Observation of α-Synuclein Preformed Fibrils Interacting with SH-SY5Y Neuroblastoma Cell Membranes Using Scanning Ion Conductance MicroscopyClick to copy article linkArticle link copied!
- Christina FengChristina FengDepartment of Chemistry and Biochemistry, California State University, Los Angeles, Los Angeles, California 90032, United StatesMore by Christina Feng
- Marisol FloresMarisol FloresDepartment of Chemistry and Biochemistry, California State University, Los Angeles, Los Angeles, California 90032, United StatesMore by Marisol Flores
- Christina DhojChristina DhojDepartment of Chemistry and Biochemistry, California State University, Los Angeles, Los Angeles, California 90032, United StatesMore by Christina Dhoj
- Adaly GarciaAdaly GarciaDepartment of Chemistry and Biochemistry, California State University, Los Angeles, Los Angeles, California 90032, United StatesMore by Adaly Garcia
- Sheehan BellecaSheehan BellecaDepartment of Chemistry and Biochemistry, California State University, Los Angeles, Los Angeles, California 90032, United StatesMore by Sheehan Belleca
- Dana Abou AbbasDana Abou AbbasDepartment of Biological Sciences, California State University, Los Angeles, Los Angeles, California 90032, United StatesMore by Dana Abou Abbas
- Jacob Parres-GoldJacob Parres-GoldDepartment of Chemistry and Biochemistry, California State University, Los Angeles, Los Angeles, California 90032, United StatesMore by Jacob Parres-Gold
- Aimee AnguianoAimee AnguianoDepartment of Chemistry and Biochemistry, California State University, Los Angeles, Los Angeles, California 90032, United StatesMore by Aimee Anguiano
- Edith PorterEdith PorterDepartment of Biological Sciences, California State University, Los Angeles, Los Angeles, California 90032, United StatesMore by Edith Porter
- Yixian Wang*Yixian Wang*Email: [email protected]. Telephone: +1-323-343-2353.Department of Chemistry and Biochemistry, California State University, Los Angeles, Los Angeles, California 90032, United StatesMore by Yixian Wang
Abstract
Parkinson’s disease (PD) is the second-most prevalent neurodegenerative disorder in the U.S. α-Synuclein (α-Syn) preformed fibrils (PFFs) have been shown to propagate PD pathology in neuronal populations. However, little work has directly characterized the morphological changes on membranes associated with α-Syn PFFs at a cellular level. Scanning ion conductance microscopy (SICM) is a noninvasive in situ cell imaging technique and therefore uniquely advantageous to investigate PFF-induced membrane changes in neuroblastoma cells. The present work used SICM to monitor cytoplasmic membrane changes of SH-SY5Y neuroblastoma cells after incubation with varying concentrations of α-Syn PFFs. Cell membrane roughness significantly increased as the concentration of α-Syn PFFs increased. Noticeable protrusions that assumed a more crystalline appearance at higher α-Syn PFF concentrations were also observed. Cell viability was only slightly reduced, though statistically significantly, to about 80% but independent of the dose. These observations indicate that within the 48 h treatment period, PFFs continue to accumulate on the cell membranes, leading to membrane roughness increase without causing prominent cell death. Since PFFs did not induce major cell death, these data suggest that early interventions targeting fibrils before further aggregation may prevent the progression of neuron loss in Parkinson’s disease.
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License Summary*
You are free to share(copy and redistribute) this article in any medium or format and to adapt(remix, transform, and build upon) the material for any purpose, even commercially within the parameters below:
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Figure 1
Figure 1. (A) Schematic of SICM. (B) Flowchart illustrating the SICM experiment procedure. PFFs (red) are added to wells containing SH-SY5Y cells and incubated for 48 h. SICM was then used to image cells, starting with a coarse whole-cell scan (64 × 64 pixels) and followed by multiple fine scans (128 × 128 pixels).
Figure 2
Figure 2. AFM characterization of PFFs. (A, B) Representative AFM images (2 μm × 2 μm) of α- Syn PFFs adhered to a mica substrate. Images were first-order flattened using XEI. (C) Histogram of the lengths of PFFs imaged with AFM (n = 3290).
Figure 3
Figure 3. Representative SICM images of SH-SY5Y cell membranes after PFF treatment for 48 h. Images a–p show flattened fine scans of 5 μm × 5 μm sections of the cell membrane, with pixels colored by their deviation from the mean z-height. Images q–t show the coarse scans of selected cells. Four experimental conditions are included: no treatment (control) and treatment with three different concentrations of α-Syn PFF (1, 5, and 10 μM). For each condition, one random coarse scan and four random sections from different cells were selected (a–d, q, control; e–h, r, 1 μM; i–l, s, 5 μM; m–p, t, 10 μM).
Figure 4
Figure 4. Quantitative analysis of the membrane roughness. (A) Roughness of cell membranes after different PFF treatments. The dark dotted line across each concentration is the mean roughness of each group. Each roughness data point is plotted to its corresponding concentration. The curves represent the frequency of the data points. The statistical significance of the differences between means was assessed via bootstrap sampling. The mean ± 95% confidence interval values are as follows: control = 1.588 ± 0.0264 (n = 570); 1 μM = 1.677 ± 0.0354 (n = 400); 5 μM = 1.775 ± 0.0291 (n = 475); 10 μM = 1.823 ± 0.0356 (n = 375). (B, C) Analysis of positive features. (B) Scatter plots of the area versus the maximum height (Zmax) of positive features extracted from cell sections under different treatments and (C) zoom-in of dashed box labeled area in (B). Numbers of features and cell sections analyzed are control, n = 138 from 5 sections; 1 μM, n = 161 from 6 sections; 5 μM, n = 721 from 9 sections; 10 μM, n = 2560 from 8 sections.
Figure 5
Figure 5. Effects of PFFs exposure on SH-SY5Y cells viability. (A) XTT assay measuring cell viability. (B) LDH release assay measuring cytotoxicity. Data are the mean ± SD, n = 3: **p < 0.01 and ***p < 0.001.
Methods
Sample Preparation
SICM
XTT and LDH Assays
Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acschemneuro.2c00478.
Additional details for methods and supplementary data (PDF)
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Acknowledgments
Prof. Jamil Momand (California State University, Los Angeles) and his student Lizbeth Flores are greatly appreciated for assisting with cell culturing. Prof. Shannon Boettcher (University of Oregon) and his student Nick D’Antona are greatly appreciated for the SEM characterization of the nanopipettes.
α-Syn | α-synuclein |
PD | Parkinson’s disease |
PBS | phosphate buffered saline |
SICM | scanning ion conductance microscopy |
XTT | sodium (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) |
LDH | lactate dehydrogenase |
References
This article references 31 other publications.
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- 4Li, Y.; Zhao, C.; Luo, F.; Liu, Z.; Gui, X.; Luo, Z.; Zhang, X.; Li, D.; Liu, C.; Li, X. Amyloid Fibril Structure of α-Synuclein Determined by Cryo-Electron Microscopy. Cell Res. 2018, 28 (9), 897– 903, DOI: 10.1038/s41422-018-0075-xGoogle Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhsVahtbvL&md5=053ffd5d6d3beca7c9a3fa322d243b83Amyloid fibril structure of α-synuclein determined by cryo-electron microscopyLi, Yaowang; Zhao, Chunyu; Luo, Feng; Liu, Zhenying; Gui, Xinrui; Luo, Zhipu; Zhang, Xiang; Li, Dan; Liu, Cong; Li, XuemingCell Research (2018), 28 (9), 897-903CODEN: CREEB6; ISSN:1001-0602. (Nature Research)α-Synuclein (α-syn) amyloid fibrils are the major component of Lewy bodies, which are the pathol. hallmark of Parkinson's disease (PD) and other synucleinopathies. High-resoln. structure of α-syn fibril is important for understanding its assembly and pathol. mechanism. Here, we detd. a fibril structure of full-length α-syn (1-140) at the resoln. of 3.07 Å by cryo-electron microscopy (cryo-EM). The fibrils are cytotoxic, and transmissible to induce endogenous α-syn aggregation in primary neurons. Based on the reconstructed cryo-EM d. map, we were able to unambiguously build the fibril structure comprising residues 37-99. The α-syn amyloid fibril structure shows two protofilaments intertwining along an approx. 21 screw axis into a left-handed helix. Each protofilament features a Greek key-like topol. Remarkably, five out of the six early-onset PD familial mutations are located at the dimer interface of the fibril (H50Q, G51D, and A53T/E) or involved in the stabilization of the protofilament (E46K). Furthermore, these PD mutations lead to the formation of fibrils with polymorphic structures distinct from that of the wild-type. Our study provides mol. insight into the fibrillar assembly of α-syn at the at. level and sheds light on the mol. pathogenesis caused by familial PD mutations of α-syn.
- 5Brundin, P.; Melki, R. Prying into the Prion Hypothesis for Parkinson’s Disease. J. Neurosci. 2017, 37 (41), 9808– 9818, DOI: 10.1523/JNEUROSCI.1788-16.2017Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtlejsb3E&md5=efe9cce8563a1e60b1ed7769c692df6cPrying into the prion hypothesis for Parkinson's diseaseBrundin, Patrik; Melki, RonaldJournal of Neuroscience (2017), 37 (41), 9808-9818CODEN: JNRSDS; ISSN:1529-2401. (Society for Neuroscience)In Parkinson's disease, intracellular α-synuclein inclusions form in neurons. We suggest that prion-like behavior of α-synuclein is a key component in Parkinson's disease pathogenesis. Although multiple mol. changes are involved in the triggering of the disease process, we propose that neuron-to-neuron transfer is a crucial event that is essential for Lewy pathol. to spread from one brain region to another. In this review, we describe key findings in human postmortem brains, cultured cells, and animal models of disease that support the idea that α-synuclein can act as a prion. We consider potential triggers of the α-synuclein misfolding and why the aggregates escape cellular degrdn. under disease conditions. We also discuss whether different strains of α-synuclein fibrils can underlie differences in cellular and regional distribution of aggregates in different synucleinopathies. Our conclusion is that α-synuclein probably acts as a prion in human diseases, and a deeper understanding of this step in the pathogenesis of Parkinson's disease can facilitate the development of disease-modifying therapies in the future.
