote their ability to propagate across the brain. Interestingly, the authors of a previous cell study on the role of microglia-derived EVs in neurodegeneration also reported that EVs reduce Aβ fibril length and did observe a concomitant increase in neurotoxicity. (77)
Altogether, this study explores the intersection between EVs and Aβ(1–42) peptides, focusing specifically on the biophysical effects of EVs on the process of Aβ(1–42) fibril formation. The finding that EVs, from two different cell sources, potently and equally slow Aβ(1–42) aggregation kinetics in vitro may, at first, suggest that they could confer neuroprotective effects in the context of AD pathology, especially to delay the onset and progression of Aβ aggregation in the extracellular space. However, the identification in this study of a fibril elongation inhibitory mechanism, which skews the balance between nucleation events and elongation (16) and the demonstration that this, indeed, leads to the formation of short and potentially cell-reactive fibrils, suggests that the consequences of EV-Aβ(1–42) interactions may in fact drive neurotoxicity and contribute to the persistent accumulation of soluble Aβ(1–42) aggregates in the brain. We thus provide a possible mechanistic explanation whereby EVs co-aggregate with Aβ(1–42) fibrils in a way that renders the elongation step significantly perturbed, and shorter fibrils form. Even though more studies will be needed to pinpoint the exact interactions between Aβ(1–42) fibrils, EVs and EV proteins, and lipids, this study contributes importantly to our current understanding of Aβ pathology and on the complex balance of neurotoxic and neuroprotective roles that cell-derived EVs may have in the onset and development of neurodegenerative disorders.

Methods

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Cell Culture and Cell Lines

SH-SY5Y cells were cultured in 1:1 medium of MEM + GlutaMAX and F-12 Nut Mix (Gibco, USA), supplemented with 10% FBS and 1% nonessential amino acids (Gibco, USA). HEK293-T cells were cultured in DMEM (Gibco, USA) supplemented with 10% FBS. Both cell lines were authenticated based on genotyping according to ANSI/ATCC standard ASN-0002 (Eurofins Genomics) and routinely verified free of mycoplasma using qPCR (Eurofins Genomics). Cells used for experiments were below passage number 20. For EV purification, cells were cultured in T75 culture flasks (Thermo Scientific) at 37 °C and 5% CO2 until they reached approximately 70% confluence. The cell medium was exchanged to 1:1 medium of MEM + GlutaMAX and F-12 Nut Mix without FBS (SH-SY5Y) or in serum-free opti-MEM (HEK293-T) for 48 h prior to collecting the conditioned medium (CM) for EV isolation.

EV Isolation

CM from SH-SY5Y or HEK293-T cells, derived as described above, was filtered through a membrane with 0.22 μm pore size (VWR) followed by low-speed centrifugation (2000g) for 20 min to remove larger particles and cell debris. This also sets a size cutoff for EV isolation. The resulting CM was then subjected to ultrafiltration using Amicon Ultra-15 10 kDa (Millipore) spin filters at 5000g for 2 h, after which the collector tube was emptied and Dulbecco’s phosphate-buffered saline (DPBS) (Thermo Fisher Scientific) was added, and a second centrifugation at the same speed was performed. (39) The EV samples in DPBS were recovered and stored at 4 °C for a maximum of 3 days for use in aggregation experiments to avoid degradation or aggregation of the EVs. EV samples for western blot were frozen and stored at −80 °C for a maximum of 1.5 months before analysis.

Nanoparticle Tracking Analysis

EV size and concentration of all EV samples were determined by NTA using a NanoSight LM14 instrument with NTA 3.3 software (Malvern Instruments). Five 60 s videos were recorded per sample using the light scatter mode and a camera setting of 13. All samples were diluted in 0.22 μm filtered DPBS to proper concentrations prior to analysis, and the software settings were kept constant for all measurements to obtain comparable results. A detection threshold of 2 and 3 was used for EVs from SH-SY5Y and HEK293-T, respectively.

Protein Content Analysis

The protein mass in the EV samples was quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Prior to the analysis, the EV samples were lysed in 1× RIPA buffer, and the lysates were incubated for 30 min on ice and intermittently mixed by tapping the tubes. The Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) was used to quantify the total amount of protein in the EV samples.

Western Blot

EV samples were prepared as described in the EV isolation. Laemmli buffer was used to denature proteins and lyse the EV samples. All samples were boiled for 10 min with gentle shaking prior to loading onto 8–16% criterion TGX precast midi protein gels (Bio-Rad) that were run in Tris–glycine SDS buffer (1×) for 1.5 h at 120 V. Equal amounts of EV particles and cellular protein extract from both cell lines were loaded onto the gel. The precision plus protein dual color ladder (Thermo Fisher) was used for visualization of gel migration and protein size. Transfer to a 0.2 μM nitrocellulose membrane was performed for 7 min by using a Trans-Blot Turbo device (Bio-Rad). LI-COR PBS blocking buffer (LI-COR Biosciences) was used to block the membrane for 1 h under agitation and at RT. Thereafter, the membrane was incubated with primary antibodies anti-Calnexin (1:20,000, Abcam), anti-Flotillin-1 (1:1000, BD Biosciences), anti-Alix (1:1000, Cell Signaling), and anti-GAPDH (1:10,000, Abcam) overnight at 4 °C and with shaking. The primary antibodies were diluted in the blocking buffer, together with 1% Tween 20. The blots were washed three times in PBS-T before incubation with secondary antibodies goat antimouse and goat antirabbit (1:20,000, LI-COR) for 1 h and 15 min at RT and with agitation. The LI-COR Odyssey Infrared scanner was used for visualization of the bands on the membrane.

Aβ(1–42) Expression and Purification

A plasmid encoding for Aβ(1–42) fused to the NT solubility tag (78) was transformed into Escherichia coli (BL21) cells and expressed overnight. The cells were centrifuged and dissolved in 20 mM Tris–HCl, 8 M urea, pH 8.0 buffer, and frozen at −20 °C for further use. For purification of the Aβ(1–42) peptide, the bacterial cells were sonicated, centrifuged, and filtered with a 0.45 μm syringe filter and thereafter loaded onto a HisPrep FF 16/10 column (GE Healthcare) equilibrated with 20 mM Tris–HCl, 8 M urea, 15 mM imidazole, pH 8.0 buffer. (43) The NT- Aβ(1–42) fusion protein, which carries a His-tag, was eluted in the same buffer supplemented with 300 mM imidazole and thereafter dialyzed against 20 mM Tris–HCl, pH 8.0 buffer for 2 h. This was followed by 1:20 mol equiv of TEV (tobacco etch virus) protease in the presence of 0.5 mM EDTA and 1.5 mM DTT and dialysis overnight at 4 °C to cleave off the NT tag. Next, the solution was loaded onto a HiLoad 16/600 Superdex 30 pg (GE Healthcare) size exclusion column equilibrated with a 20 mM sodium phosphate buffer, pH 8.0, and the monomeric Aβ(1–42) was eluted, aliquoted directly, and freeze-dried for storage at −20 °C for further use.

Aβ(1–42) Aggregation Kinetics Assays

Lyophilized Aβ(1–42) was dissolved in 6 M guanidine hydrochloride and incubated on ice for 20 min. Aβ(1–42) monomers were purified just prior to each kinetics assay by size-exclusion chromatography (SEC) in 20 mM sodium phosphate buffer (pH 8.0) using a 10/300 Superdex 75 column (GE Healthcare). The Aβ(1–42) monomer concentration was determined during SEC, from the integrated area under the collected peak in the chromatogram, using an extinction coefficient of ε280 = 1280 M–1 cm–1. The samples for aggregation assays were prepared on ice to avoid initiating aggregation. Each sample contained 5 μM ThT (Sigma) and 2 μM Aβ(1–42) together with the indicated concentration (particles/mL) of the EVs. DPBS was added to maintain a constant salt level in all samples. [EVs were purified in DPBS, whereas the Aβ(1–42) peptide was purified without salt]. All samples were thereafter added in triplicate to nonbinding, transparent bottom, half-area 96-well microplates (Corning) and sealed with a plastic film to prevent sample evaporation. ThT emission was measured with bottom-optics using a Fluostar Optima or Fluostar Omega plate reader (BMG Labtech) and a 440 ± 10 nm band-pass filter for excitation and a 485 ± 10 nm band-pass filter for emission. All aggregation kinetic assays were performed at 37 °C and under quiescent conditions. For seeded kinetics experiments, fibril seeds prepared in the absence of EVs were collected directly from the wells of a plate used in a previous aggregation experiment and mixed with new monomer solution at indicated seed concentrations determined by the volumetric ratio of the seed and monomer solutions. The seeds were formed under the same experimental conditions as described in this section (Methods). The end-point maximum was defined as the mean ThT signal calculated from data points collected over a 3 h period in the plateau phase (corresponding to 37 data points).

Analysis and Fitting of ThT Kinetic Curves

The acquired kinetic data were analyzed to determine the dominant mechanism of aggregation and extract rate constants. We used a secondary nucleation dominated model (12) and the online fitting platform AmyloFit. (38) First, the experimental data for Aβ(1–42) with no additives were fitted. This enabled the estimation of compounded rate constants for elongation and primary nucleation or elongation and secondary nucleation; k+kn and k+k2, respectively, and the determination of KM (which was kept constant to all further analysis). These values were used as initial values when analyzing Aβ(1–42) fibril formation in the presence of EVs and without fibril seeds. Thereafter, we used the same model for the seeded experiments and determined the rate constants for primary nucleation (kn), secondary nucleation (k2), or elongation (k+) independently, allowing one rate constant at the time to be probed as a fitting parameter while keeping the other two as global parameters, allowing them each to change with EV concentration but not with seed concentration. The goodness of fits was evaluated based on mean residual errors.

Atomic Force Microscopy

AFM samples were prepared by depositing 10 μL samples to freshly cleaved mica surfaces that had been positively functionalized with 10 μL of (3-aminopropyl)triethoxysilane for 30 s, washed with Milli Q water, and dried with nitrogen gas. After 10 min of incubation to let the fibrils settle, the sample solutions were removed, and the mica was washed with MQ followed by drying with nitrogen gas. The ready samples were stored in closed containers until imaging was performed. AFM images were recorded using an NTEGRA system with a gold-covered crystal cantilever (NT-MDT, NSG01, force constant ∼5.1 N/m, resonance frequency ∼ 150 Hz). 256 × 256 pixel, 5 × 5 μm images were recorded for length and height calculations, and 512 × 512 pixel images (across the same sample area) were acquired for visualization purposes. All images were analyzed using Gwyddion software. (79) Data leveling by mean plane subtraction, correction of horizontal aberrations, and minimum value shift to zero was applied to all images before manual measurement of the heights and lengths of 100–120 fibrils over 10 individual images per sample. The data were analyzed by performing a one-way ANOVA followed by statistical means comparison by two-sample t-test using Bonferroni’s correction [OriginPro 2020 software (OriginLab)].

Cryogenic Electron Microscopy

Samples of Aβ(1–42) fibrils alone, EVs alone, and Aβ(1–42) fibrils formed in the presence of EVs were prepared according to the aggregation kinetics assay and EV isolation procedures described above. Small sample volumes of 4 μL were added as thin liquid films on carbon-coated copper grids, blotted with filter paper, and plunged into liquid ethane at −180 °C in an automatic plunge freezer (Leica). This freezing procedure prevents the formation of water crystals and preserves samples in their original structure. The samples were then kept in liquid N2 and transported in a cryoholder (Fischione model 2550) to the electron microscope (JEM 2200FS) for imaging. Zero-loss images were recorded with a TVIPS F416 camera at 200 kV acceleration voltage.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acschemneuro.3c00655.

  • EV particle size determined by NTA; mean residual monomer content at the end point of aggregation; protein amount per EV particle; aggregation kinetics of medium control; fitting of ThT kinetic curves with AmyloFit and corresponding kinetic parameters; additional AFM and cryo-TEM images of Aβ(1–42) fibrils; estimation of fibril and vesicle concentrations; and cell viability assay (PDF)

Terms & Conditions

Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Author Information

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  • Corresponding Author
  • Authors
    • Vesa Halipi - Division of Chemical Biology, Department of Life Sciences, Chalmers University of Technology, Kemivägen 10, S-412 96 Gothenburg, Sweden
    • Nima Sasanian - Division of Chemical Biology, Department of Life Sciences, Chalmers University of Technology, Kemivägen 10, S-412 96 Gothenburg, Sweden
    • Julia Feng - Division of Chemical Biology, Department of Life Sciences, Chalmers University of Technology, Kemivägen 10, S-412 96 Gothenburg, Sweden
    • Jing Hu - Division of Physical Chemistry, Department of Chemistry, Lund University, SE-22100 Lund, Sweden;  Orcidhttps://orcid.org/0009-0000-3605-2185
    • Quentin Lubart - Division of Chemical Biology, Department of Life Sciences, Chalmers University of Technology, Kemivägen 10, S-412 96 Gothenburg, Sweden
    • David Bernson - Division of Chemical Biology, Department of Life Sciences, Chalmers University of Technology, Kemivägen 10, S-412 96 Gothenburg, Sweden
    • Daniel van Leeuwen - Division of Chemical Biology, Department of Life Sciences, Chalmers University of Technology, Kemivägen 10, S-412 96 Gothenburg, Sweden
    • Doryaneh Ahmadpour - Division of Chemical Biology, Department of Life Sciences, Chalmers University of Technology, Kemivägen 10, S-412 96 Gothenburg, Sweden
    • Emma Sparr - Division of Physical Chemistry, Department of Chemistry, Lund University, SE-22100 Lund, Sweden
  • Author Contributions

    E.K.E., Q.L., and D.B. initially conceptualized the idea, with later inputs from V.H. and N.S. The methodology was established by V.H., J.F., N.S., Q.L., and D.B. The majority of the experiments were carried out by V.H. and J.F., and pilot experiments were performed by Q.L., N.S., and D.B. J.H. and E.S. contributed to the cryo-TEM part. V.H., D.vL., and D.A set up the EV purification and characterization. V.H. and N.S. did the formal data analysis. V.H. prepared all figures. V.H. and E.K.E. wrote the paper. All coauthors reviewed and commented on the final draft. E.K.E. provided the funding and supervision.

  • Notes
    The authors declare no competing financial interest.
    D.vL. is currently an employee of AstraZeneca, and Q.L is currently an employee of Abbelight. Their contributions to this work were done during their respective employments at Chalmers.

Acknowledgments

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The authors acknowledge funding to E.K.E. from the Knut and Alice Wallenberg Foundation Academy Fellow (2019.0238) and project grant (2022.0134) programs, the Wenner-Gren Foundation, the Swedish Research Council (VR) (grant nos. 2016-03902 and 2020-05303), the Swedish Foundation for Strategic Research (IRC15-0065), the Åhlén Foundation, and Chalmers Area of Advance for Nanoscience and Nanotechnology.