- 6Alam, P.; Bousset, L.; Melki, R.; Otzen, D. E. α-Synuclein Oligomers and Fibrils: A Spectrum of Species, a Spectrum of Toxicities. J. Neurochem. 2019, 150 (5), 522– 534, DOI: 10.1111/jnc.14808Google Scholar6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhsFSgsrnF&md5=8e80c1d5f545bb95f162791356ad7e35α-synuclein oligomers and fibrils: a spectrum of species, a spectrum of toxicitiesAlam, Parvez; Bousset, Luc; Melki, Ronald; Otzen, Daniel E.Journal of Neurochemistry (2019), 150 (5), 522-534CODEN: JONRA9; ISSN:0022-3042. (Wiley-Blackwell)A review. This review article provides an overview of the different species that α-synuclein aggregates can populate. It also attempts to reconcile conflicting views regarding the cytotoxic roles of oligomers vs. fibrils. α-synuclein, while highly dynamic in the monomeric state, can access a large no. of different assembly states. Depending on assembly conditions, these states can interconvert over different timescales. The fibrillar state is the most thermodynamically favored due to the many stabilizing interactions formed between each monomeric unit, but different fibrillar types form at different rates. The end distribution is likely to reflect kinetic partitioning as much as thermodn. equilibra. In addn., metastable oligomeric species, some of which are on-pathway and others off-pathway, can be populated for remarkably long periods of time. Chem. modifications (phosphorylation, oxidn., covalent links to ligands, etc.) perturb these phys. interconversions and invariably destabilize the fibrillar state, leading to small prefibrillar assemblies which can coalesce into amorphous states. Both oligomeric and fibrillar species have been shown to be cytotoxic although firm conclusions require very careful evaluation of particle concns. and is complicated by the great variety and heterogeneity of different exptl. obsd. states. The mechanistic relationship between oligomers and fibrils remains to be clarified, both in terms of assembly of oligomers into fibrils and potential dissoln. of fibrils into oligomers. While oligomers are possibly implicated in the collapse of neuronal homeostasis, the fibrillar state(s) appears to be the most efficient at propagating itself both in vitro and in vivo, pointing to crit. roles for multiple different aggregate species in the progression of Parkinson's disease ().
- 7Aulić, S.; Le, T. T. N.; Moda, F.; Abounit, S.; Corvaglia, S.; Casalis, L.; Gustincich, S.; Zurzolo, C.; Tagliavini, F.; Legname, G. Defined α-Synuclein Prion-like Molecular Assemblies Spreading in Cell Culture. BMC Neurosci. 2014, 15, 69, DOI: 10.1186/1471-2202-15-69Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhslamtL3J&md5=ec139c86cdc3566511da680852341fa2Defined α-synuclein prion-like molecular assemblies spreading in cell cultureAulic, Suzana; Le, Tran Thanh Nhat; Moda, Fabio; Abounit, Saida; Corvaglia, Stefania; Casalis, Loredana; Gustincich, Stefano; Zurzolo, Chiara; Tagliavini, Fabrizio; Legname, GiuseppeBMC Neuroscience (2014), 15 (), 69/1-69/12CODEN: BNMEA6; ISSN:1471-2202. (BioMed Central Ltd.)Background: α-Synuclein (α-syn) plays a central role in the pathogenesis of synucleinopathies, a group of neurodegenerative disorders that includes Parkinson disease, dementia with Lewy bodies and multiple system atrophy. Several findings from cell culture and mouse expts. suggest intercellular α-syn transfer. Results: Through a methodol. used to obtain synthetic mammalian prions, we tested whether recombinant human α-syn amyloids can promote prion-like accumulation in neuronal cell lines in vitro. A single exposure to amyloid fibrils of human α-syn was sufficient to induce aggregation of endogenous α-syn in human neuroblastoma SH-SY5Y cells. Remarkably, endogenous wild-type α-syn was sufficient for the formation of these aggregates, and overexpression of the protein was not required. Conclusions: Our results provide compelling evidence that endogenous α-syn can accumulate in cell culture after a single exposure to exogenous α-syn short amyloid fibrils. Importantly, using α-syn short amyloid fibrils as seed, endogenous α-syn aggregates and accumulates over several passages in cell culture, providing an excellent tool for potential therapeutic screening of pathogenic α-syn aggregates.
- 8Polinski, N. K.; Volpicelli-Daley, L. A.; Sortwell, C. E.; Luk, K. C.; Cremades, N.; Gottler, L. M.; Froula, J.; Duffy, M. F.; Lee, V. M. Y.; Martinez, T. N.; Dave, K. D. Best Practices for Generating and Using Alpha-Synuclein Pre-Formed Fibrils to Model Parkinson’s Disease in Rodents. J. Parkinson's Dis. 2018, 8 (2), 303– 322, DOI: 10.3233/JPD-171248Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1MvntlKnsw%253D%253D&md5=dffad050b44a3827be3288103fce42a8Best Practices for Generating and Using Alpha-Synuclein Pre-Formed Fibrils to Model Parkinson's Disease in RodentsPolinski Nicole K; Martinez Terina N; Dave Kuldip D; Volpicelli-Daley Laura A; Froula Jessica; Sortwell Caryl E; Duffy Megan F; Luk Kelvin C; Lee Virginia M Y; Cremades Nunilo; Gottler Lindsey MJournal of Parkinson's disease (2018), 8 (2), 303-322 ISSN:.Parkinson's disease (PD) is the second most common neurodegenerative disease, affecting approximately one-percent of the population over the age of sixty. Although many animal models have been developed to study this disease, each model presents its own advantages and caveats. A unique model has arisen to study the role of alpha-synuclein (aSyn) in the pathogenesis of PD. This model involves the conversion of recombinant monomeric aSyn protein to a fibrillar form-the aSyn pre-formed fibril (aSyn PFF)-which is then injected into the brain or introduced to the media in culture. Although many groups have successfully adopted and replicated the aSyn PFF model, issues with generating consistent pathology have been reported by investigators. To improve the replicability of this model and diminish these issues, The Michael J. Fox Foundation for Parkinson's Research (MJFF) has enlisted the help of field leaders who performed key experiments to establish the aSyn PFF model to provide the research community with guidelines and practical tips for improving the robustness and success of this model. Specifically, we identify key pitfalls and suggestions for avoiding these mistakes as they relate to generating the aSyn PFFs from monomeric protein, validating the formation of pathogenic aSyn PFFs, and using the aSyn PFFs in vivo or in vitro to model PD. With this additional information, adoption and use of the aSyn PFF model should present fewer challenges, resulting in a robust and widely available model of PD.
- 9Volpicelli-Daley, L. A.; Luk, K. C.; Patel, T. P.; Tanik, S. A.; Riddle, D. M.; Stieber, A.; Meaney, D. F.; Trojanowski, J. Q.; Lee, V. M.-Y. Exogenous α-Synuclein Fibrils Induce Lewy Body Pathology Leading to Synaptic Dysfunction and Neuron Death. Neuron 2011, 72 (1), 57– 71, DOI: 10.1016/j.neuron.2011.08.033Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXht12kt7bP&md5=771dc7412f14fc4d30d15db30cf96309Exogenous α-Synuclein Fibrils Induce Lewy Body Pathology Leading to Synaptic Dysfunction and Neuron DeathVolpicelli-Daley, Laura A.; Luk, Kelvin C.; Patel, Tapan P.; Tanik, Selcuk A.; Riddle, Dawn M.; Stieber, Anna; Meaney, David F.; Trojanowski, John Q.; Lee, Virginia M.-Y.Neuron (2011), 72 (1), 57-71CODEN: NERNET; ISSN:0896-6273. (Cell Press)Inclusions composed of α-synuclein (α-syn), i.e., Lewy bodies (LBs) and Lewy neurites (LNs), define synucleinopathies including Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Here, we demonstrate that preformed fibrils generated from full-length and truncated recombinant α-syn enter primary neurons, probably by adsorptive-mediated endocytosis, and promote recruitment of sol. endogenous α-syn into insol. PD-like LBs and LNs. Remarkably, endogenous α-syn was sufficient for formation of these aggregates, and overexpression of wild-type or mutant α-syn was not required. LN-like pathol. first developed in axons and propagated to form LB-like inclusions in perikarya. Accumulation of pathol. α-syn led to selective decreases in synaptic proteins, progressive impairments in neuronal excitability and connectivity, and, eventually, neuron death. Thus, our data contribute important insights into the etiol. and pathogenesis of PD-like α-syn inclusions and their impact on neuronal functions, and they provide a model for discovering therapeutics targeting pathol. α-syn-mediated neurodegeneration.
- 10Henrich, M. T.; Geibl, F. F.; Lakshminarasimhan, H.; Stegmann, A.; Giasson, B. I.; Mao, X.; Dawson, V. L.; Dawson, T. M.; Oertel, W. H.; Surmeier, D. J. Determinants of Seeding and Spreading of α-Synuclein Pathology in the Brain. Sci. Adv. 2020, 6 (46), eabc2487 DOI: 10.1126/sciadv.abc2487Google ScholarThere is no corresponding record for this reference.
- 11Duffy, M. F.; Collier, T. J.; Patterson, J. R.; Kemp, C. J.; Fischer, D. L.; Stoll, A. C.; Sortwell, C. E. Quality Over Quantity: Advantages of Using Alpha-Synuclein Preformed Fibril Triggered Synucleinopathy to Model Idiopathic Parkinson’s Disease. Front. Neurosci. 2018, 12 DOI: 10.3389/fnins.2018.00621 .Google ScholarThere is no corresponding record for this reference.
- 12Luk, K. C.; Song, C.; O’Brien, P.; Stieber, A.; Branch, J. R.; Brunden, K. R.; Trojanowski, J. Q.; Lee, V. M.-Y. Exogenous Alpha-Synuclein Fibrils Seed the Formation of Lewy Body-like Intracellular Inclusions in Cultured Cells. Proc. Natl. Acad. Sci. U. S. A. 2009, 106 (47), 20051– 20056, DOI: 10.1073/pnas.0908005106Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhsFGjtLvL&md5=f82637d83eb1900f0a9f604e675b7d43Exogenous α-synuclein fibrils seed the formation of lewy body-like intracellular inclusions in cultured cellsLuk, Kelvin C.; Song, Cheng; O'Brien, Patrick; Stieber, Anna; Branch, Jonathan R.; Brunden, Kurt R.; Trojanowski, John Q.; Lee, Virginia M.-Y.Proceedings of the National Academy of Sciences of the United States of America (2009), 106 (47), 20051-20056, S20051/1-S20051/13CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Cytoplasmic inclusions contg. α-synuclein (α-Syn) fibrils, referred to as Lewy bodies (LBs), are the signature neuropathol. hallmarks of Parkinson's disease (PD). Although α-Syn fibrils can be generated from recombinant α-Syn protein in vitro, the prodn. of fibrillar α-Syn inclusions similar to authentic LBs in cultured cells has not been achieved. We show here that intracellular α-Syn aggregation can be triggered by the introduction of exogenously produced recombinant α-Syn fibrils into cultured cells engineered to overexpress α-Syn. Unlike unassembled α-Syn, these α-Syn fibrils "seeded" recruitment of endogenous sol. α-Syn protein and their conversion into insol., hyperphosphorylated, and ubiquitinated pathol. species. Thus, this cell model recapitulates key features of LBs in human PD brains. Also, these findings support the concept that intracellular α-Syn aggregation is normally limited by the no. of active nucleation sites present in the cytoplasm and that small quantities of α-Syn fibrils can alter this balance by acting as seeds for aggregation.
- 13Wu, Q.; Takano, H.; Riddle, D. M.; Trojanowski, J. Q.; Coulter, D. A.; Lee, V. M.-Y. α-Synuclein (ΑSyn) Preformed Fibrils Induce Endogenous ΑSyn Aggregation, Compromise Synaptic Activity and Enhance Synapse Loss in Cultured Excitatory Hippocampal Neurons. J. Neurosci. 2019, 39 (26), 5080– 5094, DOI: 10.1523/JNEUROSCI.0060-19.2019Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhvFWns7fP&md5=2017b718a5e4a30120190dd52f93ded9α-synuclein (αSyn) preformed fibrils induce endogenous αSyn aggregation, compromise synaptic activity and enhance synapse loss in cultured excitatory hippocampal neuronsWu, Qihui; Takano, Hajime; Riddle, Dawn M.; Trojanowski, John Q.; Coulter, Douglas A.; Lee, Virginia M.-Y.Journal of Neuroscience (2019), 39 (26), 5080-5094CODEN: JNRSDS; ISSN:1529-2401. (Society for Neuroscience)Synucleinopathies are characterized by the accumulation of insol. α-synuclein (αSyn). To test whether αSyn aggregates modulate synaptic activity, we used a recently developed model in primary neurons for inducing αSyn pathol. We demonstrated that preformed fibrils (PFFs) generated with recombinant human αSyn compromised synaptic activity in a time- and dose-dependent manner and that the magnitude of these deficits correlated with the formation of αSyn pathol. in cultured excitatory hippocampal neurons from both sexes of mice. Remarkably, acute passive infusion of αSyn PFFs from whole-cell patch-clamp pipet decreased mEPSC frequency within 10 min followed by induction of αSyn pathol. within 1 d. Moreover, by direct addn. of αSyn PFFs into culture medium, the formation of misfolded αSyn inclusions dramatically compromised the colocalization of synaptic markers and altered dynamic changes of dendritic spines, but the viability of neurons was not affected up to 7 d post-treatment with αSyn PFFs. Our data indicate that intraneuronal αSyn fibrils impaired the initiation of synaptogenesis and their physiol. functions, thereby suggesting that targeting synaptic dysfunction in synucleinopathies may provide a promising therapeutic direction.
- 14Hellstrand, 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.0077235Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhs1Cqu7bI&md5=390689802588f5a8d437d89cc060796fMembrane lipid co-aggregation with α-synuclein fibrilsHellstrand, Erik; Nowacka, Agnieszka; Topgaard, Daniel; Linse, Sara; Sparr, EmmaPLoS 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.
- 15Fusco, G.; De Simone, A.; Gopinath, T.; Vostrikov, V.; Vendruscolo, M.; Dobson, C. M.; Veglia, G. Direct Observation of the Three Regions in α-Synuclein That Determine Its Membrane-Bound Behaviour. Nat. Commun. 2014, 5 (1), 3827, DOI: 10.1038/ncomms4827Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXitVShsbbI&md5=2c351e0102c181a33055bbcf337b3abcDirect observation of the three regions in α-synuclein that determine its membrane-bound behaviourFusco, Giuliana; De Simone, Alfonso; Gopinath, Tata; Vostrikov, Vitaly; Vendruscolo, Michele; Dobson, Christopher M.; Veglia, GianluigiNature Communications (2014), 5 (), 3827CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)α-Synuclein (αS) is a protein involved in neurotransmitter release in presynaptic terminals, and whose aberrant aggregation is assocd. with Parkinson's disease. In dopaminergic neurons, αS exists in a tightly regulated equil. between water-sol. and membrane-assocd. forms. Here we use a combination of solid-state and soln. NMR spectroscopy to characterize the conformations of αS bound to lipid membranes mimicking the compn. and phys. properties of synaptic vesicles. The study shows three αS regions possessing distinct structural and dynamical properties, including an N-terminal helical segment having a role of membrane anchor, an unstructured C-terminal region that is weakly assocd. with the membrane and a central region acting as a sensor of the lipid properties and detg. the affinity of αS membrane binding. Taken together, our data define the nature of the interactions of αS with biol. membranes and provide insights into their roles in the function of this protein and in the mol. processes leading to its aggregation.
- 16van Maarschalkerweerd, A.; Vetri, V.; Langkilde, A. E.; Foderà, V.; Vestergaard, B. Protein/Lipid Coaggregates Are Formed During α-Synuclein-Induced Disruption of Lipid Bilayers. Biomacromolecules 2014, 15 (10), 3643– 3654, DOI: 10.1021/bm500937pGoogle Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhsFahtLnM&md5=65bb7045c384262a690880a4aeb7a031Protein/lipid coaggregates are formed during α-synuclein-induced disruption of lipid bilayersvan Maarschalkerweerd, Andreas; Vetri, Valeria; Langkilde, Annette Eva; Fodera, Vito; Vestergaard, BenteBiomacromolecules (2014), 15 (10), 3643-3654CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)Amyloid formation is assocd. with neurodegenerative diseases such as Parkinson's disease (PD). Significant α-synuclein (αSN) deposition in lipid-rich Lewy bodies is a hallmark of PD. Nonetheless, an unraveling of the connection between neurodegeneration and amyloid fibrils, including the mol. mechanisms behind potential amyloid-mediated toxic effects, is still missing. Interaction between amyloid aggregates and the lipid cell membrane is expected to play a key role in the disease progress. Here, the authors present exptl. data based on hybrid anal. of two-photon-microscopy, soln. SAXS, and CD data. The data showed in real time changes in liposome morphol. and stability upon protein addn. and revealed that membrane disruption mediated by amyloidogenic αSN was assocd. with dehydration of anionic lipid membranes and stimulation of protein secondary structure. As a result of membrane fragmentation, sol. αSN-lipid coaggregates were formed, hence, suggesting a novel mol. mechanism behind PD amyloid cytotoxicity.
- 17Pan, J.; Dalzini, A.; Khadka, N. K.; Aryal, C. M.; Song, L. Lipid Extraction by α-Synuclein Generates Semi-Transmembrane Defects and Lipoprotein Nanoparticles. ACS Omega 2018, 3 (8), 9586– 9597, DOI: 10.1021/acsomega.8b01462Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhsFCjtbnF&md5=f8e75d6db1099f7aab11874fa0a343f0Lipid Extraction by α-Synuclein Generates Semi-Transmembrane Defects and Lipoprotein NanoparticlesPan, Jianjun; Dalzini, Annalisa; Khadka, Nawal K.; Aryal, Chinta M.; Song, LikaiACS Omega (2018), 3 (8), 9586-9597CODEN: ACSODF; ISSN:2470-1343. (American Chemical Society)Membrane interactions play an essential role in physiol. and pathol. functions of the presynaptic protein, α-synuclein (αSyn). Here, we used AFM and ESR spectroscopy to investigate membrane modulations caused by αSyn. Specifically, we used several lipid bilayers to explore how different lipid species regulate αSyn-membrane interactions. We found that at a protein-to-lipid (P/L) ratio of ∼1/9, αSyn perturbed lipid bilayers by generating semi-transmembrane defects that only spanned one leaflet. In addn., αSyn co-aggregated with lipid mols. to produce ∼10-nm-sized lipoprotein nanoparticles. The obtained AFM data were consistent with the apolipoprotein characteristic of αSyn. The role of anionic lipids was elucidated by comparing results from zwitterionic and anionic lipid bilayers. Specifically, our AFM measurements showed that anionic bilayers had a larger tendency of forming bilayer defects; similarly, our ESR measurements revealed that anionic bilayers exhibited more substantial changes in lipid mobility and bilayer polarity. We also studied the effect of cholesterol. We found that cholesterol increased the capability of αSyn in inducing bilayer defects and altering lipid mobility and bilayer polarity. These data could be explained by an increase in the lipid headgroup-headgroup spacing or specific cholesterol-αSyn interactions. Interestingly, we found an inhibitory effect of the cone-shaped phosphatidylethanolamine lipids on αSyn-induced bilayer remodeling. We explained our data by considering interlipid H-bonding that can stabilize the bilayer organization and suppress lipid extn. The results of lipid-dependent membrane interactions are likely relevant to αSyn functioning.
- 18Gaspar, R.; Idini, I.; Carlström, G.; Linse, S.; Sparr, E. Transient Lipid-Protein Structures and Selective Ganglioside Uptake During α-Synuclein-Lipid Co-Aggregation. Front. Cell Dev. Biol. 2021, 9 DOI: 10.3389/fcell.2021.622764 .Google ScholarThere is no corresponding record for this reference.
- 19Liu, B.-C.; Lu, X.-Y.; Song, X.; Lei, K.-Y.; Alli, A. A.; Bao, H.-F.; Eaton, D. C.; Ma, H.-P. Scanning Ion Conductance Microscopy: A Nanotechnology for Biological Studies in Live Cells. Front. Physiol. 2013, 3, 483, DOI: 10.3389/fphys.2012.00483Google ScholarThere is no corresponding record for this reference.
- 20Zhu, C.; Huang, K.; Siepser, N. P.; Baker, L. A. Scanning Ion Conductance Microscopy. Chem. Rev. 2021, 121 (19), 11726– 11768, DOI: 10.1021/acs.chemrev.0c00962Google Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXisFSitbjF&md5=db477ec7fef9de60b4d5a9a287bf399cScanning Ion Conductance MicroscopyZhu, Cheng; Huang, Kaixiang; Siepser, Natasha P.; Baker, Lane A.Chemical Reviews (Washington, DC, United States) (2021), 121 (19), 11726-11768CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. Scanning ion conductance microscopy (SICM) has emerged as a versatile tool for studies of interfaces in biol. and materials science with notable utility in biophys. and electrochem. measurements. The heart of the SICM is a nanometer-scale electrolyte filled glass pipet that serves as a scanning probe. In the initial conception, manipulations of ion currents through the tip of the pipet and appropriate positioning hardware provided a route to recording micro- and nanoscopic mapping of the topog. of surfaces. Subsequent advances in instrumentation, probe design, and methods significantly increased opportunities for SICM beyond recording topog. Hybridization of SICM with coincident characterization techniques such as optical microscopy and faradaic electrodes have brought SICM to the forefront as a tool for nanoscale chem. measurement for a wide range of applications. Modern approaches to SICM realize an important tool in anal., bioanal., biophys., and materials measurements, where significant opportunities remain for further exploration. In this , we chronicle the development of SICM from the perspective of both the development of instrumentation and methods and the breadth of measurements performed.