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    We have characterized amyloid beta peptide (Abeta) concentration, Abeta deposition, paired helical filament formation, cerebrovascular amyloid angiopathy, apolipoprotein E (ApoE) allotype, and synaptophysin concentration in entorhinal cortex and superior frontal gyrus of normal elderly control (ND) patients, Alzheimer's disease (AD) patients, and high pathology control (HPC) patients who meet pathological criteria for AD but show no synapse loss or overt antemortem symptoms of dementia. The measures of Abeta deposition, Abeta-immunoreactive plaques with and without cores, thioflavin histofluorescent plaques, and concentrations of insoluble Abeta, failed to distinguish HPC from AD patients and were poor correlates of synaptic change. By contrast, concentrations of soluble Abeta clearly distinguished HPC from AD patients and were a strong inverse correlate of synapse loss. Further investigation revealed that Abeta40, whether in soluble or insoluble form, was a particularly useful measure for classifying ND, HPC, and AD patients compared with Abeta42. Abeta40 is known to be elevated in cerebrovascular amyloid deposits, and Abeta40 (but not Abeta42) levels, cerebrovascular amyloid angiopathy, and ApoE4 allele frequency were all highly correlated with each other. Although paired helical filaments in the form of neurofibrillary tangles or a penumbra of neurites surrounding amyloid cores also distinguished HPC from AD patients, they were less robust predictors of synapse change compared with soluble Abeta, particularly soluble Abeta40. Previous experiments attempting to relate Abeta deposition to the neurodegeneration that underlies AD dementia may have failed because they assayed the classical, visible forms of the molecule, insoluble neuropil plaques, rather than the soluble, unseen forms of the molecule.
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  7. 7
    van Dyck, C. H.; Sabbagh, M.; Cohen, S. Lecanemab in Early Alzheimer’s Disease. Reply. N. Engl. J. Med. 2023, 388 (17), 1630– 1632,  DOI: 10.1056/NEJMc2301380
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  8. 8
    Zhang, X.; Wesen, E.; Kumar, R.; Bernson, D.; Gallud, A.; Paul, A.; Wittung-Stafshede, P.; Esbjörner, E. K. Correlation between Cellular Uptake and Cytotoxicity of Fragmented α-Synuclein Amyloid Fibrils Suggests Intracellular Basis for Toxicity. ACS Chem. Neurosci. 2020, 11 (3), 233– 241,  DOI: 10.1021/acschemneuro.9b00562
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    8
    Correlation between Cellular Uptake and Cytotoxicity of Fragmented α-Synuclein Amyloid Fibrils Suggests Intracellular Basis for Toxicity
    Zhang, Xiaolu; Wesen, Emelie; Kumar, Ranjeet; Bernson, David; Gallud, Audrey; Paul, Alexandra; Wittung-Stafshede, Pernilla; Esbjoerner, Elin K.
    ACS Chemical Neuroscience (2020), 11 (3), 233-241CODEN: ACNCDM; ISSN:1948-7193. (American Chemical Society)
    Aggregation and intracellular deposition of the protein α-synuclein is an underlying characteristic of Parkinson's disease. α-Synuclein assemblies also undergo cell-cell spreading, facilitating propagation of their cellular pathol. Understanding how cellular interactions and uptake of extracellular α-synuclein assemblies depend on their phys. attributes is therefore important. We prepd. fragmented fluorescently labeled α-synuclein amyloid fibrils of different av. lengths (∼80 nm to >1μm) and compared their interactions with SH-SY5Y cells. We report that fibrils of all lengths, but not monomers, bind avidly to the cell surface. Their uptake is inversely dependent on their av. size, occurs via a heparan sulfate dependent endocytic route, and appears to have a size cutoff of ∼400 nm. The uptake of α-synuclein fibrils, but not monomers, correlates with their cytotoxicity as measured by redn. in metabolic activity, strongly suggesting an intracellular basis for α-synuclein fibril toxicity, likely involving endolysosomes.
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  9. 9
    Joshi, P.; Benussi, L.; Furlan, R.; Ghidoni, R.; Verderio, C. Extracellular vesicles in Alzheimer’s disease: friends or foes? Focus on abeta-vesicle interaction. Int. J. Mol. Sci. 2015, 16 (3), 4800– 4813,  DOI: 10.3390/ijms16034800
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    9
    Extracellular vesicles in Alzheimer's disease: friends or foes? Focus on Aβ-vesicle interaction
    Joshi, Pooja; Benussi, Luisa; Furlan, Roberto; Ghidoni, Roberta; Verderio, Claudia
    International Journal of Molecular Sciences (2015), 16 (3), 4800-4813CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)
    The intercellular transfer of amyloid-β (Aβ) and tau proteins has received increasing attention in Alzheimer's disease (AD). Among other transfer modes, Aβ and tau dissemination has been suggested to occur through release of Extracellular Vesicles (EVs), which may facilitate delivery of pathogenic proteins over large distances. Recent evidence indicates that EVs carry on their surface, specific mols. which bind to extracellular Aβ, opening the possibility that EVs may also influence Aβ assembly and synaptotoxicity. In this review we focus on studies which investigated the impact of EVs in Aβ-mediated neurodegeneration and showed either detrimental or protective role for EVs in the pathol.
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  10. 10
    Jarrett, J. T.; Berger, E. P.; Lansbury, P. T. The carboxy terminus of the .beta. amyloid protein is critical for the seeding of amyloid formation: Implications for the pathogenesis of Alzheimer’s disease. Biochemistry 1993, 32 (18), 4693– 4697,  DOI: 10.1021/bi00069a001
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    10
    The carboxy terminus of the β amyloid protein is critical for the seeding of amyloid formation: Implications for the pathogenesis of Alzheimer's disease
    Jarrett, Joseph T.; Berger, Elizabeth P.; Lansbury, Peter T., Jr.
    Biochemistry (1993), 32 (18), 4693-7CODEN: BICHAW; ISSN:0006-2960.
    Several variants of the β amyloid protein, differing only at their carboxy terminus (β1-39, β1-40, β1-42, and β1-43), have been identified as the major components of the cerebral amyloid deposits which are characteristic of Alzheimer's disease. Kinetic studies of aggregation by three naturally occurring β protein variants (β1-39, β1-40, β1-42) and four model peptides (β26-39, β26-40, β26-42, β26-43) demonstrate that amyloid formation, like crystn., is a nucleation-dependent phenomenon. This discovery has practical consequences for studies of the β amyloid protein. The length of the C-terminus is a crit. determinant of the rate of amyloid formation ("kinetic soly.") but has only a minor effect on the thermodn. soly. Amyloid formation by the kinetically sol. peptides (e.g., β1-39, β1-40, β26-39, β26-40) can be nucleated, or "seeded", by peptides which include the crit. C-terminal residues (β1-42, β26-42, β26-43, β34-42). These results suggest that nucleation may be the rate-detg. step of in vivo amyloidogenesis and that β1-42 and/or β1-43, rather than β1-40, may be the pathogenic protein(s) in AD.
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  11. 11
    Jan, A.; Adolfsson, O.; Allaman, I.; Buccarello, A. L.; Magistretti, P. J.; Pfeifer, A.; Muhs, A.; Lashuel, H. A. Aβ42 Neurotoxicity Is Mediated by Ongoing Nucleated Polymerization Process Rather than by Discrete Aβ42 Species. J. Biol. Chem. 2011, 286 (10), 8585– 8596,  DOI: 10.1074/jbc.M110.172411
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    11
    Aβ42 Neurotoxicity is Mediated by Ongoing Nucleated Polymerization Process Rather than by Discrete Aβ42 Species
    Jan, Asad; Adolfsson, Oskar; Allaman, Igor; Buccarello, Anna-Lucia; Magistretti, Pierre J.; Pfeifer, Andrea; Muhs, Andreas; Lashuel, Hilal A.
    Journal of Biological Chemistry (2011), 286 (10), 8585-8596CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    The identification of toxic Aβ species and/or the process of their formation is crucial for understanding the mechanism(s) of Aβ neurotoxicity in Alzheimer disease and also for the development of effective diagnostic and therapeutic interventions. To elucidate the structural basis of Aβ toxicity, we developed different procedures to isolate Aβ species of defined size and morphol. distribution, and we investigated their toxicity in different cell lines and primary neurons. We obsd. that crude Aβ42 prepns., contg. a monomeric and heterogeneous mixt. of Aβ42 oligomers, were more toxic than purified monomeric, protofibrillar fractions, or fibrils. The toxicity of protofibrils was directly linked to their interactions with monomeric Aβ42 and strongly dependent on their ability to convert into amyloid fibrils. Subfractionation of protofibrils diminished their fibrillization and toxicity, whereas reintroduction of monomeric Aβ42 into purified protofibril fractions restored amyloid formation and enhanced their toxicity. Selective removal of monomeric Aβ42 from these prepns., using insulin-degrading enzyme, reversed the toxicity of Aβ42 protofibrils. Together, our findings demonstrate that Aβ42 toxicity is not linked to specific prefibrillar aggregate(s) but rather to the ability of these species to grow and undergo fibril formation, which depends on the presence of monomeric Aβ42. These findings contribute significantly to the understanding of amyloid formation and toxicity in Alzheimer disease, provide novel insight into mechanisms of Aβ protofibril toxicity, and important implications for designing anti-amyloid therapies.
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  12. 12
    Cohen, S. I.; Linse, S.; Luheshi, L. M.; Hellstrand, E.; White, D. A.; Rajah, L.; Otzen, D. E.; Vendruscolo, M.; Dobson, C. M.; Knowles, T. P. Proliferation of amyloid-β42 aggregates occurs through a secondary nucleation mechanism. Proc. Natl. Acad. Sci. U.S.A. 2013, 110 (24), 9758– 9763,  DOI: 10.1073/pnas.1218402110
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    12
    Proliferation of amyloid-β42 aggregates occurs through a secondary nucleation mechanism
    Cohen, Samuel I. A.; Linse, Sara; Luheshi, Leila M.; Hellstrand, Erik; White, Duncan A.; Rajah, Luke; Otzen, Daniel E.; Vendruscolo, Michele; Dobson, Christopher M.; Knowles, Tuomas P. J.
    Proceedings of the National Academy of Sciences of the United States of America (2013), 110 (24), 9758-9763, S9758/1-S9758/11CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    The generation of toxic oligomers during the aggregation of the amyloid-β (Aβ) peptide Aβ42 into amyloid fibrils and plaques has emerged as a central feature of the onset and progression of Alzheimer's disease, but the mol. pathways that control pathol. aggregation have proved challenging to identify. Here, the authors used a combination of kinetic studies, selective radiolabeling expts., and cell viability assays to detect directly the rates of formation of both fibrils and oligomers and the resulting cytotoxic effects. The results showed that once a small but crit. concn. of amyloid fibrils had accumulated, the toxic oligomeric species were predominantly formed from monomeric peptide mols. through a fibril-catalyzed secondary nucleation reaction, rather than through a classical mechanism of homogeneous primary nucleation. This catalytic mechanism coupled together the growth of insol. amyloid fibrils and the generation of diffusible oligomeric aggregates that are implicated as neurotoxic agents in Alzheimer's disease. These results revealed that the aggregation of Aβ42 is promoted by a pos. feedback loop that originates from the interactions between the monomeric and fibrillar forms of this peptide. These findings bring together the main mol. species implicated in the Aβ aggregation cascade and suggest that perturbation of the secondary nucleation pathway identified in this study could be an effective strategy to control the proliferation of neurotoxic Aβ42 oligomers.
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    Meisl, G.; Yang, X.; Frohm, B.; Knowles, T. P.; Linse, S. Quantitative analysis of intrinsic and extrinsic factors in the aggregation mechanism of Alzheimer-associated Aβ-peptide. Sci. Rep. 2016, 6, 18728,  DOI: 10.1038/srep18728
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    Quantitative analysis of intrinsic and extrinsic factors in the aggregation mechanism of Alzheimer-associated Aβ-peptide
    Meisl, Georg; Yang, Xiaoting; Frohm, Birgitta; Knowles, Tuomas P. J.; Linse, Sara
    Scientific Reports (2016), 6 (), 18728CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)
    Disease related mutations and environmental factors are key determinants of the aggregation mechanism of the amyloid-β peptide implicated in Alzheimer's disease. Here we present an approach to investigate these factors through acquisition of highly reproducible data and global kinetic anal. to det. the mechanistic influence of intrinsic and extrinsic factors on the Aβ aggregation network. This allows us to translate the shift in macroscopic aggregation behavior into effects on the individual underlying microscopic steps. We apply this work-flow to the disease-assocd. Aβ42-A2V variant, and to a variation in pH as examples of an intrinsic and an extrinsic perturbation. In both cases, our data reveal a shift towards a mechanism in which a larger fraction of the reactive flux goes via a pathway that generates potentially toxic oligomeric species in a fibril-catalyzed reaction. This is in agreement with the finding that Aβ42-A2V leads to early-onset Alzheimer's disease and enhances neurotoxicity.
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  14. 14
    Bolognesi, B.; Cohen, S. I.; Aran Terol, P.; Esbjorner, E. K.; Giorgetti, S.; Mossuto, M. F.; Natalello, A.; Brorsson, A. C.; Knowles, T. P.; Dobson, C. M. Single Point Mutations Induce a Switch in the Molecular Mechanism of the Aggregation of the Alzheimer’s Disease Associated Aβ42 Peptide. ACS Chem. Biol. 2014, 9 (2), 378– 382,  DOI: 10.1021/cb400616y
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    14
    Single point mutations induce a switch in the molecular mechanism of the aggregation of the Alzheimer's disease associated Aβ42 peptide
    Bolognesi, Benedetta; Cohen, Samuel I. A.; Aran Terol, Pablo; Esbjorner, Elin K.; Giorgetti, Sofia; Mossuto, Maria F.; Natalello, Antonino; Brorsson, Ann-Christin; Knowles, Tuomas P. J.; Dobson, Christopher M.; Luheshi, Leila M.
    ACS Chemical Biology (2014), 9 (2), 378-382CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
    Single point mutations in the Alzheimer's disease assocd. Aβ42 peptide are found to alter significantly its neurotoxic properties in vivo and have been assocd. with early onset forms of this devastating condition. We show that such mutations can induce structural changes in Aβ42 fibrils and are assocd. with a dramatic switch in the fibril-dependent mechanism by which Aβ42 aggregates. These observations reveal how subtle perturbations to the physicochem. properties of the Aβ peptide, and the structural properties of fibrils that it forms, can have profound effects on the mechanism of its aggregation and pathogenicity.
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  15. 15
    Cohen, S. I. A.; Arosio, P.; Presto, J.; Kurudenkandy, F. R.; Biverstal, H.; Dolfe, L.; Dunning, C.; Yang, X.; Frohm, B.; Vendruscolo, M. A molecular chaperone breaks the catalytic cycle that generates toxic Aβ oligomers. Nat. Struct. Mol. Biol. 2015, 22 (3), 207– 213,  DOI: 10.1038/nsmb.2971
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    15
    A molecular chaperone breaks the catalytic cycle that generates toxic Aβ oligomers
    Cohen, Samuel I. A.; Arosio, Paolo; Presto, Jenny; Kurudenkandy, Firoz Roshan; Biverstal, Henrik; Dolfe, Lisa; Dunning, Christopher; Yang, Xiaoting; Frohm, Birgitta; Vendruscolo, Michele; Johansson, Jan; Dobson, Christopher M.; Fisahn, Andre; Knowles, Tuomas P. J.; Linse, Sara
    Nature Structural & Molecular Biology (2015), 22 (3), 207-213CODEN: NSMBCU; ISSN:1545-9993. (Nature Publishing Group)
    Alzheimer's disease is an increasingly prevalent neurodegenerative disorder whose pathogenesis has been assocd. with aggregation of the amyloid-β peptide (Aβ42). Recent studies have revealed that once Aβ42 fibrils are generated, their surfaces effectively catalyze the formation of neurotoxic oligomers. Here we show that a mol. chaperone, a human Brichos domain, can specifically inhibit this catalytic cycle and limit human Aβ42 toxicity. We demonstrate in vitro that Brichos achieves this inhibition by binding to the surfaces of fibrils, thereby redirecting the aggregation reaction to a pathway that involves minimal formation of toxic oligomeric intermediates. We verify that this mechanism occurs in living mouse brain tissue by cytotoxicity and electrophysiol. expts. These results reveal that mol. chaperones can help maintain protein homeostasis by selectively suppressing crit. microscopic steps within the complex reaction pathways responsible for the toxic effects of protein misfolding and aggregation.
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  16. 16
    Munke, A.; Persson, J.; Weiffert, T.; De Genst, E.; Meisl, G.; Arosio, P.; Carnerup, A.; Dobson, C. M.; Vendruscolo, M.; Knowles, T. P. J. Phage display and kinetic selection of antibodies that specifically inhibit amyloid self-replication. Proc. Natl. Acad. Sci. U.S.A. 2017, 114 (25), 6444– 6449,  DOI: 10.1073/pnas.1700407114
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    16
    Phage display and kinetic selection of antibodies that specifically inhibit amyloid self-replication
    Munke, Anna; Persson, Jonas; Weiffert, Tanja; De Genst, Erwin; Meisl, Georg; Arosio, Paolo; Carnerup, Anna; Dobson, Christopher M.; Vendruscolo, Michele; Knowles, Tuomas P. J.; Linse, Sara
    Proceedings of the National Academy of Sciences of the United States of America (2017), 114 (25), 6444-6449CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    The aggregation of the amyloid β peptide (Aβ) into amyloid fibrils is a defining characteristic of Alzheimer's disease. Because of the complexity of this aggregation process, effective therapeutic inhibitors will need to target the specific microscopic steps that lead to the prodn. of neurotoxic species. We introduce a strategy for generating fibril-specific antibodies that selectively suppress fibril-dependent secondary nucleation of the 42-residue form of Aβ (Aβ42). We target this step because it has been shown to produce the majority of neurotoxic species during aggregation of Aβ42. Starting from large phage display libraries of single-chain antibody fragments (scFvs), the three-stage approach that we describe includes (1) selection of scFvs with high affinity for Aβ42 fibrils after removal of scFvs that bind Aβ42 in its monomeric form; (2) ranking, by surface plasmon resonance affinity measurements, of the resulting candidate scFvs that bind to the Aβ42 fibrils; and (3) kinetic screening and anal. to find the scFvs that inhibit selectively the fibril-catalyzed secondary nucleation process in Aβ42 aggregation. By applying this approach, we have identified four scFvs that inhibit specifically the fibril-dependent secondary nucleation process. Our method also makes it possible to discard antibodies that inhibit elongation, an important factor because the suppression of elongation does not target directly the prodn. of toxic oligomers and may even lead to its increase. On the basis of our results, we suggest that the method described here could form the basis for rationally designed immunotherapy strategies to combat Alzheimer's and related neurodegenerative diseases.
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    Zhang, T.; Ma, S.; Lv, J.; Wang, X.; Afewerky, H. K.; Li, H.; Lu, Y. The emerging role of exosomes in Alzheimer’s disease. Ageing Res. Rev. 2021, 68, 101321,  DOI: 10.1016/j.arr.2021.101321
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    The emerging role of exosomes in Alzheimer's disease
    Zhang, Tongmei; Ma, Sehui; Lv, Junkai; Wang, Xinyuan; Afewerky, Henok Kessete; Li, Hao; Lu, Youming
    Ageing Research Reviews (2021), 68 (), 101321CODEN: ARRGAK; ISSN:1568-1637. (Elsevier B.V.)
    A review. Alzheimer's disease (AD), manifested by memory loss and a decline in cognitive functions, is the most prevalent neurodegenerative disease accounting for 60-80% of dementia cases. But, to-date, there is no effective treatment available to slow or stop the progression of AD. Exosomes are small extracellular vesicles that carry constituents, such as functional mRNAs, non-coding RNAs, proteins, lipids, DNA, and other bioactive substances of their source cells. In the brain, exosomes are likely to be sourced by almost all cell types and involve in cell communication to regulate cellular functions. The yet, accumulated evidence on the roles of exosomes and their constituents in the AD pathol. process suggests their significance as addnl. biomarkers and therapeutic targets for AD. This summarizes the current reported research findings on exosomes roles in the pathogenesis, diagnosis, and treatment of AD.
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    Kalluri, R.; LeBleu, V. S. The biology, function, and biomedical applications of exosomes. Science 2020, 367 (6478), eaau6977  DOI: 10.1126/science.aau6977
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    Ramos-Zaldivar, H. M.; Polakovicova, I.; Salas-Huenuleo, E.; Corvalan, A. H.; Kogan, M. J.; Yefi, C. P.; Andia, M. E. Extracellular vesicles through the blood-brain barrier: a review. Fluids Barriers CNS 2022, 19 (1), 60,  DOI: 10.1186/s12987-022-00359-3
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    Extracellular vesicles through the blood-brain barrier: a review
    Ramos-Zaldivar Hector M; Polakovicova Iva; Corvalan Alejandro H; Kogan Marcelo J; Polakovicova Iva; Corvalan Alejandro H; Salas-Huenuleo Edison; Kogan Marcelo J; Yefi Claudia P; Andia Marcelo E; Andia Marcelo E
    Fluids and barriers of the CNS (2022), 19 (1), 60 ISSN:.
    Extracellular vesicles (EVs) are particles naturally released from cells that are delimited by a lipid bilayer and are unable to replicate. How the EVs cross the Blood-Brain barrier (BBB) in a bidirectional manner between the bloodstream and brain parenchyma remains poorly understood. Most in vitro models that have evaluated this event have relied on monolayer transwell or microfluidic organ-on-a-chip techniques that do not account for the combined effect of all cellular layers that constitute the BBB at different sites of the Central Nervous System. There has not been direct transcytosis visualization through the BBB in mammals in vivo, and evidence comes from in vivo experiments in zebrafish. Literature is scarce on this topic, and techniques describing the mechanisms of EVs motion through the BBB are inconsistent. This review will focus on in vitro and in vivo methodologies used to evaluate EVs transcytosis, how EVs overcome this fundamental structure, and discuss potential methodological approaches for future analyses to clarify these issues. Understanding how EVs cross the BBB will be essential for their future use as vehicles in pharmacology and therapeutics.
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    Colombo, M.; Raposo, G.; Thery, C. Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles. Annu. Rev. Cell Dev. Biol. 2014, 30, 255– 289,  DOI: 10.1146/annurev-cellbio-101512-122326
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    Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles
    Colombo, Marina; Raposo, Graca; Thery, Clotilde
    Annual Review of Cell and Developmental Biology (2014), 30 (), 255-289CODEN: ARDBF8; ISSN:1081-0706. (Annual Reviews)
    A review. In the 1980s, exosomes were described as vesicles of endosomal origin secreted from reticulocytes. Interest increased around these extracellular vesicles, as they appeared to participate in several cellular processes. Exosomes bear proteins, lipids, and RNAs, mediating intercellular communication between different cell types in the body, and thus affecting normal and pathol. conditions. Only recently, scientists acknowledged the difficulty of sepg. exosomes from other types of extracellular vesicles, which precludes a clear attribution of a particular function to the different types of secreted vesicles. To shed light into this complex but expanding field of science, this review focuses on the definition of exosomes and other secreted extracellular vesicles. Their biogenesis, their secretion, and their subsequent fate are discussed, as their functions rely on these important processes.
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    Pan, B. T.; Johnstone, R. M. Fate of the transferrin receptor during maturation of sheep reticulocytes in vitro: selective externalization of the receptor. Cell 1983, 33 (3), 967– 978,  DOI: 10.1016/0092-8674(83)90040-5
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    Fate of the transferrin receptor during maturation of sheep reticulocytes in vitro: selective externalization of the receptor
    Pan, Bin Tao; Johnstone, Rose M.
    Cell (Cambridge, MA, United States) (1983), 33 (3), 967-78CODEN: CELLB5; ISSN:0092-8674.
    The fate of the transferrin receptor during in vitro maturation of sheep reticulocytes was followed using FITC- and 125I-labeled antitransferrin-receptor antibodies. Vesicles contg. peptides that comigrate with the transferrin receptor on polyacrylamide gels are released during incubation of sheep reticulocytes, tagged with antitransferrin-receptor antibodies. Vesicle formation does not require the presence of the antitransferrin-receptor antibodies. Using 125I-surface-labeled reticulocytes, it can be shown that the 125I-labeled material which is released is retained by an immunoaffinity column of the antitransferrin-receptor antibody. With reticulocytes tagged with 125I-labeled antitransferrin-receptor antibodies to follow the formation of vesicles, it can be shown that at 0° or in phosphate-buffered saline the rate of vesicle release is less than that at 37° in culture medium. There is selective externalization of the antibody-receptor complex, since few other membrane proteins are found in the externalized vesicles. The antitransferrin-receptor antibodies cause redistribution of the receptor into patches that do not appear to be required for vesicle formation.
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    Grey, M.; Dunning, C. J.; Gaspar, R.; Grey, C.; Brundin, P.; Sparr, E.; Linse, S. Acceleration of α-Synuclein Aggregation by Exosomes. J. Biol. Chem. 2015, 290 (5), 2969– 2982,  DOI: 10.1074/jbc.M114.585703
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    Acceleration of α-synuclein aggregation by exosomes
    Grey, Marie; Dunning, Christopher J.; Gaspar, Ricardo; Grey, Carl; Brundin, Patrik; Sparr, Emma; Linse, Sara
    Journal of Biological Chemistry (2015), 290 (5), 2969-2982CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Exosomes are small vesicles released from cells into extracellular space. Here, the authors isolated exosomes from neuroblastoma cells and investigated their influence on the aggregation of α-synuclein, a protein assocd. with Parkinson disease pathol. Using cryo-transmission electron microscopy of exosomes, the authors found spherical unilamellar vesicles with a significant protein content, and Western blot anal. revealed that they contained, as expected, the proteins flotillin-1 and alix. Using thioflavin T fluorescence to monitor aggregation kinetics, the authors found that exosomes catalyzed the process in a similar manner as a low concn. of preformed α-synuclein fibrils. The exosomes reduced the lag time indicating that they provide catalytic environments for nucleation. The catalytic effects of exosomes derived from native cells and cells that overexpressed α-synuclein did not differ. Vesicles prepd. from extd. exosome lipids accelerated aggregation, suggesting that the lipids in exosomes were sufficient for the catalytic effect to arise. Using mass spectrometry, the authors found several phospholipid classes in the exosomes, including phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, and gangliosides GM2 and GM3. Within each class, several species with different acyl chains were identified. The authors then prepd. vesicles from corresponding pure lipids or defined mixts., most of which were found to retard α-synuclein aggregation. As a striking exception, vesicles contg. ganglioside lipids GM1 or GM3 accelerated the process. Understanding how α-synuclein interacts with biol. membranes to promote neurol. disease might lead to the identification of novel therapeutic targets.
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    Calvani, R.; Picca, A.; Guerra, F.; Coelho-Junior, H. J.; Bucci, C.; Marzetti, E. Circulating extracellular vesicles: friends and foes in neurodegeneration. Neural Regener. Res. 2022, 17 (3), 534– 542,  DOI: 10.4103/1673-5374.320972
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    Circulating extracellular vesicles: friends and foes in neurodegeneration
    Picca, Anna; Guerra, Flora; Calvani, Riccardo; Coelho-Junior, Helio Jose; Bucci, Cecilia; Marzetti, Emanuele
    Neural Regeneration Research (2022), 17 (3), 534-542CODEN: NRREBM; ISSN:1673-5374. (Publishing House of Neural Regeneration Research)
    A review. Extracellular vesicles have been identified as pivotal mediators of intercellular communication with crit. roles in physiol. and pathol. conditions. Via this route, several mols. (e.g., nucleic acids, proteins, metabolites) can be transferred to proximal and distant targets to convey specific information. Extracellular vesicle-assocd. cargo mols. have been proposed as markers of several disease conditions for their potential of tracking down the generating cell. Indeed, circulating extracellular vesicles may represent biomarkers of dysfunctional cellular quality control systems esp. in conditions characterized by the accrual of intracellular misfolded proteins. Furthermore, the identification of extracellular vesicles as tools for the delivery of nucleic acids or other cargo mols. to diseased tissues makes these circulating shuttles possible targets for therapeutic development. The increasing interest in the study of extracellular vesicles as biomarkers resides mainly in the fact that the identification of peripheral levels of extracellular vesicle-assocd. proteins might reflect mol. events occurring in hardly accessible tissues, such as the brain, thereby serving as a "brain liq. biopsy". The exploitation of extracellular vesicles for diagnostic and therapeutic purposed might offer unprecedented opportunities to develop personalized approaches. Here, we discuss the bright and dark sides of extracellular vesicles in the setting of two main neurodegenerative diseases (i.e., Parkinson's and Alzheimer's diseases). A special focus will be placed on the possibility of using extracellular vesicles as biomarkers for the two conditions to enable disease tracking and treatment monitoring.
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  24. 24
    Beretta, C.; Nikitidou, E.; Streubel-Gallasch, L.; Ingelsson, M.; Sehlin, D.; Erlandsson, A. Extracellular vesicles from amyloid-β exposed cell cultures induce severe dysfunction in cortical neurons. Sci. Rep. 2020, 10 (1), 19656,  DOI: 10.1038/s41598-020-72355-2
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    Extracellular vesicles from amyloid-beta exposed cell cultures induce severe dysfunction in cortical neurons
    Beretta, Chiara; Nikitidou, Elisabeth; Streubel-Gallasch, Linn; Ingelsson, Martin; Sehlin, Dag; Erlandsson, Anna
    Scientific Reports (2020), 10 (1), 19656CODEN: SRCEC3; ISSN:2045-2322. (Nature Research)
    Alzheimer's disease (AD) is characterized by a substantial loss of neurons and synapses throughout the brain. The exact mechanism behind the neurodegeneration is still unclear, but recent data suggests that spreading of amyloid-beta (Abeta) pathol. via extracellular vesicles (EVs) may contribute to disease progression. We have previously shown that an incomplete degrdn. of Abeta42 protofibrils by astrocytes results in the release of EVs contg. neurotoxic Aβ. Here, we describe the cellular mechanisms behind EV-assocd. neurotoxicity in detail. EVs were isolated from untreated and Abeta42 protofibril exposed neuroglial co-cultures, consisting mainly of astrocytes. The EVs were added to cortical neurons for 2 or 4 days and the neurodegenerative processes were followed with immunocytochem., time-lapse imaging and transmission electron microscopy (TEM). Addn. of EVs from A beta42 protofibril exposed co-cultures resulted in synaptic loss, severe mitochondrial impairment and apoptosis. TEM anal. demonstrated that the EVs induced axonal swelling and vacuolization of the neuronal cell bodies. Interestingly, EV exposed neurons also displayed pathol. lamellar bodies of cholesterol deposits in lysosomal compartments. Taken together, our data show that the secretion of EVs from A beta exposed cells induces neuronal dysfunction in several ways, indicating a central role for EVs in the progression of Aβ-induced pathol.
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  25. 25
    Jiang, L.; Dong, H.; Cao, H.; Ji, X.; Luan, S.; Liu, J. Exosomes in Pathogenesis, Diagnosis, and Treatment of Alzheimer’s Disease. Med. Sci. Monit. 2019, 25, 3329– 3335,  DOI: 10.12659/MSM.914027
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    25
    Exosomes in pathogenesis, diagnosis, and treatment of Alzheimer's disease
    Jiang, Liqun; Dong, Huijie; Cao, Hua; Ji, Xiaofei; Luan, Siyu; Liu, Jing
    Medical Science Monitor (2019), 25 (), 3329-3335CODEN: MSMOFR; ISSN:1643-3750. (International Scientific Information, Inc.)
    Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the accumulation of β-amyloid peptide 1-42 and phosphorylation of tau protein in the brain. Thus far, the transfer mechanism of these cytotoxic proteins between nerve cells remains unclear. Recent studies have shown that nanoscale extracellular vesicles (exosomes) originating from cells may play important roles in this transfer process. In addn., several genetic materials and proteins are also involved in intercellular communication by the secretion of the exosomes. That proposes novel avenues for early diagnosis and biol. treatment in AD, based on exosome detection and intervention. In this review, exosome-related pathways of cytotoxic protein intercellular transfer in AD, and the effect of membrane proteins on exosomes targeting cells are first introduced. The advances in exosome-related biomarker detection in AD are summarized. Finally, the advantages and challenges of reducing cytotoxic protein accumulation via exosomal intervention for AD treatment are discussed. It is envisaged that future research in exosomes may well provide new insights into the pathogenesis, diagnosis, and treatment of AD.
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    Rajendran, L.; Honsho, M.; Zahn, T. R.; Keller, P.; Geiger, K. D.; Verkade, P.; Simons, K. Alzheimer’s disease β-amyloid peptides are released in association with exosomes. Proc. Natl. Acad. Sci. U.S.A. 2006, 103 (30), 11172– 11177,  DOI: 10.1073/pnas.0603838103
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    26
    Alzheimer's disease β-amyloid peptides are released in association with exosomes
    Rajendran, Lawrence; Honsho, Masanori; Zahn, Tobias R.; Keller, Patrick; Geiger, Kethrin D.; Verkade, Paul; Simons, Kai
    Proceedings of the National Academy of Sciences of the United States of America (2006), 103 (30), 11172-11177CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Although the exact etiol. of Alzheimer's disease (AD) is a topic of debate, the consensus is that the accumulation of β-amyloid (AP) peptides in the senile plaques is one of the hallmarks of the progression of the disease. The AO peptide is formed by the amyloidogenic cleavage of the amyloid precursor protein (APP) by β- and γ-secretases. The endocytic system has been implicated in the cleavages leading to the formation of Aβ. However, the identity of the intracellular compartment where the amyloidogenic secretases cleave and the mechanism by which the intracellularly generated Aβ is released into the extracellular milieu are not clear. Here, we show that β-cleavage occurs in early endosomes followed by routing of Aβ to multivesicular bodies (MVBs) in HeLa and N2a cells. Subsequently, a minute fraction of Aβ peptides can be secreted from the cells in assocn. with exosomes, intraluminal vesicles of MVBs that are released into the extracellular space as a result of fusion of MVBs with the plasma membrane. Exosomal proteins were found to accumulate in the plaques of AD patient brains, suggesting a role in the pathogenesis of AD.
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    Soudy, R.; Kimura, R.; Fu, W.; Patel, A.; Jhamandas, J. Extracellular vesicles enriched with amylin receptor are cytoprotective against the Aß toxicity in vitro. PLoS One 2022, 17 (4), e0267164  DOI: 10.1371/journal.pone.0267164
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    27
    Extracellular vesicles enriched with amylin receptor are cytoprotective against the Ass toxicity in vitro
    Soudy, Rania; Kimura, Ryoichi; Fu, Wen; Patel, Aarti; Jhamandas, Jack
    PLoS One (2022), 17 (4), e0267164CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
    Extracellular vesicles (EVs) are double membrane structures released by all cell types with identified roles in the generation, transportation, and degrdn. of amyloid-β protein (Aβ) oligomers in Alzheimer's disease (AD). EVs are thus increasingly recognized to play a neuroprotective role in AD, through their ability to counteract the neurotoxic effects of Aβ, possibly through interactions with specific receptors on cell membranes. Our previous studies have identified the amylin receptor (AMY), particularly AMY3 subtype, as a mediator of the deleterious actions of Aβ in vitro and in vivo exptl. paradigms. In the present study, we demonstrate that AMY3 enriched EVs can bind sol. oligomers of Ass and protect N2a cells against toxic effects of this peptide. The effect was specific to amylin receptor as it was blocked in the presence of amylin receptor antagonist AC253. This notion was supported by reduced Aβ binding to EVs from AMY depleted mice compared to those from wild type (Wt) mice. Finally, application of AMY3, but not Wt derived, EVs to hippocampal brain slices improved Aβ-induced redn. of long-term potentiation, a cellular surrogate of memory. Collectively, our observations support the role of AMY receptors, particularly AMY3, in EVs as a potential therapeutic target for AD.
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    Ribeiro, D.; Horvath, I.; Heath, N.; Hicks, R.; Forslow, A.; Wittung-Stafshede, P. Extracellular vesicles from human pancreatic islets suppress human islet amyloid polypeptide amyloid formation. Proc. Natl. Acad. Sci. U.S.A. 2017, 114 (42), 11127– 11132,  DOI: 10.1073/pnas.1711389114
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    28
    Extracellular vesicles from human pancreatic islets suppress human islet amyloid polypeptide amyloid formation
    Ribeiro, Diana; Horvath, Istvan; Heath, Nikki; Hicks, Ryan; Forsloew, Anna; Wittung-Stafshede, Pernilla
    Proceedings of the National Academy of Sciences of the United States of America (2017), 114 (42), 11127-11132CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Extracellular vesicles (EVs) are small vesicles released by cells to aid cell-cell communication and tissue homeostasis. Human islet amyloid polypeptide (IAPP) is the major component of amyloid deposits found in pancreatic islets of patients with type 2 diabetes (T2D). IAPP is secreted in conjunction with insulin from pancreatic β cells to regulate glucose metab. Here, using a combination of anal. and biophys. methods in vitro, we tested whether EVs isolated from pancreatic islets of healthy patients and patients with T2D modulate IAPP amyloid formation. We discovered that pancreatic EVs from healthy patients reduce IAPP amyloid formation by peptide scavenging, but T2D pancreatic and human serum EVs have no effect. In accordance with these differential effects, the insulin:C-peptide ratio and lipid compn. differ between EVs from healthy pancreas and EVs from T2D pancreas and serum. It appears that healthy pancreatic EVs limit IAPP amyloid formation via direct binding as a tissue-specific control mechanism.
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    Haass, C.; Kaether, C.; Thinakaran, G.; Sisodia, S. Trafficking and proteolytic processing of APP. Cold Spring Harbor Perspect. Med. 2012, 2 (5), a006270,  DOI: 10.1101/cshperspect.a006270
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    Trafficking and proteolytic processing of APP
    Haass, Christian; Kaether, Christoph; Thinakaran, Copal; Sisodia, Sangram
    Cold Spring Harbor Perspectives in Medicine (2012), 2 (5), a006270/1-a006270/25CODEN: CSHPFV; ISSN:2157-1422. (Cold Spring Harbor Laboratory Press)
    A review. Accumulations of insol. deposits of amyloid β-peptide are major pathol. hallmarks of Alzheimer disease. Amyloid β-peptide is derived by sequential proteolytic processing from a large type 1 trans-membrane protein, the β-amyloid precursor protein. The proteolytic enzymes involved in its processing are named secretases. β- And γ-secretase liberate by sequential cleavage the neurotoxic amyloid β-peptide, whereas α-secretase prevents its generation by cleaving within the middle of the amyloid domain. In this chapter we describe the cell biol. and biochem. characteristics of the three secretase activities involved in the proteolytic processing of the precursor protein. In addn. we outline how the precursor protein maturates and traffics through the secretory pathway to reach the subcellular locations where the individual secretases are preferentially active. Furthermore, we illuminate how neuronal activity and mutations which cause familial Alzheimer disease affect amyloid β-peptide generation and therefore disease onset and progression.
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    Haass, C.; Koo, E. H.; Mellon, A.; Hung, A. Y.; Selkoe, D. J. Targeting of cell-surface β-amyloid precursor protein to lysosomes: alternative processing into amyloid-bearing fragments. Nature 1992, 357 (6378), 500– 503,  DOI: 10.1038/357500a0
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    30
    Targeting of cell-surface β-amyloid precursor protein to lysosomes: alternative processing into amyloid-bearing fragments
    Haass, Christian; Koo, Edward H.; Mellon, Angela; Hung, Albert Y.; Selkoe, Dennis J.
    Nature (London, United Kingdom) (1992), 357 (6378), 500-3CODEN: NATUAS; ISSN:0028-0836.
    Progressive cerebral deposition of the amyloid β-peptide is an early and invariant feature of Alzheimer's disease. The β-peptide is released by proteolytic cleavages from the β-amyloid precursor protein (βAPP), a membrane-spanning glycoprotein expressed in most mammalian cells. Normal secretion of βAPP involves a cleavage in the β-peptide region, releasing the sol. extramembranous portion and retaining a 10 kDa C-terminal fragment in the membrane. Because this secretory pathway precludes β-amyloid formation, the authors searched for an alternative proteolytic processing pathway that can generate β-peptide-bearing fragments from full-length βAPP. Incubation of living human endothelial cells with a βAPP antibody revealed reinternalization of mature βAPP from the cell surface and its targeting to endosomes/lysosomes. After cell-surface biotinylation, full-length biotinylated βAPP was recovered inside the cells. Purifn. of lysosomes directly demonstrated the presence of mature βAPP and an extensive array of β-peptide-contg. proteolytic products. The results define a second processing pathway for βAPP and suggest that it may be responsible for generating amyloid-bearing fragments in Alzheimer's disease.
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    Gabrielli, M.; Prada, I.; Joshi, P.; Falcicchia, C.; D’Arrigo, G.; Rutigliano, G.; Battocchio, E.; Zenatelli, R.; Tozzi, F.; Radeghieri, A. Microglial large extracellular vesicles propagate early synaptic dysfunction in Alzheimer’s disease. Brain 2022, 145 (8), 2849– 2868,  DOI: 10.1093/brain/awac083
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    Microglial large extracellular vesicles propagate early synaptic dysfunction in Alzheimer's disease
    Gabrielli Martina; Prada Ilaria; Joshi Pooja; D'Arrigo Giulia; Battocchio Elisabetta; Verderio Claudia; Falcicchia Chiara; Origlia Nicola; Rutigliano Grazia; Rutigliano Grazia; Battocchio Elisabetta; Zenatelli Rossella; Radeghieri Annalisa; Tozzi Francesca; Radeghieri Annalisa; Arancio Ottavio; Arancio Ottavio; Arancio Ottavio