- 21Chen, C.-C.; Zhou, Y.; Baker, L. A. Scanning Ion Conductance Microscopy. Annu. Rev. Anal. Chem. 2012, 5 (1), 207– 228, DOI: 10.1146/annurev-anchem-062011-143203Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xht1GmtLjP&md5=55fd8cac7508bb73dc073fe98465b9bfScanning ion conductance microscopyChen, Chiao-Chen; Zhou, Yi; Baker, Lane A.Annual Review of Analytical Chemistry (2012), 5 (), 207-228CODEN: ARACFU; ISSN:1936-1327. (Annual Reviews Inc.)A review. Scanning ion conductance microscopy (SICM) is a versatile type of scanning probe microscopy for studies in mol. biol. and materials science. Recent advances in feedback and probe fabrication have greatly increased the resoln., stability, and speed of imaging. Noncontact imaging and the ability to deliver materials to localized areas have made SICM esp. fruitful for studies of mol. biol., and many examples of such use have been reported. In this review, we highlight new developments in the operation of SICM and describe some of the most exciting recent studies from this growing field.
- 22Klenerman, D.; Shevchuk, A.; Novak, P.; Korchev, Y. E.; Davis, S. J. Imaging the Cell Surface and Its Organization down to the Level of Single Molecules. Philos. Trans. R. Soc. B Biol. Sci. 2013, 368 (1611), 20120027, DOI: 10.1098/rstb.2012.0027Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC3s3ksFGhtg%253D%253D&md5=e84d23d999591e4be8212a5dabb1d01fImaging the cell surface and its organization down to the level of single moleculesKlenerman David; Shevchuk Andrew; Novak Pavel; Korchev Yuri E; Davis Simon JPhilosophical transactions of the Royal Society of London. Series B, Biological sciences (2013), 368 (1611), 20120027 ISSN:.Determining the organization of key molecules on the surface of live cells in two dimensions and how this changes during biological processes, such as signalling, is a major challenge in cell biology and requires methods with nanoscale spatial resolution and high temporal resolution. Here, we review biophysical tools, based on scanning ion conductance microscopy and single-molecule fluorescence and the combination of both of these methods, which have recently been developed to address these issues. We then give examples of how these methods have been be applied to provide new insights into cell membrane organization and function, and discuss some of the issues that will need to be addressed to further exploit these methods in the future.
- 23Zhu, C.; Shi, W.; Daleke, D. L.; Baker, L. A. Monitoring Dynamic Spiculation in Red Blood Cells with Scanning Ion Conductance Microscopy. Analyst 2018, 143 (5), 1087– 1093, DOI: 10.1039/C7AN01986FGoogle Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXntVKntg%253D%253D&md5=e6ee856e020daccea918e6afdf89f1f3Monitoring dynamic spiculation in red blood cells with scanning ion conductance microscopyZhu, Cheng; Shi, Wenqing; Daleke, David L.; Baker, Lane A.Analyst (Cambridge, United Kingdom) (2018), 143 (5), 1087-1093CODEN: ANALAO; ISSN:0003-2654. (Royal Society of Chemistry)Phospholipids are crit. structural components of the membrane of human erythrocytes and their asym. transbilayer distribution is essential for normal cell functions. Phospholipid asymmetry is maintained by transporters that shuttle phospholipids between the inner leaflet and the outer leaflet of the membrane bilayer. When an exogenous, short acyl chain, phosphatidylcholine (PC) or phosphatidylserine (PS) is incorporated into erythrocytes, a discocyte-to-echinocyte shape change is induced. PC treated cells remain echinocytic, while PS treated cells return to discocytes, and eventually stomatocytes, due to the action of an inwardly directed transporter. These morphol. changes have been well studied by light microscopy and SEM in the past few decades. However, most of this research is based on the glutaraldehyde fixed cells, which limits the dynamic study in discrete time points instead of continuous single cell measurements. Scanning ion conductance microscopy (SICM) is a scanning probe technique which is ideal for live cell imaging due to high resoln., in situ and non-contact scanning. To better understand these phospholipid-induced morphol. changes, SICM was used to scan the morphol. change of human erythrocytes after the incorporation of exogenous dilauroylphosphatidylserine (DLPS) and the results revealed single cell dynamic morphol. changes and the movement of spicules on the membrane surface.
- 24Rubfiaro, A. S.; Tsegay, P. S.; Lai, Y.; Cabello, E.; Shaver, M.; Hutcheson, J.; Liu, Y.; He, J. Scanning Ion Conductance Microscopy Study Reveals the Disruption of the Integrity of the Human Cell Membrane Structure by Oxidative DNA Damage. ACS Appl. Bio Mater. 2021, 4 (2), 1632– 1639, DOI: 10.1021/acsabm.0c01461Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhtVOgt7s%253D&md5=80d72aa6c13b7d77bb1dd2db492b4b1aScanning Ion Conductance Microscopy Study Reveals the Disruption of the Integrity of the Human Cell Membrane Structure by Oxidative DNA DamageRubfiaro, Alberto S.; Tsegay, Pawlos S.; Lai, Yanhao; Cabello, Emmanuel; Shaver, Mohammad; Hutcheson, Joshua; Liu, Yuan; He, JinACS Applied Bio Materials (2021), 4 (2), 1632-1639CODEN: AABMCB; ISSN:2576-6422. (American Chemical Society)Oxidative stress can damage organs, tissues, and cells through reactive oxygen species (ROS) by oxidizing DNA, proteins, and lipids, thereby resulting in diseases. However, the underlying mol. mechanisms remain to be elucidated. Employing scanning ion conductance microscopy (SICM), the authors explored the early responses of human embryonic kidney (HEK293H) cells to oxidative DNA damage induced by potassium chromate (K2CrO4). The short term (1-2 h) exposure to a low concn. (10μM) of K2CrO4 damaged the lipid membrane of HEK293H cells, resulting in structural defects and depolarization of the cell membrane and reducing cellular secretion activity shortly after the treatment. Further the K2CrO4 treatment decreased the expression of the cytoskeleton protein, β-actin, by inducing oxidative DNA damage in the exon 4 of the β-actin gene. These results suggest that K2CrO4 caused oxidative DNA damage in cytoskeleton genes such as β-actin and reduced their expression, thereby disrupting the organization of the cytoskeleton beneath the cell membrane and inducing cell membrane damages. The authors' study provides direct evidence that oxidative DNA damage disrupted human cell membrane integrity by deregulating cytoskeleton gene expression.
- 25Parres-Gold, J.; Chieng, A.; Wong Su, S.; Wang, Y. Real-Time Characterization of Cell Membrane Disruption by α-Synuclein Oligomers in Live SH-SY5Y Neuroblastoma Cells. ACS Chem. Neurosci. 2020, 11 (17), 2528– 2534, DOI: 10.1021/acschemneuro.0c00309Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsFaqtrrJ&md5=ce8faca3d9088e072cee2c942983f1a7Real-Time Characterization of Cell Membrane Disruption by α-Synuclein Oligomers in Live SH-SY5Y Neuroblastoma CellsParres-Gold, Jacob; Chieng, Andy; Wong Su, Stephanie; Wang, YixianACS Chemical Neuroscience (2020), 11 (17), 2528-2534CODEN: ACNCDM; ISSN:1948-7193. (American Chemical Society)Aggregation of the natively unfolded protein α-synuclein (α-Syn) has been widely correlated to the neuronal death assocd. with Parkinson's disease. Mutations and protein overaccumulation can promote the aggregation of α-Syn into oligomers and fibrils. Recent work has suggested that α-Syn oligomers can permeabilize the neuronal membrane, promoting calcium influx and cell death. However, the mechanism of this permeabilization is still uncertain and has yet to be characterized in live cells. This work uses scanning ion conductance microscopy (SICM) to image, in real time and without using chem. probes, the topogs. of live SH-SY5Y neuroblastoma cells after exposure to α-Syn oligomers. Substantial morphol. changes were obsd., with micrometer-scale hills and troughs obsd. at lower α-Syn concns. (1.00μM) and large, transient pores obsd. at higher α-Syn concns. (6.0μM). These findings suggest that α-Syn oligomers may permeabilize the neuronal membrane by destabilizing the lipid bilayer and opening transient pores.
- 26Bridi, J. C.; Hirth, F. Mechanisms of α-Synuclein Induced Synaptopathy in Parkinson’s Disease. Front. Neurosci. 2018, 12, 80, DOI: 10.3389/fnins.2018.00080Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1MroslGhtg%253D%253D&md5=c6ea0fd85d1ead1c07397d2b29afd4f9Mechanisms of α-Synuclein Induced Synaptopathy in Parkinson's DiseaseBridi Jessika C; Hirth FrankFrontiers in neuroscience (2018), 12 (), 80 ISSN:1662-4548.Parkinson's disease (PD) is characterized by intracellular inclusions of aggregated and misfolded α-Synuclein (α-Syn), and the loss of dopaminergic (DA) neurons in the brain. The resulting motor abnormalities mark the progression of PD, while non-motor symptoms can already be identified during early, prodromal stages of disease. Recent studies provide evidence that during this early prodromal phase, synaptic and axonal abnormalities occur before the degenerative loss of neuronal cell bodies. These early phenotypes can be attributed to synaptic accumulation of toxic α-Syn. Under physiological conditions, α-Syn functions in its native conformation as a soluble monomer. However, PD patient brains are characterized by intracellular inclusions of insoluble fibrils. Yet, oligomers and protofibrils of α-Syn have been identified to be the most toxic species, with their accumulation at presynaptic terminals affecting several steps of neurotransmitter release. First, high levels of α-Syn alter the size of synaptic vesicle pools and impair their trafficking. Second, α-Syn overexpression can either misregulate or redistribute proteins of the presynaptic SNARE complex. This leads to deficient tethering, docking, priming and fusion of synaptic vesicles at the active zone (AZ). Third, α-Syn inclusions are found within the presynaptic AZ, accompanied by a decrease in AZ protein levels. Furthermore, α-Syn overexpression reduces the endocytic retrieval of synaptic vesicle membranes during vesicle recycling. These presynaptic alterations mediated by accumulation of α-Syn, together impair neurotransmitter exocytosis and neuronal communication. Although α-Syn is expressed throughout the brain and enriched at presynaptic terminals, DA neurons are the most vulnerable in PD, likely because α-Syn directly regulates dopamine levels. Indeed, evidence suggests that α-Syn is a negative modulator of dopamine by inhibiting enzymes responsible for its synthesis. In addition, α-Syn is able to interact with and reduce the activity of VMAT2 and DAT. The resulting dysregulation of dopamine levels directly contributes to the formation of toxic α-Syn oligomers. Together these data suggest a vicious cycle of accumulating α-Syn and deregulated dopamine that triggers synaptic dysfunction and impaired neuronal communication, ultimately causing synaptopathy and progressive neurodegeneration in Parkinson's disease.