    Brain : a journal of neurology (2022), 145 (8), 2849-2868 ISSN:.
    Synaptic dysfunction is an early mechanism in Alzheimer's disease that involves progressively larger areas of the brain over time. However, how it starts and propagates is unknown. Here we show that amyloid-β released by microglia in association with large extracellular vesicles (Aβ-EVs) alters dendritic spine morphology in vitro, at the site of neuron interaction, and impairs synaptic plasticity both in vitro and in vivo in the entorhinal cortex-dentate gyrus circuitry. One hour after Aβ-EV injection into the mouse entorhinal cortex, long-term potentiation was impaired in the entorhinal cortex but not in the dentate gyrus, its main target region, while 24 h later it was also impaired in the dentate gyrus, revealing a spreading of long-term potentiation deficit between the two regions. Similar results were obtained upon injection of extracellular vesicles carrying Aβ naturally secreted by CHO7PA2 cells, while neither Aβ42 alone nor inflammatory extracellular vesicles devoid of Aβ were able to propagate long-term potentiation impairment. Using optical tweezers combined to time-lapse imaging to study Aβ-EV-neuron interaction, we show that Aβ-EVs move anterogradely at the axon surface and that their motion can be blocked through annexin-V coating. Importantly, when Aβ-EV motility was inhibited, no propagation of long-term potentiation deficit occurred along the entorhinal-hippocampal circuit, implicating large extracellular vesicle motion at the neuron surface in the spreading of long-term potentiation impairment. Our data indicate the involvement of large microglial extracellular vesicles in the rise and propagation of early synaptic dysfunction in Alzheimer's disease and suggest a new mechanism controlling the diffusion of large extracellular vesicles and their pathogenic signals in the brain parenchyma, paving the way for novel therapeutic strategies to delay the disease.
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    Sardar Sinha, M.; Ansell-Schultz, A.; Civitelli, L.; Hildesjo, C.; Larsson, M.; Lannfelt, L.; Ingelsson, M.; Hallbeck, M. Alzheimer’s disease pathology propagation by exosomes containing toxic amyloid-beta oligomers. Acta Neuropathol. 2018, 136 (1), 41– 56,  DOI: 10.1007/s00401-018-1868-1
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    Alzheimer's disease pathology propagation by exosomes containing toxic amyloid-beta oligomers
    Sardar Sinha, Maitrayee; Ansell-Schultz, Anna; Civitelli, Livia; Hildesjoe, Camilla; Larsson, Max; Lannfelt, Lars; Ingelsson, Martin; Hallbeck, Martin
    Acta Neuropathologica (2018), 136 (1), 41-56CODEN: ANPTAL; ISSN:0001-6322. (Springer)
    The gradual deterioration of cognitive functions in Alzheimer's disease is paralleled by a hierarchical progression of amyloid-beta and tau brain pathol. Recent findings indicate that toxic oligomers of amyloid-beta may cause propagation of pathol. in a prion-like manner, although the underlying mechanisms are incompletely understood. Here we show that small extracellular vesicles, exosomes, from Alzheimer patients' brains contain increased levels of amyloid-beta oligomers and can act as vehicles for the neuron-to-neuron transfer of such toxic species in recipient neurons in culture. Moreover, blocking the formation, secretion or uptake of exosomes was found to reduce both the spread of oligomers and the related toxicity. Taken together, our results imply that exosomes are centrally involved in Alzheimer's disease and that they could serve as targets for development of new diagnostic and therapeutic principles.
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    Gustafsson, G.; Loov, C.; Persson, E.; Lazaro, D. F.; Takeda, S.; Bergstrom, J.; Erlandsson, A.; Sehlin, D.; Balaj, L.; Gyorgy, B. Secretion and Uptake of α-Synuclein Via Extracellular Vesicles in Cultured Cells. Cell. Mol. Neurobiol. 2018, 38 (8), 1539– 1550,  DOI: 10.1007/s10571-018-0622-5
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    33
    Secretion and Uptake of α-Synuclein Via Extracellular Vesicles in Cultured Cells
    Gustafsson, Gabriel; Loeoev, Camilla; Persson, Emma; Lazaro, Diana F.; Takeda, Shuko; Bergstroem, Joakim; Erlandsson, Anna; Sehlin, Dag; Balaj, Leonora; Gyoergy, Bence; Hallbeck, Martin; Outeiro, Tiago F.; Breakefield, Xandra O.; Hyman, Bradley T.; Ingelsson, Martin
    Cellular and Molecular Neurobiology (2018), 38 (8), 1539-1550CODEN: CMNEDI; ISSN:0272-4340. (Springer)
    In Parkinson's disease and other Lewy body disorders, the propagation of pathol. has been accredited to the spreading of extracellular α-synuclein (a-syn). Although the pathogenic mechanisms are not fully understood, cell-to-cell transfer of α-syn via exosomes and other extracellular vesicles (EVs) has been reported. Here, we investigated whether altered mol. properties of α-syn can influence the distribution and secretion of α-syn in human neuroblastoma cells. Different a-syn variants, including α-syn:hemi-Venus and disease-causing mutants, were overexpressed and EVs were isolated from the conditioned medium. Of the secreted α-syn, 0.1-2% was assocd. with vesicles. The major part of EV α-syn was attached to the outer membrane of vesicles, whereas a smaller fraction was found in their lumen. For α-syn expressed with N-terminal hemi-Venus, the relative levels assocd. with EVs were higher than for WT α-syn. Moreover, such EV-assocd. α-syn:hemi-Venus species were internalized in recipient cells to a higher degree than the corresponding free-floating forms. Among the disease-causing mutants, A53T α-syn displayed an increased assocn. with EVs. Taken together, our data suggest that α-syn species with presumably lost physiol. functions or altered aggregation properties may shift the cellular processing towards vesicular secretion. Our findings thus lend further support to the tenet that EVs can mediate spreading of harmful a-syn species and thereby contribute to the pathol. in α-synucleinopathies.
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    Lim, C. Z. J.; Zhang, Y.; Chen, Y.; Zhao, H.; Stephenson, M. C.; Ho, N. R. Y.; Chen, Y.; Chung, J.; Reilhac, A.; Loh, T. P. Subtyping of circulating exosome-bound amyloid β reflects brain plaque deposition. Nat. Commun. 2019, 10 (1), 1144,  DOI: 10.1038/s41467-019-09030-2
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    Subtyping of circulating exosome-bound amyloid β reflects brain plaque deposition
    Lim Carine Z J; Zhang Yan; Zhao Haitao; Chen Yuan; Shao Huilin; Lim Carine Z J; Zhang Yan; Zhao Haitao; Ho Nicholas R Y; Chen Yuan; Loh Tze Ping; Shao Huilin; Chen Yu; Chung Jaehoon; Stephenson Mary C; Reilhac Anthonin; Ho Nicholas R Y; Shao Huilin; Loh Tze Ping; Chen Christopher L H; Chen Christopher L H; Shao Huilin
    Nature communications (2019), 10 (1), 1144 ISSN:.
    Despite intense interests in developing blood measurements of Alzheimer's disease (AD), the progress has been confounded by limited sensitivity and poor correlation to brain pathology. Here, we present a dedicated analytical platform for measuring different populations of circulating amyloid β (Aβ) proteins - exosome-bound vs. unbound - directly from blood. The technology, termed amplified plasmonic exosome (APEX), leverages in situ enzymatic conversion of localized optical deposits and double-layered plasmonic nanostructures to enable sensitive, multiplexed population analysis. It demonstrates superior sensitivity (~200 exosomes), and enables diverse target co-localization in exosomes. Employing the platform, we find that prefibrillar Aβ aggregates preferentially bind with exosomes. We thus define a population of Aβ as exosome-bound (Aβ42+ CD63+) and measure its abundance directly from AD and control blood samples. As compared to the unbound or total circulating Aβ, the exosome-bound Aβ measurement could better reflect PET imaging of brain amyloid plaques and differentiate various clinical groups.
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    Yuyama, K.; Igarashi, Y. Exosomes as Carriers of Alzheimer’s Amyloid-ß. Front. Neurosci. 2017, 11, 229,  DOI: 10.3389/fnins.2017.00229
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    Exosomes as Carriers of Alzheimer's Amyloid-ss
    Yuyama Kohei; Igarashi Yasuyuki
    Frontiers in neuroscience (2017), 11 (), 229 ISSN:1662-4548.
    The intracerebral level of the aggregation-prone peptide, amyloid-ss (Ass), is constantly maintained by multiple clearance mechanisms, including several degradation enzymes, and brain efflux. Disruption of the clearance machinery and the resultant Ass accumulation gives rise to neurotoxic assemblies, leading to the pathogenesis of Alzheimer's disease (AD). In addition to the classic mechanisms of Ass clearance, the protein may be processed by secreted vesicles, although this possibility has not been extensively investigated. We showed that neuronal exosomes, a subtype of extracellular nanovesicles, enwrap, or trap Ass and transport it into microglia for degradation. Here, we review Ass sequestration and elimination by exosomes, and discuss how this clearance machinery might contribute to AD pathogenesis and how it might be exploited for effective AD therapy.
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    Bordanaba-Florit, G.; Royo, F.; Kruglik, S. G.; Falcon-Perez, J. M. Using single-vesicle technologies to unravel the heterogeneity of extracellular vesicles. Nat. Protoc. 2021, 16 (7), 3163– 3185,  DOI: 10.1038/s41596-021-00551-z
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    Using single-vesicle technologies to unravel the heterogeneity of extracellular vesicles
    Bordanaba-Florit, Guillermo; Royo, Felix; Kruglik, Sergei G.; Falcon-Perez, Juan M.
    Nature Protocols (2021), 16 (7), 3163-3185CODEN: NPARDW; ISSN:1750-2799. (Nature Portfolio)
    Abstr.: Extracellular vesicles (EVs) are heterogeneous lipid containers with a complex mol. cargo comprising several populations with unique roles in biol. processes. These vesicles are closely assocd. with specific physiol. features, which makes them invaluable in the detection and monitoring of various diseases. EVs play a key role in pathophysiol. processes by actively triggering genetic or metabolic responses. However, the heterogeneity of their structure and compn. hinders their application in medical diagnosis and therapies. This diversity makes it difficult to establish their exact physiol. roles, and the functions and compn. of different EV (sub)populations. Ensemble averaging approaches currently employed for EV characterization, such as western blotting or 'omics' technologies, tend to obscure rather than reveal these heterogeneities. Recent developments in single-vesicle anal. have made it possible to overcome these limitations and have facilitated the development of practical clin. applications. In this review, we discuss the benefits and challenges inherent to the current methods for the anal. of single vesicles and review the contribution of these approaches to the understanding of EV biol. We describe the contributions of these recent technol. advances to the characterization and phenotyping of EVs, examn. of the role of EVs in cell-to-cell communication pathways and the identification and validation of EVs as disease biomarkers. Finally, we discuss the potential of innovative single-vesicle imaging and anal. methodologies using microfluidic devices, which promise to deliver rapid and effective basic and practical applications for minimally invasive prognosis systems.
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    Thery, C.; Witwer, K. W.; Aikawa, E.; Alcaraz, M. J.; Anderson, J. D.; Andriantsitohaina, R.; Antoniou, A.; Arab, T.; Archer, F.; Atkin-Smith, G. K. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J. Extracell. Vesicles 2018, 7 (1), 1535750,  DOI: 10.1080/20013078.2018.1535750
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    Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines
    Thery Clotilde; Lavieu Gregory; Martin-Jaular Lorena; Mathieu Mathilde; Tkach Mercedes; Witwer Kenneth W; Huang Yiyao; Muth Dillon C; Powell Bonita H; Schoyen Tine Hiorth; Zhao Zezhou; Witwer Kenneth W; Datta Chaudhuri Amrita; Aikawa Elena; Aikawa Elena; Alcaraz Maria Jose; Anderson Johnathon D; Andriantsitohaina Ramaroson; Le Lay Soazig; Martinez M Carmen; Antoniou Anna; Antoniou Anna; Arab Tanina; Archer Fabienne; Atkin-Smith Georgia K; Baxter Amy A; Caruso Sarah; Cheng Lesley; Greening David W; Hill Andrew F; Jiang Lanzhou; Mathivanan Suresh; Poon Ivan Kh; Tixeira Rochelle; Ayre D Craig; Ayre D Craig; Bach Jean-Marie; Bosch Steffi; Bachurski Daniel; Baharvand Hossein; Shekari Faezeh; Baharvand Hossein; Balaj Leonora; Baldacchino Shawn; Bauer Natalie N; Bebawy Mary; Beckham Carla; Bedina Zavec Apolonija; Benmoussa Abderrahim; Boilard Eric; Gilbert Caroline; Berardi Anna C; Bergese Paolo; Radeghieri Annalisa; Bergese Paolo; Bergese Paolo; Busatto Sara; Radeghieri Annalisa; Bielska Ewa; Blenkiron Cherie; Bobis-Wozowicz Sylwia; Zuba-Surma Ewa K; Boireau Wilfrid; Elie-Caille Celine; Frelet-Barrand Annie; Bongiovanni Antonella; Borras Francesc E; Gamez-Valero Ana; Borras Francesc E; Borras Francesc E; Boulanger Chantal M; Loyer Xavier; Boulanger Chantal M; Loyer Xavier; Breakefield Xandra; Breglio Andrew M; Breglio Andrew M; Brennan Meadhbh A; Brennan Meadhbh A; Brennan Meadhbh A; Brigstock David R; Brigstock David R; Brisson Alain; Broekman Marike Ld; Broekman Marike Ld; Broekman Marike Ld; Bromberg Jacqueline F; Bromberg Jacqueline F; Bryl-Gorecka Paulina; Buch Shilpa; Hu Guoku; Liao Ke; Buck Amy H; Burger Dylan; Vinas Jose L; Burger Dylan; Vinas Jose L; Burger Dylan; Vinas Jose L; Busatto Sara; Buschmann Dominik; Mussack Veronika; Pfaffl Michael W; Bussolati Benedetta; Buzas Edit I; Buzas Edit I; Forsonits Andras; Hegyesi Hargita; Khamari Delaram; Kovacs Arpad Ferenc; Sodar Barbara W; Visnovitz Tamas; Vukman Krisztina V; Wiener Zoltan; Byrd James Bryan; Camussi Giovanni; Carter David Rf; Pink Ryan C; Chamley Lawrence W; Chang Yu-Ting; Chen Chihchen; Chen Chihchen; Chen Shuai; Chin Andrew R; Di Vizio Dolores; Mariscal Javier; Clayton Aled; Cocks Alex; Webber Jason P; Clerici Stefano P; Cocucci Emanuele; Cocucci Emanuele; Coffey Robert J; Cordeiro-da-Silva Anabela; Couch Yvonne; Coumans Frank Aw; Nieuwland Rienk; Coyle Beth; Jackson Hannah K; Crescitelli Rossella; Lasser Cecilia; Lotvall Jan; Shelke Ganesh Vilas; Criado Miria Ferreira; D'Souza-Schorey Crislyn; Das Saumya; de Candia Paola; De Santana Eliezer F; De Wever Olivier; Dhondt Bert; Hendrix An; Van Deun Jan; De Wever Olivier; Dhondt Bert; Hendrix An; Van Deun Jan; Del Portillo Hernando A; Del Portillo Hernando A; Del Portillo Hernando A; Demaret Tanguy; Lombard Catherine A; Deville Sarah; Deville Sarah; Mertens Inge; Devitt Andrew; Dhondt Bert; Dieterich Lothar C; Dolo Vincenza; Giusti Ilaria; Dominguez Rubio Ana Paula; Dominici Massimo; Dominici Massimo; Dourado Mauricio R; Dourado Mauricio R; Driedonks Tom Ap; Nolte-'t Hoen Esther Nm; Stoorvogel Willem; van der Grein Susanne G; van Herwijnen Martijn Jc; Wauben Marca Hm; Duarte Filipe V; Rodrigues Silvia C; Duncan Heather M; Duncan Heather M; Eichenberger Ramon M; Sotillo Javier; Ekstrom Karin; El Andaloussi Samir; Gorgens Andre; El Andaloussi Samir; Erdbrugger Uta; Musante Luca; Falcon-Perez Juan M; Falcon-Perez Juan M; Fatima Farah; Fish Jason E; Fish Jason E; Gustafson Dakota; Schneider Raphael; Flores-Bellver Miguel; Fricke Fabia; Fricke Fabia; Fuhrmann Gregor; Fuhrmann Gregor; Fuhrmann Gregor; Gabrielsson Susanne; Gamez-Valero Ana; Gardiner Chris; Gartner Kathrin; Gaudin Raphael; Gaudin Raphael; Gho Yong Song; Park Jaesung; Giebel Bernd; Gorgens Andre; Gimona Mario; Rohde Eva; Goberdhan Deborah Ci; Gorgens Andre; Gorski Sharon M; Xu Jing; Gorski Sharon M; Xu Jing; Gross Julia Christina; Linnemannstons Karen; Witte Leonie; Gross Julia Christina; Linnemannstons Karen; Witte Leonie; Gualerzi Alice; Gupta Gopal N; Handberg Aase; Handberg Aase; Haraszti Reka A; Khvorova Anastasia; Harrison Paul; Hochberg Fred H; Nolan John P; Hochberg Fred H; Hoffmann Karl F; Holder Beth; Holder Beth; Holthofer Harry; Hosseinkhani Baharak; Huang Yiyao; Zheng Lei; Huber Veronica; Shahaj Eriomina; Hunt Stuart; Ibrahim Ahmed Gamal-Eldin; Ikezu Tsuneya; Inal Jameel M; Isin Mustafa; Ivanova Alena; Jacobsen Soren; Jacobsen Soren; Jay Steven M; Jayachandran Muthuvel; Jenster Guido; Martens-Uzunova Elena S; Johnson Suzanne M; Jones Jennifer C; Morales-Kastresana Aizea; Welsh Joshua A; Jong Ambrose; Jong Ambrose; Jovanovic-Talisman Tijana; Jung Stephanie; Kalluri Raghu; Kano Shin-Ichi; Kaur Sukhbir; Roberts David D; Kawamura Yumi; Kawamura Yumi; Keller Evan T; Tewari Muneesh; Keller Evan T; Khomyakova Elena; Khomyakova Elena; Kierulf Peter; Kim Kwang Pyo; Kislinger Thomas; Kislinger Thomas; Klingeborn Mikael; Klinke David J 2nd; Klinke David J 2nd; Kornek Miroslaw; Kornek Miroslaw; Kosanovic Maja M; Kramer-Albers Eva-Maria; Krasemann Susanne; Krause Mirja; Kusuma Gina D; Lim Rebecca; Kurochkin Igor V; Kusuma Gina D; Lim Rebecca; Kuypers Soren; Laitinen Saara; Langevin Scott M; Langevin Scott M; Languino Lucia R; Lannigan Joanne; Laurent Louise C; Lazaro-Ibanez Elisa; Shatnyeva Olga; Lee Myung-Shin; Lee Yi Xin Fiona; Lemos Debora S; Lenassi Metka; Leszczynska Aleksandra; Li Isaac Ts; Libregts Sten F; Ligeti Erzsebet; Lorincz Akos M; Lim Sai Kiang; Line Aija; Llorente Alicia; Lorenowicz Magdalena J; Lovett Jason; Myburgh Kathryn H; Lowry Michelle C; O'Driscoll Lorraine; Lu Quan; Lukomska Barbara; Lunavat Taral R; Maas Sybren Ln; Maas Sybren Ln; Malhi Harmeet; Marcilla Antonio; Marcilla Antonio; Mariani Jacopo; Martins Vilma Regina; Maugeri Marco; Nawaz Muhammad; McGinnis Lynda K; McVey Mark J; McVey Mark J; Meckes David G Jr; Meehan Katie L; Mertens Inge; Minciacchi Valentina R; Moller Andreas; Soekmadji Carolina; Moller Jorgensen Malene; Moller Jorgensen Malene; Morhayim Jess; Mullier Francois; Mullier Francois; Muraca Maurizio; Najrana Tanbir; Nazarenko Irina; Nazarenko Irina; Nejsum Peter; Neri Christian; Neri Tommaso; Nimrichter Leonardo; Noren Hooten Nicole; O'Grady Tina; O'Loghlen Ana; Ochiya Takahiro; Olivier Martin; Rak Janusz; Ortiz Alberto; Ortiz Alberto; Ortiz Alberto; Ortiz Luis A; Osteikoetxea Xabier; Ostergaard Ole; Ostergaard Ole; Ostrowski Matias; Pegtel D Michiel; Peinado Hector; Perut Francesca; Phinney Donald G; Pieters Bartijn Ch; Pisetsky David S; Pisetsky David S; Pogge von Strandmann Elke; Polakovicova Iva; Polakovicova Iva; Prada Ilaria; Pulliam Lynn; Raffai Robert L; Pulliam Lynn; Quesenberry Peter; Raffai Robert L; Raimondo Stefania; Rak Janusz; Ramirez Marcel I; Ramirez Marcel I; Raposo Graca; Rayyan Morsi S; Zheutlin Alexander R; Regev-Rudzki Neta; Ricklefs Franz L; Robbins Paul D; Rodrigues Silvia C; Rohde Eva; Rohde Eva; Rome Sophie; Rouschop Kasper Ma; Rughetti Aurelia; Russell Ashley E; Saa Paula; Sahoo Susmita; Salas-Huenuleo Edison; Salas-Huenuleo Edison; Sanchez Catherine; Saugstad Julie A; Saul Meike J; Schiffelers Raymond M; Vader Pieter; Schneider Raphael; Scott Aaron; Sharma Shivani; Sharma Shivani; Sharma Shivani; Shelke Ganesh Vilas; Shetty Ashok K; Shetty Ashok K; Shiba Kiyotaka; Siljander Pia R-M; Siljander Pia R-M; Silva Andreia M; Silva Andreia M; Vasconcelos M Helena; Silva Andreia M; Skowronek Agata; Snyder Orman L 2nd; Soares Rodrigo Pedro; Soekmadji Carolina; Stahl Philip D; Stott Shannon L; Stott Shannon L; Strasser Erwin F; Swift Simon; Tahara Hidetoshi; Tewari Muneesh; Tewari Muneesh; Timms Kate; Tiwari Swasti; Tiwari Swasti; Toh Wei Seong; Tomasini Richard; Torrecilhas Ana Claudia; Tosar Juan Pablo; Tosar Juan Pablo; Toxavidis Vasilis; Urbanelli Lorena; van Balkom Bas Wm; Van Keuren-Jensen Kendall; van Niel Guillaume; Verweij Frederik J; van Royen Martin E; van Wijnen Andre J; Vasconcelos M Helena; Vasconcelos M Helena; Vechetti Ivan J Jr; Veit Tiago D; Vella Laura J; Vella Laura J; Velot Emilie; Vestad Beate; Vestad Beate; Vestad Beate; Wahlgren Jessica; Watson Dionysios C; Watson Dionysios C; Weaver Alissa; Weber Viktoria; Wehman Ann M; Weiss Daniel J; Wendt Sebastian; Wheelock Asa M; Wolfram Joy; Wolfram Joy; Wolfram Joy; Xagorari Angeliki; Xander Patricia; Yan Xiaomei; Yanez-Mo Maria; Yanez-Mo Maria; Yin Hang; Yuana Yuana; Zappulli Valentina; Zarubova Jana; Zarubova Jana; Zarubova Jana; Zekas Vytautas; Zhang Jian-Ye; Zickler Antje M; Zimmermann Pascale; Zimmermann Pascale; Zivkovic Angela M; Zocco Davide
    Journal of extracellular vesicles (2018), 7 (1), 1535750 ISSN:2001-3078.
    The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles ("MISEV") guidelines for the field in 2014. We now update these "MISEV2014" guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.
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    Meisl, G.; Kirkegaard, J. B.; Arosio, P.; Michaels, T. C.; Vendruscolo, M.; Dobson, C. M.; Linse, S.; Knowles, T. P. Molecular mechanisms of protein aggregation from global fitting of kinetic models. Nat. Protoc. 2016, 11 (2), 252– 272,  DOI: 10.1038/nprot.2016.010
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    Molecular mechanisms of protein aggregation from global fitting of kinetic models
    Meisl, Georg; Kirkegaard, Julius B.; Arosio, Paolo; Michaels, Thomas C. T.; Vendruscolo, Michele; Dobson, Christopher M.; Linse, Sara; Knowles, Tuomas P. J.
    Nature Protocols (2016), 11 (2), 252-272CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)
    The elucidation of the mol. mechanisms by which sol. proteins convert into their amyloid forms is a fundamental prerequisite for understanding and controlling disorders that are linked to protein aggregation, such as Alzheimer's and Parkinson's diseases. However, because of the complexity assocd. with aggregation reaction networks, the anal. of kinetic data of protein aggregation to obtain the underlying mechanisms represents a complex task. Here we describe a framework, using quant. kinetic assays and global fitting, to det. and to verify a mol. mechanism for aggregation reactions that is compatible with exptl. kinetic data. We implement this approach in a web-based software, AmyloFit. Our procedure starts from the results of kinetic expts. that measure the concn. of aggregate mass as a function of time. We illustrate the approach with results from the aggregation of the β-amyloid (Aβ) peptides measured using thioflavin T, but the method is suitable for data from any similar kinetic expt. measuring the accumulation of aggregate mass as a function of time; the input data are in the form of a tab-sepd. text file. We also outline general exptl. strategies and practical considerations for obtaining kinetic data of sufficient quality to draw detailed mechanistic conclusions, and the procedure starts with instructions for extensive data quality control. For the core part of the anal., we provide an online platform (http://www.amylofit.ch.cam.ac.uk) that enables robust global anal. of kinetic data without the need for extensive programming or detailed math. knowledge. The software automates repetitive tasks and guides users through the key steps of kinetic anal.: detn. of constraints to be placed on the aggregation mechanism based on the concn. dependence of the aggregation reaction, choosing from several fundamental models describing assembly into linear aggregates and fitting the chosen models using an advanced minimization algorithm to yield the reaction orders and rate consts. Finally, we outline how to use this approach to investigate which targets potential inhibitors of amyloid formation bind to and where in the reaction mechanism they act. The protocol, from processing data to detg. mechanisms, can be completed in <1 d.
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    Spackova, B.; Klein Moberg, H.; Fritzsche, J.; Tenghamn, J.; Sjosten, G.; Sipova-Jungova, H.; Albinsson, D.; Lubart, Q.; van Leeuwen, D.; Westerlund, F. Label-free nanofluidic scattering microscopy of size and mass of single diffusing molecules and nanoparticles. Nat. Methods 2022, 19 (6), 751– 758,  DOI: 10.1038/s41592-022-01491-6
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    Label-free nanofluidic scattering microscopy of size and mass of single diffusing molecules and nanoparticles
    Spackova, Barbora; Klein Moberg, Henrik; Fritzsche, Joachim; Tenghamn, Johan; Sjoesten, Gustaf; Sipova-Jungova, Hana; Albinsson, David; Lubart, Quentin; van Leeuwen, Daniel; Westerlund, Fredrik; Midtvedt, Daniel; Esbjoerner, Elin K.; Kaell, Mikael; Volpe, Giovanni; Langhammer, Christoph
    Nature Methods (2022), 19 (6), 751-758CODEN: NMAEA3; ISSN:1548-7091. (Nature Portfolio)
    Label-free characterization of single biomols. aims to complement fluorescence microscopy in situations where labeling compromises data interpretation, is tech. challenging or even impossible. However, existing methods require the investigated species to bind to a surface to be visible, thereby leaving a large fraction of analytes undetected. Here, we present nanofluidic scattering microscopy (NSM), which overcomes these limitations by enabling label-free, real-time imaging of single biomols. diffusing inside a nanofluidic channel. NSM facilitates accurate detn. of mol. wt. from the measured optical contrast and of the hydrodynamic radius from the measured diffusivity, from which information about the conformational state can be inferred. Furthermore, we demonstrate its applicability to the anal. of a complex biofluid, using conditioned cell culture medium contg. extracellular vesicles as an example. We foresee the application of NSM to monitor conformational changes, aggregation and interactions of single biomols., and to analyze single-cell secretomes.
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    Howard, J.; Browne, J.; Bollard, S.; Peters, S.; Sweeney, C.; Wynne, K.; Potter, S.; McCann, A.; Kelly, P. The protein and miRNA profile of plasma extracellular vesicles (EVs) can distinguish feline mammary adenocarcinoma patients from healthy feline controls. Sci. Rep. 2023, 13 (1), 9178,  DOI: 10.1038/s41598-023-36110-7
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    The protein and miRNA profile of plasma extracellular vesicles (EVs) can distinguish feline mammary adenocarcinoma patients from healthy feline controls
    Howard, Jane; Browne, John; Bollard, Stephanie; Peters, Susan; Sweeney, Ciara; Wynne, Kieran; Potter, Shirley; McCann, Amanda; Kelly, Pamela
    Scientific Reports (2023), 13 (1), 9178CODEN: SRCEC3; ISSN:2045-2322. (Nature Portfolio)
    Feline mammary adenocarcinomas (FMA) are aggressive tumors with metastatic capability and limited treatment options. This study aims to investigate whether miRNAs assocd. with FMA tumors are secreted in extracellular vesicles (EVs) and whether they can potentially be used as a cancer biomarker in EVs from feline plasma. Tumors and matched tumor free margins from 10 felines with FMA were selected. Following a detailed literature search, RT-qPCR analyses of 90 miRNAs identified 8 miRNAs of interest for further investigation. Tumor tissue, margins and plasma were subsequently collected from a further 10 felines with FMA. EVs were isolated from the plasma. RT-qPCR expression analyses of the 8 miRNAs of interest were carried out in tumor tissue, margins, FMA EVs and control EVs. Addnl., proteomic anal. of both control and FMA plasma derived EVs was undertaken. RT-qPCR revealed significantly increased miR-20a and miR-15b in tumors compared to margins. A significant decrease in miR-15b and miR-20a was detected in EVs from FMAs compared to healthy feline EVs. The proteomic content of EVs distinguished FMAs from controls, with the protein targets of miR-20a and miR-15b also displaying lower levels in the EVs from patients with FMA. This study has demonstrated that miRNAs are readily detectable in both the tissue and plasma derived EVs from patients with FMA. These miRNAs and their protein targets are a detectable panel of markers in circulating plasma EVs that may inform future diagnostic tests for FMA in a non-invasive manner. Moreover, the clin. relevance of miR-20a and miR-15b warrants further investigation.
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    Alvarez-Erviti, L.; Seow, Y.; Schapira, A. H.; Gardiner, C.; Sargent, I. L.; Wood, M. J.; Cooper, J. M. Lysosomal dysfunction increases exosome-mediated alpha-synuclein release and transmission. Neurobiol. Dis. 2011, 42 (3), 360– 367,  DOI: 10.1016/j.nbd.2011.01.029
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    Lysosomal dysfunction increases exosome-mediated alpha-synuclein release and transmission
    Alvarez-Erviti, Lydia; Seow, Yiqi; Schapira, Anthony H.; Gardiner, Chris; Sargent, Ian L.; Wood, Matthew J. A.; Cooper, J. Mark
    Neurobiology of Disease (2011), 42 (3), 360-367CODEN: NUDIEM; ISSN:0969-9961. (Elsevier B.V.)
    Alpha-synuclein aggregation plays a central role in Parkinson's disease pathol. Direct transmission of alpha-synuclein from pathol. affected to healthy unaffected neurons may be important in the anatomical spread of the disease through the nervous system. We have demonstrated that exosomes released from alpha-synuclein over-expressing SH-SY5Y cells contained alpha-synuclein and these exosomes were capable of efficiently transferring alpha-synuclein protein to normal SH-SY5Y cells. Moreover, the incubation of cells with ammonium chloride or bafilomycin A1 to produce the lysosomal dysfunction recently reported in Parkinson's disease led to an increase in the release of alpha-synuclein in exosomes and a concomitant increase in alpha-synuclein transmission to recipient cells. This study clearly demonstrates the importance of exosomes in both the release of alpha synuclein and its transmission between cells and suggests that factors assocd. with PD pathol. accelerate this process. These mechanisms may play an important role in PD pathol. and provide a suitable target for therapeutic intervention.
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    Biancalana, M.; Koide, S. Molecular mechanism of Thioflavin-T binding to amyloid fibrils. Biochim. Biophys. Acta 2010, 1804 (7), 1405– 1412,  DOI: 10.1016/j.bbapap.2010.04.001
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    Molecular mechanism of thioflavin-T binding to amyloid fibrils
    Biancalana, Matthew; Koide, Shohei
    Biochimica et Biophysica Acta, Proteins and Proteomics (2010), 1804 (7), 1405-1412CODEN: BBAPBW; ISSN:1570-9639. (Elsevier B. V.)
    A review. Intense efforts to detect, diagnose, and analyze the kinetic and structural properties of amyloid fibrils have generated a powerful toolkit of amyloid-specific mol. probes. Since its 1st description in 1959, the fluorescent dye, thioflavin-T (ThT), has become among the most widely used "gold stds." for selectively staining and identifying amyloid fibrils both in vivo and in vitro. The large enhancement of its fluorescence emission upon its binding to fibrils makes ThT a particularly powerful and convenient tool. Despite its widespread use in clin. and basic science applications, the mol. mechanism for the ability of ThT to recognize diverse types of amyloid fibrils and for the dye's characteristic fluorescence has only begun to be elucidated. Here, the authors review recent progress in the understanding of ThT-fibril interactions at at. resoln. These studies have yielded important insights into amyloid structures and the processes of fibril formation, and they also offer guidance for designing the next generation of amyloid assembly diagnostics, inhibitors, and therapeutics.
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    Sasanian, N.; Bernson, D.; Horvath, I.; Wittung-Stafshede, P.; Esbjörner, E. K. Redox-Dependent Copper Ion Modulation of Amyloid-β (1-42) Aggregation In Vitro. Biomolecules 2020, 10 (6), 924,  DOI: 10.3390/biom10060924
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    Redox-dependent copper ion modulation of amyloid-β (1-42) aggregation in vitro
    Sasanian, Nima; Bernson, David; Horvath, Istvan; Wittung-Stafshede, Pernilla; Esbjoerner, Elin K.
    Biomolecules (2020), 10 (6), 924CODEN: BIOMHC; ISSN:2218-273X. (MDPI AG)
    Plaque deposits composed of amyloid-β (Aβ) fibrils are pathol. hallmarks of Alzheimer's disease (AD). Although copper ion dyshomeostasis is apparent in AD brains and copper ions are found co-deposited with Aβ peptides in patients' plaques, the mol. effects of copper ion interactions and redox-state dependence on Aβ aggregation remain elusive. By combining biophys. and theor. approaches, we here show that Cu2+ (oxidized) and Cu+ (reduced) ions have opposite effects on the assembly kinetics of recombinant Aβ(1-42) into amyloid fibrils in vitro. Cu2+ inhibits both the unseeded and seeded aggregation of Aβ(1-42) at pH 8.0. Using math. models to fit the kinetic data, we find that Cu2+ prevents fibril elongation. The Cu2+-mediated inhibition of Aβ aggregation shows the largest effect around pH 6.0 but is lost at pH 5.0, which corresponds to the pH in lysosomes. In contrast to Cu2+, Cu+ ion binding mildly catalyzes the Aβ(1-42) aggregation via a mechanism that accelerates primary nucleation, possibly via the formation of Cu+-bridged Aβ(1-42) dimers. Taken together, our study emphasizes redox-dependent copper ion effects on Aβ(1-42) aggregation and thereby provides further knowledge of putative copper-dependent mechanisms resulting in AD.
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    Longobardi, A.; Nicsanu, R.; Bellini, S.; Squitti, R.; Catania, M.; Tiraboschi, P.; Saraceno, C.; Ferrari, C.; Zanardini, R.; Binetti, G. Cerebrospinal Fluid EV Concentration and Size Are Altered in Alzheimer’s Disease and Dementia with Lewy Bodies. Cells 2022, 11 (3), 462,  DOI: 10.3390/cells11030462
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    Cerebrospinal Fluid EV Concentration and Size Are Altered in Alzheimer's Disease and Dementia with Lewy Bodies
    Longobardi, Antonio; Nicsanu, Roland; Bellini, Sonia; Squitti, Rosanna; Catania, Marcella; Tiraboschi, Pietro; Saraceno, Claudia; Ferrari, Clarissa; Zanardini, Roberta; Binetti, Giuliano; Di Fede, Giuseppe; Benussi, Luisa; Ghidoni, Roberta
    Cells (2022), 11 (3), 462CODEN: CELLC6; ISSN:2073-4409. (MDPI AG)
    Alzheimer's disease (AD), dementia with Lewy bodies (DLB) and frontotemporal dementia (FTD) represent the three major neurodegenerative dementias characterized by abnormal brain protein accumulation. In this study, we investigated extracellular vesicles (EVs) and neurotrophic factors in the cerebrospinal fluid (CSF) of 120 subjects: 36 with AD, 30 with DLB, 34 with FTD and 20 controls. Specifically, CSF EVs were analyzed by Nanoparticle Tracking Anal. and neurotrophic factors were measured with ELISA. We found higher EV concn. and lower EV size in AD and DLB groups compared to the controls. Classification tree anal. demonstrated EV size as the best parameter able to discriminate the patients from the controls (96.7% vs. 3.3%, resp.). The diagnostic performance of the EV concn./size ratio resulted in a fair discrimination level with an area under the curve of 0.74. Moreover, the EV concn./size ratio was assocd. with the p-Tau181/Aβ42 ratio in AD patients. In addn., we described altered levels of cystatin C and progranulin in the DLB and AD groups. We did not find any correlation between neurotrophic factors and EV parameters. In conclusion, the results of this study suggest a common involvement of the endosomal pathway in neurodegenerative dementias, giving important insight into the mol. mechanisms underlying these pathologies.
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  45. 45
    Pait, M. C.; Kaye, S. D.; Su, Y.; Kumar, A.; Singh, S.; Gironda, S. C.; Vincent, S.; Anwar, M.; Carroll, C. M.; Snipes, J. A. Novel method for collecting hippocampal interstitial fluid extracellular vesicles (EV-ISF) reveals sex-dependent changes in microglial EV proteome in response to Aβ pathology. bioRxiv 2023,  DOI: 10.1101/2023.03.10.532133
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    Limbocker, R.; Chia, S.; Ruggeri, F. S.; Perni, M.; Cascella, R.; Heller, G. T.; Meisl, G.; Mannini, B.; Habchi, J.; Michaels, T. C. T. Trodusquemine enhances Aβ42 aggregation but suppresses its toxicity by displacing oligomers from cell membranes. Nat. Commun. 2019, 10 (1), 225,  DOI: 10.1038/s41467-018-07699-5
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    Trodusquemine enhances Aβ42 aggregation but suppresses its toxicity by displacing oligomers from cell membranes
    Limbocker, Ryan; Chia, Sean; Ruggeri, Francesco S.; Perni, Michele; Cascella, Roberta; Heller, Gabriella T.; Meisl, Georg; Mannini, Benedetta; Habchi, Johnny; Michaels, Thomas C. T.; Challa, Pavan K.; Ahn, Minkoo; Casford, Samuel T.; Fernando, Nilumi; Xu, Catherine K.; Kloss, Nina D.; Cohen, Samuel I. A.; Kumita, Janet R.; Cecchi, Cristina; Zasloff, Michael; Linse, Sara; Knowles, Tuomas P. J.; Chiti, Fabrizio; Vendruscolo, Michele; Dobson, Christopher M.
    Nature Communications (2019), 10 (1), 225CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
    Transient oligomeric species formed during the aggregation process of the 42-residue form of the amyloid-β peptide (Aβ42) are key pathogenic agents in Alzheimer's disease (AD). To investigate the relationship between Aβ42 aggregation and its cytotoxicity and the influence of a potential drug on both phenomena, we have studied the effects of trodusquemine. This aminosterol enhances the rate of aggregation by promoting monomer-dependent secondary nucleation, but significantly reduces the toxicity of the resulting oligomers to neuroblastoma cells by inhibiting their binding to the cellular membranes. When administered to a C. elegans model of AD, we again observe an increase in aggregate formation alongside the suppression of Aβ42-induced toxicity. In addn. to oligomer displacement, the reduced toxicity could also point towards an increased rate of conversion of oligomers to less toxic fibrils. The ability of a small mol. to reduce the toxicity of oligomeric species represents a potential therapeutic strategy against AD.
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    Arosio, P.; Cukalevski, R.; Frohm, B.; Knowles, T. P.; Linse, S. Quantification of the Concentration of Aβ42 Propagons during the Lag Phase by an Amyloid Chain Reaction Assay. J. Am. Chem. Soc. 2014, 136 (1), 219– 225,  DOI: 10.1021/ja408765u
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    Quantification of the Concentration of Aβ42 Propagons during the Lag Phase by an Amyloid Chain Reaction Assay
    Arosio, Paolo; Cukalevski, Risto; Frohm, Birgitta; Knowles, Tuomas P. J.; Linse, Sara
    Journal of the American Chemical Society (2014), 136 (1), 219-225CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    The aggregation of the amyloid beta peptide, Aβ42, implicated in Alzheimer's disease, was characterized by a lag phase followed by a rapid growth phase. Conventional methods to study this reaction are not sensitive to events taking place early in the lag phase promoting the assumption that only monomeric or oligomeric species are present at early stages and that the lag time is defined by the primary nucleation rate only. Here the authors exploit the high sensitivity of chem. chain reactions to the reagent compn. to develop an assay which improves by 2 orders of magnitude the detection limit of conventional bulk techniques and allows the concn. of fibrillar Aβ42 propagons to be detected and quantified even during the lag time. The method relies on the chain reaction multiplication of a small no. of initial fibrils by secondary nucleation on the fibril surface in the presence of monomeric peptides, allowing the quantification of the no. of initial propagons by comparing the multiplication reaction kinetics with controlled seeding data. The quant. results of the chain reaction assay are confirmed by qual. TEM anal. The results demonstrate the nonlinearity of the aggregation process which involves both primary and secondary nucleation events even at the early stages of the reaction during the lag-phase.
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    Meisl, G.; Yang, X.; Hellstrand, E.; Frohm, B.; Kirkegaard, J. B.; Cohen, S. I.; Dobson, C. M.; Linse, S.; Knowles, T. P. Differences in nucleation behavior underlie the contrasting aggregation kinetics of the Aβ40 and Aβ42 peptides. Proc. Natl. Acad. Sci. U.S.A. 2014, 111 (26), 9384– 9389,  DOI: 10.1073/pnas.1401564111
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    48
    Differences in nucleation behavior underlie the contrasting aggregation kinetics of the Aβ40 and Aβ42 peptides
    Meisl, Georg; Yang, Xiaoting; Hellstrand, Erik; Frohm, Birgitta; Kirkegaard, Julius B.; Cohen, Samuel I. A.; Dobson, Christopher M.; Linse, Sara; Knowles, Tuomas P. J.
    Proceedings of the National Academy of Sciences of the United States of America (2014), 111 (26), 9384-9389CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    The two major forms of the amyloid-beta (Aβ) peptide found in plaques in patients suffering from Alzheimer's disease, Aβ40 and Aβ42, only differ by two amino acids in the C-terminal region, yet they display markedly different aggregation behavior. The origins of these differences have remained challenging to connect to specific mol.-level processes underlying the aggregation reaction. In this paper we use a general strategy to apply the conventional workflow of chem. kinetics to the aggregation of the Aβ40 peptide to identify the differences between Aβ40 and Aβ42 in terms of the microscopic determinants of the aggregation reaction. Our results reveal that the major source of aggregates in the case of Aβ40 is a fibril-catalyzed nucleation process, the multistep nature of which is evident through its satn. behavior. Moreover, our results show that the significant differences in the obsd. behavior of the two proteins originate not simply from a uniform increase in all microscopic rates for Aβ42 compared with Aβ40, but rather are due to a shift of more than one order of magnitude in the relative importance of primary nucleation vs. fibril-catalyzed secondary nucleation processes. This anal. sheds light on the microscopic determinants of the aggregation behavior of the principal forms of Aβ and outlines a general approach toward achieving an understanding at the mol. level of the aberrant deposition of insol. peptides in neurodegenerative disorders.
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    Xue, W. F.; Hellewell, A. L.; Gosal, W. S.; Homans, S. W.; Hewitt, E. W.; Radford, S. E. Fibril fragmentation enhances amyloid cytotoxicity. J. Biol. Chem. 2009, 284 (49), 34272– 34282,  DOI: 10.1074/jbc.M109.049809
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    Fibril Fragmentation Enhances Amyloid Cytotoxicity
    Xue, Wei-Feng; Hellewell, Andrew L.; Gosal, Walraj S.; Homans, Steve W.; Hewitt, Eric W.; Radford, Sheena E.
    Journal of Biological Chemistry (2009), 284 (49), 34272-34282CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Fibrils assocd. with amyloid disease are mol. assemblies of key biol. importance, yet how cells respond to the presence of amyloid remains unclear. Cellular responses may not only depend on the chem. compn. or mol. properties of the amyloid fibrils, but their phys. attributes such as length, width, or surface area may also play important roles. Here, we report a systematic investigation of the effect of fragmentation on the structural and biol. properties of amyloid fibrils. In addn. to the expected relationship between fragmentation and the ability to seed, we show a striking finding that fibril length correlates with the ability to disrupt membranes and to reduce cell viability. Thus, despite otherwise unchanged mol. architecture, shorter fibrillar samples show enhanced cytotoxic potential than their longer counterparts. The results highlight the importance of fibril length in amyloid disease, with fragmentation not only providing a mechanism by which fibril load can be rapidly increased but also creating fibrillar species of different dimensions that can endow new or enhanced biol. properties such as amyloid cytotoxicity.
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    Colvin, M. T.; Silvers, R.; Ni, Q. Z.; Can, T. V.; Sergeyev, I.; Rosay, M.; Donovan, K. J.; Michael, B.; Wall, J.; Linse, S. Atomic Resolution Structure of Monomorphic Aβ42 Amyloid Fibrils. J. Am. Chem. Soc. 2016, 138 (30), 9663– 9674,  DOI: 10.1021/jacs.6b05129
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    Atomic Resolution Structure of Monomorphic Aβ42 Amyloid Fibrils
    Colvin, Michael T.; Silvers, Robert; Ni, Qing Zhe; Can, Thach V.; Sergeyev, Ivan; Rosay, Melanie; Donovan, Kevin J.; Michael, Brian; Wall, Joseph; Linse, Sara; Griffin, Robert G.
    Journal of the American Chemical Society (2016), 138 (30), 9663-9674CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Amyloid-β (Aβ) is a 39-42 residue protein produced by the cleavage of the amyloid precursor protein (APP), which subsequently aggregates to form cross-β amyloid fibrils that are a hallmark of Alzheimer's disease (AD). The most prominent forms of Aβ are Aβ1-40 and Aβ1-42, which differ by two amino acids (I and A) at the C-terminus. However, Aβ42 is more neurotoxic and essential to the etiol. of AD. Here, we present an at. resoln. structure of a monomorphic form of AβM01-42 amyloid fibrils derived from over 500 13C-13C, 13C-15N distance and backbone angle structural constraints obtained from high field magic angle spinning NMR spectra. The structure (PDB ID: 5KK3) shows that the fibril core consists of a dimer of Aβ42 mols., each contg. four β-strands in a S-shaped amyloid fold, and arranged in a manner that generates two hydrophobic cores that are capped at the end of the chain by a salt bridge. The outer surface of the monomers presents hydrophilic side chains to the solvent. The interface between the monomers of the dimer shows clear contacts between M35 of one mol. and L17 and Q15 of the second. Intermol. 13C-15N constraints demonstrate that the amyloid fibrils are parallel in register. The RMSD of the backbone structure (Q15-A42) is 0.71 ± 0.12 Å and of all heavy atoms is 1.07 ± 0.08 Å. The structure provides a point of departure for the design of drugs that bind to the fibril surface and therefore interfere with secondary nucleation and for other therapeutic approaches to mitigate Aβ42 aggregation.
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    Lansbury, P. T.; Costa, P. R.; Griffiths, J. M.; Simon, E. J.; Auger, M.; Halverson, K. J.; Kocisko, D. A.; Hendsch, Z. S.; Ashburn, T. T.; Spencer, R. G. Structural model for the β-amyloid fibril based on interstrand alignment of an antiparallel-sheet comprising a C-terminal peptide. Nat. Struct. Biol. 1995, 2 (11), 990– 998,  DOI: 10.1038/nsb1195-990
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    Structural model for the β-amyloid fibril based on interstrand alignment of an antiparallel-sheet comprising a C-terminal peptide
    Lansbury, Peter T., Jr.; Costa, Philip R.; Griffiths, Janet M.; Simon, Eric J.; Auger, Michele; Halverson, Kurt J.; Kocisko, David A.; Hendsch, Zachary S.; Ashburn, Ted T.; et al.
    Nature Structural Biology (1995), 2 (11), 990-8CODEN: NSBIEW; ISSN:1072-8368. (Nature Publishing Co.)
    Amyloids are a class of noncryst., yet ordered, protein aggregates. A new approach was used to provide the initial structural data on an amyloid fibril-comprising a peptide (β34-42) from the C-terminus of the β-amyloid protein-based on measurement of intramol. 13C-13C distances and 13C chem. shifts by solid-state 13C NMR and individual amide absorption frequencies by isotope-edited IR spectroscopy. Intermol. orientation and alignment within the amyloid sheet was detd. by fitting models to obsd. intermol. 13C-13C couplings. Although the structural model the authors present is defined to relatively low resoln., it nevertheless shows a pleated antiparallel β-sheet characterized by a specific intermol. alignment.
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    Meinhardt, J.; Sachse, C.; Hortschansky, P.; Grigorieff, N.; Fandrich, M. Aβ(1-40) Fibril Polymorphism Implies Diverse Interaction Patterns in Amyloid Fibrils. J. Mol. Biol. 2009, 386 (3), 869– 877,  DOI: 10.1016/j.jmb.2008.11.005
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    Aβ(1-40) Fibril Polymorphism Implies Diverse Interaction Patterns in Amyloid Fibrils
    Meinhardt, Jessica; Sachse, Carsten; Hortschansky, Peter; Grigorieff, Nikolaus; Faendrich, Marcus
    Journal of Molecular Biology (2009), 386 (3), 869-877CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
    Amyloid fibrils characterize a diverse group of human diseases that includes Alzheimer's disease, Creutzfeldt-Jakob, and type II diabetes. Alzheimer's amyloid fibrils consist of amyloid-β (Aβ) peptide and occur in a range of structurally different fibril morphologies. The structural characteristics of 12 single Aβ(1-40) amyloid fibrils, all formed under the same soln. conditions, were detd. by electron cryo-microscopy and three-dimensional reconstruction. The majority of analyzed fibrils form a range of morphologies that show almost continuously altering structural properties. The obsd. fibril polymorphism implies that for the same polypeptide sequence, amyloid formation can lead to many different patterns of inter- or intra-residue interactions. This property differs significantly from native, monomeric protein folding reactions that for one protein sequence, produce only one ordered conformation and only one set of inter-residue interactions.
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    Hellstrand, E.; Nowacka, A.; Topgaard, D.; Linse, S.; Sparr, E. Membrane Lipid Co-Aggregation with α-Synuclein Fibrils. PLoS One 2013, 8 (10), e77235  DOI: 10.1371/journal.pone.0077235
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    Membrane lipid co-aggregation with α-synuclein fibrils
    Hellstrand, Erik; Nowacka, Agnieszka; Topgaard, Daniel; Linse, Sara; Sparr, Emma
    PLoS One (2013), 8 (10), e77235CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
    Amyloid deposits from several human diseases have been found to contain membrane lipids. Co-aggregation of lipids and amyloid proteins in amyloid aggregates, and the related extn. of lipids from cellular membranes, can influence structure and function in both the membrane and the formed amyloid deposit. Co-aggregation can therefore have important implications for the pathol. consequences of amyloid formation. Still, very little is known about the mechanism behind co-aggregation and mol. structure in the formed aggregates. To address this, we study in vitro co-aggregation by incubating phospholipid model membranes with the Parkinson's disease-assocd. protein, α-synuclein, in monomeric form. After aggregation, we find spontaneous uptake of phospholipids from anionic model membranes into the amyloid fibrils. Phospholipid quantification, polarization transfer solid-state NMR and cryo-TEM together reveal co-aggregation of phospholipids and α-synuclein in a saturable manner with a strong dependence on lipid compn. At low lipid to protein ratios, there is a close assocn. of phospholipids to the fibril structure, which is apparent from reduced phospholipid mobility and morphol. changes in fibril bundling. At higher lipid to protein ratios, addnl. vesicles adsorb along the fibrils. While interactions between lipids and amyloid-protein are generally discussed within the perspective of different protein species adsorbing to and perturbing the lipid membrane, the current work reveals amyloid formation in the presence of lipids as a co-aggregation process. The interaction leads to the formation of lipid-protein co-aggregates with distinct structure, dynamics and morphol. compared to assemblies formed by either lipid or protein alone.
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    Yuyama, K.; Sun, H.; Mitsutake, S.; Igarashi, Y. Sphingolipid-modulated Exosome Secretion Promotes Clearance of Amyloid-β by Microglia. J. Biol. Chem. 2012, 287 (14), 10977– 10989,  DOI: 10.1074/jbc.M111.324616
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    54
    Sphingolipid-modulated exosome secretion promotes clearance of amyloid-β by microglia
    Yuyama, Kohei; Sun, Hui; Mitsutake, Susumu; Igarashi, Yasuyuki
    Journal of Biological Chemistry (2012), 287 (14), 10977-10989CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Amyloid β-peptide (Aβ), the pathogenic agent of Alzheimer disease, is a physiol. metabolite whose levels are constantly controlled in normal brain. Recent studies have demonstrated that a fraction of extracellular Aβ is assocd. with exosomes, small membrane vesicles of endosomal origin, although the fate of Aβ in assocn. with exosome is largely unknown. In this study, we identified novel roles for neuron-derived exosomes acting on extracellular Aβ, i.e. exosomes drive conformational changes in Aβ to form nontoxic amyloid fibrils and promote uptake of Aβ by microglia. The Aβ internalized together with exosomes was further transported to lysosomes and degraded. We also found that blockade of phosphatidylserine on the surface of exosomes by annexin V not only prevented exosome uptake but also suppressed Aβ incorporation into microglia. In addn., we demonstrated that secretion of neuron-derived exosomes was modulated by the activities of sphingolipid-metabolizing enzymes, including neutral sphingomyelinase 2 (nSMase2) and sphingomyelin synthase 2 (SMS2). In transwell expts., up-regulation of exosome secretion from neuronal cells by treatment with SMS2 siRNA enhanced Aβ uptake into microglial cells and significantly decreased extracellular levels of Aβ. Our findings indicate a novel mechanism responsible for clearance of Aβ through its assocn. with exosomes. The modulation of the vesicle release and/or elimination may alter the risk of AD.
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    Perez-Gonzalez, R.; Gauthier, S. A.; Kumar, A.; Levy, E. The exosome secretory pathway transports amyloid precursor protein carboxyl-terminal fragments from the cell into the brain extracellular space. J. Biol. Chem. 2012, 287 (51), 43108– 43115,  DOI: 10.1074/jbc.M112.404467
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    The exosome secretory pathway transports amyloid precursor protein Carboxyl-terminal fragments from the cell into the brain extracellular space
    Perez-Gonzalez, Rocio; Gauthier, Sebastien A.; Kumar, Asok; Levy, Efrat
    Journal of Biological Chemistry (2012), 287 (51), 43108-43115CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    In vitro studies have shown that neuronal cell cultures secrete exosomes contg. amyloid-β precursor protein (APP) and the APP-processing products, C-terminal fragments (CTFs) and amyloid-β (Aβ). We investigated the secretion of full-length APP (flAPP) and APP CTFs via the exosome secretory pathway in vivo. To this end, we developed a novel protocol designed to isolate exosomes secreted into mouse brain extracellular space. Exosomes with typical morphol. were isolated from freshly removed mouse brains and from frozen mouse and human brain tissues, demonstrating that exosomes can be isolated from post-mortem tissue frozen for long periods of time. flAPP, APP CTFs, and enzymes that cleave both flAPP and APP CTFs were identified in brain exosomes. Although higher levels of both flAPP and APP CTFs were obsd. in exosomes isolated from the brains of transgenic mice overexpressing human APP (Tg2576) compared with wild-type control mice, there was no difference in the no. of secreted brain exosomes. These data indicate that the levels of flAPP and APP CTFs assocd. with exosomes mirror the cellular levels of flAPP and APP CTFs. Interestingly, exosomes isolated from the brains of both Tg2576 and wild-type mice are enriched with APP CTFs relative to flAPP. Thus, we hypothesize that the exosome secretory pathway plays a pleiotropic role in the brain: exosome secretion is beneficial to the cell, acting as a specific releasing system of neurotoxic APP CTFs and Aβ, but the secretion of exosomes enriched with APP CTFs, neurotoxic proteins that are also a source of secreted Aβ, is harmful to the brain.
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    Musteikyte, G.; Jayaram, A. K.; Xu, C. K.; Vendruscolo, M.; Krainer, G.; Knowles, T. P. J. Interactions of α-synuclein oligomers with lipid membranes. Biochim. Biophys. Acta, Biomembr. 2021, 1863 (4), 183536,  DOI: 10.1016/j.bbamem.2020.183536
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    Interactions of α-synuclein oligomers with lipid membranes
    Musteikyte, Greta; Jayaram, Akhila K.; Xu, Catherine K.; Vendruscolo, Michele; Krainer, Georg; Knowles, Tuomas P. J.
    Biochimica et Biophysica Acta, Biomembranes (2021), 1863 (4), 183536CODEN: BBBMBS; ISSN:0005-2736. (Elsevier B.V.)
    A review. Parkinson's disease is an increasingly prevalent and currently incurable neurodegenerative disorder. At the mol. level, this disease is characterized by the formation of aberrant intracellular protein deposits known as Lewy bodies. Oligomeric forms of the protein α-synuclein (αS), which are believed to be both intermediates and byproducts of Lewy body formation, are considered to be the main pathogenic species. Interactions of such oligomers with lipid membranes are increasingly emerging as a major mol. pathway underpinning their toxicity. Here author review recent progress in author understanding of the interactions of αS oligomers with lipid membranes. Author highlight key structural and biophys. features of αS oligomers, the effects of these features on αS oligomer membrane binding properties, and resultant implications for understanding the etiol. of Parkinson's disease. Author discuss mechanistic modes of αS oligomer-lipid membrane interactions and the effects of environmental factors to such modes. Finally, author provide an overview of the current understanding of the main mol. determinants of αS oligomer toxicity in vivo.
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    Skotland, T.; Sagini, K.; Sandvig, K.; Llorente, A. An emerging focus on lipids in extracellular vesicles. Adv. Drug Delivery Rev. 2020, 159, 308– 321,  DOI: 10.1016/j.addr.2020.03.002
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    An emerging focus on lipids in extracellular vesicles
    Skotland, Tore; Sagini, Krizia; Sandvig, Kirsten; Llorente, Alicia
    Advanced Drug Delivery Reviews (2020), 159 (), 308-321CODEN: ADDREP; ISSN:0169-409X. (Elsevier B.V.)
    A review. Extracellular vesicles contain a lipid bilayer membrane that protects the encapsulated material, such as proteins, nucleic acids, lipids and metabolites, from the extracellular environment. These vesicles are released from cells via different mechanisms. During recent years extracellular vesicles have been studied as possible biomarkers for different diseases, as biol. nanoparticles for drug delivery, and in basic studies as a tool to understand the structure of biol. membranes and the mechanisms involved in vesicular trafficking. Lipids are essential mol. components of extracellular vesicles, but at the moment our knowledge about the lipid compn. and the function of lipids in these vesicles is limited. However, the interest of the research community in these mols. is increasing as their role in extracellular vesicles is starting to be acknowledged. In this review, we will present the status of the field and describe what is needed to bring it forward.
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    Skotland, T.; Hessvik, N. P.; Sandvig, K.; Llorente, A. Exosomal lipid composition and the role of ether lipids and phosphoinositides in exosome biology. J. Lipid Res. 2019, 60 (1), 9– 18,  DOI: 10.1194/jlr.R084343
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    Exosomal lipid composition and the role of ether lipids and phosphoinositides in exosome biology
    Skotland, Tore; Hessvik, Nina P.; Sandvig, Kirsten; Llorente, Alicia
    Journal of Lipid Research (2019), 60 (1), 9-18CODEN: JLPRAW; ISSN:0022-2275. (American Society for Biochemistry and Molecular Biology)
    A review. Exosomes are a type of extracellular vesicle released from cells after fusion of multivesicular bodies with the plasma membrane. These vesicles are often enriched in cholesterol, SM, glycosphingolipids, and phosphatidylserine. Lipids not only have a structural role in exosomal membranes but also are essential players in exosome formation and release to the extracellular environment. Our knowledge about the importance of lipids in exosome biol. is increasing due to recent technol. developments in lipidomics and a stronger focus on the biol. functions of these mols. Here, we review the available information about the lipid compn. of exosomes. Special attention is given to ether lipids, a relatively unexplored type of lipids involved in membrane trafficking and abundant in some exosomes. Moreover, we discuss how the lipid compn. of exosome prepns. may provide useful information about their purity. Finally, we discuss the role of phosphoinositides, membrane phospholipids that help to regulate membrane dynamics, in exosome release and how this process may be linked to secretory autophagy. Knowledge about exosome lipid compn. is important to understand the biol. of these vesicles and to investigate possible medical applications.
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    Ghadami, S.; Dellinger, K. The lipid composition of extracellular vesicles: applications in diagnostics and therapeutic delivery. Front. Mol. Biosci. 2023, 10, 1198044,  DOI: 10.3389/fmolb.2023.1198044
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    The lipid composition of extracellular vesicles: applications in diagnostics and therapeutic delivery
    Ghadami, Samaneh; Dellinger, Kristen
    Frontiers in Molecular Biosciences (2023), 10 (), 1198044CODEN: FMBRBS; ISSN:2296-889X. (Frontiers Media S.A.)
    A review. Extracellular vesicles (EVs), including exosomes, with nanoscale sizes, biol. origins, various functions, and unique lipid and protein compns. have been introduced as versatile tools for diagnostic and therapeutic medical applications. Numerous studies have reported the importance of the lipid compn. of EVs and its influence on their mechanism of action. For example, changes in the lipidomic profile of EVs have been shown to influence the progression of various diseases, including ovarian malignancies and prostate cancer. In this review, we endeavored to examine differences in the lipid content of EV membranes derived from different cell types to characterize their capabilities as diagnostic tools and treatments for diseases like cancer and Alzheimer's disease. We addnl. discuss designing functionalized vesicles, whether synthetically by hybrid methods or by changing the lipid compn. of natural EVs. Lastly, we provide an overview of current and potential biomedical applications and perspectives on the future of this growing field.
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  60. 60
    Lindberg, D. J.; Wesen, E.; Bjorkeroth, J.; Rocha, S.; Esbjörner, E. K. Lipid membranes catalyse the fibril formation of the amyloid-β (1–42) peptide through lipid-fibril interactions that reinforce secondary pathways. Biochim. Biophys. Acta, Biomembr. 2017, 1859 (10), 1921– 1929,  DOI: 10.1016/j.bbamem.2017.05.012
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    Lipid membranes catalyze the fibril formation of the amyloid-β(1-42) peptide through lipid-fibril interactions that reinforce secondary pathways
    Lindberg, David J.; Wesen, Emelie; Bjoerkeroth, Johan; Rocha, Sandra; Esbjoerner, Elin K.
    Biochimica et Biophysica Acta, Biomembranes (2017), 1859 (10), 1921-1929CODEN: BBBMBS; ISSN:0005-2736. (Elsevier B.V.)
    Alzheimer's disease is assocd. with the aggregation of amyloid-β (Aβ) peptides into oligomers and fibrils. We have explored how model lipid membranes modulate the rate and mechanisms of Aβ(1-42) self-assembly, in order to shed light on how this pathol. reaction may occur in the lipid-rich environments that the peptide encounters in the brain. Using a combination of in vitro biophys. expts. and theor. approaches, we show that zwitterionic DOPC lipid vesicles accelerate the Aβ(1-42) fibril growth rate by interacting specifically with the growing fibrils. We probed this interaction with help of a purpose-developed FRET assay that monitors the proximity between a fibril-specific dye and fluorescent lipids in the lipid vesicle membrane. To further rationalize these findings, we used math. models to fit the aggregation kinetics of Aβ(1-42) and found that lipid vesicles altered specific mechanistic steps in the aggregation reaction; they augmented monomer-dependent secondary nucleation at the surface of existing fibrils and facilitated monomer-independent catalytic processes consistent with fibril fragmentation. We further showed that DOPC vesicles had no effect on primary nucleation. This finding was consistent with expts. showing that Aβ(1-42) monomers do not directly bind to the lipid bilayer. Taken together, these results show that plain lipid membranes with charge and compn. that is representative of outer cell membranes can significantly augment autocatalytic steps in the self-assembly of Aβ(1-42) into fibrils. This new insight suggests that strategies to reduce fibril-lipid interactions in the brain may have therapeutic value.
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    Habchi, J.; Chia, S.; Galvagnion, C.; Michaels, T. C. T.; Bellaiche, M. M. J.; Ruggeri, F. S.; Sanguanini, M.; Idini, I.; Kumita, J. R.; Sparr, E. Cholesterol catalyses Aβ42 aggregation through a heterogeneous nucleation pathway in the presence of lipid membranes. Nat. Chem. 2018, 10 (6), 673– 683,  DOI: 10.1038/s41557-018-0031-x
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    Cholesterol catalyzes Aβ42 aggregation through a heterogeneous nucleation pathway in the presence of lipid membranes
    Habchi, Johnny; Chia, Sean; Galvagnion, Celine; Michaels, Thomas C. T.; Bellaiche, Mathias M. J.; Ruggeri, Francesco Simone; Sanguanini, Michele; Idini, Ilaria; Kumita, Janet R.; Sparr, Emma; Linse, Sara; Dobson, Christopher M.; Knowles, Tuomas P. J.; Vendruscolo, Michele
    Nature Chemistry (2018), 10 (6), 673-683CODEN: NCAHBB; ISSN:1755-4330. (Nature Research)
    Alzheimer's disease is a neurodegenerative disorder assocd. with the aberrant aggregation of the amyloid-β peptide. Although increasing evidence implicates cholesterol in the pathogenesis of Alzheimer's disease, the detailed mechanistic link between this lipid mol. and the disease process remains to be fully established. To address this problem, we adopted a kinetics-based strategy that revealed a specific catalytic role of cholesterol in the aggregation of Aβ42 (the 42-residue form of the amyloid-β peptide). More specifically, we demonstrated that lipid membranes contg. cholesterol promoted Aβ42 aggregation by enhancing its primary nucleation rate by up to 20-fold through a heterogeneous nucleation pathway. We further showed that this process occurred as a result of cooperativity in the interaction of multiple cholesterol mols. with Aβ42. Thus. these results identify a specific microscopic pathway by which cholesterol dramatically enhances the onset of Aβ42 aggregation, thereby helping rationalize the link between Alzheimer's disease and the impairment of cholesterol homeostasis.
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    Amaro, M.; Sachl, R.; Aydogan, G.; Mikhalyov, I. I.; Vacha, R.; Hof, M. GM1 Ganglioside Inhibits β-Amyloid Oligomerization Induced by Sphingomyelin. Angew. Chem., Int. Ed. Engl. 2016, 55 (32), 9411– 9415,  DOI: 10.1002/anie.201603178
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    GM1 Ganglioside Inhibits β-Amyloid Oligomerization Induced by Sphingomyelin
    Amaro Mariana; Sachl Radek; Aydogan Gokcan; Hof Martin; Mikhalyov Ilya I; Vacha Robert
    Angewandte Chemie (International ed. in English) (2016), 55 (32), 9411-5 ISSN:.
    β-Amyloid (Aβ) oligomers are neurotoxic and implicated in Alzheimer's disease. Neuronal plasma membranes may mediate formation of Aβ oligomers in vivo. Membrane components sphingomyelin and GM1 have been shown to promote aggregation of Aβ; however, these studies were performed under extreme, non-physiological conditions. We demonstrate that physiological levels of GM1 , organized in nanodomains do not seed oligomerization of Aβ40 monomers. We show that sphingomyelin triggers oligomerization of Aβ40 and that GM1 is counteractive thus preventing oligomerization. We propose a molecular explanation that is supported by all-atom molecular dynamics simulations. The preventive role of GM1 in the oligomerization of Aβ40 suggests that decreasing levels of GM1 in the brain, for example, due to aging, could reduce protection against Aβ oligomerization and contribute to the onset of Alzheimer's disease.
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    Sanguanini, M.; Baumann, K. N.; Preet, S.; Chia, S.; Habchi, J.; Knowles, T. P. J.; Vendruscolo, M. Complexity in Lipid Membrane Composition Induces Resilience to Aβ42 Aggregation. ACS Chem. Neurosci. 2020, 11 (9), 1347– 1352,  DOI: 10.1021/acschemneuro.0c00101
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    63
    Complexity in Lipid Membrane Composition Induces Resilience to Aβ42 Aggregation
    Sanguanini, Michele; Baumann, Kevin N.; Preet, Swapan; Chia, Sean; Habchi, Johnny; Knowles, Tuomas P. J.; Vendruscolo, Michele
    ACS Chemical Neuroscience (2020), 11 (9), 1347-1352CODEN: ACNCDM; ISSN:1948-7193. (American Chemical Society)
    The mol. origins of Alzheimer's disease are assocd. with the aggregation of the amyloid-β peptide (Aβ). This process is controlled by a complex cellular homeostasis system, which involves a variety of components, including proteins, metabolites, and lipids. It has been shown in particular that certain components of lipid membranes can speed up Aβ aggregation. This observation prompts the question of whether there are protective cellular mechanisms to counterbalance this effect. Here, to address this issue, we investigate the role of the compn. of lipid membranes in modulating the aggregation process of Aβ. By adopting a chem. kinetics approach, we first identify a panel of lipids that affect the aggregation of the 42-residue form of Aβ (Aβ42), ranging from enhancement to inhibition. We then show that these effects tend to av. out in mixts. of these lipids, as such mixts. buffer extreme aggregation behaviors as the no. of components increases. These results indicate that a degree of quality control on protein aggregation can be achieved through a mechanism by which an increase in the mol. complexity of lipid membranes balances opposite effects and creates resilience to aggregation.
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    Noori, L.; Filip, K.; Nazmara, Z.; Mahakizadeh, S.; Hassanzadeh, G.; Caruso Bavisotto, C.; Bucchieri, F.; Marino Gammazza, A.; Cappello, F.; Wnuk, M. Contribution of Extracellular Vesicles and Molecular Chaperones in Age-Related Neurodegenerative Disorders of the CNS. Int. J. Mol. Sci. 2023, 24 (2), 927,  DOI: 10.3390/ijms24020927
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    Contribution of Extracellular Vesicles and Molecular Chaperones in Age-Related Neurodegenerative Disorders of the CNS
    Noori, Leila; Filip, Kamila; Nazmara, Zohreh; Mahakizadeh, Simin; Hassanzadeh, Gholamreza; Caruso Bavisotto, Celeste; Bucchieri, Fabio; Marino Gammazza, Antonella; Cappello, Francesco; Wnuk, Maciej; Scalia, Federica
    International Journal of Molecular Sciences (2023), 24 (2), 927CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)
    A review. Many neurodegenerative disorders are characterized by the abnormal aggregation of misfolded proteins that form amyloid deposits which possess prion-like behavior such as self-replication, intercellular transmission, and consequent induction of native forms of the same protein in surrounding cells. The distribution of the accumulated proteins and their correlated toxicity seem to be involved in the progression of nervous system degeneration. Mol. chaperones are known to maintain proteostasis, contribute to protein refolding to protect their function, and eliminate fatally misfolded proteins, prohibiting harmful effects. However, chaperone network efficiency declines during aging, prompting the onset and the development of neurol. disorders. Extracellular vesicles (EVs) are tiny membranous structures produced by a wide range of cells under physiol. and pathol. conditions, suggesting their significant role in fundamental processes particularly in cellular communication. They modulate the behavior of nearby and distant cells through their biol. cargo. In the pathol. context, EVs transport disease-causing entities, including prions, α-syn, and tau, helping to spread damage to non-affected areas and accelerating the progression of neurodegeneration. However, EVs are considered effective for delivering therapeutic factors to the nervous system, since they are capable of crossing the blood-brain barrier (BBB) and are involved in the transportation of a variety of cellular entities. Here, we review the neurodegeneration process caused mainly by the inefficiency of chaperone systems as well as EV performance in neuropathies, their potential as diagnostic biomarkers and a promising EV-based therapeutic approach.
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    Evans, C. G.; Wisen, S.; Gestwicki, J. E. Heat Shock Proteins 70 and 90 Inhibit Early Stages of Amyloid β-(1–42) Aggregation in Vitro. J. Biol. Chem. 2006, 281 (44), 33182– 33191,  DOI: 10.1074/jbc.M606192200
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    Heat shock proteins 70 and 90 inhibit early stages of amyloid β-(1-42) aggregation in Vitro
    Evans, Christopher G.; Wisen, Susanne; Gestwicki, Jason E.
    Journal of Biological Chemistry (2006), 281 (44), 33182-33191CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Alzheimer disease is a neurol. disorder that is characterized by the presence of fibrils and oligomers composed of the amyloid β (Aβ) peptide. In models of Alzheimer disease, overexpression of mol. chaperones, specifically heat shock protein 70 (Hsp70), suppresses phenotypes related to Aβ aggregation. These observations led to the hypothesis that chaperones might interact with Aβ and block self-assocn. However, although biochem. evidence to support this model has been collected in other neurodegenerative systems, the interaction between chaperones and Aβ has not been similarly explored. Here, we examine the effects of Hsp70/40 and Hsp90 on Aβ aggregation in vitro. We found that recombinant Hsp70/40 and Hsp90 block Aβ self-assembly and that these chaperones are effective at substoichiometric concns. (∼1:50). The anti-aggregation activity of Hsp70 can be inhibited by a nonhydrolyzable nucleotide analog and encouraged by pharmacol. stimulation of its ATPase activity. Finally, we were interested in discerning what type of amyloid structures can be acted upon by these chaperones. To address this question, we added Hsp70/40 and Hsp90 to pre-formed oligomers and fibrils. Based on thioflavin T reactivity, the combination of Hsp70/40 and Hsp90 caused structural changes in oligomers but had little effect on fibrils. These results suggest that if these chaperones are present in the same cellular compartment in which Aβ is produced, Hsp70/40 and Hsp90 may suppress the early stages of self-assembly. Thus, these results are consistent with a model in which pharmacol. activation of chaperones might have a favorable therapeutic effect on Alzheimer disease.
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    Arosio, P.; Michaels, T. C.; Linse, S.; Mansson, C.; Emanuelsson, C.; Presto, J.; Johansson, J.; Vendruscolo, M.; Dobson, C. M.; Knowles, T. P. Kinetic analysis reveals the diversity of microscopic mechanisms through which molecular chaperones suppress amyloid formation. Nat. Commun. 2016, 7, 10948,  DOI: 10.1038/ncomms10948
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    Kinetic analysis reveals the diversity of microscopic mechanisms through which molecular chaperones suppress amyloid formation
    Arosio, Paolo; Michaels, Thomas C. T.; Linse, Sara; Mansson, Cecilia; Emanuelsson, Cecilia; Presto, Jenny; Johansson, Jan; Vendruscolo, Michele; Dobson, Christopher M.; Knowles, Tuomas P. J.
    Nature Communications (2016), 7 (), 10948CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)
    It is increasingly recognized that mol. chaperones play a key role in modulating the formation of amyloid fibrils, a process assocd. with a wide range of human disorders. Understanding the detailed mechanisms by which they perform this function, however, has been challenging because of the great complexity of the protein aggregation process itself. In this work, we build on a previous kinetic approach and develop a model that considers pairwise interactions between mol. chaperones and different protein species to identify the protein components targeted by the chaperones and the corresponding microscopic reaction steps that are inhibited. We show that these interactions conserve the topol. of the unperturbed reaction network but modify the connectivity wts. between the different microscopic steps. Moreover, by analyzing several protein-mol. chaperone systems, we reveal the striking diversity in the microscopic mechanisms by which mol. chaperones act to suppress amyloid formation.
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    Shammas, S. L.; Waudby, C. A.; Wang, S.; Buell, A. K.; Knowles, T. P.; Ecroyd, H.; Welland, M. E.; Carver, J. A.; Dobson, C. M.; Meehan, S. Binding of the Molecular Chaperone αB-Crystallin to Aβ Amyloid Fibrils Inhibits Fibril Elongation. Biophys. J. 2011, 101 (7), 1681– 1689,  DOI: 10.1016/j.bpj.2011.07.056
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    Binding of the Molecular Chaperone αB-Crystallin to Aβ Amyloid Fibrils Inhibits Fibril Elongation
    Shammas, Sarah L.; Waudby, Christopher A.; Wang, Shuyu; Buell, Alexander K.; Knowles, Tuomas P. J.; Ecroyd, Heath; Welland, Mark E.; Carver, John A.; Dobson, Christopher M.; Meehan, Sarah
    Biophysical Journal (2011), 101 (7), 1681-1689CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
    The mol. chaperone αB-crystallin is a small heat-shock protein that is upregulated in response to a multitude of stress stimuli, and is found colocalized with Aβ amyloid fibrils in the extracellular plaques that are characteristic of Alzheimer's disease. We investigated whether this archetypical small heat-shock protein has the ability to interact with Aβ fibrils in vitro. We find that αB-crystallin binds to wild-type Aβ42 fibrils with micromolar affinity, and also binds to fibrils formed from the E22G Arctic mutation of Aβ42. Immunoelectron microscopy confirms that binding occurs along the entire length and ends of the fibrils. Investigations into the effect of αB-crystallin on the seeded growth of Aβ fibrils, both in soln. and on the surface of a quartz crystal microbalance biosensor, reveal that the binding of αB-crystallin to seed fibrils strongly inhibits their elongation. Because the lag phase in sigmoidal fibril assembly kinetics is dominated by elongation and fragmentation rates, the chaperone mechanism identified here represents a highly effective means to inhibit fibril proliferation. Together with previous observations of αB-crystallin interaction with α-synuclein and insulin fibrils, the results suggest that this mechanism is a generic means of providing mol. chaperone protection against amyloid fibril formation.
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    Abelein, A.; Graslund, A.; Danielsson, J. Zinc as chaperone-mimicking agent for retardation of amyloid β peptide fibril formation. Proc. Natl. Acad. Sci. U.S.A. 2015, 112 (17), 5407– 5412,  DOI: 10.1073/pnas.1421961112
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    Zinc as chaperone-mimicking agent for retardation of amyloid β peptide fibril formation
    Abelein, Axel; Graeslund, Astrid; Danielsson, Jens
    Proceedings of the National Academy of Sciences of the United States of America (2015), 112 (17), 5407-5412CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Metal ions have emerged to play a key role in the aggregation process of a amyloid-β (Aβ) peptide that is closely related to the pathogenesis of Alzheimer's disease. A detailed understanding of the underlying mechanistic process of peptide-metal ion interactions, however, has been challenging to obtain. Here, by applying a combination of NMR relaxation dispersion and fluorescence kinetic methods the authors investigated quant. thermodn. Aβ-Zn2+ binding features as well as how Zn2+ modulates the nucleation mechanism of the aggregation process. The results showed that, under near-physiol. conditions, substoichiometric amts. of Zn2+ effectively retarded the generation of amyloid fibrils. A global kinetic profile anal. revealed that in the absence of Zn2+, Aβ40 aggregation was driven by a monomer-dependent secondary nucleation process in addn. to fibril-end elongation. In the presence of Zn2+, the elongation rate was reduced, resulting in redn. of the aggregation rate, but not a complete inhibition of amyloid formation. The authors showed that Zn2+ transiently bound to residues in the N-terminus of the monomeric peptide. A thermodn. anal. supported a model where the N-terminus is folded around the Zn2+ ion, forming a marginally stable, short-lived folded Aβ40 species. This conformation was highly dynamic and only a few percent of the peptide mols. adopted this structure at any given time point. These findings suggested that the folded Aβ40-Zn2+ complex modulates the fibril ends, where elongation takes place, which efficiently retards fibril formation. In this conceptual framework, the authors propose that Zn2+ adopts the role of a minimal anti-aggregation chaperone for Aβ40.
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    Ghalebani, L.; Wahlstrom, A.; Danielsson, J.; Warmlander, S. K.; Graslund, A. pH-dependence of the specific binding of Cu(II) and Zn(II) ions to the amyloid-β peptide. Biochem. Biophys. Res. Commun. 2012, 421 (3), 554– 560,  DOI: 10.1016/j.bbrc.2012.04.043
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    pH-dependence of the specific binding of Cu(II) and Zn(II) ions to the amyloid-β peptide
    Ghalebani, Leila; Wahlstroem, Anna; Danielsson, Jens; Waermlaender, Sebastian K. T. S.; Graeslund, Astrid
    Biochemical and Biophysical Research Communications (2012), 421 (3), 554-560CODEN: BBRCA9; ISSN:0006-291X. (Elsevier B.V.)
    Metal ions like Cu(II) and Zn(II) are accumulated in Alzheimer's disease amyloid plaques. The amyloid-β (Aβ) peptide involved in the disease interacts with these metal ions at neutral pH via ligands provided by the N-terminal histidines and the N-terminus. The present study uses high-resoln. NMR spectroscopy to monitor the residue-specific interactions of Cu(II) and Zn(II) with 15N- and 13C,15N-labeled Aβ(1-40) peptides at varying pH levels. At pH 7.4 both ions bind to the specific ligands, competing with one another. At pH 5.5 Cu(II) retains its specific histidine ligands, while Zn(II) seems to lack residue-specific interactions. The low pH mimics acidosis which is linked to inflammatory processes in vivo. The results suggest that the cell toxic effects of redox active Cu(II) binding to Aβ may be reversed by the protective activity of non-redox active Zn(II) binding to the same major binding site under non-acidic conditions. Under acidic conditions, the protective effect of Zn(II) may be decreased or changed, since Zn(II) is less able to compete with Cu(II) for the specific binding site on the Aβ peptide under these conditions.
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    Lindberg, D. J.; Wranne, M. S.; Gilbert Gatty, M.; Westerlund, F.; Esbjörner, E. K. Steady-state and time-resolved Thioflavin-T fluorescence can report on morphological differences in amyloid fibrils formed by Aβ(1-40) and Aβ(1-42). Biochem. Biophys. Res. Commun. 2015, 458 (2), 418– 423,  DOI: 10.1016/j.bbrc.2015.01.132
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    Steady-state and time-resolved Thioflavin-T fluorescence can report on morphological differences in amyloid fibrils formed by Aβ(1-40) and Aβ(1-42)
    Lindberg, David J.; Wranne, Moa S.; Gilbert Gatty, Melina; Westerlund, Fredrik; Esbjoerner, Elin K.
    Biochemical and Biophysical Research Communications (2015), 458 (2), 418-423CODEN: BBRCA9; ISSN:0006-291X. (Elsevier B.V.)
    Thioflavin-T (ThT) is one of the most commonly used dyes for amyloid detection, but the origin of its fluorescence enhancement is not fully understood. Herein we have characterised the ThT fluorescence response upon binding to the Aβ(1-40) and Aβ(1-42) variants of the Alzheimer's-related peptide amyloid-β, in order to explore how the photophys. properties of this dye relates to structural and morphol. properties of two amyloid fibril types formed by peptides with a high degree of sequence homol. We show that the steady-state ThT fluorescence is 1.7 times more intense with Aβ(1-40) compared to Aβ(1-42) fibrils in concn. matched samples prepd. under quiescent conditions. By measuring the excited state lifetime of bound ThT, we also demonstrate a distinct difference between the two fibril isoforms, with Aβ(1-42) fibrils producing a longer ThT fluorescence lifetime compared to Aβ(1-40). The substantial steady-state intensity difference is therefore not explained by differences in fluorescence quantum yield. Further, we find that the ThT fluorescence intensity, but not the fluorescence lifetime, is dependent on the fibril prepn. method (quiescent vs. agitated conditions). We therefore propose that the fluorescence lifetime is inherent to each isoform and sensitively reports on fibril microstructure in the protofilament whereas the total fluorescence intensity relates to the amt. of exposed β-sheet in the mature Aβ fibrils and hence to differences in their morphol. Our results highlight the complexity of ThT fluorescence, and demonstrate its extended use in amyloid fibril characterization.
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    Pallbo, J.; Olsson, U.; Sparr, E. Strong inhibition of peptide amyloid formation by a fatty acid. Biophys. J. 2021, 120 (20), 4536– 4546,  DOI: 10.1016/j.bpj.2021.08.035
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    Strong inhibition of peptide amyloid formation by a fatty acid
    Pallbo, Jon; Olsson, Ulf; Sparr, Emma
    Biophysical Journal (2021), 120 (20), 4536-4546CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
    The aggregation of peptides into amyloid fibrils is assocd. with several diseases, including Alzheimer's and Parkinson's disease. Because hydrophobic interactions often play an important role in amyloid formation, the presence of various hydrophobic or amphiphilic mols., such as lipids, may influence the aggregation process. We have studied the effect of a fatty acid, linoleic acid, on the fibrillation process of the amyloid-forming model peptide NACore (GAVVTGVTAVA). NACore is a peptide fragment spanning residue 68-78 of the protein α-synuclein involved in Parkinson's disease. Based primarily on CD measurements, we found that even a very small amt. of linoleic acid can substantially inhibit the fibrillation of NACore. This inhibitory effect manifests itself through a prolongation of the lag phase of the peptide fibrillation. The effect is greatest when the fatty acid is present from the beginning of the process together with the monomeric peptide. Cryogenic transmission electron microscopy revealed the presence of nonfibrillar clusters among NACore fibrils formed in the presence of linoleic acid. We argue that the obsd. inhibitory effect on fibrillation is due to co-assocn. of peptide oligomers and fatty acid aggregates at the early stage of the process. An important aspect of this mechanism is that it is nonmonomeric peptide structures that assoc. with the fatty acid aggregates. Similar mechanisms of action could be relevant in amyloid formation occurring in vivo, where the aggregation takes place in a lipid-rich environment.
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    Sturchio, A.; Dwivedi, A. K.; Young, C. B.; Malm, T.; Marsili, L.; Sharma, J. S.; Mahajan, A.; Hill, E. J.; Andaloussi, S. E.; Poston, K. L. High cerebrospinal amyloid-β 42 is associated with normal cognition in individuals with brain amyloidosis. EClinicalMedicine 2021, 38, 100988,  DOI: 10.1016/j.eclinm.2021.100988
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    High cerebrospinal amyloid-β 42 is associated with normal cognition in individuals with brain amyloidosis
    Sturchio Andrea; Marsili Luca; Sharma Jennifer S; Mahajan Abhimanyu; Hill Emily J; Espay Alberto J; Sturchio Andrea; Dwivedi Alok K; Young Christina B; Poston Kathleen L; Malm Tarja; Andaloussi Samir El; Ezzat Kariem; Manfredsson Fredric P; Schneider Lon S
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  1. Mohsin Shafiq, Andreu Matamoros-Angles, Sussane Caroline Meister, Markus Glatzel. Comment on “Extracellular Vesicles Slow Down Aβ(1–42) Aggregation by Interfering with the Amyloid Fibril Elongation Step”. ACS Chemical Neuroscience 2024, 15 (21) , 3791-3793. https://doi.org/10.1021/acschemneuro.4c00601
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  • Abstract