- 27Winner, B.; Jappelli, R.; Maji, S. K.; Desplats, P. A.; Boyer, L.; Aigner, S.; Hetzer, C.; Loher, T.; Vilar, M.; Campioni, S.; Tzitzilonis, C.; Soragni, A.; Jessberger, S.; Mira, H.; Consiglio, A.; Pham, E.; Masliah, E.; Gage, F. H.; Riek, R. In Vivo Demonstration That α-Synuclein Oligomers Are Toxic. Proc. Natl. Acad. Sci. U. S. A. 2011, 108 (10), 4194– 4199, DOI: 10.1073/pnas.1100976108Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXjsF2nu78%253D&md5=7af57c2b6e3726b17c5cb2e035f92ff5In vivo demonstration that α-synuclein oligomers are toxicWinner, Beate; Jappelli, Roberto; Maji, Samir K.; Desplats, Paula A.; Boyer, Leah; Aigner, Stefan; Hetzer, Claudia; Loher, Thomas; Vilar, Marial; Campioni, Silvia; Tzitzilonis, Christos; Soragni, Alice; Jessberger, Sebastian; Mira, Helena; Consiglio, Antonella; Pham, Emiley; Masliah, Eliezer; Gage, Fred H.; Riek, RolandProceedings of the National Academy of Sciences of the United States of America (2011), 108 (10), 4194-4199, S4194/1-S4194/12CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The aggregation of proteins into oligomers and amyloid fibrils is characteristic of several neurodegenerative diseases, including Parkinson disease (PD). In PD, the process of aggregation of α-synuclein (α-syn) from monomers, via oligomeric intermediates, into amyloid fibrils is considered the disease-causative toxic mechanism. We developed α-syn mutants that promote oligomer or fibril formation and tested the toxicity of these mutants by using a rat lentivirus system to investigate loss of dopaminergic neurons in the substantia nigra. The most severe dopaminergic loss in the substantia nigra is obsd. in animals with the α-syn variants that form oligomers (i.e., E57K and E35K), whereas the α-syn variants that form fibrils very quickly are less toxic. We show that α-syn oligomers are toxic in vivo and that α-syn oligomers might interact with and potentially disrupt membranes.
- 28Bigi, A.; Ermini, E.; Chen, S. W.; Cascella, R.; Cecchi, C. Exploring the Release of Toxic Oligomers from α-Synuclein Fibrils with Antibodies and STED Microscopy. Life 2021, 11 (5), 431, DOI: 10.3390/life11050431Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXis1Cis7bK&md5=95009700ddba46421a6db0da96886037Exploring the release of toxic oligomers from α-synuclein fibrils with antibodies and STED microscopyBigi, Alessandra; Ermini, Emilio; Chen, Serene W.; Cascella, Roberta; Cecchi, CristinaLife (Basel, Switzerland) (2021), 11 (5), 431CODEN: LBSIB7; ISSN:2075-1729. (MDPI AG)α-Synuclein (αS) is an intrinsically disordered and highly dynamic protein involved in dopamine release at presynaptic terminals. The abnormal aggregation of αS as mature fibrils into intraneuronal inclusion bodies is directly linked to Parkinson's disease. Increasing exptl. evidence suggests that sol. oligomers formed early during the aggregation process are the most cytotoxic forms of αS. This study investigated the uptake by neuronal cells of pathol. relevant αS oligomers and fibrils exploiting a range of conformation-sensitive antibodies, and the super-resoln. stimulated emission depletion (STED) microscopy. We found that prefibrillar oligomers promptly penetrate neuronal membranes, thus resulting in cell dysfunction. By contrast, fibril docking to the phospholipid bilayer is accompanied by αS conformational changes with a progressive release of A11-reactive oligomers, which can enter into the neurons and trigger cell impairment. Our data provide important evidence on the role of αS fibrils as a source of harmful oligomers, which resemble the intermediate conformers formed de novo during aggregation, underling the dynamic and reversible nature of protein aggregates responsible for α-synucleinopathies.
- 29Volpicelli-Daley, L. A.; Luk, K. C.; Lee, V. M.-Y. Addition of Exogenous α-Synuclein Preformed Fibrils to Primary Neuronal Cultures to Seed Recruitment of Endogenous α-Synuclein to Lewy Body and Lewy Neurite-like Aggregates. Nat. Protoc. 2014, 9 (9), 2135– 2146, DOI: 10.1038/nprot.2014.143Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhtlGntb3K&md5=1273f2338024b4321357a3dfa8420ffcAddition of exogenous α-synuclein preformed fibrils to primary neuronal cultures to seed recruitment of endogenous α-synuclein to Lewy body and Lewy neurite-like aggregatesVolpicelli-Daley, Laura A.; Luk, Kelvin C.; Lee, Virginia M-Y.Nature Protocols (2014), 9 (9), 2135-2146CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)This protocol describes a primary neuronal model of formation of α-synuclein (α-syn) aggregates that recapitulate features of the Lewy bodies and Lewy neurites found in Parkinson's disease brains and other synucleinopathies. This model allows investigation of aggregate formation, their impact on neuron function, and development of therapeutics. Addn. of preformed fibrils (PFFs) synthesized from recombinant α-syn to neurons seeds the recruitment of endogenous α-syn into aggregates characterized by detergent insoly. and hyperphosphorylation. Aggregate formation follows a lag phase of 2-3 d, followed by formation in axons by days 4-7, spread to somatodendritic compartments by days 7-10 and neuron death ∼14 d after PFF addn. Here we provide methods and highlight the crucial steps for PFF formation, PFF addn. to cultured hippocampal neurons and confirmation of aggregate formation. Neurons derived from various brain regions from nontransgenic and genetically engineered mice and rats can be used, allowing interrogation of the effect of specific genes on aggregate formation.
- 30Shimozawa, A.; Fujita, Y.; Kondo, H.; Takimoto, Y.; Terada, M.; Sanagi, M.; Hisanaga, S.; Hasegawa, M. Effect of L-DOPA/Benserazide on Propagation of Pathological α-Synuclein. Front. Neurosci. 2019, 13, 595, DOI: 10.3389/fnins.2019.00595Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3Mzis1GqtA%253D%253D&md5=8478f3d4503c417cc5a618f840deb2d3Effect of L-DOPA/Benserazide on Propagation of Pathological α-SynucleinShimozawa Aki; Fujita Yuuki; Kondo Hiromi; Terada Makoto; Hasegawa Masato; Shimozawa Aki; Fujita Yuuki; Hisanaga Shin-Ichi; Takimoto Yu; Sanagi MasanaoFrontiers in neuroscience (2019), 13 (), 595 ISSN:1662-4548.Parkinson's disease (PD) and related disorders are characterized by filamentous or fibrous structures consisting of abnormal α-synuclein in the brains of patients, and the distributions and spread of these pathologies are closely correlated with disease progression. L-DOPA (a dopamine precursor) is the most effective therapy for PD, but it remains unclear whether the drug has any effect on the formation and propagation of pathogenic abnormal α-synuclein in vivo. Here, we tested whether or not L-DOPA influences the prion-like spread of α-synuclein pathologies in a wild-type (WT) mouse model of α-synuclein propagation. To quantitative the pathological α-synuclein in mice, we prepared brain sections stained with an anti-phosphoSer129 (PS129) antibody after pretreatments with autoclaving and formic acid, and carefully analyzed positive aggregates on multiple sections covering the areas of interest using a microscope. Notably, a significant reduction in the accumulation of phosphorylated α-synuclein was detected in substantia nigra of L-DOPA/benserazide (a dopamine decarboxylase inhibitor)-treated mice, compared with control mice. These results suggest that L-DOPA may slow the progression of PD in vivo by suppressing the aggregation of α-synuclein in dopaminergic neurons and the cell-to-cell propagation of abnormal α-synuclein. This is the first report describing the suppressing effect of L-DOPA/benserazide on the propagation of pathological α-synuclein. The experimental protocols and detection methods in this study are expected to be useful for evaluation of drug candidates or new therapies targeting the propagation of α-synuclein.
- 31Conway, K. A.; Rochet, J.-C.; Bieganski, R. M.; Lansbury, P. T. Kinetic Stabilization of the α-Synuclein Protofibril by a Dopamine-α-Synuclein Adduct. Science 2001, 294 (5545), 1346– 1349, DOI: 10.1126/science.1063522Google Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXotlKmurk%253D&md5=ff9705bd276af2c78db588b7beb321cbKinetic stabilization of the α-synuclein protofibril by a dopamine-α-synuclein adductConway, Kelly A.; Rachet, Jean-Christophe; Bieganski, Robert M.; Lansbury, Peter T., Jr.Science (Washington, DC, United States) (2001), 294 (5545), 1346-1349CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)The substantia nigra in Parkinson's disease (PD) is depleted of dopaminergic neurons and contains fibrillar Lewy bodies comprising primarily α-synuclein. We screened a library t6 identify drug-like mols. to probe the relation between neurodegeneration and α-synuclein fibrilization. All but one of 15 fibril inhibitors were catecholamines related to dopamine. The inhibitory activity of dopamine depended on its oxidative ligation to α-synuclein and was selective for the protofibril-to-fibril conversion, causing accumulation of the α-synuclein protofibril. Adduct formation provides an explanation for the dopaminergic selectivity of α-synuclein-assocd. neurotoxicity in PD and has implications for current and future PD therapeutic and diagnostic strategies.