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    Figure 1

    Figure 1. Size and molecular identity of EVs. (a) Size distributions and particle concentrations of EVs isolated from SH-SY5Y (black) and HEK293-T (blue), as determined by NTA. Mean EV diameters ± standard deviations are given in the legend. (b) Western blots showing the presence of different protein markers in the EV samples and whole cell lysates. Abbreviations: SH = SH-SY5Y and HEK = HEK293-T.

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    Figure 2

    Figure 2. Aβ(1–42) aggregation kinetics in the presence of EVs. (a,b) Change in ThT fluorescence as a function of time, representing the aggregation kinetics of 2 μM Aβ(1–42) into amyloid fibrils in the presence of increasing concentrations of EVs purified from (a) SH-SY5Y and (b) HEK293-T cells. The EV concentrations are given in particles/mL, as indicated by the legend in (a). Three replicate kinetic curves are overlaid for each condition. (c) Change in end-point ThT fluorescence (defined as the mean ThT signal over the final 3 h of the plateau phase, which corresponds to 37 data points) as a function of increasing EV concentration. (d) Reaction half-times and (e) reaction growth-times, extracted from the data in (a,b). The error bars represent standard deviation (n = 3).

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    Figure 3

    Figure 3. Effect of EVs on the seeded aggregation of Aβ(1–42). (a–f) Normalized Aβ(1–42) aggregation kinetic curves showing the effects of EVs in the absence (a,d) and presence (b,c and e,f) of 5 or 25% preformed Aβ(1–42) fibril seeds. Panels (a–c) and (d–f) show data for SH-SY5Y and HEK293-T EVs, respectively. The solid lines were fitted to the data using a multistep secondary nucleation model of amyloid formation setting the rate constant for elongation (k+) as a free parameter as described in the main text. The parameters underlying these fits are given in Tables S4 and S5. (g,h) Reaction half-times as a function of EV and seed concentration, derived from the data in, respectively, (a–c and d–f). The error bars represent the standard deviation (n = 3). (i) Change in the elongation rate constant (k+) as a function of EV concentration, as determined by the fitting of the data in a–f. The elongation rates are reported relative to that of 2 μM Aβ(1–42) aggregating in the absence of EVs.

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    Figure 4

    Figure 4. Morphological characterization of Aβ(1–42) fibrils formed in the absence and presence of EVs. (a–c) AFM images of Aβ(1–42) fibrils formed (a) in phosphate buffer with DPBS (see Methods) and (b,c) in the presence of SH-SY5Y and HEK293-T EVs. Scale bars = 2 μm. (d,e) AFM-based analysis of the distributions of (d) fibril lengths (e) and cross-sectional heights of the Aβ(1–42) fibrils formed in the absence and presence of EVs (n = 100–120 per condition, *** denotes p < 0.001 by one-way ANOVA). (f–k) Cryo-TEM images of Aβ(1–42) fibrils formed in the absence of EVs (f,g) and in the presence of EVs from, respectively, SH-SY5Y(h,i) and HEK293-T (j,k) cells. The Aβ(1–42) fibrils formed in the presence of EVs contained small dark dots, indicated by the white arrows in (i) and (k), suggestive of the dense association of EV components. Scale bars = 250 nm. All analyses have an EV concentration of 7.2 × 109 particles/mL.

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      Lue L F; Kuo Y M; Roher A E; Brachova L; Shen Y; Sue L; Beach T; Kurth J H; Rydel R E; Rogers J
      The American journal of pathology (1999), 155 (3), 853-62 ISSN:0002-9440.
      We have characterized amyloid beta peptide (Abeta) concentration, Abeta deposition, paired helical filament formation, cerebrovascular amyloid angiopathy, apolipoprotein E (ApoE) allotype, and synaptophysin concentration in entorhinal cortex and superior frontal gyrus of normal elderly control (ND) patients, Alzheimer's disease (AD) patients, and high pathology control (HPC) patients who meet pathological criteria for AD but show no synapse loss or overt antemortem symptoms of dementia. The measures of Abeta deposition, Abeta-immunoreactive plaques with and without cores, thioflavin histofluorescent plaques, and concentrations of insoluble Abeta, failed to distinguish HPC from AD patients and were poor correlates of synaptic change. By contrast, concentrations of soluble Abeta clearly distinguished HPC from AD patients and were a strong inverse correlate of synapse loss. Further investigation revealed that Abeta40, whether in soluble or insoluble form, was a particularly useful measure for classifying ND, HPC, and AD patients compared with Abeta42. Abeta40 is known to be elevated in cerebrovascular amyloid deposits, and Abeta40 (but not Abeta42) levels, cerebrovascular amyloid angiopathy, and ApoE4 allele frequency were all highly correlated with each other. Although paired helical filaments in the form of neurofibrillary tangles or a penumbra of neurites surrounding amyloid cores also distinguished HPC from AD patients, they were less robust predictors of synapse change compared with soluble Abeta, particularly soluble Abeta40. Previous experiments attempting to relate Abeta deposition to the neurodegeneration that underlies AD dementia may have failed because they assayed the classical, visible forms of the molecule, insoluble neuropil plaques, rather than the soluble, unseen forms of the molecule.
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    7. 7
      van Dyck, C. H.; Sabbagh, M.; Cohen, S. Lecanemab in Early Alzheimer’s Disease. Reply. N. Engl. J. Med. 2023, 388 (17), 1630– 1632,  DOI: 10.1056/NEJMc2301380
      There is no corresponding record for this reference.
    8. 8
      Zhang, X.; Wesen, E.; Kumar, R.; Bernson, D.; Gallud, A.; Paul, A.; Wittung-Stafshede, P.; Esbjörner, E. K. Correlation between Cellular Uptake and Cytotoxicity of Fragmented α-Synuclein Amyloid Fibrils Suggests Intracellular Basis for Toxicity. ACS Chem. Neurosci. 2020, 11 (3), 233– 241,  DOI: 10.1021/acschemneuro.9b00562
      8
      Correlation between Cellular Uptake and Cytotoxicity of Fragmented α-Synuclein Amyloid Fibrils Suggests Intracellular Basis for Toxicity
      Zhang, Xiaolu; Wesen, Emelie; Kumar, Ranjeet; Bernson, David; Gallud, Audrey; Paul, Alexandra; Wittung-Stafshede, Pernilla; Esbjoerner, Elin K.
      ACS Chemical Neuroscience (2020), 11 (3), 233-241CODEN: ACNCDM; ISSN:1948-7193. (American Chemical Society)
      Aggregation and intracellular deposition of the protein α-synuclein is an underlying characteristic of Parkinson's disease. α-Synuclein assemblies also undergo cell-cell spreading, facilitating propagation of their cellular pathol. Understanding how cellular interactions and uptake of extracellular α-synuclein assemblies depend on their phys. attributes is therefore important. We prepd. fragmented fluorescently labeled α-synuclein amyloid fibrils of different av. lengths (∼80 nm to >1μm) and compared their interactions with SH-SY5Y cells. We report that fibrils of all lengths, but not monomers, bind avidly to the cell surface. Their uptake is inversely dependent on their av. size, occurs via a heparan sulfate dependent endocytic route, and appears to have a size cutoff of ∼400 nm. The uptake of α-synuclein fibrils, but not monomers, correlates with their cytotoxicity as measured by redn. in metabolic activity, strongly suggesting an intracellular basis for α-synuclein fibril toxicity, likely involving endolysosomes.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXitFOmug%253D%253D&md5=16f3be12c04a1dcd45bc5b418aefbfee
    9. 9
      Joshi, P.; Benussi, L.; Furlan, R.; Ghidoni, R.; Verderio, C. Extracellular vesicles in Alzheimer’s disease: friends or foes? Focus on abeta-vesicle interaction. Int. J. Mol. Sci. 2015, 16 (3), 4800– 4813,  DOI: 10.3390/ijms16034800
      9
      Extracellular vesicles in Alzheimer's disease: friends or foes? Focus on Aβ-vesicle interaction
      Joshi, Pooja; Benussi, Luisa; Furlan, Roberto; Ghidoni, Roberta; Verderio, Claudia
      International Journal of Molecular Sciences (2015), 16 (3), 4800-4813CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)
      The intercellular transfer of amyloid-β (Aβ) and tau proteins has received increasing attention in Alzheimer's disease (AD). Among other transfer modes, Aβ and tau dissemination has been suggested to occur through release of Extracellular Vesicles (EVs), which may facilitate delivery of pathogenic proteins over large distances. Recent evidence indicates that EVs carry on their surface, specific mols. which bind to extracellular Aβ, opening the possibility that EVs may also influence Aβ assembly and synaptotoxicity. In this review we focus on studies which investigated the impact of EVs in Aβ-mediated neurodegeneration and showed either detrimental or protective role for EVs in the pathol.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXlvVKrt7g%253D&md5=8fbb46bae29b650902f3fa5323ce3dc9
    10. 10
      Jarrett, J. T.; Berger, E. P.; Lansbury, P. T. The carboxy terminus of the .beta. amyloid protein is critical for the seeding of amyloid formation: Implications for the pathogenesis of Alzheimer’s disease. Biochemistry 1993, 32 (18), 4693– 4697,  DOI: 10.1021/bi00069a001
      10
      The carboxy terminus of the β amyloid protein is critical for the seeding of amyloid formation: Implications for the pathogenesis of Alzheimer's disease
      Jarrett, Joseph T.; Berger, Elizabeth P.; Lansbury, Peter T., Jr.
      Biochemistry (1993), 32 (18), 4693-7CODEN: BICHAW; ISSN:0006-2960.
      Several variants of the β amyloid protein, differing only at their carboxy terminus (β1-39, β1-40, β1-42, and β1-43), have been identified as the major components of the cerebral amyloid deposits which are characteristic of Alzheimer's disease. Kinetic studies of aggregation by three naturally occurring β protein variants (β1-39, β1-40, β1-42) and four model peptides (β26-39, β26-40, β26-42, β26-43) demonstrate that amyloid formation, like crystn., is a nucleation-dependent phenomenon. This discovery has practical consequences for studies of the β amyloid protein. The length of the C-terminus is a crit. determinant of the rate of amyloid formation ("kinetic soly.") but has only a minor effect on the thermodn. soly. Amyloid formation by the kinetically sol. peptides (e.g., β1-39, β1-40, β26-39, β26-40) can be nucleated, or "seeded", by peptides which include the crit. C-terminal residues (β1-42, β26-42, β26-43, β34-42). These results suggest that nucleation may be the rate-detg. step of in vivo amyloidogenesis and that β1-42 and/or β1-43, rather than β1-40, may be the pathogenic protein(s) in AD.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3sXksVKktL0%253D&md5=30847ab3946acd7c0d3f8816e3a1be8a
    11. 11
      Jan, A.; Adolfsson, O.; Allaman, I.; Buccarello, A. L.; Magistretti, P. J.; Pfeifer, A.; Muhs, A.; Lashuel, H. A. Aβ42 Neurotoxicity Is Mediated by Ongoing Nucleated Polymerization Process Rather than by Discrete Aβ42 Species. J. Biol. Chem. 2011, 286 (10), 8585– 8596,  DOI: 10.1074/jbc.M110.172411
      11
      Aβ42 Neurotoxicity is Mediated by Ongoing Nucleated Polymerization Process Rather than by Discrete Aβ42 Species
      Jan, Asad; Adolfsson, Oskar; Allaman, Igor; Buccarello, Anna-Lucia; Magistretti, Pierre J.; Pfeifer, Andrea; Muhs, Andreas; Lashuel, Hilal A.
      Journal of Biological Chemistry (2011), 286 (10), 8585-8596CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      The identification of toxic Aβ species and/or the process of their formation is crucial for understanding the mechanism(s) of Aβ neurotoxicity in Alzheimer disease and also for the development of effective diagnostic and therapeutic interventions. To elucidate the structural basis of Aβ toxicity, we developed different procedures to isolate Aβ species of defined size and morphol. distribution, and we investigated their toxicity in different cell lines and primary neurons. We obsd. that crude Aβ42 prepns., contg. a monomeric and heterogeneous mixt. of Aβ42 oligomers, were more toxic than purified monomeric, protofibrillar fractions, or fibrils. The toxicity of protofibrils was directly linked to their interactions with monomeric Aβ42 and strongly dependent on their ability to convert into amyloid fibrils. Subfractionation of protofibrils diminished their fibrillization and toxicity, whereas reintroduction of monomeric Aβ42 into purified protofibril fractions restored amyloid formation and enhanced their toxicity. Selective removal of monomeric Aβ42 from these prepns., using insulin-degrading enzyme, reversed the toxicity of Aβ42 protofibrils. Together, our findings demonstrate that Aβ42 toxicity is not linked to specific prefibrillar aggregate(s) but rather to the ability of these species to grow and undergo fibril formation, which depends on the presence of monomeric Aβ42. These findings contribute significantly to the understanding of amyloid formation and toxicity in Alzheimer disease, provide novel insight into mechanisms of Aβ protofibril toxicity, and important implications for designing anti-amyloid therapies.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXislylu7s%253D&md5=7f89314657c353a8707d35d0153a9581
    12. 12
      Cohen, S. I.; Linse, S.; Luheshi, L. M.; Hellstrand, E.; White, D. A.; Rajah, L.; Otzen, D. E.; Vendruscolo, M.; Dobson, C. M.; Knowles, T. P. Proliferation of amyloid-β42 aggregates occurs through a secondary nucleation mechanism. Proc. Natl. Acad. Sci. U.S.A. 2013, 110 (24), 9758– 9763,  DOI: 10.1073/pnas.1218402110
      12
      Proliferation of amyloid-β42 aggregates occurs through a secondary nucleation mechanism
      Cohen, Samuel I. A.; Linse, Sara; Luheshi, Leila M.; Hellstrand, Erik; White, Duncan A.; Rajah, Luke; Otzen, Daniel E.; Vendruscolo, Michele; Dobson, Christopher M.; Knowles, Tuomas P. J.
      Proceedings of the National Academy of Sciences of the United States of America (2013), 110 (24), 9758-9763, S9758/1-S9758/11CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      The generation of toxic oligomers during the aggregation of the amyloid-β (Aβ) peptide Aβ42 into amyloid fibrils and plaques has emerged as a central feature of the onset and progression of Alzheimer's disease, but the mol. pathways that control pathol. aggregation have proved challenging to identify. Here, the authors used a combination of kinetic studies, selective radiolabeling expts., and cell viability assays to detect directly the rates of formation of both fibrils and oligomers and the resulting cytotoxic effects. The results showed that once a small but crit. concn. of amyloid fibrils had accumulated, the toxic oligomeric species were predominantly formed from monomeric peptide mols. through a fibril-catalyzed secondary nucleation reaction, rather than through a classical mechanism of homogeneous primary nucleation. This catalytic mechanism coupled together the growth of insol. amyloid fibrils and the generation of diffusible oligomeric aggregates that are implicated as neurotoxic agents in Alzheimer's disease. These results revealed that the aggregation of Aβ42 is promoted by a pos. feedback loop that originates from the interactions between the monomeric and fibrillar forms of this peptide. These findings bring together the main mol. species implicated in the Aβ aggregation cascade and suggest that perturbation of the secondary nucleation pathway identified in this study could be an effective strategy to control the proliferation of neurotoxic Aβ42 oligomers.
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    13. 13
      Meisl, G.; Yang, X.; Frohm, B.; Knowles, T. P.; Linse, S. Quantitative analysis of intrinsic and extrinsic factors in the aggregation mechanism of Alzheimer-associated Aβ-peptide. Sci. Rep. 2016, 6, 18728,  DOI: 10.1038/srep18728
      13
      Quantitative analysis of intrinsic and extrinsic factors in the aggregation mechanism of Alzheimer-associated Aβ-peptide
      Meisl, Georg; Yang, Xiaoting; Frohm, Birgitta; Knowles, Tuomas P. J.; Linse, Sara
      Scientific Reports (2016), 6 (), 18728CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)
      Disease related mutations and environmental factors are key determinants of the aggregation mechanism of the amyloid-β peptide implicated in Alzheimer's disease. Here we present an approach to investigate these factors through acquisition of highly reproducible data and global kinetic anal. to det. the mechanistic influence of intrinsic and extrinsic factors on the Aβ aggregation network. This allows us to translate the shift in macroscopic aggregation behavior into effects on the individual underlying microscopic steps. We apply this work-flow to the disease-assocd. Aβ42-A2V variant, and to a variation in pH as examples of an intrinsic and an extrinsic perturbation. In both cases, our data reveal a shift towards a mechanism in which a larger fraction of the reactive flux goes via a pathway that generates potentially toxic oligomeric species in a fibril-catalyzed reaction. This is in agreement with the finding that Aβ42-A2V leads to early-onset Alzheimer's disease and enhances neurotoxicity.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xos1CisA%253D%253D&md5=27cfdd4cbb3b8cfcff1dab473078c5c4
    14. 14
      Bolognesi, B.; Cohen, S. I.; Aran Terol, P.; Esbjorner, E. K.; Giorgetti, S.; Mossuto, M. F.; Natalello, A.; Brorsson, A. C.; Knowles, T. P.; Dobson, C. M. Single Point Mutations Induce a Switch in the Molecular Mechanism of the Aggregation of the Alzheimer’s Disease Associated Aβ42 Peptide. ACS Chem. Biol. 2014, 9 (2), 378– 382,  DOI: 10.1021/cb400616y
      14
      Single point mutations induce a switch in the molecular mechanism of the aggregation of the Alzheimer's disease associated Aβ42 peptide
      Bolognesi, Benedetta; Cohen, Samuel I. A.; Aran Terol, Pablo; Esbjorner, Elin K.; Giorgetti, Sofia; Mossuto, Maria F.; Natalello, Antonino; Brorsson, Ann-Christin; Knowles, Tuomas P. J.; Dobson, Christopher M.; Luheshi, Leila M.
      ACS Chemical Biology (2014), 9 (2), 378-382CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
      Single point mutations in the Alzheimer's disease assocd. Aβ42 peptide are found to alter significantly its neurotoxic properties in vivo and have been assocd. with early onset forms of this devastating condition. We show that such mutations can induce structural changes in Aβ42 fibrils and are assocd. with a dramatic switch in the fibril-dependent mechanism by which Aβ42 aggregates. These observations reveal how subtle perturbations to the physicochem. properties of the Aβ peptide, and the structural properties of fibrils that it forms, can have profound effects on the mechanism of its aggregation and pathogenicity.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhslersrvP&md5=4d97eed88020ffb3ddecca43979c8017
    15. 15
      Cohen, S. I. A.; Arosio, P.; Presto, J.; Kurudenkandy, F. R.; Biverstal, H.; Dolfe, L.; Dunning, C.; Yang, X.; Frohm, B.; Vendruscolo, M. A molecular chaperone breaks the catalytic cycle that generates toxic Aβ oligomers. Nat. Struct. Mol. Biol. 2015, 22 (3), 207– 213,  DOI: 10.1038/nsmb.2971
      15
      A molecular chaperone breaks the catalytic cycle that generates toxic Aβ oligomers
      Cohen, Samuel I. A.; Arosio, Paolo; Presto, Jenny; Kurudenkandy, Firoz Roshan; Biverstal, Henrik; Dolfe, Lisa; Dunning, Christopher; Yang, Xiaoting; Frohm, Birgitta; Vendruscolo, Michele; Johansson, Jan; Dobson, Christopher M.; Fisahn, Andre; Knowles, Tuomas P. J.; Linse, Sara
      Nature Structural & Molecular Biology (2015), 22 (3), 207-213CODEN: NSMBCU; ISSN:1545-9993. (Nature Publishing Group)
      Alzheimer's disease is an increasingly prevalent neurodegenerative disorder whose pathogenesis has been assocd. with aggregation of the amyloid-β peptide (Aβ42). Recent studies have revealed that once Aβ42 fibrils are generated, their surfaces effectively catalyze the formation of neurotoxic oligomers. Here we show that a mol. chaperone, a human Brichos domain, can specifically inhibit this catalytic cycle and limit human Aβ42 toxicity. We demonstrate in vitro that Brichos achieves this inhibition by binding to the surfaces of fibrils, thereby redirecting the aggregation reaction to a pathway that involves minimal formation of toxic oligomeric intermediates. We verify that this mechanism occurs in living mouse brain tissue by cytotoxicity and electrophysiol. expts. These results reveal that mol. chaperones can help maintain protein homeostasis by selectively suppressing crit. microscopic steps within the complex reaction pathways responsible for the toxic effects of protein misfolding and aggregation.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXivVSitLY%253D&md5=03c46f97e7f2361193667db1d4b94f07
    16. 16
      Munke, A.; Persson, J.; Weiffert, T.; De Genst, E.; Meisl, G.; Arosio, P.; Carnerup, A.; Dobson, C. M.; Vendruscolo, M.; Knowles, T. P. J. Phage display and kinetic selection of antibodies that specifically inhibit amyloid self-replication. Proc. Natl. Acad. Sci. U.S.A. 2017, 114 (25), 6444– 6449,  DOI: 10.1073/pnas.1700407114
      16
      Phage display and kinetic selection of antibodies that specifically inhibit amyloid self-replication
      Munke, Anna; Persson, Jonas; Weiffert, Tanja; De Genst, Erwin; Meisl, Georg; Arosio, Paolo; Carnerup, Anna; Dobson, Christopher M.; Vendruscolo, Michele; Knowles, Tuomas P. J.; Linse, Sara
      Proceedings of the National Academy of Sciences of the United States of America (2017), 114 (25), 6444-6449CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      The aggregation of the amyloid β peptide (Aβ) into amyloid fibrils is a defining characteristic of Alzheimer's disease. Because of the complexity of this aggregation process, effective therapeutic inhibitors will need to target the specific microscopic steps that lead to the prodn. of neurotoxic species. We introduce a strategy for generating fibril-specific antibodies that selectively suppress fibril-dependent secondary nucleation of the 42-residue form of Aβ (Aβ42). We target this step because it has been shown to produce the majority of neurotoxic species during aggregation of Aβ42. Starting from large phage display libraries of single-chain antibody fragments (scFvs), the three-stage approach that we describe includes (1) selection of scFvs with high affinity for Aβ42 fibrils after removal of scFvs that bind Aβ42 in its monomeric form; (2) ranking, by surface plasmon resonance affinity measurements, of the resulting candidate scFvs that bind to the Aβ42 fibrils; and (3) kinetic screening and anal. to find the scFvs that inhibit selectively the fibril-catalyzed secondary nucleation process in Aβ42 aggregation. By applying this approach, we have identified four scFvs that inhibit specifically the fibril-dependent secondary nucleation process. Our method also makes it possible to discard antibodies that inhibit elongation, an important factor because the suppression of elongation does not target directly the prodn. of toxic oligomers and may even lead to its increase. On the basis of our results, we suggest that the method described here could form the basis for rationally designed immunotherapy strategies to combat Alzheimer's and related neurodegenerative diseases.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXpt1Onsbc%253D&md5=d74bbc04df48705d2c989dbf6ef4b5b4
    17. 17
      Zhang, T.; Ma, S.; Lv, J.; Wang, X.; Afewerky, H. K.; Li, H.; Lu, Y. The emerging role of exosomes in Alzheimer’s disease. Ageing Res. Rev. 2021, 68, 101321,  DOI: 10.1016/j.arr.2021.101321
      17
      The emerging role of exosomes in Alzheimer's disease
      Zhang, Tongmei; Ma, Sehui; Lv, Junkai; Wang, Xinyuan; Afewerky, Henok Kessete; Li, Hao; Lu, Youming
      Ageing Research Reviews (2021), 68 (), 101321CODEN: ARRGAK; ISSN:1568-1637. (Elsevier B.V.)
      A review. Alzheimer's disease (AD), manifested by memory loss and a decline in cognitive functions, is the most prevalent neurodegenerative disease accounting for 60-80% of dementia cases. But, to-date, there is no effective treatment available to slow or stop the progression of AD. Exosomes are small extracellular vesicles that carry constituents, such as functional mRNAs, non-coding RNAs, proteins, lipids, DNA, and other bioactive substances of their source cells. In the brain, exosomes are likely to be sourced by almost all cell types and involve in cell communication to regulate cellular functions. The yet, accumulated evidence on the roles of exosomes and their constituents in the AD pathol. process suggests their significance as addnl. biomarkers and therapeutic targets for AD. This summarizes the current reported research findings on exosomes roles in the pathogenesis, diagnosis, and treatment of AD.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhsVSmsL3P&md5=e07e92517a7e52490ab735e4939564bd
    18. 18
      Kalluri, R.; LeBleu, V. S. The biology, function, and biomedical applications of exosomes. Science 2020, 367 (6478), eaau6977  DOI: 10.1126/science.aau6977
      There is no corresponding record for this reference.
    19. 19
      Ramos-Zaldivar, H. M.; Polakovicova, I.; Salas-Huenuleo, E.; Corvalan, A. H.; Kogan, M. J.; Yefi, C. P.; Andia, M. E. Extracellular vesicles through the blood-brain barrier: a review. Fluids Barriers CNS 2022, 19 (1), 60,  DOI: 10.1186/s12987-022-00359-3
      19
      Extracellular vesicles through the blood-brain barrier: a review
      Ramos-Zaldivar Hector M; Polakovicova Iva; Corvalan Alejandro H; Kogan Marcelo J; Polakovicova Iva; Corvalan Alejandro H; Salas-Huenuleo Edison; Kogan Marcelo J; Yefi Claudia P; Andia Marcelo E; Andia Marcelo E
      Fluids and barriers of the CNS (2022), 19 (1), 60 ISSN:.
      Extracellular vesicles (EVs) are particles naturally released from cells that are delimited by a lipid bilayer and are unable to replicate. How the EVs cross the Blood-Brain barrier (BBB) in a bidirectional manner between the bloodstream and brain parenchyma remains poorly understood. Most in vitro models that have evaluated this event have relied on monolayer transwell or microfluidic organ-on-a-chip techniques that do not account for the combined effect of all cellular layers that constitute the BBB at different sites of the Central Nervous System. There has not been direct transcytosis visualization through the BBB in mammals in vivo, and evidence comes from in vivo experiments in zebrafish. Literature is scarce on this topic, and techniques describing the mechanisms of EVs motion through the BBB are inconsistent. This review will focus on in vitro and in vivo methodologies used to evaluate EVs transcytosis, how EVs overcome this fundamental structure, and discuss potential methodological approaches for future analyses to clarify these issues. Understanding how EVs cross the BBB will be essential for their future use as vehicles in pharmacology and therapeutics.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB2MbivF2mug%253D%253D&md5=b65bb18442c771c1126be803b9661a32
    20. 20
      Colombo, M.; Raposo, G.; Thery, C. Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles. Annu. Rev. Cell Dev. Biol. 2014, 30, 255– 289,  DOI: 10.1146/annurev-cellbio-101512-122326
      20
      Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles
      Colombo, Marina; Raposo, Graca; Thery, Clotilde
      Annual Review of Cell and Developmental Biology (2014), 30 (), 255-289CODEN: ARDBF8; ISSN:1081-0706. (Annual Reviews)
      A review. In the 1980s, exosomes were described as vesicles of endosomal origin secreted from reticulocytes. Interest increased around these extracellular vesicles, as they appeared to participate in several cellular processes. Exosomes bear proteins, lipids, and RNAs, mediating intercellular communication between different cell types in the body, and thus affecting normal and pathol. conditions. Only recently, scientists acknowledged the difficulty of sepg. exosomes from other types of extracellular vesicles, which precludes a clear attribution of a particular function to the different types of secreted vesicles. To shed light into this complex but expanding field of science, this review focuses on the definition of exosomes and other secreted extracellular vesicles. Their biogenesis, their secretion, and their subsequent fate are discussed, as their functions rely on these important processes.
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    21. 21
      Pan, B. T.; Johnstone, R. M. Fate of the transferrin receptor during maturation of sheep reticulocytes in vitro: selective externalization of the receptor. Cell 1983, 33 (3), 967– 978,  DOI: 10.1016/0092-8674(83)90040-5
      21
      Fate of the transferrin receptor during maturation of sheep reticulocytes in vitro: selective externalization of the receptor
      Pan, Bin Tao; Johnstone, Rose M.
      Cell (Cambridge, MA, United States) (1983), 33 (3), 967-78CODEN: CELLB5; ISSN:0092-8674.
      The fate of the transferrin receptor during in vitro maturation of sheep reticulocytes was followed using FITC- and 125I-labeled antitransferrin-receptor antibodies. Vesicles contg. peptides that comigrate with the transferrin receptor on polyacrylamide gels are released during incubation of sheep reticulocytes, tagged with antitransferrin-receptor antibodies. Vesicle formation does not require the presence of the antitransferrin-receptor antibodies. Using 125I-surface-labeled reticulocytes, it can be shown that the 125I-labeled material which is released is retained by an immunoaffinity column of the antitransferrin-receptor antibody. With reticulocytes tagged with 125I-labeled antitransferrin-receptor antibodies to follow the formation of vesicles, it can be shown that at 0° or in phosphate-buffered saline the rate of vesicle release is less than that at 37° in culture medium. There is selective externalization of the antibody-receptor complex, since few other membrane proteins are found in the externalized vesicles. The antitransferrin-receptor antibodies cause redistribution of the receptor into patches that do not appear to be required for vesicle formation.
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    22. 22
      Grey, M.; Dunning, C. J.; Gaspar, R.; Grey, C.; Brundin, P.; Sparr, E.; Linse, S. Acceleration of α-Synuclein Aggregation by Exosomes. J. Biol. Chem. 2015, 290 (5), 2969– 2982,  DOI: 10.1074/jbc.M114.585703
      22
      Acceleration of α-synuclein aggregation by exosomes
      Grey, Marie; Dunning, Christopher J.; Gaspar, Ricardo; Grey, Carl; Brundin, Patrik; Sparr, Emma; Linse, Sara
      Journal of Biological Chemistry (2015), 290 (5), 2969-2982CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Exosomes are small vesicles released from cells into extracellular space. Here, the authors isolated exosomes from neuroblastoma cells and investigated their influence on the aggregation of α-synuclein, a protein assocd. with Parkinson disease pathol. Using cryo-transmission electron microscopy of exosomes, the authors found spherical unilamellar vesicles with a significant protein content, and Western blot anal. revealed that they contained, as expected, the proteins flotillin-1 and alix. Using thioflavin T fluorescence to monitor aggregation kinetics, the authors found that exosomes catalyzed the process in a similar manner as a low concn. of preformed α-synuclein fibrils. The exosomes reduced the lag time indicating that they provide catalytic environments for nucleation. The catalytic effects of exosomes derived from native cells and cells that overexpressed α-synuclein did not differ. Vesicles prepd. from extd. exosome lipids accelerated aggregation, suggesting that the lipids in exosomes were sufficient for the catalytic effect to arise. Using mass spectrometry, the authors found several phospholipid classes in the exosomes, including phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, and gangliosides GM2 and GM3. Within each class, several species with different acyl chains were identified. The authors then prepd. vesicles from corresponding pure lipids or defined mixts., most of which were found to retard α-synuclein aggregation. As a striking exception, vesicles contg. ganglioside lipids GM1 or GM3 accelerated the process. Understanding how α-synuclein interacts with biol. membranes to promote neurol. disease might lead to the identification of novel therapeutic targets.
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    23. 23
      Calvani, R.; Picca, A.; Guerra, F.; Coelho-Junior, H. J.; Bucci, C.; Marzetti, E. Circulating extracellular vesicles: friends and foes in neurodegeneration. Neural Regener. Res. 2022, 17 (3), 534– 542,  DOI: 10.4103/1673-5374.320972
      23
      Circulating extracellular vesicles: friends and foes in neurodegeneration
      Picca, Anna; Guerra, Flora; Calvani, Riccardo; Coelho-Junior, Helio Jose; Bucci, Cecilia; Marzetti, Emanuele
      Neural Regeneration Research (2022), 17 (3), 534-542CODEN: NRREBM; ISSN:1673-5374. (Publishing House of Neural Regeneration Research)
      A review. Extracellular vesicles have been identified as pivotal mediators of intercellular communication with crit. roles in physiol. and pathol. conditions. Via this route, several mols. (e.g., nucleic acids, proteins, metabolites) can be transferred to proximal and distant targets to convey specific information. Extracellular vesicle-assocd. cargo mols. have been proposed as markers of several disease conditions for their potential of tracking down the generating cell. Indeed, circulating extracellular vesicles may represent biomarkers of dysfunctional cellular quality control systems esp. in conditions characterized by the accrual of intracellular misfolded proteins. Furthermore, the identification of extracellular vesicles as tools for the delivery of nucleic acids or other cargo mols. to diseased tissues makes these circulating shuttles possible targets for therapeutic development. The increasing interest in the study of extracellular vesicles as biomarkers resides mainly in the fact that the identification of peripheral levels of extracellular vesicle-assocd. proteins might reflect mol. events occurring in hardly accessible tissues, such as the brain, thereby serving as a "brain liq. biopsy". The exploitation of extracellular vesicles for diagnostic and therapeutic purposed might offer unprecedented opportunities to develop personalized approaches. Here, we discuss the bright and dark sides of extracellular vesicles in the setting of two main neurodegenerative diseases (i.e., Parkinson's and Alzheimer's diseases). A special focus will be placed on the possibility of using extracellular vesicles as biomarkers for the two conditions to enable disease tracking and treatment monitoring.
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    24. 24
      Beretta, C.; Nikitidou, E.; Streubel-Gallasch, L.; Ingelsson, M.; Sehlin, D.; Erlandsson, A. Extracellular vesicles from amyloid-β exposed cell cultures induce severe dysfunction in cortical neurons. Sci. Rep. 2020, 10 (1), 19656,  DOI: 10.1038/s41598-020-72355-2
      24
      Extracellular vesicles from amyloid-beta exposed cell cultures induce severe dysfunction in cortical neurons
      Beretta, Chiara; Nikitidou, Elisabeth; Streubel-Gallasch, Linn; Ingelsson, Martin; Sehlin, Dag; Erlandsson, Anna
      Scientific Reports (2020), 10 (1), 19656CODEN: SRCEC3; ISSN:2045-2322. (Nature Research)
      Alzheimer's disease (AD) is characterized by a substantial loss of neurons and synapses throughout the brain. The exact mechanism behind the neurodegeneration is still unclear, but recent data suggests that spreading of amyloid-beta (Abeta) pathol. via extracellular vesicles (EVs) may contribute to disease progression. We have previously shown that an incomplete degrdn. of Abeta42 protofibrils by astrocytes results in the release of EVs contg. neurotoxic Aβ. Here, we describe the cellular mechanisms behind EV-assocd. neurotoxicity in detail. EVs were isolated from untreated and Abeta42 protofibril exposed neuroglial co-cultures, consisting mainly of astrocytes. The EVs were added to cortical neurons for 2 or 4 days and the neurodegenerative processes were followed with immunocytochem., time-lapse imaging and transmission electron microscopy (TEM). Addn. of EVs from A beta42 protofibril exposed co-cultures resulted in synaptic loss, severe mitochondrial impairment and apoptosis. TEM anal. demonstrated that the EVs induced axonal swelling and vacuolization of the neuronal cell bodies. Interestingly, EV exposed neurons also displayed pathol. lamellar bodies of cholesterol deposits in lysosomal compartments. Taken together, our data show that the secretion of EVs from A beta exposed cells induces neuronal dysfunction in several ways, indicating a central role for EVs in the progression of Aβ-induced pathol.
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    25. 25
      Jiang, L.; Dong, H.; Cao, H.; Ji, X.; Luan, S.; Liu, J. Exosomes in Pathogenesis, Diagnosis, and Treatment of Alzheimer’s Disease. Med. Sci. Monit. 2019, 25, 3329– 3335,  DOI: 10.12659/MSM.914027
      25
      Exosomes in pathogenesis, diagnosis, and treatment of Alzheimer's disease
      Jiang, Liqun; Dong, Huijie; Cao, Hua; Ji, Xiaofei; Luan, Siyu; Liu, Jing
      Medical Science Monitor (2019), 25 (), 3329-3335CODEN: MSMOFR; ISSN:1643-3750. (International Scientific Information, Inc.)
      Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the accumulation of β-amyloid peptide 1-42 and phosphorylation of tau protein in the brain. Thus far, the transfer mechanism of these cytotoxic proteins between nerve cells remains unclear. Recent studies have shown that nanoscale extracellular vesicles (exosomes) originating from cells may play important roles in this transfer process. In addn., several genetic materials and proteins are also involved in intercellular communication by the secretion of the exosomes. That proposes novel avenues for early diagnosis and biol. treatment in AD, based on exosome detection and intervention. In this review, exosome-related pathways of cytotoxic protein intercellular transfer in AD, and the effect of membrane proteins on exosomes targeting cells are first introduced. The advances in exosome-related biomarker detection in AD are summarized. Finally, the advantages and challenges of reducing cytotoxic protein accumulation via exosomal intervention for AD treatment are discussed. It is envisaged that future research in exosomes may well provide new insights into the pathogenesis, diagnosis, and treatment of AD.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXisVGlsL%252FI&md5=27788b7d55740579c2ecc1878ef428c5
    26. 26
      Rajendran, L.; Honsho, M.; Zahn, T. R.; Keller, P.; Geiger, K. D.; Verkade, P.; Simons, K. Alzheimer’s disease β-amyloid peptides are released in association with exosomes. Proc. Natl. Acad. Sci. U.S.A. 2006, 103 (30), 11172– 11177,  DOI: 10.1073/pnas.0603838103
      26
      Alzheimer's disease β-amyloid peptides are released in association with exosomes
      Rajendran, Lawrence; Honsho, Masanori; Zahn, Tobias R.; Keller, Patrick; Geiger, Kethrin D.; Verkade, Paul; Simons, Kai
      Proceedings of the National Academy of Sciences of the United States of America (2006), 103 (30), 11172-11177CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Although the exact etiol. of Alzheimer's disease (AD) is a topic of debate, the consensus is that the accumulation of β-amyloid (AP) peptides in the senile plaques is one of the hallmarks of the progression of the disease. The AO peptide is formed by the amyloidogenic cleavage of the amyloid precursor protein (APP) by β- and γ-secretases. The endocytic system has been implicated in the cleavages leading to the formation of Aβ. However, the identity of the intracellular compartment where the amyloidogenic secretases cleave and the mechanism by which the intracellularly generated Aβ is released into the extracellular milieu are not clear. Here, we show that β-cleavage occurs in early endosomes followed by routing of Aβ to multivesicular bodies (MVBs) in HeLa and N2a cells. Subsequently, a minute fraction of Aβ peptides can be secreted from the cells in assocn. with exosomes, intraluminal vesicles of MVBs that are released into the extracellular space as a result of fusion of MVBs with the plasma membrane. Exosomal proteins were found to accumulate in the plaques of AD patient brains, suggesting a role in the pathogenesis of AD.
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    27. 27
      Soudy, R.; Kimura, R.; Fu, W.; Patel, A.; Jhamandas, J. Extracellular vesicles enriched with amylin receptor are cytoprotective against the Aß toxicity in vitro. PLoS One 2022, 17 (4), e0267164  DOI: 10.1371/journal.pone.0267164
      27
      Extracellular vesicles enriched with amylin receptor are cytoprotective against the Ass toxicity in vitro
      Soudy, Rania; Kimura, Ryoichi; Fu, Wen; Patel, Aarti; Jhamandas, Jack
      PLoS One (2022), 17 (4), e0267164CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
      Extracellular vesicles (EVs) are double membrane structures released by all cell types with identified roles in the generation, transportation, and degrdn. of amyloid-β protein (Aβ) oligomers in Alzheimer's disease (AD). EVs are thus increasingly recognized to play a neuroprotective role in AD, through their ability to counteract the neurotoxic effects of Aβ, possibly through interactions with specific receptors on cell membranes. Our previous studies have identified the amylin receptor (AMY), particularly AMY3 subtype, as a mediator of the deleterious actions of Aβ in vitro and in vivo exptl. paradigms. In the present study, we demonstrate that AMY3 enriched EVs can bind sol. oligomers of Ass and protect N2a cells against toxic effects of this peptide. The effect was specific to amylin receptor as it was blocked in the presence of amylin receptor antagonist AC253. This notion was supported by reduced Aβ binding to EVs from AMY depleted mice compared to those from wild type (Wt) mice. Finally, application of AMY3, but not Wt derived, EVs to hippocampal brain slices improved Aβ-induced redn. of long-term potentiation, a cellular surrogate of memory. Collectively, our observations support the role of AMY receptors, particularly AMY3, in EVs as a potential therapeutic target for AD.
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    28. 28
      Ribeiro, D.; Horvath, I.; Heath, N.; Hicks, R.; Forslow, A.; Wittung-Stafshede, P. Extracellular vesicles from human pancreatic islets suppress human islet amyloid polypeptide amyloid formation. Proc. Natl. Acad. Sci. U.S.A. 2017, 114 (42), 11127– 11132,  DOI: 10.1073/pnas.1711389114
      28
      Extracellular vesicles from human pancreatic islets suppress human islet amyloid polypeptide amyloid formation
      Ribeiro, Diana; Horvath, Istvan; Heath, Nikki; Hicks, Ryan; Forsloew, Anna; Wittung-Stafshede, Pernilla
      Proceedings of the National Academy of Sciences of the United States of America (2017), 114 (42), 11127-11132CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Extracellular vesicles (EVs) are small vesicles released by cells to aid cell-cell communication and tissue homeostasis. Human islet amyloid polypeptide (IAPP) is the major component of amyloid deposits found in pancreatic islets of patients with type 2 diabetes (T2D). IAPP is secreted in conjunction with insulin from pancreatic β cells to regulate glucose metab. Here, using a combination of anal. and biophys. methods in vitro, we tested whether EVs isolated from pancreatic islets of healthy patients and patients with T2D modulate IAPP amyloid formation. We discovered that pancreatic EVs from healthy patients reduce IAPP amyloid formation by peptide scavenging, but T2D pancreatic and human serum EVs have no effect. In accordance with these differential effects, the insulin:C-peptide ratio and lipid compn. differ between EVs from healthy pancreas and EVs from T2D pancreas and serum. It appears that healthy pancreatic EVs limit IAPP amyloid formation via direct binding as a tissue-specific control mechanism.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsFyqs7vM&md5=531acf23e32b8c8f405425174d42343b
    29. 29
      Haass, C.; Kaether, C.; Thinakaran, G.; Sisodia, S. Trafficking and proteolytic processing of APP. Cold Spring Harbor Perspect. Med. 2012, 2 (5), a006270,  DOI: 10.1101/cshperspect.a006270
      29
      Trafficking and proteolytic processing of APP
      Haass, Christian; Kaether, Christoph; Thinakaran, Copal; Sisodia, Sangram
      Cold Spring Harbor Perspectives in Medicine (2012), 2 (5), a006270/1-a006270/25CODEN: CSHPFV; ISSN:2157-1422. (Cold Spring Harbor Laboratory Press)
      A review. Accumulations of insol. deposits of amyloid β-peptide are major pathol. hallmarks of Alzheimer disease. Amyloid β-peptide is derived by sequential proteolytic processing from a large type 1 trans-membrane protein, the β-amyloid precursor protein. The proteolytic enzymes involved in its processing are named secretases. β- And γ-secretase liberate by sequential cleavage the neurotoxic amyloid β-peptide, whereas α-secretase prevents its generation by cleaving within the middle of the amyloid domain. In this chapter we describe the cell biol. and biochem. characteristics of the three secretase activities involved in the proteolytic processing of the precursor protein. In addn. we outline how the precursor protein maturates and traffics through the secretory pathway to reach the subcellular locations where the individual secretases are preferentially active. Furthermore, we illuminate how neuronal activity and mutations which cause familial Alzheimer disease affect amyloid β-peptide generation and therefore disease onset and progression.
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    30. 30
      Haass, C.; Koo, E. H.; Mellon, A.; Hung, A. Y.; Selkoe, D. J. Targeting of cell-surface β-amyloid precursor protein to lysosomes: alternative processing into amyloid-bearing fragments. Nature 1992, 357 (6378), 500– 503,  DOI: 10.1038/357500a0
      30
      Targeting of cell-surface β-amyloid precursor protein to lysosomes: alternative processing into amyloid-bearing fragments
      Haass, Christian; Koo, Edward H.; Mellon, Angela; Hung, Albert Y.; Selkoe, Dennis J.
      Nature (London, United Kingdom) (1992), 357 (6378), 500-3CODEN: NATUAS; ISSN:0028-0836.
      Progressive cerebral deposition of the amyloid β-peptide is an early and invariant feature of Alzheimer's disease. The β-peptide is released by proteolytic cleavages from the β-amyloid precursor protein (βAPP), a membrane-spanning glycoprotein expressed in most mammalian cells. Normal secretion of βAPP involves a cleavage in the β-peptide region, releasing the sol. extramembranous portion and retaining a 10 kDa C-terminal fragment in the membrane. Because this secretory pathway precludes β-amyloid formation, the authors searched for an alternative proteolytic processing pathway that can generate β-peptide-bearing fragments from full-length βAPP. Incubation of living human endothelial cells with a βAPP antibody revealed reinternalization of mature βAPP from the cell surface and its targeting to endosomes/lysosomes. After cell-surface biotinylation, full-length biotinylated βAPP was recovered inside the cells. Purifn. of lysosomes directly demonstrated the presence of mature βAPP and an extensive array of β-peptide-contg. proteolytic products. The results define a second processing pathway for βAPP and suggest that it may be responsible for generating amyloid-bearing fragments in Alzheimer's disease.
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    31. 31
      Gabrielli, M.; Prada, I.; Joshi, P.; Falcicchia, C.; D’Arrigo, G.; Rutigliano, G.; Battocchio, E.; Zenatelli, R.; Tozzi, F.; Radeghieri, A. Microglial large extracellular vesicles propagate early synaptic dysfunction in Alzheimer’s disease. Brain 2022, 145 (8), 2849– 2868,  DOI: 10.1093/brain/awac083
      31
      Microglial large extracellular vesicles propagate early synaptic dysfunction in Alzheimer's disease
      Gabrielli Martina; Prada Ilaria; Joshi Pooja; D'Arrigo Giulia; Battocchio Elisabetta; Verderio Claudia; Falcicchia Chiara; Origlia Nicola; Rutigliano Grazia; Rutigliano Grazia; Battocchio Elisabetta; Zenatelli Rossella; Radeghieri Annalisa; Tozzi Francesca; Radeghieri Annalisa; Arancio Ottavio; Arancio Ottavio; Arancio Ottavio
      Brain : a journal of neurology (2022), 145 (8), 2849-2868 ISSN:.
      Synaptic dysfunction is an early mechanism in Alzheimer's disease that involves progressively larger areas of the brain over time. However, how it starts and propagates is unknown. Here we show that amyloid-β released by microglia in association with large extracellular vesicles (Aβ-EVs) alters dendritic spine morphology in vitro, at the site of neuron interaction, and impairs synaptic plasticity both in vitro and in vivo in the entorhinal cortex-dentate gyrus circuitry. One hour after Aβ-EV injection into the mouse entorhinal cortex, long-term potentiation was impaired in the entorhinal cortex but not in the dentate gyrus, its main target region, while 24 h later it was also impaired in the dentate gyrus, revealing a spreading of long-term potentiation deficit between the two regions. Similar results were obtained upon injection of extracellular vesicles carrying Aβ naturally secreted by CHO7PA2 cells, while neither Aβ42 alone nor inflammatory extracellular vesicles devoid of Aβ were able to propagate long-term potentiation impairment. Using optical tweezers combined to time-lapse imaging to study Aβ-EV-neuron interaction, we show that Aβ-EVs move anterogradely at the axon surface and that their motion can be blocked through annexin-V coating. Importantly, when Aβ-EV motility was inhibited, no propagation of long-term potentiation deficit occurred along the entorhinal-hippocampal circuit, implicating large extracellular vesicle motion at the neuron surface in the spreading of long-term potentiation impairment. Our data indicate the involvement of large microglial extracellular vesicles in the rise and propagation of early synaptic dysfunction in Alzheimer's disease and suggest a new mechanism controlling the diffusion of large extracellular vesicles and their pathogenic signals in the brain parenchyma, paving the way for novel therapeutic strategies to delay the disease.
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    32. 32
      Sardar Sinha, M.; Ansell-Schultz, A.; Civitelli, L.; Hildesjo, C.; Larsson, M.; Lannfelt, L.; Ingelsson, M.; Hallbeck, M. Alzheimer’s disease pathology propagation by exosomes containing toxic amyloid-beta oligomers. Acta Neuropathol. 2018, 136 (1), 41– 56,  DOI: 10.1007/s00401-018-1868-1
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      Alzheimer's disease pathology propagation by exosomes containing toxic amyloid-beta oligomers
      Sardar Sinha, Maitrayee; Ansell-Schultz, Anna; Civitelli, Livia; Hildesjoe, Camilla; Larsson, Max; Lannfelt, Lars; Ingelsson, Martin; Hallbeck, Martin
      Acta Neuropathologica (2018), 136 (1), 41-56CODEN: ANPTAL; ISSN:0001-6322. (Springer)
      The gradual deterioration of cognitive functions in Alzheimer's disease is paralleled by a hierarchical progression of amyloid-beta and tau brain pathol. Recent findings indicate that toxic oligomers of amyloid-beta may cause propagation of pathol. in a prion-like manner, although the underlying mechanisms are incompletely understood. Here we show that small extracellular vesicles, exosomes, from Alzheimer patients' brains contain increased levels of amyloid-beta oligomers and can act as vehicles for the neuron-to-neuron transfer of such toxic species in recipient neurons in culture. Moreover, blocking the formation, secretion or uptake of exosomes was found to reduce both the spread of oligomers and the related toxicity. Taken together, our results imply that exosomes are centrally involved in Alzheimer's disease and that they could serve as targets for development of new diagnostic and therapeutic principles.