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Abstract
Figure 1
Figure 1. (A) Schematic of SICM. (B) Flowchart illustrating the SICM experiment procedure. PFFs (red) are added to wells containing SH-SY5Y cells and incubated for 48 h. SICM was then used to image cells, starting with a coarse whole-cell scan (64 × 64 pixels) and followed by multiple fine scans (128 × 128 pixels).
Figure 2
Figure 2. AFM characterization of PFFs. (A, B) Representative AFM images (2 μm × 2 μm) of α- Syn PFFs adhered to a mica substrate. Images were first-order flattened using XEI. (C) Histogram of the lengths of PFFs imaged with AFM (n = 3290).
Figure 3
Figure 3. Representative SICM images of SH-SY5Y cell membranes after PFF treatment for 48 h. Images a–p show flattened fine scans of 5 μm × 5 μm sections of the cell membrane, with pixels colored by their deviation from the mean z-height. Images q–t show the coarse scans of selected cells. Four experimental conditions are included: no treatment (control) and treatment with three different concentrations of α-Syn PFF (1, 5, and 10 μM). For each condition, one random coarse scan and four random sections from different cells were selected (a–d, q, control; e–h, r, 1 μM; i–l, s, 5 μM; m–p, t, 10 μM).
Figure 4
Figure 4. Quantitative analysis of the membrane roughness. (A) Roughness of cell membranes after different PFF treatments. The dark dotted line across each concentration is the mean roughness of each group. Each roughness data point is plotted to its corresponding concentration. The curves represent the frequency of the data points. The statistical significance of the differences between means was assessed via bootstrap sampling. The mean ± 95% confidence interval values are as follows: control = 1.588 ± 0.0264 (n = 570); 1 μM = 1.677 ± 0.0354 (n = 400); 5 μM = 1.775 ± 0.0291 (n = 475); 10 μM = 1.823 ± 0.0356 (n = 375). (B, C) Analysis of positive features. (B) Scatter plots of the area versus the maximum height (Zmax) of positive features extracted from cell sections under different treatments and (C) zoom-in of dashed box labeled area in (B). Numbers of features and cell sections analyzed are control, n = 138 from 5 sections; 1 μM, n = 161 from 6 sections; 5 μM, n = 721 from 9 sections; 10 μM, n = 2560 from 8 sections.
Figure 5
Figure 5. Effects of PFFs exposure on SH-SY5Y cells viability. (A) XTT assay measuring cell viability. (B) LDH release assay measuring cytotoxicity. Data are the mean ± SD, n = 3: **p < 0.01 and ***p < 0.001.
References
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- 3Cascella, R.; Chen, S. W.; Bigi, A.; Camino, J. D.; Xu, C. K.; Dobson, C. M.; Chiti, F.; Cremades, N.; Cecchi, C. The Release of Toxic Oligomers from α-Synuclein Fibrils Induces Dysfunction in Neuronal Cells. Nat. Commun. 2021, 12 (1), 1814, DOI: 10.1038/s41467-021-21937-33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXntl2qtbg%253D&md5=ea3bf353d1308262961c9670f8612989The release of toxic oligomers from α-synuclein fibrils induces dysfunction in neuronal cellsCascella, Roberta; Chen, Serene W.; Bigi, Alessandra; Camino, Jose D.; Xu, Catherine K.; Dobson, Christopher M.; Chiti, Fabrizio; Cremades, Nunilo; Cecchi, CristinaNature Communications (2021), 12 (1), 1814CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)The self-assembly of α-synuclein (αS) into intraneuronal inclusion bodies is a key characteristic of Parkinsons disease. To define the nature of the species giving rise to neuronal damage, we have investigated the mechanism of action of the main αS populations that have been obsd. to form progressively during fibril growth. The αS fibrils release sol. prefibrillar oligomeric species with cross-β structure and solvent-exposed hydrophobic clusters αS prefibrillar oligomers are efficient in crossing and permeabilize neuronal membranes, causing cellular insults. Short fibrils are more neurotoxic than long fibrils due to the higher proportion of fibrillar ends, resulting in a rapid release of oligomers. The kinetics of released αS oligomers match the obsd. kinetics of toxicity in cellular systems. In addn. to previous evidence that αS fibrils can spread in different brain areas, our in vitro results reveal that αS fibrils can also release oligomeric species responsible for an immediate dysfunction of the neurons in the vicinity of these species.
- 4Li, Y.; Zhao, C.; Luo, F.; Liu, Z.; Gui, X.; Luo, Z.; Zhang, X.; Li, D.; Liu, C.; Li, X. Amyloid Fibril Structure of α-Synuclein Determined by Cryo-Electron Microscopy. Cell Res. 2018, 28 (9), 897– 903, DOI: 10.1038/s41422-018-0075-x4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhsVahtbvL&md5=053ffd5d6d3beca7c9a3fa322d243b83Amyloid fibril structure of α-synuclein determined by cryo-electron microscopyLi, Yaowang; Zhao, Chunyu; Luo, Feng; Liu, Zhenying; Gui, Xinrui; Luo, Zhipu; Zhang, Xiang; Li, Dan; Liu, Cong; Li, XuemingCell Research (2018), 28 (9), 897-903CODEN: CREEB6; ISSN:1001-0602. (Nature Research)α-Synuclein (α-syn) amyloid fibrils are the major component of Lewy bodies, which are the pathol. hallmark of Parkinson's disease (PD) and other synucleinopathies. High-resoln. structure of α-syn fibril is important for understanding its assembly and pathol. mechanism. Here, we detd. a fibril structure of full-length α-syn (1-140) at the resoln. of 3.07 Å by cryo-electron microscopy (cryo-EM). The fibrils are cytotoxic, and transmissible to induce endogenous α-syn aggregation in primary neurons. Based on the reconstructed cryo-EM d. map, we were able to unambiguously build the fibril structure comprising residues 37-99. The α-syn amyloid fibril structure shows two protofilaments intertwining along an approx. 21 screw axis into a left-handed helix. Each protofilament features a Greek key-like topol. Remarkably, five out of the six early-onset PD familial mutations are located at the dimer interface of the fibril (H50Q, G51D, and A53T/E) or involved in the stabilization of the protofilament (E46K). Furthermore, these PD mutations lead to the formation of fibrils with polymorphic structures distinct from that of the wild-type. Our study provides mol. insight into the fibrillar assembly of α-syn at the at. level and sheds light on the mol. pathogenesis caused by familial PD mutations of α-syn.
- 5Brundin, P.; Melki, R. Prying into the Prion Hypothesis for Parkinson’s Disease. J. Neurosci. 2017, 37 (41), 9808– 9818, DOI: 10.1523/JNEUROSCI.1788-16.20175https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtlejsb3E&md5=efe9cce8563a1e60b1ed7769c692df6cPrying into the prion hypothesis for Parkinson's diseaseBrundin, Patrik; Melki, RonaldJournal of Neuroscience (2017), 37 (41), 9808-9818CODEN: JNRSDS; ISSN:1529-2401. (Society for Neuroscience)In Parkinson's disease, intracellular α-synuclein inclusions form in neurons. We suggest that prion-like behavior of α-synuclein is a key component in Parkinson's disease pathogenesis. Although multiple mol. changes are involved in the triggering of the disease process, we propose that neuron-to-neuron transfer is a crucial event that is essential for Lewy pathol. to spread from one brain region to another. In this review, we describe key findings in human postmortem brains, cultured cells, and animal models of disease that support the idea that α-synuclein can act as a prion. We consider potential triggers of the α-synuclein misfolding and why the aggregates escape cellular degrdn. under disease conditions. We also discuss whether different strains of α-synuclein fibrils can underlie differences in cellular and regional distribution of aggregates in different synucleinopathies. Our conclusion is that α-synuclein probably acts as a prion in human diseases, and a deeper understanding of this step in the pathogenesis of Parkinson's disease can facilitate the development of disease-modifying therapies in the future.
- 6Alam, P.; Bousset, L.; Melki, R.; Otzen, D. E. α-Synuclein Oligomers and Fibrils: A Spectrum of Species, a Spectrum of Toxicities. J. Neurochem. 2019, 150 (5), 522– 534, DOI: 10.1111/jnc.148086https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhsFSgsrnF&md5=8e80c1d5f545bb95f162791356ad7e35α-synuclein oligomers and fibrils: a spectrum of species, a spectrum of toxicitiesAlam, Parvez; Bousset, Luc; Melki, Ronald; Otzen, Daniel E.Journal of Neurochemistry (2019), 150 (5), 522-534CODEN: JONRA9; ISSN:0022-3042. (Wiley-Blackwell)A review. This review article provides an overview of the different species that α-synuclein aggregates can populate. It also attempts to reconcile conflicting views regarding the cytotoxic roles of oligomers vs. fibrils. α-synuclein, while highly dynamic in the monomeric state, can access a large no. of different assembly states. Depending on assembly conditions, these states can interconvert over different timescales. The fibrillar state is the most thermodynamically favored due to the many stabilizing interactions formed between each monomeric unit, but different fibrillar types form at different rates. The end distribution is likely to reflect kinetic partitioning as much as thermodn. equilibra. In addn., metastable oligomeric species, some of which are on-pathway and others off-pathway, can be populated for remarkably long periods of time. Chem. modifications (phosphorylation, oxidn., covalent links to ligands, etc.) perturb these phys. interconversions and invariably destabilize the fibrillar state, leading to small prefibrillar assemblies which can coalesce into amorphous states. Both oligomeric and fibrillar species have been shown to be cytotoxic although firm conclusions require very careful evaluation of particle concns. and is complicated by the great variety and heterogeneity of different exptl. obsd. states. The mechanistic relationship between oligomers and fibrils remains to be clarified, both in terms of assembly of oligomers into fibrils and potential dissoln. of fibrils into oligomers. While oligomers are possibly implicated in the collapse of neuronal homeostasis, the fibrillar state(s) appears to be the most efficient at propagating itself both in vitro and in vivo, pointing to crit. roles for multiple different aggregate species in the progression of Parkinson's disease ().