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    33. 33
      Gustafsson, G.; Loov, C.; Persson, E.; Lazaro, D. F.; Takeda, S.; Bergstrom, J.; Erlandsson, A.; Sehlin, D.; Balaj, L.; Gyorgy, B. Secretion and Uptake of α-Synuclein Via Extracellular Vesicles in Cultured Cells. Cell. Mol. Neurobiol. 2018, 38 (8), 1539– 1550,  DOI: 10.1007/s10571-018-0622-5
      33
      Secretion and Uptake of α-Synuclein Via Extracellular Vesicles in Cultured Cells
      Gustafsson, Gabriel; Loeoev, Camilla; Persson, Emma; Lazaro, Diana F.; Takeda, Shuko; Bergstroem, Joakim; Erlandsson, Anna; Sehlin, Dag; Balaj, Leonora; Gyoergy, Bence; Hallbeck, Martin; Outeiro, Tiago F.; Breakefield, Xandra O.; Hyman, Bradley T.; Ingelsson, Martin
      Cellular and Molecular Neurobiology (2018), 38 (8), 1539-1550CODEN: CMNEDI; ISSN:0272-4340. (Springer)
      In Parkinson's disease and other Lewy body disorders, the propagation of pathol. has been accredited to the spreading of extracellular α-synuclein (a-syn). Although the pathogenic mechanisms are not fully understood, cell-to-cell transfer of α-syn via exosomes and other extracellular vesicles (EVs) has been reported. Here, we investigated whether altered mol. properties of α-syn can influence the distribution and secretion of α-syn in human neuroblastoma cells. Different a-syn variants, including α-syn:hemi-Venus and disease-causing mutants, were overexpressed and EVs were isolated from the conditioned medium. Of the secreted α-syn, 0.1-2% was assocd. with vesicles. The major part of EV α-syn was attached to the outer membrane of vesicles, whereas a smaller fraction was found in their lumen. For α-syn expressed with N-terminal hemi-Venus, the relative levels assocd. with EVs were higher than for WT α-syn. Moreover, such EV-assocd. α-syn:hemi-Venus species were internalized in recipient cells to a higher degree than the corresponding free-floating forms. Among the disease-causing mutants, A53T α-syn displayed an increased assocn. with EVs. Taken together, our data suggest that α-syn species with presumably lost physiol. functions or altered aggregation properties may shift the cellular processing towards vesicular secretion. Our findings thus lend further support to the tenet that EVs can mediate spreading of harmful a-syn species and thereby contribute to the pathol. in α-synucleinopathies.
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    34. 34
      Lim, C. Z. J.; Zhang, Y.; Chen, Y.; Zhao, H.; Stephenson, M. C.; Ho, N. R. Y.; Chen, Y.; Chung, J.; Reilhac, A.; Loh, T. P. Subtyping of circulating exosome-bound amyloid β reflects brain plaque deposition. Nat. Commun. 2019, 10 (1), 1144,  DOI: 10.1038/s41467-019-09030-2
      34
      Subtyping of circulating exosome-bound amyloid β reflects brain plaque deposition
      Lim Carine Z J; Zhang Yan; Zhao Haitao; Chen Yuan; Shao Huilin; Lim Carine Z J; Zhang Yan; Zhao Haitao; Ho Nicholas R Y; Chen Yuan; Loh Tze Ping; Shao Huilin; Chen Yu; Chung Jaehoon; Stephenson Mary C; Reilhac Anthonin; Ho Nicholas R Y; Shao Huilin; Loh Tze Ping; Chen Christopher L H; Chen Christopher L H; Shao Huilin
      Nature communications (2019), 10 (1), 1144 ISSN:.
      Despite intense interests in developing blood measurements of Alzheimer's disease (AD), the progress has been confounded by limited sensitivity and poor correlation to brain pathology. Here, we present a dedicated analytical platform for measuring different populations of circulating amyloid β (Aβ) proteins - exosome-bound vs. unbound - directly from blood. The technology, termed amplified plasmonic exosome (APEX), leverages in situ enzymatic conversion of localized optical deposits and double-layered plasmonic nanostructures to enable sensitive, multiplexed population analysis. It demonstrates superior sensitivity (~200 exosomes), and enables diverse target co-localization in exosomes. Employing the platform, we find that prefibrillar Aβ aggregates preferentially bind with exosomes. We thus define a population of Aβ as exosome-bound (Aβ42+ CD63+) and measure its abundance directly from AD and control blood samples. As compared to the unbound or total circulating Aβ, the exosome-bound Aβ measurement could better reflect PET imaging of brain amyloid plaques and differentiate various clinical groups.
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    35. 35
      Yuyama, K.; Igarashi, Y. Exosomes as Carriers of Alzheimer’s Amyloid-ß. Front. Neurosci. 2017, 11, 229,  DOI: 10.3389/fnins.2017.00229
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      Exosomes as Carriers of Alzheimer's Amyloid-ss
      Yuyama Kohei; Igarashi Yasuyuki
      Frontiers in neuroscience (2017), 11 (), 229 ISSN:1662-4548.
      The intracerebral level of the aggregation-prone peptide, amyloid-ss (Ass), is constantly maintained by multiple clearance mechanisms, including several degradation enzymes, and brain efflux. Disruption of the clearance machinery and the resultant Ass accumulation gives rise to neurotoxic assemblies, leading to the pathogenesis of Alzheimer's disease (AD). In addition to the classic mechanisms of Ass clearance, the protein may be processed by secreted vesicles, although this possibility has not been extensively investigated. We showed that neuronal exosomes, a subtype of extracellular nanovesicles, enwrap, or trap Ass and transport it into microglia for degradation. Here, we review Ass sequestration and elimination by exosomes, and discuss how this clearance machinery might contribute to AD pathogenesis and how it might be exploited for effective AD therapy.
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    36. 36
      Bordanaba-Florit, G.; Royo, F.; Kruglik, S. G.; Falcon-Perez, J. M. Using single-vesicle technologies to unravel the heterogeneity of extracellular vesicles. Nat. Protoc. 2021, 16 (7), 3163– 3185,  DOI: 10.1038/s41596-021-00551-z
      36
      Using single-vesicle technologies to unravel the heterogeneity of extracellular vesicles
      Bordanaba-Florit, Guillermo; Royo, Felix; Kruglik, Sergei G.; Falcon-Perez, Juan M.
      Nature Protocols (2021), 16 (7), 3163-3185CODEN: NPARDW; ISSN:1750-2799. (Nature Portfolio)
      Abstr.: Extracellular vesicles (EVs) are heterogeneous lipid containers with a complex mol. cargo comprising several populations with unique roles in biol. processes. These vesicles are closely assocd. with specific physiol. features, which makes them invaluable in the detection and monitoring of various diseases. EVs play a key role in pathophysiol. processes by actively triggering genetic or metabolic responses. However, the heterogeneity of their structure and compn. hinders their application in medical diagnosis and therapies. This diversity makes it difficult to establish their exact physiol. roles, and the functions and compn. of different EV (sub)populations. Ensemble averaging approaches currently employed for EV characterization, such as western blotting or 'omics' technologies, tend to obscure rather than reveal these heterogeneities. Recent developments in single-vesicle anal. have made it possible to overcome these limitations and have facilitated the development of practical clin. applications. In this review, we discuss the benefits and challenges inherent to the current methods for the anal. of single vesicles and review the contribution of these approaches to the understanding of EV biol. We describe the contributions of these recent technol. advances to the characterization and phenotyping of EVs, examn. of the role of EVs in cell-to-cell communication pathways and the identification and validation of EVs as disease biomarkers. Finally, we discuss the potential of innovative single-vesicle imaging and anal. methodologies using microfluidic devices, which promise to deliver rapid and effective basic and practical applications for minimally invasive prognosis systems.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhtleqs7fN&md5=a377b8440458aa015c7a555c511942d3
    37. 37
      Thery, C.; Witwer, K. W.; Aikawa, E.; Alcaraz, M. J.; Anderson, J. D.; Andriantsitohaina, R.; Antoniou, A.; Arab, T.; Archer, F.; Atkin-Smith, G. K. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J. Extracell. Vesicles 2018, 7 (1), 1535750,  DOI: 10.1080/20013078.2018.1535750
      37
      Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines
      Thery Clotilde; Lavieu Gregory; Martin-Jaular Lorena; Mathieu Mathilde; Tkach Mercedes; Witwer Kenneth W; Huang Yiyao; Muth Dillon C; Powell Bonita H; Schoyen Tine Hiorth; Zhao Zezhou; Witwer Kenneth W; Datta Chaudhuri Amrita; Aikawa Elena; Aikawa Elena; Alcaraz Maria Jose; Anderson Johnathon D; Andriantsitohaina Ramaroson; Le Lay Soazig; Martinez M Carmen; Antoniou Anna; Antoniou Anna; Arab Tanina; Archer Fabienne; Atkin-Smith Georgia K; Baxter Amy A; Caruso Sarah; Cheng Lesley; Greening David W; Hill Andrew F; Jiang Lanzhou; Mathivanan Suresh; Poon Ivan Kh; Tixeira Rochelle; Ayre D Craig; Ayre D Craig; Bach Jean-Marie; Bosch Steffi; Bachurski Daniel; Baharvand Hossein; Shekari Faezeh; Baharvand Hossein; Balaj Leonora; Baldacchino Shawn; Bauer Natalie N; Bebawy Mary; Beckham Carla; Bedina Zavec Apolonija; Benmoussa Abderrahim; Boilard Eric; Gilbert Caroline; Berardi Anna C; Bergese Paolo; Radeghieri Annalisa; Bergese Paolo; Bergese Paolo; Busatto Sara; Radeghieri Annalisa; Bielska Ewa; Blenkiron Cherie; Bobis-Wozowicz Sylwia; Zuba-Surma Ewa K; Boireau Wilfrid; Elie-Caille Celine; Frelet-Barrand Annie; Bongiovanni Antonella; Borras Francesc E; Gamez-Valero Ana; Borras Francesc E; Borras Francesc E; Boulanger Chantal M; Loyer Xavier; Boulanger Chantal M; Loyer Xavier; Breakefield Xandra; Breglio Andrew M; Breglio Andrew M; Brennan Meadhbh A; Brennan Meadhbh A; Brennan Meadhbh A; Brigstock David R; Brigstock David R; Brisson Alain; Broekman Marike Ld; Broekman Marike Ld; Broekman Marike Ld; Bromberg Jacqueline F; Bromberg Jacqueline F; Bryl-Gorecka Paulina; Buch Shilpa; Hu Guoku; Liao Ke; Buck Amy H; Burger Dylan; Vinas Jose L; Burger Dylan; Vinas Jose L; Burger Dylan; Vinas Jose L; Busatto Sara; Buschmann Dominik; Mussack Veronika; Pfaffl Michael W; Bussolati Benedetta; Buzas Edit I; Buzas Edit I; Forsonits Andras; Hegyesi Hargita; Khamari Delaram; Kovacs Arpad Ferenc; Sodar Barbara W; Visnovitz Tamas; Vukman Krisztina V; Wiener Zoltan; Byrd James Bryan; Camussi Giovanni; Carter David Rf; Pink Ryan C; Chamley Lawrence W; Chang Yu-Ting; Chen Chihchen; Chen Chihchen; Chen Shuai; Chin Andrew R; Di Vizio Dolores; Mariscal Javier; Clayton Aled; Cocks Alex; Webber Jason P; Clerici Stefano P; Cocucci Emanuele; Cocucci Emanuele; Coffey Robert J; Cordeiro-da-Silva Anabela; Couch Yvonne; Coumans Frank Aw; Nieuwland Rienk; Coyle Beth; Jackson Hannah K; Crescitelli Rossella; Lasser Cecilia; Lotvall Jan; Shelke Ganesh Vilas; Criado Miria Ferreira; D'Souza-Schorey Crislyn; Das Saumya; de Candia Paola; De Santana Eliezer F; De Wever Olivier; Dhondt Bert; Hendrix An; Van Deun Jan; De Wever Olivier; Dhondt Bert; Hendrix An; Van Deun Jan; Del Portillo Hernando A; Del Portillo Hernando A; Del Portillo Hernando A; Demaret Tanguy; Lombard Catherine A; Deville Sarah; Deville Sarah; Mertens Inge; Devitt Andrew; Dhondt Bert; Dieterich Lothar C; Dolo Vincenza; Giusti Ilaria; Dominguez Rubio Ana Paula; Dominici Massimo; Dominici Massimo; Dourado Mauricio R; Dourado Mauricio R; Driedonks Tom Ap; Nolte-'t Hoen Esther Nm; Stoorvogel Willem; van der Grein Susanne G; van Herwijnen Martijn Jc; Wauben Marca Hm; Duarte Filipe V; Rodrigues Silvia C; Duncan Heather M; Duncan Heather M; Eichenberger Ramon M; Sotillo Javier; Ekstrom Karin; El Andaloussi Samir; Gorgens Andre; El Andaloussi Samir; Erdbrugger Uta; Musante Luca; Falcon-Perez Juan M; Falcon-Perez Juan M; Fatima Farah; Fish Jason E; Fish Jason E; Gustafson Dakota; Schneider Raphael; Flores-Bellver Miguel; Fricke Fabia; Fricke Fabia; Fuhrmann Gregor; Fuhrmann Gregor; Fuhrmann Gregor; Gabrielsson Susanne; Gamez-Valero Ana; Gardiner Chris; Gartner Kathrin; Gaudin Raphael; Gaudin Raphael; Gho Yong Song; Park Jaesung; Giebel Bernd; Gorgens Andre; Gimona Mario; Rohde Eva; Goberdhan Deborah Ci; Gorgens Andre; Gorski Sharon M; Xu Jing; Gorski Sharon M; Xu Jing; Gross Julia Christina; Linnemannstons Karen; Witte Leonie; Gross Julia Christina; Linnemannstons Karen; Witte Leonie; Gualerzi Alice; Gupta Gopal N; Handberg Aase; Handberg Aase; Haraszti Reka A; Khvorova Anastasia; Harrison Paul; Hochberg Fred H; Nolan John P; Hochberg Fred H; Hoffmann Karl F; Holder Beth; Holder Beth; Holthofer Harry; Hosseinkhani Baharak; Huang Yiyao; Zheng Lei; Huber Veronica; Shahaj Eriomina; Hunt Stuart; Ibrahim Ahmed Gamal-Eldin; Ikezu Tsuneya; Inal Jameel M; Isin Mustafa; Ivanova Alena; Jacobsen Soren; Jacobsen Soren; Jay Steven M; Jayachandran Muthuvel; Jenster Guido; Martens-Uzunova Elena S; Johnson Suzanne M; Jones Jennifer C; Morales-Kastresana Aizea; Welsh Joshua A; Jong Ambrose; Jong Ambrose; Jovanovic-Talisman Tijana; Jung Stephanie; Kalluri Raghu; Kano Shin-Ichi; Kaur Sukhbir; Roberts David D; Kawamura Yumi; Kawamura Yumi; Keller Evan T; Tewari Muneesh; Keller Evan T; Khomyakova Elena; Khomyakova Elena; Kierulf Peter; Kim Kwang Pyo; Kislinger Thomas; Kislinger Thomas; Klingeborn Mikael; Klinke David J 2nd; Klinke David J 2nd; Kornek Miroslaw; Kornek Miroslaw; Kosanovic Maja M; Kramer-Albers Eva-Maria; Krasemann Susanne; Krause Mirja; Kusuma Gina D; Lim Rebecca; Kurochkin Igor V; Kusuma Gina D; Lim Rebecca; Kuypers Soren; Laitinen Saara; Langevin Scott M; Langevin Scott M; Languino Lucia R; Lannigan Joanne; Laurent Louise C; Lazaro-Ibanez Elisa; Shatnyeva Olga; Lee Myung-Shin; Lee Yi Xin Fiona; Lemos Debora S; Lenassi Metka; Leszczynska Aleksandra; Li Isaac Ts; Libregts Sten F; Ligeti Erzsebet; Lorincz Akos M; Lim Sai Kiang; Line Aija; Llorente Alicia; Lorenowicz Magdalena J; Lovett Jason; Myburgh Kathryn H; Lowry Michelle C; O'Driscoll Lorraine; Lu Quan; Lukomska Barbara; Lunavat Taral R; Maas Sybren Ln; Maas Sybren Ln; Malhi Harmeet; Marcilla Antonio; Marcilla Antonio; Mariani Jacopo; Martins Vilma Regina; Maugeri Marco; Nawaz Muhammad; McGinnis Lynda K; McVey Mark J; McVey Mark J; Meckes David G Jr; Meehan Katie L; Mertens Inge; Minciacchi Valentina R; Moller Andreas; Soekmadji Carolina; Moller Jorgensen Malene; Moller Jorgensen Malene; Morhayim Jess; Mullier Francois; Mullier Francois; Muraca Maurizio; Najrana Tanbir; Nazarenko Irina; Nazarenko Irina; Nejsum Peter; Neri Christian; Neri Tommaso; Nimrichter Leonardo; Noren Hooten Nicole; O'Grady Tina; O'Loghlen Ana; Ochiya Takahiro; Olivier Martin; Rak Janusz; Ortiz Alberto; Ortiz Alberto; Ortiz Alberto; Ortiz Luis A; Osteikoetxea Xabier; Ostergaard Ole; Ostergaard Ole; Ostrowski Matias; Pegtel D Michiel; Peinado Hector; Perut Francesca; Phinney Donald G; Pieters Bartijn Ch; Pisetsky David S; Pisetsky David S; Pogge von Strandmann Elke; Polakovicova Iva; Polakovicova Iva; Prada Ilaria; Pulliam Lynn; Raffai Robert L; Pulliam Lynn; Quesenberry Peter; Raffai Robert L; Raimondo Stefania; Rak Janusz; Ramirez Marcel I; Ramirez Marcel I; Raposo Graca; Rayyan Morsi S; Zheutlin Alexander R; Regev-Rudzki Neta; Ricklefs Franz L; Robbins Paul D; Rodrigues Silvia C; Rohde Eva; Rohde Eva; Rome Sophie; Rouschop Kasper Ma; Rughetti Aurelia; Russell Ashley E; Saa Paula; Sahoo Susmita; Salas-Huenuleo Edison; Salas-Huenuleo Edison; Sanchez Catherine; Saugstad Julie A; Saul Meike J; Schiffelers Raymond M; Vader Pieter; Schneider Raphael; Scott Aaron; Sharma Shivani; Sharma Shivani; Sharma Shivani; Shelke Ganesh Vilas; Shetty Ashok K; Shetty Ashok K; Shiba Kiyotaka; Siljander Pia R-M; Siljander Pia R-M; Silva Andreia M; Silva Andreia M; Vasconcelos M Helena; Silva Andreia M; Skowronek Agata; Snyder Orman L 2nd; Soares Rodrigo Pedro; Soekmadji Carolina; Stahl Philip D; Stott Shannon L; Stott Shannon L; Strasser Erwin F; Swift Simon; Tahara Hidetoshi; Tewari Muneesh; Tewari Muneesh; Timms Kate; Tiwari Swasti; Tiwari Swasti; Toh Wei Seong; Tomasini Richard; Torrecilhas Ana Claudia; Tosar Juan Pablo; Tosar Juan Pablo; Toxavidis Vasilis; Urbanelli Lorena; van Balkom Bas Wm; Van Keuren-Jensen Kendall; van Niel Guillaume; Verweij Frederik J; van Royen Martin E; van Wijnen Andre J; Vasconcelos M Helena; Vasconcelos M Helena; Vechetti Ivan J Jr; Veit Tiago D; Vella Laura J; Vella Laura J; Velot Emilie; Vestad Beate; Vestad Beate; Vestad Beate; Wahlgren Jessica; Watson Dionysios C; Watson Dionysios C; Weaver Alissa; Weber Viktoria; Wehman Ann M; Weiss Daniel J; Wendt Sebastian; Wheelock Asa M; Wolfram Joy; Wolfram Joy; Wolfram Joy; Xagorari Angeliki; Xander Patricia; Yan Xiaomei; Yanez-Mo Maria; Yanez-Mo Maria; Yin Hang; Yuana Yuana; Zappulli Valentina; Zarubova Jana; Zarubova Jana; Zarubova Jana; Zekas Vytautas; Zhang Jian-Ye; Zickler Antje M; Zimmermann Pascale; Zimmermann Pascale; Zivkovic Angela M; Zocco Davide
      Journal of extracellular vesicles (2018), 7 (1), 1535750 ISSN:2001-3078.
      The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles ("MISEV") guidelines for the field in 2014. We now update these "MISEV2014" guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.
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    38. 38
      Meisl, G.; Kirkegaard, J. B.; Arosio, P.; Michaels, T. C.; Vendruscolo, M.; Dobson, C. M.; Linse, S.; Knowles, T. P. Molecular mechanisms of protein aggregation from global fitting of kinetic models. Nat. Protoc. 2016, 11 (2), 252– 272,  DOI: 10.1038/nprot.2016.010
      38
      Molecular mechanisms of protein aggregation from global fitting of kinetic models
      Meisl, Georg; Kirkegaard, Julius B.; Arosio, Paolo; Michaels, Thomas C. T.; Vendruscolo, Michele; Dobson, Christopher M.; Linse, Sara; Knowles, Tuomas P. J.
      Nature Protocols (2016), 11 (2), 252-272CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)
      The elucidation of the mol. mechanisms by which sol. proteins convert into their amyloid forms is a fundamental prerequisite for understanding and controlling disorders that are linked to protein aggregation, such as Alzheimer's and Parkinson's diseases. However, because of the complexity assocd. with aggregation reaction networks, the anal. of kinetic data of protein aggregation to obtain the underlying mechanisms represents a complex task. Here we describe a framework, using quant. kinetic assays and global fitting, to det. and to verify a mol. mechanism for aggregation reactions that is compatible with exptl. kinetic data. We implement this approach in a web-based software, AmyloFit. Our procedure starts from the results of kinetic expts. that measure the concn. of aggregate mass as a function of time. We illustrate the approach with results from the aggregation of the β-amyloid (Aβ) peptides measured using thioflavin T, but the method is suitable for data from any similar kinetic expt. measuring the accumulation of aggregate mass as a function of time; the input data are in the form of a tab-sepd. text file. We also outline general exptl. strategies and practical considerations for obtaining kinetic data of sufficient quality to draw detailed mechanistic conclusions, and the procedure starts with instructions for extensive data quality control. For the core part of the anal., we provide an online platform (http://www.amylofit.ch.cam.ac.uk) that enables robust global anal. of kinetic data without the need for extensive programming or detailed math. knowledge. The software automates repetitive tasks and guides users through the key steps of kinetic anal.: detn. of constraints to be placed on the aggregation mechanism based on the concn. dependence of the aggregation reaction, choosing from several fundamental models describing assembly into linear aggregates and fitting the chosen models using an advanced minimization algorithm to yield the reaction orders and rate consts. Finally, we outline how to use this approach to investigate which targets potential inhibitors of amyloid formation bind to and where in the reaction mechanism they act. The protocol, from processing data to detg. mechanisms, can be completed in <1 d.
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    39. 39
      Spackova, B.; Klein Moberg, H.; Fritzsche, J.; Tenghamn, J.; Sjosten, G.; Sipova-Jungova, H.; Albinsson, D.; Lubart, Q.; van Leeuwen, D.; Westerlund, F. Label-free nanofluidic scattering microscopy of size and mass of single diffusing molecules and nanoparticles. Nat. Methods 2022, 19 (6), 751– 758,  DOI: 10.1038/s41592-022-01491-6
      39
      Label-free nanofluidic scattering microscopy of size and mass of single diffusing molecules and nanoparticles
      Spackova, Barbora; Klein Moberg, Henrik; Fritzsche, Joachim; Tenghamn, Johan; Sjoesten, Gustaf; Sipova-Jungova, Hana; Albinsson, David; Lubart, Quentin; van Leeuwen, Daniel; Westerlund, Fredrik; Midtvedt, Daniel; Esbjoerner, Elin K.; Kaell, Mikael; Volpe, Giovanni; Langhammer, Christoph
      Nature Methods (2022), 19 (6), 751-758CODEN: NMAEA3; ISSN:1548-7091. (Nature Portfolio)
      Label-free characterization of single biomols. aims to complement fluorescence microscopy in situations where labeling compromises data interpretation, is tech. challenging or even impossible. However, existing methods require the investigated species to bind to a surface to be visible, thereby leaving a large fraction of analytes undetected. Here, we present nanofluidic scattering microscopy (NSM), which overcomes these limitations by enabling label-free, real-time imaging of single biomols. diffusing inside a nanofluidic channel. NSM facilitates accurate detn. of mol. wt. from the measured optical contrast and of the hydrodynamic radius from the measured diffusivity, from which information about the conformational state can be inferred. Furthermore, we demonstrate its applicability to the anal. of a complex biofluid, using conditioned cell culture medium contg. extracellular vesicles as an example. We foresee the application of NSM to monitor conformational changes, aggregation and interactions of single biomols., and to analyze single-cell secretomes.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XhsVWjsLvE&md5=af8a3f9d1fb228dfce611d9b28e59790
    40. 40
      Howard, J.; Browne, J.; Bollard, S.; Peters, S.; Sweeney, C.; Wynne, K.; Potter, S.; McCann, A.; Kelly, P. The protein and miRNA profile of plasma extracellular vesicles (EVs) can distinguish feline mammary adenocarcinoma patients from healthy feline controls. Sci. Rep. 2023, 13 (1), 9178,  DOI: 10.1038/s41598-023-36110-7
      40
      The protein and miRNA profile of plasma extracellular vesicles (EVs) can distinguish feline mammary adenocarcinoma patients from healthy feline controls
      Howard, Jane; Browne, John; Bollard, Stephanie; Peters, Susan; Sweeney, Ciara; Wynne, Kieran; Potter, Shirley; McCann, Amanda; Kelly, Pamela
      Scientific Reports (2023), 13 (1), 9178CODEN: SRCEC3; ISSN:2045-2322. (Nature Portfolio)
      Feline mammary adenocarcinomas (FMA) are aggressive tumors with metastatic capability and limited treatment options. This study aims to investigate whether miRNAs assocd. with FMA tumors are secreted in extracellular vesicles (EVs) and whether they can potentially be used as a cancer biomarker in EVs from feline plasma. Tumors and matched tumor free margins from 10 felines with FMA were selected. Following a detailed literature search, RT-qPCR analyses of 90 miRNAs identified 8 miRNAs of interest for further investigation. Tumor tissue, margins and plasma were subsequently collected from a further 10 felines with FMA. EVs were isolated from the plasma. RT-qPCR expression analyses of the 8 miRNAs of interest were carried out in tumor tissue, margins, FMA EVs and control EVs. Addnl., proteomic anal. of both control and FMA plasma derived EVs was undertaken. RT-qPCR revealed significantly increased miR-20a and miR-15b in tumors compared to margins. A significant decrease in miR-15b and miR-20a was detected in EVs from FMAs compared to healthy feline EVs. The proteomic content of EVs distinguished FMAs from controls, with the protein targets of miR-20a and miR-15b also displaying lower levels in the EVs from patients with FMA. This study has demonstrated that miRNAs are readily detectable in both the tissue and plasma derived EVs from patients with FMA. These miRNAs and their protein targets are a detectable panel of markers in circulating plasma EVs that may inform future diagnostic tests for FMA in a non-invasive manner. Moreover, the clin. relevance of miR-20a and miR-15b warrants further investigation.
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    41. 41
      Alvarez-Erviti, L.; Seow, Y.; Schapira, A. H.; Gardiner, C.; Sargent, I. L.; Wood, M. J.; Cooper, J. M. Lysosomal dysfunction increases exosome-mediated alpha-synuclein release and transmission. Neurobiol. Dis. 2011, 42 (3), 360– 367,  DOI: 10.1016/j.nbd.2011.01.029
      41
      Lysosomal dysfunction increases exosome-mediated alpha-synuclein release and transmission
      Alvarez-Erviti, Lydia; Seow, Yiqi; Schapira, Anthony H.; Gardiner, Chris; Sargent, Ian L.; Wood, Matthew J. A.; Cooper, J. Mark
      Neurobiology of Disease (2011), 42 (3), 360-367CODEN: NUDIEM; ISSN:0969-9961. (Elsevier B.V.)
      Alpha-synuclein aggregation plays a central role in Parkinson's disease pathol. Direct transmission of alpha-synuclein from pathol. affected to healthy unaffected neurons may be important in the anatomical spread of the disease through the nervous system. We have demonstrated that exosomes released from alpha-synuclein over-expressing SH-SY5Y cells contained alpha-synuclein and these exosomes were capable of efficiently transferring alpha-synuclein protein to normal SH-SY5Y cells. Moreover, the incubation of cells with ammonium chloride or bafilomycin A1 to produce the lysosomal dysfunction recently reported in Parkinson's disease led to an increase in the release of alpha-synuclein in exosomes and a concomitant increase in alpha-synuclein transmission to recipient cells. This study clearly demonstrates the importance of exosomes in both the release of alpha synuclein and its transmission between cells and suggests that factors assocd. with PD pathol. accelerate this process. These mechanisms may play an important role in PD pathol. and provide a suitable target for therapeutic intervention.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXkvVGlur0%253D&md5=de46764fe9cbf14695df7e90fe185704
    42. 42
      Biancalana, M.; Koide, S. Molecular mechanism of Thioflavin-T binding to amyloid fibrils. Biochim. Biophys. Acta 2010, 1804 (7), 1405– 1412,  DOI: 10.1016/j.bbapap.2010.04.001
      42
      Molecular mechanism of thioflavin-T binding to amyloid fibrils
      Biancalana, Matthew; Koide, Shohei
      Biochimica et Biophysica Acta, Proteins and Proteomics (2010), 1804 (7), 1405-1412CODEN: BBAPBW; ISSN:1570-9639. (Elsevier B. V.)
      A review. Intense efforts to detect, diagnose, and analyze the kinetic and structural properties of amyloid fibrils have generated a powerful toolkit of amyloid-specific mol. probes. Since its 1st description in 1959, the fluorescent dye, thioflavin-T (ThT), has become among the most widely used "gold stds." for selectively staining and identifying amyloid fibrils both in vivo and in vitro. The large enhancement of its fluorescence emission upon its binding to fibrils makes ThT a particularly powerful and convenient tool. Despite its widespread use in clin. and basic science applications, the mol. mechanism for the ability of ThT to recognize diverse types of amyloid fibrils and for the dye's characteristic fluorescence has only begun to be elucidated. Here, the authors review recent progress in the understanding of ThT-fibril interactions at at. resoln. These studies have yielded important insights into amyloid structures and the processes of fibril formation, and they also offer guidance for designing the next generation of amyloid assembly diagnostics, inhibitors, and therapeutics.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXmsVGlsbs%253D&md5=81e2da6940732c11dd04e4ae078c5741
    43. 43
      Sasanian, N.; Bernson, D.; Horvath, I.; Wittung-Stafshede, P.; Esbjörner, E. K. Redox-Dependent Copper Ion Modulation of Amyloid-β (1-42) Aggregation In Vitro. Biomolecules 2020, 10 (6), 924,  DOI: 10.3390/biom10060924
      43
      Redox-dependent copper ion modulation of amyloid-β (1-42) aggregation in vitro
      Sasanian, Nima; Bernson, David; Horvath, Istvan; Wittung-Stafshede, Pernilla; Esbjoerner, Elin K.
      Biomolecules (2020), 10 (6), 924CODEN: BIOMHC; ISSN:2218-273X. (MDPI AG)
      Plaque deposits composed of amyloid-β (Aβ) fibrils are pathol. hallmarks of Alzheimer's disease (AD). Although copper ion dyshomeostasis is apparent in AD brains and copper ions are found co-deposited with Aβ peptides in patients' plaques, the mol. effects of copper ion interactions and redox-state dependence on Aβ aggregation remain elusive. By combining biophys. and theor. approaches, we here show that Cu2+ (oxidized) and Cu+ (reduced) ions have opposite effects on the assembly kinetics of recombinant Aβ(1-42) into amyloid fibrils in vitro. Cu2+ inhibits both the unseeded and seeded aggregation of Aβ(1-42) at pH 8.0. Using math. models to fit the kinetic data, we find that Cu2+ prevents fibril elongation. The Cu2+-mediated inhibition of Aβ aggregation shows the largest effect around pH 6.0 but is lost at pH 5.0, which corresponds to the pH in lysosomes. In contrast to Cu2+, Cu+ ion binding mildly catalyzes the Aβ(1-42) aggregation via a mechanism that accelerates primary nucleation, possibly via the formation of Cu+-bridged Aβ(1-42) dimers. Taken together, our study emphasizes redox-dependent copper ion effects on Aβ(1-42) aggregation and thereby provides further knowledge of putative copper-dependent mechanisms resulting in AD.
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    44. 44
      Longobardi, A.; Nicsanu, R.; Bellini, S.; Squitti, R.; Catania, M.; Tiraboschi, P.; Saraceno, C.; Ferrari, C.; Zanardini, R.; Binetti, G. Cerebrospinal Fluid EV Concentration and Size Are Altered in Alzheimer’s Disease and Dementia with Lewy Bodies. Cells 2022, 11 (3), 462,  DOI: 10.3390/cells11030462
      44
      Cerebrospinal Fluid EV Concentration and Size Are Altered in Alzheimer's Disease and Dementia with Lewy Bodies
      Longobardi, Antonio; Nicsanu, Roland; Bellini, Sonia; Squitti, Rosanna; Catania, Marcella; Tiraboschi, Pietro; Saraceno, Claudia; Ferrari, Clarissa; Zanardini, Roberta; Binetti, Giuliano; Di Fede, Giuseppe; Benussi, Luisa; Ghidoni, Roberta
      Cells (2022), 11 (3), 462CODEN: CELLC6; ISSN:2073-4409. (MDPI AG)
      Alzheimer's disease (AD), dementia with Lewy bodies (DLB) and frontotemporal dementia (FTD) represent the three major neurodegenerative dementias characterized by abnormal brain protein accumulation. In this study, we investigated extracellular vesicles (EVs) and neurotrophic factors in the cerebrospinal fluid (CSF) of 120 subjects: 36 with AD, 30 with DLB, 34 with FTD and 20 controls. Specifically, CSF EVs were analyzed by Nanoparticle Tracking Anal. and neurotrophic factors were measured with ELISA. We found higher EV concn. and lower EV size in AD and DLB groups compared to the controls. Classification tree anal. demonstrated EV size as the best parameter able to discriminate the patients from the controls (96.7% vs. 3.3%, resp.). The diagnostic performance of the EV concn./size ratio resulted in a fair discrimination level with an area under the curve of 0.74. Moreover, the EV concn./size ratio was assocd. with the p-Tau181/Aβ42 ratio in AD patients. In addn., we described altered levels of cystatin C and progranulin in the DLB and AD groups. We did not find any correlation between neurotrophic factors and EV parameters. In conclusion, the results of this study suggest a common involvement of the endosomal pathway in neurodegenerative dementias, giving important insight into the mol. mechanisms underlying these pathologies.
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    45. 45
      Pait, M. C.; Kaye, S. D.; Su, Y.; Kumar, A.; Singh, S.; Gironda, S. C.; Vincent, S.; Anwar, M.; Carroll, C. M.; Snipes, J. A. Novel method for collecting hippocampal interstitial fluid extracellular vesicles (EV-ISF) reveals sex-dependent changes in microglial EV proteome in response to Aβ pathology. bioRxiv 2023,  DOI: 10.1101/2023.03.10.532133
      There is no corresponding record for this reference.
    46. 46
      Limbocker, R.; Chia, S.; Ruggeri, F. S.; Perni, M.; Cascella, R.; Heller, G. T.; Meisl, G.; Mannini, B.; Habchi, J.; Michaels, T. C. T. Trodusquemine enhances Aβ42 aggregation but suppresses its toxicity by displacing oligomers from cell membranes. Nat. Commun. 2019, 10 (1), 225,  DOI: 10.1038/s41467-018-07699-5
      46
      Trodusquemine enhances Aβ42 aggregation but suppresses its toxicity by displacing oligomers from cell membranes
      Limbocker, Ryan; Chia, Sean; Ruggeri, Francesco S.; Perni, Michele; Cascella, Roberta; Heller, Gabriella T.; Meisl, Georg; Mannini, Benedetta; Habchi, Johnny; Michaels, Thomas C. T.; Challa, Pavan K.; Ahn, Minkoo; Casford, Samuel T.; Fernando, Nilumi; Xu, Catherine K.; Kloss, Nina D.; Cohen, Samuel I. A.; Kumita, Janet R.; Cecchi, Cristina; Zasloff, Michael; Linse, Sara; Knowles, Tuomas P. J.; Chiti, Fabrizio; Vendruscolo, Michele; Dobson, Christopher M.
      Nature Communications (2019), 10 (1), 225CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
      Transient oligomeric species formed during the aggregation process of the 42-residue form of the amyloid-β peptide (Aβ42) are key pathogenic agents in Alzheimer's disease (AD). To investigate the relationship between Aβ42 aggregation and its cytotoxicity and the influence of a potential drug on both phenomena, we have studied the effects of trodusquemine. This aminosterol enhances the rate of aggregation by promoting monomer-dependent secondary nucleation, but significantly reduces the toxicity of the resulting oligomers to neuroblastoma cells by inhibiting their binding to the cellular membranes. When administered to a C. elegans model of AD, we again observe an increase in aggregate formation alongside the suppression of Aβ42-induced toxicity. In addn. to oligomer displacement, the reduced toxicity could also point towards an increased rate of conversion of oligomers to less toxic fibrils. The ability of a small mol. to reduce the toxicity of oligomeric species represents a potential therapeutic strategy against AD.
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    47. 47
      Arosio, P.; Cukalevski, R.; Frohm, B.; Knowles, T. P.; Linse, S. Quantification of the Concentration of Aβ42 Propagons during the Lag Phase by an Amyloid Chain Reaction Assay. J. Am. Chem. Soc. 2014, 136 (1), 219– 225,  DOI: 10.1021/ja408765u
      47
      Quantification of the Concentration of Aβ42 Propagons during the Lag Phase by an Amyloid Chain Reaction Assay
      Arosio, Paolo; Cukalevski, Risto; Frohm, Birgitta; Knowles, Tuomas P. J.; Linse, Sara
      Journal of the American Chemical Society (2014), 136 (1), 219-225CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      The aggregation of the amyloid beta peptide, Aβ42, implicated in Alzheimer's disease, was characterized by a lag phase followed by a rapid growth phase. Conventional methods to study this reaction are not sensitive to events taking place early in the lag phase promoting the assumption that only monomeric or oligomeric species are present at early stages and that the lag time is defined by the primary nucleation rate only. Here the authors exploit the high sensitivity of chem. chain reactions to the reagent compn. to develop an assay which improves by 2 orders of magnitude the detection limit of conventional bulk techniques and allows the concn. of fibrillar Aβ42 propagons to be detected and quantified even during the lag time. The method relies on the chain reaction multiplication of a small no. of initial fibrils by secondary nucleation on the fibril surface in the presence of monomeric peptides, allowing the quantification of the no. of initial propagons by comparing the multiplication reaction kinetics with controlled seeding data. The quant. results of the chain reaction assay are confirmed by qual. TEM anal. The results demonstrate the nonlinearity of the aggregation process which involves both primary and secondary nucleation events even at the early stages of the reaction during the lag-phase.
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    48. 48
      Meisl, G.; Yang, X.; Hellstrand, E.; Frohm, B.; Kirkegaard, J. B.; Cohen, S. I.; Dobson, C. M.; Linse, S.; Knowles, T. P. Differences in nucleation behavior underlie the contrasting aggregation kinetics of the Aβ40 and Aβ42 peptides. Proc. Natl. Acad. Sci. U.S.A. 2014, 111 (26), 9384– 9389,  DOI: 10.1073/pnas.1401564111
      48
      Differences in nucleation behavior underlie the contrasting aggregation kinetics of the Aβ40 and Aβ42 peptides
      Meisl, Georg; Yang, Xiaoting; Hellstrand, Erik; Frohm, Birgitta; Kirkegaard, Julius B.; Cohen, Samuel I. A.; Dobson, Christopher M.; Linse, Sara; Knowles, Tuomas P. J.
      Proceedings of the National Academy of Sciences of the United States of America (2014), 111 (26), 9384-9389CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      The two major forms of the amyloid-beta (Aβ) peptide found in plaques in patients suffering from Alzheimer's disease, Aβ40 and Aβ42, only differ by two amino acids in the C-terminal region, yet they display markedly different aggregation behavior. The origins of these differences have remained challenging to connect to specific mol.-level processes underlying the aggregation reaction. In this paper we use a general strategy to apply the conventional workflow of chem. kinetics to the aggregation of the Aβ40 peptide to identify the differences between Aβ40 and Aβ42 in terms of the microscopic determinants of the aggregation reaction. Our results reveal that the major source of aggregates in the case of Aβ40 is a fibril-catalyzed nucleation process, the multistep nature of which is evident through its satn. behavior. Moreover, our results show that the significant differences in the obsd. behavior of the two proteins originate not simply from a uniform increase in all microscopic rates for Aβ42 compared with Aβ40, but rather are due to a shift of more than one order of magnitude in the relative importance of primary nucleation vs. fibril-catalyzed secondary nucleation processes. This anal. sheds light on the microscopic determinants of the aggregation behavior of the principal forms of Aβ and outlines a general approach toward achieving an understanding at the mol. level of the aberrant deposition of insol. peptides in neurodegenerative disorders.
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    49. 49
      Xue, W. F.; Hellewell, A. L.; Gosal, W. S.; Homans, S. W.; Hewitt, E. W.; Radford, S. E. Fibril fragmentation enhances amyloid cytotoxicity. J. Biol. Chem. 2009, 284 (49), 34272– 34282,  DOI: 10.1074/jbc.M109.049809
      49
      Fibril Fragmentation Enhances Amyloid Cytotoxicity
      Xue, Wei-Feng; Hellewell, Andrew L.; Gosal, Walraj S.; Homans, Steve W.; Hewitt, Eric W.; Radford, Sheena E.
      Journal of Biological Chemistry (2009), 284 (49), 34272-34282CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Fibrils assocd. with amyloid disease are mol. assemblies of key biol. importance, yet how cells respond to the presence of amyloid remains unclear. Cellular responses may not only depend on the chem. compn. or mol. properties of the amyloid fibrils, but their phys. attributes such as length, width, or surface area may also play important roles. Here, we report a systematic investigation of the effect of fragmentation on the structural and biol. properties of amyloid fibrils. In addn. to the expected relationship between fragmentation and the ability to seed, we show a striking finding that fibril length correlates with the ability to disrupt membranes and to reduce cell viability. Thus, despite otherwise unchanged mol. architecture, shorter fibrillar samples show enhanced cytotoxic potential than their longer counterparts. The results highlight the importance of fibril length in amyloid disease, with fragmentation not only providing a mechanism by which fibril load can be rapidly increased but also creating fibrillar species of different dimensions that can endow new or enhanced biol. properties such as amyloid cytotoxicity.
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    50. 50
      Colvin, M. T.; Silvers, R.; Ni, Q. Z.; Can, T. V.; Sergeyev, I.; Rosay, M.; Donovan, K. J.; Michael, B.; Wall, J.; Linse, S. Atomic Resolution Structure of Monomorphic Aβ42 Amyloid Fibrils. J. Am. Chem. Soc. 2016, 138 (30), 9663– 9674,  DOI: 10.1021/jacs.6b05129
      50
      Atomic Resolution Structure of Monomorphic Aβ42 Amyloid Fibrils
      Colvin, Michael T.; Silvers, Robert; Ni, Qing Zhe; Can, Thach V.; Sergeyev, Ivan; Rosay, Melanie; Donovan, Kevin J.; Michael, Brian; Wall, Joseph; Linse, Sara; Griffin, Robert G.
      Journal of the American Chemical Society (2016), 138 (30), 9663-9674CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Amyloid-β (Aβ) is a 39-42 residue protein produced by the cleavage of the amyloid precursor protein (APP), which subsequently aggregates to form cross-β amyloid fibrils that are a hallmark of Alzheimer's disease (AD). The most prominent forms of Aβ are Aβ1-40 and Aβ1-42, which differ by two amino acids (I and A) at the C-terminus. However, Aβ42 is more neurotoxic and essential to the etiol. of AD. Here, we present an at. resoln. structure of a monomorphic form of AβM01-42 amyloid fibrils derived from over 500 13C-13C, 13C-15N distance and backbone angle structural constraints obtained from high field magic angle spinning NMR spectra. The structure (PDB ID: 5KK3) shows that the fibril core consists of a dimer of Aβ42 mols., each contg. four β-strands in a S-shaped amyloid fold, and arranged in a manner that generates two hydrophobic cores that are capped at the end of the chain by a salt bridge. The outer surface of the monomers presents hydrophilic side chains to the solvent. The interface between the monomers of the dimer shows clear contacts between M35 of one mol. and L17 and Q15 of the second. Intermol. 13C-15N constraints demonstrate that the amyloid fibrils are parallel in register. The RMSD of the backbone structure (Q15-A42) is 0.71 ± 0.12 Å and of all heavy atoms is 1.07 ± 0.08 Å. The structure provides a point of departure for the design of drugs that bind to the fibril surface and therefore interfere with secondary nucleation and for other therapeutic approaches to mitigate Aβ42 aggregation.
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    51. 51
      Lansbury, P. T.; Costa, P. R.; Griffiths, J. M.; Simon, E. J.; Auger, M.; Halverson, K. J.; Kocisko, D. A.; Hendsch, Z. S.; Ashburn, T. T.; Spencer, R. G. Structural model for the β-amyloid fibril based on interstrand alignment of an antiparallel-sheet comprising a C-terminal peptide. Nat. Struct. Biol. 1995, 2 (11), 990– 998,  DOI: 10.1038/nsb1195-990
      51
      Structural model for the β-amyloid fibril based on interstrand alignment of an antiparallel-sheet comprising a C-terminal peptide
      Lansbury, Peter T., Jr.; Costa, Philip R.; Griffiths, Janet M.; Simon, Eric J.; Auger, Michele; Halverson, Kurt J.; Kocisko, David A.; Hendsch, Zachary S.; Ashburn, Ted T.; et al.
      Nature Structural Biology (1995), 2 (11), 990-8CODEN: NSBIEW; ISSN:1072-8368. (Nature Publishing Co.)
      Amyloids are a class of noncryst., yet ordered, protein aggregates. A new approach was used to provide the initial structural data on an amyloid fibril-comprising a peptide (β34-42) from the C-terminus of the β-amyloid protein-based on measurement of intramol. 13C-13C distances and 13C chem. shifts by solid-state 13C NMR and individual amide absorption frequencies by isotope-edited IR spectroscopy. Intermol. orientation and alignment within the amyloid sheet was detd. by fitting models to obsd. intermol. 13C-13C couplings. Although the structural model the authors present is defined to relatively low resoln., it nevertheless shows a pleated antiparallel β-sheet characterized by a specific intermol. alignment.
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    52. 52
      Meinhardt, J.; Sachse, C.; Hortschansky, P.; Grigorieff, N.; Fandrich, M. Aβ(1-40) Fibril Polymorphism Implies Diverse Interaction Patterns in Amyloid Fibrils. J. Mol. Biol. 2009, 386 (3), 869– 877,  DOI: 10.1016/j.jmb.2008.11.005
      52
      Aβ(1-40) Fibril Polymorphism Implies Diverse Interaction Patterns in Amyloid Fibrils
      Meinhardt, Jessica; Sachse, Carsten; Hortschansky, Peter; Grigorieff, Nikolaus; Faendrich, Marcus
      Journal of Molecular Biology (2009), 386 (3), 869-877CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
      Amyloid fibrils characterize a diverse group of human diseases that includes Alzheimer's disease, Creutzfeldt-Jakob, and type II diabetes. Alzheimer's amyloid fibrils consist of amyloid-β (Aβ) peptide and occur in a range of structurally different fibril morphologies. The structural characteristics of 12 single Aβ(1-40) amyloid fibrils, all formed under the same soln. conditions, were detd. by electron cryo-microscopy and three-dimensional reconstruction. The majority of analyzed fibrils form a range of morphologies that show almost continuously altering structural properties. The obsd. fibril polymorphism implies that for the same polypeptide sequence, amyloid formation can lead to many different patterns of inter- or intra-residue interactions. This property differs significantly from native, monomeric protein folding reactions that for one protein sequence, produce only one ordered conformation and only one set of inter-residue interactions.
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    53. 53
      Hellstrand, E.; Nowacka, A.; Topgaard, D.; Linse, S.; Sparr, E. Membrane Lipid Co-Aggregation with α-Synuclein Fibrils. PLoS One 2013, 8 (10), e77235  DOI: 10.1371/journal.pone.0077235
      53
      Membrane lipid co-aggregation with α-synuclein fibrils
      Hellstrand, Erik; Nowacka, Agnieszka; Topgaard, Daniel; Linse, Sara; Sparr, Emma
      PLoS One (2013), 8 (10), e77235CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
      Amyloid deposits from several human diseases have been found to contain membrane lipids. Co-aggregation of lipids and amyloid proteins in amyloid aggregates, and the related extn. of lipids from cellular membranes, can influence structure and function in both the membrane and the formed amyloid deposit. Co-aggregation can therefore have important implications for the pathol. consequences of amyloid formation. Still, very little is known about the mechanism behind co-aggregation and mol. structure in the formed aggregates. To address this, we study in vitro co-aggregation by incubating phospholipid model membranes with the Parkinson's disease-assocd. protein, α-synuclein, in monomeric form. After aggregation, we find spontaneous uptake of phospholipids from anionic model membranes into the amyloid fibrils. Phospholipid quantification, polarization transfer solid-state NMR and cryo-TEM together reveal co-aggregation of phospholipids and α-synuclein in a saturable manner with a strong dependence on lipid compn. At low lipid to protein ratios, there is a close assocn. of phospholipids to the fibril structure, which is apparent from reduced phospholipid mobility and morphol. changes in fibril bundling. At higher lipid to protein ratios, addnl. vesicles adsorb along the fibrils. While interactions between lipids and amyloid-protein are generally discussed within the perspective of different protein species adsorbing to and perturbing the lipid membrane, the current work reveals amyloid formation in the presence of lipids as a co-aggregation process. The interaction leads to the formation of lipid-protein co-aggregates with distinct structure, dynamics and morphol. compared to assemblies formed by either lipid or protein alone.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhs1Cqu7bI&md5=390689802588f5a8d437d89cc060796f
    54. 54
      Yuyama, K.; Sun, H.; Mitsutake, S.; Igarashi, Y. Sphingolipid-modulated Exosome Secretion Promotes Clearance of Amyloid-β by Microglia. J. Biol. Chem. 2012, 287 (14), 10977– 10989,  DOI: 10.1074/jbc.M111.324616
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      Sphingolipid-modulated exosome secretion promotes clearance of amyloid-β by microglia
      Yuyama, Kohei; Sun, Hui; Mitsutake, Susumu; Igarashi, Yasuyuki
      Journal of Biological Chemistry (2012), 287 (14), 10977-10989CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Amyloid β-peptide (Aβ), the pathogenic agent of Alzheimer disease, is a physiol. metabolite whose levels are constantly controlled in normal brain. Recent studies have demonstrated that a fraction of extracellular Aβ is assocd. with exosomes, small membrane vesicles of endosomal origin, although the fate of Aβ in assocn. with exosome is largely unknown. In this study, we identified novel roles for neuron-derived exosomes acting on extracellular Aβ, i.e. exosomes drive conformational changes in Aβ to form nontoxic amyloid fibrils and promote uptake of Aβ by microglia. The Aβ internalized together with exosomes was further transported to lysosomes and degraded. We also found that blockade of phosphatidylserine on the surface of exosomes by annexin V not only prevented exosome uptake but also suppressed Aβ incorporation into microglia. In addn., we demonstrated that secretion of neuron-derived exosomes was modulated by the activities of sphingolipid-metabolizing enzymes, including neutral sphingomyelinase 2 (nSMase2) and sphingomyelin synthase 2 (SMS2). In transwell expts., up-regulation of exosome secretion from neuronal cells by treatment with SMS2 siRNA enhanced Aβ uptake into microglial cells and significantly decreased extracellular levels of Aβ. Our findings indicate a novel mechanism responsible for clearance of Aβ through its assocn. with exosomes. The modulation of the vesicle release and/or elimination may alter the risk of AD.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XkvVWls7g%253D&md5=9ace83688666fc983f358750d5028458
    55. 55
      Perez-Gonzalez, R.; Gauthier, S. A.; Kumar, A.; Levy, E. The exosome secretory pathway transports amyloid precursor protein carboxyl-terminal fragments from the cell into the brain extracellular space. J. Biol. Chem. 2012, 287 (51), 43108– 43115,  DOI: 10.1074/jbc.M112.404467
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      The exosome secretory pathway transports amyloid precursor protein Carboxyl-terminal fragments from the cell into the brain extracellular space
      Perez-Gonzalez, Rocio; Gauthier, Sebastien A.; Kumar, Asok; Levy, Efrat
      Journal of Biological Chemistry (2012), 287 (51), 43108-43115CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      In vitro studies have shown that neuronal cell cultures secrete exosomes contg. amyloid-β precursor protein (APP) and the APP-processing products, C-terminal fragments (CTFs) and amyloid-β (Aβ). We investigated the secretion of full-length APP (flAPP) and APP CTFs via the exosome secretory pathway in vivo. To this end, we developed a novel protocol designed to isolate exosomes secreted into mouse brain extracellular space. Exosomes with typical morphol. were isolated from freshly removed mouse brains and from frozen mouse and human brain tissues, demonstrating that exosomes can be isolated from post-mortem tissue frozen for long periods of time. flAPP, APP CTFs, and enzymes that cleave both flAPP and APP CTFs were identified in brain exosomes. Although higher levels of both flAPP and APP CTFs were obsd. in exosomes isolated from the brains of transgenic mice overexpressing human APP (Tg2576) compared with wild-type control mice, there was no difference in the no. of secreted brain exosomes. These data indicate that the levels of flAPP and APP CTFs assocd. with exosomes mirror the cellular levels of flAPP and APP CTFs. Interestingly, exosomes isolated from the brains of both Tg2576 and wild-type mice are enriched with APP CTFs relative to flAPP. Thus, we hypothesize that the exosome secretory pathway plays a pleiotropic role in the brain: exosome secretion is beneficial to the cell, acting as a specific releasing system of neurotoxic APP CTFs and Aβ, but the secretion of exosomes enriched with APP CTFs, neurotoxic proteins that are also a source of secreted Aβ, is harmful to the brain.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhvVeksbnI&md5=9dd8afd9aab9b4d54c688cc9409d72d9
    56. 56
      Musteikyte, G.; Jayaram, A. K.; Xu, C. K.; Vendruscolo, M.; Krainer, G.; Knowles, T. P. J. Interactions of α-synuclein oligomers with lipid membranes. Biochim. Biophys. Acta, Biomembr. 2021, 1863 (4), 183536,  DOI: 10.1016/j.bbamem.2020.183536
      56
      Interactions of α-synuclein oligomers with lipid membranes
      Musteikyte, Greta; Jayaram, Akhila K.; Xu, Catherine K.; Vendruscolo, Michele; Krainer, Georg; Knowles, Tuomas P. J.
      Biochimica et Biophysica Acta, Biomembranes (2021), 1863 (4), 183536CODEN: BBBMBS; ISSN:0005-2736. (Elsevier B.V.)
      A review. Parkinson's disease is an increasingly prevalent and currently incurable neurodegenerative disorder. At the mol. level, this disease is characterized by the formation of aberrant intracellular protein deposits known as Lewy bodies. Oligomeric forms of the protein α-synuclein (αS), which are believed to be both intermediates and byproducts of Lewy body formation, are considered to be the main pathogenic species. Interactions of such oligomers with lipid membranes are increasingly emerging as a major mol. pathway underpinning their toxicity. Here author review recent progress in author understanding of the interactions of αS oligomers with lipid membranes. Author highlight key structural and biophys. features of αS oligomers, the effects of these features on αS oligomer membrane binding properties, and resultant implications for understanding the etiol. of Parkinson's disease. Author discuss mechanistic modes of αS oligomer-lipid membrane interactions and the effects of environmental factors to such modes. Finally, author provide an overview of the current understanding of the main mol. determinants of αS oligomer toxicity in vivo.
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    57. 57
      Skotland, T.; Sagini, K.; Sandvig, K.; Llorente, A. An emerging focus on lipids in extracellular vesicles. Adv. Drug Delivery Rev. 2020, 159, 308– 321,  DOI: 10.1016/j.addr.2020.03.002
      57
      An emerging focus on lipids in extracellular vesicles
      Skotland, Tore; Sagini, Krizia; Sandvig, Kirsten; Llorente, Alicia
      Advanced Drug Delivery Reviews (2020), 159 (), 308-321CODEN: ADDREP; ISSN:0169-409X. (Elsevier B.V.)
      A review. Extracellular vesicles contain a lipid bilayer membrane that protects the encapsulated material, such as proteins, nucleic acids, lipids and metabolites, from the extracellular environment. These vesicles are released from cells via different mechanisms. During recent years extracellular vesicles have been studied as possible biomarkers for different diseases, as biol. nanoparticles for drug delivery, and in basic studies as a tool to understand the structure of biol. membranes and the mechanisms involved in vesicular trafficking. Lipids are essential mol. components of extracellular vesicles, but at the moment our knowledge about the lipid compn. and the function of lipids in these vesicles is limited. However, the interest of the research community in these mols. is increasing as their role in extracellular vesicles is starting to be acknowledged. In this review, we will present the status of the field and describe what is needed to bring it forward.
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    58. 58
      Skotland, T.; Hessvik, N. P.; Sandvig, K.; Llorente, A. Exosomal lipid composition and the role of ether lipids and phosphoinositides in exosome biology. J. Lipid Res. 2019, 60 (1), 9– 18,  DOI: 10.1194/jlr.R084343
      58
      Exosomal lipid composition and the role of ether lipids and phosphoinositides in exosome biology
      Skotland, Tore; Hessvik, Nina P.; Sandvig, Kirsten; Llorente, Alicia
      Journal of Lipid Research (2019), 60 (1), 9-18CODEN: JLPRAW; ISSN:0022-2275. (American Society for Biochemistry and Molecular Biology)
      A review. Exosomes are a type of extracellular vesicle released from cells after fusion of multivesicular bodies with the plasma membrane. These vesicles are often enriched in cholesterol, SM, glycosphingolipids, and phosphatidylserine. Lipids not only have a structural role in exosomal membranes but also are essential players in exosome formation and release to the extracellular environment. Our knowledge about the importance of lipids in exosome biol. is increasing due to recent technol. developments in lipidomics and a stronger focus on the biol. functions of these mols. Here, we review the available information about the lipid compn. of exosomes. Special attention is given to ether lipids, a relatively unexplored type of lipids involved in membrane trafficking and abundant in some exosomes. Moreover, we discuss how the lipid compn. of exosome prepns. may provide useful information about their purity. Finally, we discuss the role of phosphoinositides, membrane phospholipids that help to regulate membrane dynamics, in exosome release and how this process may be linked to secretory autophagy. Knowledge about exosome lipid compn. is important to understand the biol. of these vesicles and to investigate possible medical applications.
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    59. 59
      Ghadami, S.; Dellinger, K. The lipid composition of extracellular vesicles: applications in diagnostics and therapeutic delivery. Front. Mol. Biosci. 2023, 10, 1198044,  DOI: 10.3389/fmolb.2023.1198044
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      The lipid composition of extracellular vesicles: applications in diagnostics and therapeutic delivery
      Ghadami, Samaneh; Dellinger, Kristen
      Frontiers in Molecular Biosciences (2023), 10 (), 1198044CODEN: FMBRBS; ISSN:2296-889X. (Frontiers Media S.A.)
      A review. Extracellular vesicles (EVs), including exosomes, with nanoscale sizes, biol. origins, various functions, and unique lipid and protein compns. have been introduced as versatile tools for diagnostic and therapeutic medical applications. Numerous studies have reported the importance of the lipid compn. of EVs and its influence on their mechanism of action. For example, changes in the lipidomic profile of EVs have been shown to influence the progression of various diseases, including ovarian malignancies and prostate cancer. In this review, we endeavored to examine differences in the lipid content of EV membranes derived from different cell types to characterize their capabilities as diagnostic tools and treatments for diseases like cancer and Alzheimer's disease. We addnl. discuss designing functionalized vesicles, whether synthetically by hybrid methods or by changing the lipid compn. of natural EVs. Lastly, we provide an overview of current and potential biomedical applications and perspectives on the future of this growing field.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXhs1Gms7bO&md5=c562ed61ca54d6243ec8f098fa707570
    60. 60
      Lindberg, D. J.; Wesen, E.; Bjorkeroth, J.; Rocha, S.; Esbjörner, E. K. Lipid membranes catalyse the fibril formation of the amyloid-β (1–42) peptide through lipid-fibril interactions that reinforce secondary pathways. Biochim. Biophys. Acta, Biomembr. 2017, 1859 (10), 1921– 1929,  DOI: 10.1016/j.bbamem.2017.05.012
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      Lipid membranes catalyze the fibril formation of the amyloid-β(1-42) peptide through lipid-fibril interactions that reinforce secondary pathways
      Lindberg, David J.; Wesen, Emelie; Bjoerkeroth, Johan; Rocha, Sandra; Esbjoerner, Elin K.
      Biochimica et Biophysica Acta, Biomembranes (2017), 1859 (10), 1921-1929CODEN: BBBMBS; ISSN:0005-2736. (Elsevier B.V.)
      Alzheimer's disease is assocd. with the aggregation of amyloid-β (Aβ) peptides into oligomers and fibrils. We have explored how model lipid membranes modulate the rate and mechanisms of Aβ(1-42) self-assembly, in order to shed light on how this pathol. reaction may occur in the lipid-rich environments that the peptide encounters in the brain. Using a combination of in vitro biophys. expts. and theor. approaches, we show that zwitterionic DOPC lipid vesicles accelerate the Aβ(1-42) fibril growth rate by interacting specifically with the growing fibrils. We probed this interaction with help of a purpose-developed FRET assay that monitors the proximity between a fibril-specific dye and fluorescent lipids in the lipid vesicle membrane. To further rationalize these findings, we used math. models to fit the aggregation kinetics of Aβ(1-42) and found that lipid vesicles altered specific mechanistic steps in the aggregation reaction; they augmented monomer-dependent secondary nucleation at the surface of existing fibrils and facilitated monomer-independent catalytic processes consistent with fibril fragmentation. We further showed that DOPC vesicles had no effect on primary nucleation. This finding was consistent with expts. showing that Aβ(1-42) monomers do not directly bind to the lipid bilayer. Taken together, these results show that plain lipid membranes with charge and compn. that is representative of outer cell membranes can significantly augment autocatalytic steps in the self-assembly of Aβ(1-42) into fibrils. This new insight suggests that strategies to reduce fibril-lipid interactions in the brain may have therapeutic value.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtFCgu7fL&md5=3711a1bebb5da2ffbb33ae69302b67da
    61. 61
      Habchi, J.; Chia, S.; Galvagnion, C.; Michaels, T. C. T.; Bellaiche, M. M. J.; Ruggeri, F. S.; Sanguanini, M.; Idini, I.; Kumita, J. R.; Sparr, E. Cholesterol catalyses Aβ42 aggregation through a heterogeneous nucleation pathway in the presence of lipid membranes. Nat. Chem. 2018, 10 (6), 673– 683,  DOI: 10.1038/s41557-018-0031-x
      61
      Cholesterol catalyzes Aβ42 aggregation through a heterogeneous nucleation pathway in the presence of lipid membranes
      Habchi, Johnny; Chia, Sean; Galvagnion, Celine; Michaels, Thomas C. T.; Bellaiche, Mathias M. J.; Ruggeri, Francesco Simone; Sanguanini, Michele; Idini, Ilaria; Kumita, Janet R.; Sparr, Emma; Linse, Sara; Dobson, Christopher M.; Knowles, Tuomas P. J.; Vendruscolo, Michele
      Nature Chemistry (2018), 10 (6), 673-683CODEN: NCAHBB; ISSN:1755-4330. (Nature Research)
      Alzheimer's disease is a neurodegenerative disorder assocd. with the aberrant aggregation of the amyloid-β peptide. Although increasing evidence implicates cholesterol in the pathogenesis of Alzheimer's disease, the detailed mechanistic link between this lipid mol. and the disease process remains to be fully established. To address this problem, we adopted a kinetics-based strategy that revealed a specific catalytic role of cholesterol in the aggregation of Aβ42 (the 42-residue form of the amyloid-β peptide). More specifically, we demonstrated that lipid membranes contg. cholesterol promoted Aβ42 aggregation by enhancing its primary nucleation rate by up to 20-fold through a heterogeneous nucleation pathway. We further showed that this process occurred as a result of cooperativity in the interaction of multiple cholesterol mols. with Aβ42. Thus. these results identify a specific microscopic pathway by which cholesterol dramatically enhances the onset of Aβ42 aggregation, thereby helping rationalize the link between Alzheimer's disease and the impairment of cholesterol homeostasis.
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    62. 62
      Amaro, M.; Sachl, R.; Aydogan, G.; Mikhalyov, I. I.; Vacha, R.; Hof, M. GM1 Ganglioside Inhibits β-Amyloid Oligomerization Induced by Sphingomyelin. Angew. Chem., Int. Ed. Engl. 2016, 55 (32), 9411– 9415,  DOI: 10.1002/anie.201603178
      62
      GM1 Ganglioside Inhibits β-Amyloid Oligomerization Induced by Sphingomyelin
      Amaro Mariana; Sachl Radek; Aydogan Gokcan; Hof Martin; Mikhalyov Ilya I; Vacha Robert
      Angewandte Chemie (International ed. in English) (2016), 55 (32), 9411-5 ISSN:.
      β-Amyloid (Aβ) oligomers are neurotoxic and implicated in Alzheimer's disease. Neuronal plasma membranes may mediate formation of Aβ oligomers in vivo. Membrane components sphingomyelin and GM1 have been shown to promote aggregation of Aβ; however, these studies were performed under extreme, non-physiological conditions. We demonstrate that physiological levels of GM1 , organized in nanodomains do not seed oligomerization of Aβ40 monomers. We show that sphingomyelin triggers oligomerization of Aβ40 and that GM1 is counteractive thus preventing oligomerization. We propose a molecular explanation that is supported by all-atom molecular dynamics simulations. The preventive role of GM1 in the oligomerization of Aβ40 suggests that decreasing levels of GM1 in the brain, for example, due to aging, could reduce protection against Aβ oligomerization and contribute to the onset of Alzheimer's disease.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2s%252Fpt12rtw%253D%253D&md5=6deb9f91515a8a068fa76524aaa0a4c9
    63. 63
      Sanguanini, M.; Baumann, K. N.; Preet, S.; Chia, S.; Habchi, J.; Knowles, T. P. J.; Vendruscolo, M. Complexity in Lipid Membrane Composition Induces Resilience to Aβ42 Aggregation. ACS Chem. Neurosci. 2020, 11 (9), 1347– 1352,  DOI: 10.1021/acschemneuro.0c00101
      63
      Complexity in Lipid Membrane Composition Induces Resilience to Aβ42 Aggregation
      Sanguanini, Michele; Baumann, Kevin N.; Preet, Swapan; Chia, Sean; Habchi, Johnny; Knowles, Tuomas P. J.; Vendruscolo, Michele
      ACS Chemical Neuroscience (2020), 11 (9), 1347-1352CODEN: ACNCDM; ISSN:1948-7193. (American Chemical Society)
      The mol. origins of Alzheimer's disease are assocd. with the aggregation of the amyloid-β peptide (Aβ). This process is controlled by a complex cellular homeostasis system, which involves a variety of components, including proteins, metabolites, and lipids. It has been shown in particular that certain components of lipid membranes can speed up Aβ aggregation. This observation prompts the question of whether there are protective cellular mechanisms to counterbalance this effect. Here, to address this issue, we investigate the role of the compn. of lipid membranes in modulating the aggregation process of Aβ. By adopting a chem. kinetics approach, we first identify a panel of lipids that affect the aggregation of the 42-residue form of Aβ (Aβ42), ranging from enhancement to inhibition. We then show that these effects tend to av. out in mixts. of these lipids, as such mixts. buffer extreme aggregation behaviors as the no. of components increases. These results indicate that a degree of quality control on protein aggregation can be achieved through a mechanism by which an increase in the mol. complexity of lipid membranes balances opposite effects and creates resilience to aggregation.
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    64. 64
      Noori, L.; Filip, K.; Nazmara, Z.; Mahakizadeh, S.; Hassanzadeh, G.; Caruso Bavisotto, C.; Bucchieri, F.; Marino Gammazza, A.; Cappello, F.; Wnuk, M. Contribution of Extracellular Vesicles and Molecular Chaperones in Age-Related Neurodegenerative Disorders of the CNS. Int. J. Mol. Sci. 2023, 24 (2), 927,  DOI: 10.3390/ijms24020927
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      Contribution of Extracellular Vesicles and Molecular Chaperones in Age-Related Neurodegenerative Disorders of the CNS
      Noori, Leila; Filip, Kamila; Nazmara, Zohreh; Mahakizadeh, Simin; Hassanzadeh, Gholamreza; Caruso Bavisotto, Celeste; Bucchieri, Fabio; Marino Gammazza, Antonella; Cappello, Francesco; Wnuk, Maciej; Scalia, Federica
      International Journal of Molecular Sciences (2023), 24 (2), 927CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)
      A review. Many neurodegenerative disorders are characterized by the abnormal aggregation of misfolded proteins that form amyloid deposits which possess prion-like behavior such as self-replication, intercellular transmission, and consequent induction of native forms of the same protein in surrounding cells. The distribution of the accumulated proteins and their correlated toxicity seem to be involved in the progression of nervous system degeneration. Mol. chaperones are known to maintain proteostasis, contribute to protein refolding to protect their function, and eliminate fatally misfolded proteins, prohibiting harmful effects. However, chaperone network efficiency declines during aging, prompting the onset and the development of neurol. disorders. Extracellular vesicles (EVs) are tiny membranous structures produced by a wide range of cells under physiol. and pathol. conditions, suggesting their significant role in fundamental processes particularly in cellular communication. They modulate the behavior of nearby and distant cells through their biol. cargo. In the pathol. context, EVs transport disease-causing entities, including prions, α-syn, and tau, helping to spread damage to non-affected areas and accelerating the progression of neurodegeneration. However, EVs are considered effective for delivering therapeutic factors to the nervous system, since they are capable of crossing the blood-brain barrier (BBB) and are involved in the transportation of a variety of cellular entities. Here, we review the neurodegeneration process caused mainly by the inefficiency of chaperone systems as well as EV performance in neuropathies, their potential as diagnostic biomarkers and a promising EV-based therapeutic approach.
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    65. 65
      Evans, C. G.; Wisen, S.; Gestwicki, J. E. Heat Shock Proteins 70 and 90 Inhibit Early Stages of Amyloid β-(1–42) Aggregation in Vitro. J. Biol. Chem. 2006, 281 (44), 33182– 33191,  DOI: 10.1074/jbc.M606192200
      65
      Heat shock proteins 70 and 90 inhibit early stages of amyloid β-(1-42) aggregation in Vitro
      Evans, Christopher G.; Wisen, Susanne; Gestwicki, Jason E.
      Journal of Biological Chemistry (2006), 281 (44), 33182-33191CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Alzheimer disease is a neurol. disorder that is characterized by the presence of fibrils and oligomers composed of the amyloid β (Aβ) peptide. In models of Alzheimer disease, overexpression of mol. chaperones, specifically heat shock protein 70 (Hsp70), suppresses phenotypes related to Aβ aggregation. These observations led to the hypothesis that chaperones might interact with Aβ and block self-assocn. However, although biochem. evidence to support this model has been collected in other neurodegenerative systems, the interaction between chaperones and Aβ has not been similarly explored. Here, we examine the effects of Hsp70/40 and Hsp90 on Aβ aggregation in vitro. We found that recombinant Hsp70/40 and Hsp90 block Aβ self-assembly and that these chaperones are effective at substoichiometric concns. (∼1:50). The anti-aggregation activity of Hsp70 can be inhibited by a nonhydrolyzable nucleotide analog and encouraged by pharmacol. stimulation of its ATPase activity. Finally, we were interested in discerning what type of amyloid structures can be acted upon by these chaperones. To address this question, we added Hsp70/40 and Hsp90 to pre-formed oligomers and fibrils. Based on thioflavin T reactivity, the combination of Hsp70/40 and Hsp90 caused structural changes in oligomers but had little effect on fibrils. These results suggest that if these chaperones are present in the same cellular compartment in which Aβ is produced, Hsp70/40 and Hsp90 may suppress the early stages of self-assembly. Thus, these results are consistent with a model in which pharmacol. activation of chaperones might have a favorable therapeutic effect on Alzheimer disease.
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    66. 66
      Arosio, P.; Michaels, T. C.; Linse, S.; Mansson, C.; Emanuelsson, C.; Presto, J.; Johansson, J.; Vendruscolo, M.; Dobson, C. M.; Knowles, T. P. Kinetic analysis reveals the diversity of microscopic mechanisms through which molecular chaperones suppress amyloid formation. Nat. Commun. 2016, 7, 10948,  DOI: 10.1038/ncomms10948
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      Kinetic analysis reveals the diversity of microscopic mechanisms through which molecular chaperones suppress amyloid formation
      Arosio, Paolo; Michaels, Thomas C. T.; Linse, Sara; Mansson, Cecilia; Emanuelsson, Cecilia; Presto, Jenny; Johansson, Jan; Vendruscolo, Michele; Dobson, Christopher M.; Knowles, Tuomas P. J.
      Nature Communications (2016), 7 (), 10948CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)
      It is increasingly recognized that mol. chaperones play a key role in modulating the formation of amyloid fibrils, a process assocd. with a wide range of human disorders. Understanding the detailed mechanisms by which they perform this function, however, has been challenging because of the great complexity of the protein aggregation process itself. In this work, we build on a previous kinetic approach and develop a model that considers pairwise interactions between mol. chaperones and different protein species to identify the protein components targeted by the chaperones and the corresponding microscopic reaction steps that are inhibited. We show that these interactions conserve the topol. of the unperturbed reaction network but modify the connectivity wts. between the different microscopic steps. Moreover, by analyzing several protein-mol. chaperone systems, we reveal the striking diversity in the microscopic mechanisms by which mol. chaperones act to suppress amyloid formation.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XkvVCmsLc%253D&md5=af61b69395b916de5ec38125ab03a909
    67. 67
      Shammas, S. L.; Waudby, C. A.; Wang, S.; Buell, A. K.; Knowles, T. P.; Ecroyd, H.; Welland, M. E.; Carver, J. A.; Dobson, C. M.; Meehan, S. Binding of the Molecular Chaperone αB-Crystallin to Aβ Amyloid Fibrils Inhibits Fibril Elongation. Biophys. J. 2011, 101 (7), 1681– 1689,  DOI: 10.1016/j.bpj.2011.07.056
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      Binding of the Molecular Chaperone αB-Crystallin to Aβ Amyloid Fibrils Inhibits Fibril Elongation
      Shammas, Sarah L.; Waudby, Christopher A.; Wang, Shuyu; Buell, Alexander K.; Knowles, Tuomas P. J.; Ecroyd, Heath; Welland, Mark E.; Carver, John A.; Dobson, Christopher M.; Meehan, Sarah
      Biophysical Journal (2011), 101 (7), 1681-1689CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
      The mol. chaperone αB-crystallin is a small heat-shock protein that is upregulated in response to a multitude of stress stimuli, and is found colocalized with Aβ amyloid fibrils in the extracellular plaques that are characteristic of Alzheimer's disease. We investigated whether this archetypical small heat-shock protein has the ability to interact with Aβ fibrils in vitro. We find that αB-crystallin binds to wild-type Aβ42 fibrils with micromolar affinity, and also binds to fibrils formed from the E22G Arctic mutation of Aβ42. Immunoelectron microscopy confirms that binding occurs along the entire length and ends of the fibrils. Investigations into the effect of αB-crystallin on the seeded growth of Aβ fibrils, both in soln. and on the surface of a quartz crystal microbalance biosensor, reveal that the binding of αB-crystallin to seed fibrils strongly inhibits their elongation. Because the lag phase in sigmoidal fibril assembly kinetics is dominated by elongation and fragmentation rates, the chaperone mechanism identified here represents a highly effective means to inhibit fibril proliferation. Together with previous observations of αB-crystallin interaction with α-synuclein and insulin fibrils, the results suggest that this mechanism is a generic means of providing mol. chaperone protection against amyloid fibril formation.
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    68. 68
      Abelein, A.; Graslund, A.; Danielsson, J. Zinc as chaperone-mimicking agent for retardation of amyloid β peptide fibril formation. Proc. Natl. Acad. Sci. U.S.A. 2015, 112 (17), 5407– 5412,  DOI: 10.1073/pnas.1421961112
      68
      Zinc as chaperone-mimicking agent for retardation of amyloid β peptide fibril formation
      Abelein, Axel; Graeslund, Astrid; Danielsson, Jens
      Proceedings of the National Academy of Sciences of the United States of America (2015), 112 (17), 5407-5412CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Metal ions have emerged to play a key role in the aggregation process of a amyloid-β (Aβ) peptide that is closely related to the pathogenesis of Alzheimer's disease. A detailed understanding of the underlying mechanistic process of peptide-metal ion interactions, however, has been challenging to obtain. Here, by applying a combination of NMR relaxation dispersion and fluorescence kinetic methods the authors investigated quant. thermodn. Aβ-Zn2+ binding features as well as how Zn2+ modulates the nucleation mechanism of the aggregation process. The results showed that, under near-physiol. conditions, substoichiometric amts. of Zn2+ effectively retarded the generation of amyloid fibrils. A global kinetic profile anal. revealed that in the absence of Zn2+, Aβ40 aggregation was driven by a monomer-dependent secondary nucleation process in addn. to fibril-end elongation. In the presence of Zn2+, the elongation rate was reduced, resulting in redn. of the aggregation rate, but not a complete inhibition of amyloid formation. The authors showed that Zn2+ transiently bound to residues in the N-terminus of the monomeric peptide. A thermodn. anal. supported a model where the N-terminus is folded around the Zn2+ ion, forming a marginally stable, short-lived folded Aβ40 species. This conformation was highly dynamic and only a few percent of the peptide mols. adopted this structure at any given time point. These findings suggested that the folded Aβ40-Zn2+ complex modulates the fibril ends, where elongation takes place, which efficiently retards fibril formation. In this conceptual framework, the authors propose that Zn2+ adopts the role of a minimal anti-aggregation chaperone for Aβ40.
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    69. 69
      Ghalebani, L.; Wahlstrom, A.; Danielsson, J.; Warmlander, S. K.; Graslund, A. pH-dependence of the specific binding of Cu(II) and Zn(II) ions to the amyloid-β peptide. Biochem. Biophys. Res. Commun. 2012, 421 (3), 554– 560,  DOI: 10.1016/j.bbrc.2012.04.043
      69
      pH-dependence of the specific binding of Cu(II) and Zn(II) ions to the amyloid-β peptide
      Ghalebani, Leila; Wahlstroem, Anna; Danielsson, Jens; Waermlaender, Sebastian K. T. S.; Graeslund, Astrid
      Biochemical and Biophysical Research Communications (2012), 421 (3), 554-560CODEN: BBRCA9; ISSN:0006-291X. (Elsevier B.V.)
      Metal ions like Cu(II) and Zn(II) are accumulated in Alzheimer's disease amyloid plaques. The amyloid-β (Aβ) peptide involved in the disease interacts with these metal ions at neutral pH via ligands provided by the N-terminal histidines and the N-terminus. The present study uses high-resoln. NMR spectroscopy to monitor the residue-specific interactions of Cu(II) and Zn(II) with 15N- and 13C,15N-labeled Aβ(1-40) peptides at varying pH levels. At pH 7.4 both ions bind to the specific ligands, competing with one another. At pH 5.5 Cu(II) retains its specific histidine ligands, while Zn(II) seems to lack residue-specific interactions. The low pH mimics acidosis which is linked to inflammatory processes in vivo. The results suggest that the cell toxic effects of redox active Cu(II) binding to Aβ may be reversed by the protective activity of non-redox active Zn(II) binding to the same major binding site under non-acidic conditions. Under acidic conditions, the protective effect of Zn(II) may be decreased or changed, since Zn(II) is less able to compete with Cu(II) for the specific binding site on the Aβ peptide under these conditions.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XmsVeksL8%253D&md5=82cba63273ca9869e3daa1d3468ce16c
    70. 70
      Lindberg, D. J.; Wranne, M. S.; Gilbert Gatty, M.; Westerlund, F.; Esbjörner, E. K. Steady-state and time-resolved Thioflavin-T fluorescence can report on morphological differences in amyloid fibrils formed by Aβ(1-40) and Aβ(1-42). Biochem. Biophys. Res. Commun. 2015, 458 (2), 418– 423,  DOI: 10.1016/j.bbrc.2015.01.132
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      Steady-state and time-resolved Thioflavin-T fluorescence can report on morphological differences in amyloid fibrils formed by Aβ(1-40) and Aβ(1-42)
      Lindberg, David J.; Wranne, Moa S.; Gilbert Gatty, Melina; Westerlund, Fredrik; Esbjoerner, Elin K.
      Biochemical and Biophysical Research Communications (2015), 458 (2), 418-423CODEN: BBRCA9; ISSN:0006-291X. (Elsevier B.V.)
      Thioflavin-T (ThT) is one of the most commonly used dyes for amyloid detection, but the origin of its fluorescence enhancement is not fully understood. Herein we have characterised the ThT fluorescence response upon binding to the Aβ(1-40) and Aβ(1-42) variants of the Alzheimer's-related peptide amyloid-β, in order to explore how the photophys. properties of this dye relates to structural and morphol. properties of two amyloid fibril types formed by peptides with a high degree of sequence homol. We show that the steady-state ThT fluorescence is 1.7 times more intense with Aβ(1-40) compared to Aβ(1-42) fibrils in concn. matched samples prepd. under quiescent conditions. By measuring the excited state lifetime of bound ThT, we also demonstrate a distinct difference between the two fibril isoforms, with Aβ(1-42) fibrils producing a longer ThT fluorescence lifetime compared to Aβ(1-40). The substantial steady-state intensity difference is therefore not explained by differences in fluorescence quantum yield. Further, we find that the ThT fluorescence intensity, but not the fluorescence lifetime, is dependent on the fibril prepn. method (quiescent vs. agitated conditions). We therefore propose that the fluorescence lifetime is inherent to each isoform and sensitively reports on fibril microstructure in the protofilament whereas the total fluorescence intensity relates to the amt. of exposed β-sheet in the mature Aβ fibrils and hence to differences in their morphol. Our results highlight the complexity of ThT fluorescence, and demonstrate its extended use in amyloid fibril characterization.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXisVSrsro%253D&md5=af8329897ed1f4231703132b288413cc
    71. 71
      Pallbo, J.; Olsson, U.; Sparr, E. Strong inhibition of peptide amyloid formation by a fatty acid. Biophys. J. 2021, 120 (20), 4536– 4546,  DOI: 10.1016/j.bpj.2021.08.035
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      Strong inhibition of peptide amyloid formation by a fatty acid
      Pallbo, Jon; Olsson, Ulf; Sparr, Emma
      Biophysical Journal (2021), 120 (20), 4536-4546CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
      The aggregation of peptides into amyloid fibrils is assocd. with several diseases, including Alzheimer's and Parkinson's disease. Because hydrophobic interactions often play an important role in amyloid formation, the presence of various hydrophobic or amphiphilic mols., such as lipids, may influence the aggregation process. We have studied the effect of a fatty acid, linoleic acid, on the fibrillation process of the amyloid-forming model peptide NACore (GAVVTGVTAVA). NACore is a peptide fragment spanning residue 68-78 of the protein α-synuclein involved in Parkinson's disease. Based primarily on CD measurements, we found that even a very small amt. of linoleic acid can substantially inhibit the fibrillation of NACore. This inhibitory effect manifests itself through a prolongation of the lag phase of the peptide fibrillation. The effect is greatest when the fatty acid is present from the beginning of the process together with the monomeric peptide. Cryogenic transmission electron microscopy revealed the presence of nonfibrillar clusters among NACore fibrils formed in the presence of linoleic acid. We argue that the obsd. inhibitory effect on fibrillation is due to co-assocn. of peptide oligomers and fatty acid aggregates at the early stage of the process. An important aspect of this mechanism is that it is nonmonomeric peptide structures that assoc. with the fatty acid aggregates. Similar mechanisms of action could be relevant in amyloid formation occurring in vivo, where the aggregation takes place in a lipid-rich environment.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhvFOjsbfF&md5=c44fc2e5a6aff63514cf70e70fd460e2
    72. 72
      Sturchio, A.; Dwivedi, A. K.; Young, C. B.; Malm, T.; Marsili, L.; Sharma, J. S.; Mahajan, A.; Hill, E. J.; Andaloussi, S. E.; Poston, K. L. High cerebrospinal amyloid-β 42 is associated with normal cognition in individuals with brain amyloidosis. EClinicalMedicine 2021, 38, 100988,  DOI: 10.1016/j.eclinm.2021.100988
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      High cerebrospinal amyloid-β 42 is associated with normal cognition in individuals with brain amyloidosis
      Sturchio Andrea; Marsili Luca; Sharma Jennifer S; Mahajan Abhimanyu; Hill Emily J; Espay Alberto J; Sturchio Andrea; Dwivedi Alok K; Young Christina B; Poston Kathleen L; Malm Tarja; Andaloussi Samir El; Ezzat Kariem; Manfredsson Fredric P; Schneider Lon S
      EClinicalMedicine (2021), 38 (), 100988 ISSN:.
      BACKGROUND: Brain amyloidosis does not invariably predict dementia. We hypothesized that high soluble 42-amino acid β amyloid (Aβ42) peptide levels are associated with normal cognition and hippocampal volume despite increasing brain amyloidosis. METHODS: This cross-sectional study of 598 amyloid-positive participants in the Alzheimer's Disease Neuroimaging Initiative cohort examined whether levels of soluble Aβ42 are higher in amyloid-positive normal cognition (NC) individuals compared to mild cognitive impairment (MCI) and Alzheimer's disease (AD) and whether this relationship applies to neuropsychological assessments and hippocampal volume measured within the same year. All subjects were evaluated between June 2010 and February 2019. Brain amyloid positivity was defined as positron emission tomography-based standard uptake value ratio (SUVR) ≥1.08 for ([18]) F-florbetaben or 1.11 for ([18])F-florbetapir, with higher SUVR indicating more brain amyloidosis. Analyses were adjusted for age, sex, education, APOE4, p-tau, t-tau, and centiloids levels. FINDINGS: Higher soluble Aβ42 levels were observed in NC (864.00 pg/ml) than in MCI (768.60 pg/ml) or AD (617.46 pg/ml), with the relationship between NC, MCI, and AD maintained across all amyloid tertiles. In adjusted analysis, there was a larger absolute effect size of soluble Aβ42 than SUVR for NC (0.82 vs. 0.40) and MCI (0.60 vs. 0.26) versus AD. Each standard deviation increase in Aβ42 was associated with greater odds of NC than AD (adjusted odds ratio, 6.26; p < 0.001) or MCI (1.42; p = 0.006). Higher soluble Aβ42 levels were also associated with better neuropsychological function and larger hippocampal volume. INTERPRETATION: Normal cognition and hippocampal volume are associated with preservation of high soluble Aβ42 levels despite increasing brain amyloidosis. FUNDING: Please refer to the Funding section at the end of the article.
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    73. 73
      Raskatov, J. A. What Is the ″Relevant″ Amyloid beta42 Concentration?. Chembiochem 2019, 20 (13), 1725– 1726,  DOI: 10.1002/cbic.201900097
      73
      What Is the "Relevant" Amyloid β42 Concentration?
      Raskatov, Jevgenij A.
      ChemBioChem (2019), 20 (13), 1725-1726CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Alzheimer's amyloid beta can perform a wide variety of actions that are highly concn. dependent. This viewpoint aims to provide a framework for basic considerations on what might be considered brain-relevant concns. of the peptide. Some implications for the therapeutic implementation of the recently emerged oligomer-to-fibril strategy are discussed.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXps1Ggt7o%253D&md5=31ef4547854b0a30723ee191e1b177c3
    74. 74
      Sani, M. A.; Gehman, J. D.; Separovic, F. Lipid matrix plays a role in Abeta fibril kinetics and morphology. FEBS Lett. 2011, 585 (5), 749– 754,  DOI: 10.1016/j.febslet.2011.02.011
      74
      Lipid matrix plays a role in Abeta fibril kinetics and morphology
      Sani, Marc-Antoine; Gehman, John D.; Separovic, Frances
      FEBS Letters (2011), 585 (5), 749-754CODEN: FEBLAL; ISSN:0014-5793. (Elsevier B.V.)
      While neuronal membranes are proposed to be the primary target of amyloid plaques, the effect of phospholipids on fibril formation kinetics and morphol. has not yet been resolved. We report that interaction of various compns. with neuronal mimics promoted different processes of fibril formation; neg. charged surfaces increased the lag time and elongation rate in thioflavin T assays, while brain total lipid ext. had an opposite effect compared to that in the absence of lipid. Electron microscopy showed thin and elongated fibrils when the peptide was incubated with anionic lipids, while neutral surfaces promoted coarse and small fibrils. CD and thioflavin T assays confirmed an initially unstructured peptide, and measured its transition to an aggregated beta-sheet conformation.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXjsF2jurk%253D&md5=ac9e49ee4e1078f6f543df0eab649d10
    75. 75
      Buell, A. K. The growth of amyloid fibrils: rates and mechanisms. Biochem. J. 2019, 476 (19), 2677– 2703,  DOI: 10.1042/BCJ20160868
      75
      The growth of amyloid fibrils: rates and mechanisms
      Buell, Alexander K.
      Biochemical Journal (2019), 476 (19), 2677-2703CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)
      A review. Amyloid fibrils are β-sheet-rich linear protein polymers that can be formed by a large variety of different proteins. These assemblies have received much interest in recent decades, due to their role in a range of human disorders. However, amyloid fibrils are also found in a functional context, whereby their structural, mech. and thermodn. properties are exploited by biol. systems. Amyloid fibrils form through a nucleated polymn. mechanism with secondary processes acting in many cases to amplify the no. of fibrils. The filamentous nature of amyloid fibrils implies that the fibril growth rate is, by several orders of magnitude, the fastest step of the overall aggregation reaction. This article focusses specifically on in vitro exptl. studies of the process of amyloid fibril growth, or elongation, and summarises the state of knowledge of its kinetics and mechanisms. This work attempts to provide the most comprehensive summary, to date, of the available exptl. data on amyloid fibril elongation rate consts. and the temp. and concn. dependence of amyloid fibril elongation rates. These data are compared with those from other types of protein polymers. This comparison with data from other polymerising proteins is interesting and relevant because many of the basic ideas and concepts discussed here were first introduced for non-amyloid protein polymers, most notably by the Japanese school of Oosawa and co-workers for cytoskeletal filaments.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXitlCks73I&md5=8e586b46664361f983cdb4ace86503a7
    76. 76
      Linse, S.; Scheidt, T.; Bernfur, K.; Vendruscolo, M.; Dobson, C. M.; Cohen, S. I. A.; Sileikis, E.; Lundqvist, M.; Qian, F.; O’Malley, T. Kinetic fingerprints differentiate the mechanisms of action of anti-Aβ antibodies. Nat. Struct. Mol. Biol. 2020, 27 (12), 1125– 1133,  DOI: 10.1038/s41594-020-0505-6
      76
      Kinetic fingerprints differentiate the mechanisms of action of anti-Aβ antibodies
      Linse, Sara; Scheidt, Tom; Bernfur, Katja; Vendruscolo, Michele; Dobson, Christopher M.; Cohen, Samuel I. A.; Sileikis, Eimantas; Lundqvist, Martin; Qian, Fang; O'Malley, Tiernan; Bussiere, Thierry; Weinreb, Paul H.; Xu, Catherine K.; Meisl, Georg; Devenish, Sean R. A.; Knowles, Tuomas P. J.; Hansson, Oskar
      Nature Structural & Molecular Biology (2020), 27 (12), 1125-1133CODEN: NSMBCU; ISSN:1545-9993. (Nature Research)
      The amyloid cascade hypothesis, according to which the self-assembly of amyloid-β peptide (Aβ) is a causative process in Alzheimer's disease, has driven many therapeutic efforts for the past 20 years. Failures of clin. trials investigating Aβ-targeted therapies have been interpreted as evidence against this hypothesis, irresp. of the characteristics and mechanisms of action of the therapeutic agents, which are highly challenging to assess. Here, we combine kinetic analyses with quant. binding measurements to address the mechanism of action of four clin. stage anti-Aβ antibodies, aducanumab, gantenerumab, bapineuzumab and solanezumab. We quantify the influence of these antibodies on the aggregation kinetics and on the prodn. of oligomeric aggregates and link these effects to the affinity and stoichiometry of each antibody for monomeric and fibrillar forms of Aβ. Our results reveal that, uniquely among these four antibodies, aducanumab dramatically reduces the flux of Aβ oligomers.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhvFegurjF&md5=2adc8febabbb832a3c8db63019db83e6
    77. 77
      Joshi, P.; Turola, E.; Ruiz, A.; Bergami, A.; Libera, D. D.; Benussi, L.; Giussani, P.; Magnani, G.; Comi, G.; Legname, G. Microglia convert aggregated amyloid-β into neurotoxic forms through the shedding of microvesicles. Cell Death Differ. 2014, 21 (4), 582– 593,  DOI: 10.1038/cdd.2013.180
      77
      Microglia convert aggregated amyloid-β into neurotoxic forms through the shedding of microvesicles
      Joshi, P.; Turola, E.; Ruiz, A.; Bergami, A.; Libera, D. D.; Benussi, L.; Giussani, P.; Magnani, G.; Comi, G.; Legname, G.; Ghidoni, R.; Furlan, R.; Matteoli, M.; Verderio, C.
      Cell Death & Differentiation (2014), 21 (4), 582-593CODEN: CDDIEK; ISSN:1350-9047. (Nature Publishing Group)
      Alzheimer's disease (AD) is characterized by extracellular amyloid-β (Aβ) deposition, which activates microglia, induces neuroinflammation and drives neurodegeneration. Recent evidence indicates that sol. pre-fibrillar Aβ species, rather than insol. fibrils, are the most toxic forms of Aβ. Preventing sol. Aβ formation represents, therefore, a major goal in AD. We investigated whether microvesicles (MVs) released extracellularly by reactive microglia may contribute to AD degeneration. We found that prodn. of myeloid MVs, likely of microglial origin, is strikingly high in AD patients and in subjects with mild cognitive impairment and that AD MVs are toxic for cultured neurons. The mechanism responsible for MV neurotoxicity was defined in vitro using MVs produced by primary microglia. We demonstrated that neurotoxicity of MVs results from (i) the capability of MV lipids to promote formation of sol. Aβ species from extracellular insol. aggregates and (ii) from the presence of neurotoxic Aβ forms trafficked to MVs after Aβ internalization into microglia. MV neurotoxicity was neutralized by the Aβ-interacting protein PrP and anti-Aβ antibodies, which prevented binding to neurons of neurotoxic sol. Aβ species. This study identifies microglia-derived MVs as a novel mechanism by which microglia participate in AD degeneration, and suggest new therapeutic strategies for the treatment of the disease.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvFCqsr%252FP&md5=753debbef2534229964ff9faacba45a5
    78. 78
      Abelein, A.; Chen, G.; Kitoka, K.; Aleksis, R.; Oleskovs, F.; Sarr, M.; Landreh, M.; Pahnke, J.; Nordling, K.; Kronqvist, N. High-yield Production of Amyloid-β Peptide Enabled by a Customized Spider Silk Domain. Sci. Rep. 2020, 10 (1), 235,  DOI: 10.1038/s41598-019-57143-x
      78
      High-yield Production of Amyloid-β Peptide Enabled by a Customized Spider Silk Domain
      Abelein, Axel; Chen, Gefei; Kitoka, Kristine; Aleksis, Rihards; Oleskovs, Filips; Sarr, Medoune; Landreh, Michael; Pahnke, Jens; Nordling, Kerstin; Kronqvist, Nina; Jaudzems, Kristaps; Rising, Anna; Johansson, Jan; Biverstaal, Henrik
      Scientific Reports (2020), 10 (1), 235CODEN: SRCEC3; ISSN:2045-2322. (Nature Research)
      Abstr.: During storage in the silk gland, the N-terminal domain (NT) of spider silk proteins (spidroins) keeps the aggregation-prone repetitive region in soln. at extreme concns. We observe that NTs from different spidroins have co-evolved with their resp. repeat region, and now use an NT that is distantly related to previously used NTs, for efficient recombinant prodn. of the amyloid-β peptide (Aβ) implicated in Alzheimer's disease. A designed variant of NT from Nephila clavipes flagelliform spidroin, which in nature allows prodn. and storage of β-hairpin repeat segments, gives exceptionally high yields of different human Aβ variants as a soly. tag. This tool enables efficient prodn. of target peptides also in minimal medium and gives up to 10 times more isotope-labeled monomeric Aβ peptides per L bacterial culture than previously reported.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXivFOntro%253D&md5=90233031e7c7d85d05ee5c14d9f09629
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      Necas, D.; Klapetek, P. Gwyddion: an open-source software for SPM data analysis. Cent. Eur. J. Phys. 2012, 10 (1), 181– 188,  DOI: 10.2478/s11534-011-0096-2
      There is no corresponding record for this reference.

  • Supporting Information

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    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acschemneuro.3c00655.

    • EV particle size determined by NTA; mean residual monomer content at the end point of aggregation; protein amount per EV particle; aggregation kinetics of medium control; fitting of ThT kinetic curves with AmyloFit and corresponding kinetic parameters; additional AFM and cryo-TEM images of Aβ(1–42) fibrils; estimation of fibril and vesicle concentrations; and cell viability assay (PDF)


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