- 7Aulić, S.; Le, T. T. N.; Moda, F.; Abounit, S.; Corvaglia, S.; Casalis, L.; Gustincich, S.; Zurzolo, C.; Tagliavini, F.; Legname, G. Defined α-Synuclein Prion-like Molecular Assemblies Spreading in Cell Culture. BMC Neurosci. 2014, 15, 69, DOI: 10.1186/1471-2202-15-697https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhslamtL3J&md5=ec139c86cdc3566511da680852341fa2Defined α-synuclein prion-like molecular assemblies spreading in cell cultureAulic, Suzana; Le, Tran Thanh Nhat; Moda, Fabio; Abounit, Saida; Corvaglia, Stefania; Casalis, Loredana; Gustincich, Stefano; Zurzolo, Chiara; Tagliavini, Fabrizio; Legname, GiuseppeBMC Neuroscience (2014), 15 (), 69/1-69/12CODEN: BNMEA6; ISSN:1471-2202. (BioMed Central Ltd.)Background: α-Synuclein (α-syn) plays a central role in the pathogenesis of synucleinopathies, a group of neurodegenerative disorders that includes Parkinson disease, dementia with Lewy bodies and multiple system atrophy. Several findings from cell culture and mouse expts. suggest intercellular α-syn transfer. Results: Through a methodol. used to obtain synthetic mammalian prions, we tested whether recombinant human α-syn amyloids can promote prion-like accumulation in neuronal cell lines in vitro. A single exposure to amyloid fibrils of human α-syn was sufficient to induce aggregation of endogenous α-syn in human neuroblastoma SH-SY5Y cells. Remarkably, endogenous wild-type α-syn was sufficient for the formation of these aggregates, and overexpression of the protein was not required. Conclusions: Our results provide compelling evidence that endogenous α-syn can accumulate in cell culture after a single exposure to exogenous α-syn short amyloid fibrils. Importantly, using α-syn short amyloid fibrils as seed, endogenous α-syn aggregates and accumulates over several passages in cell culture, providing an excellent tool for potential therapeutic screening of pathogenic α-syn aggregates.
- 8Polinski, N. K.; Volpicelli-Daley, L. A.; Sortwell, C. E.; Luk, K. C.; Cremades, N.; Gottler, L. M.; Froula, J.; Duffy, M. F.; Lee, V. M. Y.; Martinez, T. N.; Dave, K. D. Best Practices for Generating and Using Alpha-Synuclein Pre-Formed Fibrils to Model Parkinson’s Disease in Rodents. J. Parkinson's Dis. 2018, 8 (2), 303– 322, DOI: 10.3233/JPD-1712488https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1MvntlKnsw%253D%253D&md5=dffad050b44a3827be3288103fce42a8Best Practices for Generating and Using Alpha-Synuclein Pre-Formed Fibrils to Model Parkinson's Disease in RodentsPolinski Nicole K; Martinez Terina N; Dave Kuldip D; Volpicelli-Daley Laura A; Froula Jessica; Sortwell Caryl E; Duffy Megan F; Luk Kelvin C; Lee Virginia M Y; Cremades Nunilo; Gottler Lindsey MJournal of Parkinson's disease (2018), 8 (2), 303-322 ISSN:.Parkinson's disease (PD) is the second most common neurodegenerative disease, affecting approximately one-percent of the population over the age of sixty. Although many animal models have been developed to study this disease, each model presents its own advantages and caveats. A unique model has arisen to study the role of alpha-synuclein (aSyn) in the pathogenesis of PD. This model involves the conversion of recombinant monomeric aSyn protein to a fibrillar form-the aSyn pre-formed fibril (aSyn PFF)-which is then injected into the brain or introduced to the media in culture. Although many groups have successfully adopted and replicated the aSyn PFF model, issues with generating consistent pathology have been reported by investigators. To improve the replicability of this model and diminish these issues, The Michael J. Fox Foundation for Parkinson's Research (MJFF) has enlisted the help of field leaders who performed key experiments to establish the aSyn PFF model to provide the research community with guidelines and practical tips for improving the robustness and success of this model. Specifically, we identify key pitfalls and suggestions for avoiding these mistakes as they relate to generating the aSyn PFFs from monomeric protein, validating the formation of pathogenic aSyn PFFs, and using the aSyn PFFs in vivo or in vitro to model PD. With this additional information, adoption and use of the aSyn PFF model should present fewer challenges, resulting in a robust and widely available model of PD.
- 9Volpicelli-Daley, L. A.; Luk, K. C.; Patel, T. P.; Tanik, S. A.; Riddle, D. M.; Stieber, A.; Meaney, D. F.; Trojanowski, J. Q.; Lee, V. M.-Y. Exogenous α-Synuclein Fibrils Induce Lewy Body Pathology Leading to Synaptic Dysfunction and Neuron Death. Neuron 2011, 72 (1), 57– 71, DOI: 10.1016/j.neuron.2011.08.0339https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXht12kt7bP&md5=771dc7412f14fc4d30d15db30cf96309Exogenous α-Synuclein Fibrils Induce Lewy Body Pathology Leading to Synaptic Dysfunction and Neuron DeathVolpicelli-Daley, Laura A.; Luk, Kelvin C.; Patel, Tapan P.; Tanik, Selcuk A.; Riddle, Dawn M.; Stieber, Anna; Meaney, David F.; Trojanowski, John Q.; Lee, Virginia M.-Y.Neuron (2011), 72 (1), 57-71CODEN: NERNET; ISSN:0896-6273. (Cell Press)Inclusions composed of α-synuclein (α-syn), i.e., Lewy bodies (LBs) and Lewy neurites (LNs), define synucleinopathies including Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Here, we demonstrate that preformed fibrils generated from full-length and truncated recombinant α-syn enter primary neurons, probably by adsorptive-mediated endocytosis, and promote recruitment of sol. endogenous α-syn into insol. PD-like LBs and LNs. Remarkably, endogenous α-syn was sufficient for formation of these aggregates, and overexpression of wild-type or mutant α-syn was not required. LN-like pathol. first developed in axons and propagated to form LB-like inclusions in perikarya. Accumulation of pathol. α-syn led to selective decreases in synaptic proteins, progressive impairments in neuronal excitability and connectivity, and, eventually, neuron death. Thus, our data contribute important insights into the etiol. and pathogenesis of PD-like α-syn inclusions and their impact on neuronal functions, and they provide a model for discovering therapeutics targeting pathol. α-syn-mediated neurodegeneration.
- 10Henrich, M. T.; Geibl, F. F.; Lakshminarasimhan, H.; Stegmann, A.; Giasson, B. I.; Mao, X.; Dawson, V. L.; Dawson, T. M.; Oertel, W. H.; Surmeier, D. J. Determinants of Seeding and Spreading of α-Synuclein Pathology in the Brain. Sci. Adv. 2020, 6 (46), eabc2487 DOI: 10.1126/sciadv.abc2487There is no corresponding record for this reference.
- 11Duffy, M. F.; Collier, T. J.; Patterson, J. R.; Kemp, C. J.; Fischer, D. L.; Stoll, A. C.; Sortwell, C. E. Quality Over Quantity: Advantages of Using Alpha-Synuclein Preformed Fibril Triggered Synucleinopathy to Model Idiopathic Parkinson’s Disease. Front. Neurosci. 2018, 12 DOI: 10.3389/fnins.2018.00621 .There is no corresponding record for this reference.
- 12Luk, K. C.; Song, C.; O’Brien, P.; Stieber, A.; Branch, J. R.; Brunden, K. R.; Trojanowski, J. Q.; Lee, V. M.-Y. Exogenous Alpha-Synuclein Fibrils Seed the Formation of Lewy Body-like Intracellular Inclusions in Cultured Cells. Proc. Natl. Acad. Sci. U. S. A. 2009, 106 (47), 20051– 20056, DOI: 10.1073/pnas.090800510612https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhsFGjtLvL&md5=f82637d83eb1900f0a9f604e675b7d43Exogenous α-synuclein fibrils seed the formation of lewy body-like intracellular inclusions in cultured cellsLuk, Kelvin C.; Song, Cheng; O'Brien, Patrick; Stieber, Anna; Branch, Jonathan R.; Brunden, Kurt R.; Trojanowski, John Q.; Lee, Virginia M.-Y.Proceedings of the National Academy of Sciences of the United States of America (2009), 106 (47), 20051-20056, S20051/1-S20051/13CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Cytoplasmic inclusions contg. α-synuclein (α-Syn) fibrils, referred to as Lewy bodies (LBs), are the signature neuropathol. hallmarks of Parkinson's disease (PD). Although α-Syn fibrils can be generated from recombinant α-Syn protein in vitro, the prodn. of fibrillar α-Syn inclusions similar to authentic LBs in cultured cells has not been achieved. We show here that intracellular α-Syn aggregation can be triggered by the introduction of exogenously produced recombinant α-Syn fibrils into cultured cells engineered to overexpress α-Syn. Unlike unassembled α-Syn, these α-Syn fibrils "seeded" recruitment of endogenous sol. α-Syn protein and their conversion into insol., hyperphosphorylated, and ubiquitinated pathol. species. Thus, this cell model recapitulates key features of LBs in human PD brains. Also, these findings support the concept that intracellular α-Syn aggregation is normally limited by the no. of active nucleation sites present in the cytoplasm and that small quantities of α-Syn fibrils can alter this balance by acting as seeds for aggregation.
- 13Wu, Q.; Takano, H.; Riddle, D. M.; Trojanowski, J. Q.; Coulter, D. A.; Lee, V. M.-Y. α-Synuclein (ΑSyn) Preformed Fibrils Induce Endogenous ΑSyn Aggregation, Compromise Synaptic Activity and Enhance Synapse Loss in Cultured Excitatory Hippocampal Neurons. J. Neurosci. 2019, 39 (26), 5080– 5094, DOI: 10.1523/JNEUROSCI.0060-19.201913https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhvFWns7fP&md5=2017b718a5e4a30120190dd52f93ded9α-synuclein (αSyn) preformed fibrils induce endogenous αSyn aggregation, compromise synaptic activity and enhance synapse loss in cultured excitatory hippocampal neuronsWu, Qihui; Takano, Hajime; Riddle, Dawn M.; Trojanowski, John Q.; Coulter, Douglas A.; Lee, Virginia M.-Y.Journal of Neuroscience (2019), 39 (26), 5080-5094CODEN: JNRSDS; ISSN:1529-2401. (Society for Neuroscience)Synucleinopathies are characterized by the accumulation of insol. α-synuclein (αSyn). To test whether αSyn aggregates modulate synaptic activity, we used a recently developed model in primary neurons for inducing αSyn pathol. We demonstrated that preformed fibrils (PFFs) generated with recombinant human αSyn compromised synaptic activity in a time- and dose-dependent manner and that the magnitude of these deficits correlated with the formation of αSyn pathol. in cultured excitatory hippocampal neurons from both sexes of mice. Remarkably, acute passive infusion of αSyn PFFs from whole-cell patch-clamp pipet decreased mEPSC frequency within 10 min followed by induction of αSyn pathol. within 1 d. Moreover, by direct addn. of αSyn PFFs into culture medium, the formation of misfolded αSyn inclusions dramatically compromised the colocalization of synaptic markers and altered dynamic changes of dendritic spines, but the viability of neurons was not affected up to 7 d post-treatment with αSyn PFFs. Our data indicate that intraneuronal αSyn fibrils impaired the initiation of synaptogenesis and their physiol. functions, thereby suggesting that targeting synaptic dysfunction in synucleinopathies may provide a promising therapeutic direction.
- 14Hellstrand, 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.007723514https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhs1Cqu7bI&md5=390689802588f5a8d437d89cc060796fMembrane lipid co-aggregation with α-synuclein fibrilsHellstrand, Erik; Nowacka, Agnieszka; Topgaard, Daniel; Linse, Sara; Sparr, EmmaPLoS 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.
- 15Fusco, G.; De Simone, A.; Gopinath, T.; Vostrikov, V.; Vendruscolo, M.; Dobson, C. M.; Veglia, G. Direct Observation of the Three Regions in α-Synuclein That Determine Its Membrane-Bound Behaviour. Nat. Commun. 2014, 5 (1), 3827, DOI: 10.1038/ncomms482715https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXitVShsbbI&md5=2c351e0102c181a33055bbcf337b3abcDirect observation of the three regions in α-synuclein that determine its membrane-bound behaviourFusco, Giuliana; De Simone, Alfonso; Gopinath, Tata; Vostrikov, Vitaly; Vendruscolo, Michele; Dobson, Christopher M.; Veglia, GianluigiNature Communications (2014), 5 (), 3827CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)α-Synuclein (αS) is a protein involved in neurotransmitter release in presynaptic terminals, and whose aberrant aggregation is assocd. with Parkinson's disease. In dopaminergic neurons, αS exists in a tightly regulated equil. between water-sol. and membrane-assocd. forms. Here we use a combination of solid-state and soln. NMR spectroscopy to characterize the conformations of αS bound to lipid membranes mimicking the compn. and phys. properties of synaptic vesicles. The study shows three αS regions possessing distinct structural and dynamical properties, including an N-terminal helical segment having a role of membrane anchor, an unstructured C-terminal region that is weakly assocd. with the membrane and a central region acting as a sensor of the lipid properties and detg. the affinity of αS membrane binding. Taken together, our data define the nature of the interactions of αS with biol. membranes and provide insights into their roles in the function of this protein and in the mol. processes leading to its aggregation.
- 16van Maarschalkerweerd, A.; Vetri, V.; Langkilde, A. E.; Foderà, V.; Vestergaard, B. Protein/Lipid Coaggregates Are Formed During α-Synuclein-Induced Disruption of Lipid Bilayers. Biomacromolecules 2014, 15 (10), 3643– 3654, DOI: 10.1021/bm500937p16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhsFahtLnM&md5=65bb7045c384262a690880a4aeb7a031Protein/lipid coaggregates are formed during α-synuclein-induced disruption of lipid bilayersvan Maarschalkerweerd, Andreas; Vetri, Valeria; Langkilde, Annette Eva; Fodera, Vito; Vestergaard, BenteBiomacromolecules (2014), 15 (10), 3643-3654CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)Amyloid formation is assocd. with neurodegenerative diseases such as Parkinson's disease (PD). Significant α-synuclein (αSN) deposition in lipid-rich Lewy bodies is a hallmark of PD. Nonetheless, an unraveling of the connection between neurodegeneration and amyloid fibrils, including the mol. mechanisms behind potential amyloid-mediated toxic effects, is still missing. Interaction between amyloid aggregates and the lipid cell membrane is expected to play a key role in the disease progress. Here, the authors present exptl. data based on hybrid anal. of two-photon-microscopy, soln. SAXS, and CD data. The data showed in real time changes in liposome morphol. and stability upon protein addn. and revealed that membrane disruption mediated by amyloidogenic αSN was assocd. with dehydration of anionic lipid membranes and stimulation of protein secondary structure. As a result of membrane fragmentation, sol. αSN-lipid coaggregates were formed, hence, suggesting a novel mol. mechanism behind PD amyloid cytotoxicity.
- 17Pan, J.; Dalzini, A.; Khadka, N. K.; Aryal, C. M.; Song, L. Lipid Extraction by α-Synuclein Generates Semi-Transmembrane Defects and Lipoprotein Nanoparticles. ACS Omega 2018, 3 (8), 9586– 9597, DOI: 10.1021/acsomega.8b0146217https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhsFCjtbnF&md5=f8e75d6db1099f7aab11874fa0a343f0Lipid Extraction by α-Synuclein Generates Semi-Transmembrane Defects and Lipoprotein NanoparticlesPan, Jianjun; Dalzini, Annalisa; Khadka, Nawal K.; Aryal, Chinta M.; Song, LikaiACS Omega (2018), 3 (8), 9586-9597CODEN: ACSODF; ISSN:2470-1343. (American Chemical Society)Membrane interactions play an essential role in physiol. and pathol. functions of the presynaptic protein, α-synuclein (αSyn). Here, we used AFM and ESR spectroscopy to investigate membrane modulations caused by αSyn. Specifically, we used several lipid bilayers to explore how different lipid species regulate αSyn-membrane interactions. We found that at a protein-to-lipid (P/L) ratio of ∼1/9, αSyn perturbed lipid bilayers by generating semi-transmembrane defects that only spanned one leaflet. In addn., αSyn co-aggregated with lipid mols. to produce ∼10-nm-sized lipoprotein nanoparticles. The obtained AFM data were consistent with the apolipoprotein characteristic of αSyn. The role of anionic lipids was elucidated by comparing results from zwitterionic and anionic lipid bilayers. Specifically, our AFM measurements showed that anionic bilayers had a larger tendency of forming bilayer defects; similarly, our ESR measurements revealed that anionic bilayers exhibited more substantial changes in lipid mobility and bilayer polarity. We also studied the effect of cholesterol. We found that cholesterol increased the capability of αSyn in inducing bilayer defects and altering lipid mobility and bilayer polarity. These data could be explained by an increase in the lipid headgroup-headgroup spacing or specific cholesterol-αSyn interactions. Interestingly, we found an inhibitory effect of the cone-shaped phosphatidylethanolamine lipids on αSyn-induced bilayer remodeling. We explained our data by considering interlipid H-bonding that can stabilize the bilayer organization and suppress lipid extn. The results of lipid-dependent membrane interactions are likely relevant to αSyn functioning.
- 18Gaspar, R.; Idini, I.; Carlström, G.; Linse, S.; Sparr, E. Transient Lipid-Protein Structures and Selective Ganglioside Uptake During α-Synuclein-Lipid Co-Aggregation. Front. Cell Dev. Biol. 2021, 9 DOI: 10.3389/fcell.2021.622764 .There is no corresponding record for this reference.
- 19Liu, B.-C.; Lu, X.-Y.; Song, X.; Lei, K.-Y.; Alli, A. A.; Bao, H.-F.; Eaton, D. C.; Ma, H.-P. Scanning Ion Conductance Microscopy: A Nanotechnology for Biological Studies in Live Cells. Front. Physiol. 2013, 3, 483, DOI: 10.3389/fphys.2012.00483There is no corresponding record for this reference.
- 20Zhu, C.; Huang, K.; Siepser, N. P.; Baker, L. A. Scanning Ion Conductance Microscopy. Chem. Rev. 2021, 121 (19), 11726– 11768, DOI: 10.1021/acs.chemrev.0c0096220https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXisFSitbjF&md5=db477ec7fef9de60b4d5a9a287bf399cScanning Ion Conductance MicroscopyZhu, Cheng; Huang, Kaixiang; Siepser, Natasha P.; Baker, Lane A.Chemical Reviews (Washington, DC, United States) (2021), 121 (19), 11726-11768CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. Scanning ion conductance microscopy (SICM) has emerged as a versatile tool for studies of interfaces in biol. and materials science with notable utility in biophys. and electrochem. measurements. The heart of the SICM is a nanometer-scale electrolyte filled glass pipet that serves as a scanning probe. In the initial conception, manipulations of ion currents through the tip of the pipet and appropriate positioning hardware provided a route to recording micro- and nanoscopic mapping of the topog. of surfaces. Subsequent advances in instrumentation, probe design, and methods significantly increased opportunities for SICM beyond recording topog. Hybridization of SICM with coincident characterization techniques such as optical microscopy and faradaic electrodes have brought SICM to the forefront as a tool for nanoscale chem. measurement for a wide range of applications. Modern approaches to SICM realize an important tool in anal., bioanal., biophys., and materials measurements, where significant opportunities remain for further exploration. In this , we chronicle the development of SICM from the perspective of both the development of instrumentation and methods and the breadth of measurements performed.
- 21Chen, C.-C.; Zhou, Y.; Baker, L. A. Scanning Ion Conductance Microscopy. Annu. Rev. Anal. Chem. 2012, 5 (1), 207– 228, DOI: 10.1146/annurev-anchem-062011-14320321https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xht1GmtLjP&md5=55fd8cac7508bb73dc073fe98465b9bfScanning ion conductance microscopyChen, Chiao-Chen; Zhou, Yi; Baker, Lane A.Annual Review of Analytical Chemistry (2012), 5 (), 207-228CODEN: ARACFU; ISSN:1936-1327. (Annual Reviews Inc.)A review. Scanning ion conductance microscopy (SICM) is a versatile type of scanning probe microscopy for studies in mol. biol. and materials science. Recent advances in feedback and probe fabrication have greatly increased the resoln., stability, and speed of imaging. Noncontact imaging and the ability to deliver materials to localized areas have made SICM esp. fruitful for studies of mol. biol., and many examples of such use have been reported. In this review, we highlight new developments in the operation of SICM and describe some of the most exciting recent studies from this growing field.
- 22Klenerman, D.; Shevchuk, A.; Novak, P.; Korchev, Y. E.; Davis, S. J. Imaging the Cell Surface and Its Organization down to the Level of Single Molecules. Philos. Trans. R. Soc. B Biol. Sci. 2013, 368 (1611), 20120027, DOI: 10.1098/rstb.2012.002722https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC3s3ksFGhtg%253D%253D&md5=e84d23d999591e4be8212a5dabb1d01fImaging the cell surface and its organization down to the level of single moleculesKlenerman David; Shevchuk Andrew; Novak Pavel; Korchev Yuri E; Davis Simon JPhilosophical transactions of the Royal Society of London. Series B, Biological sciences (2013), 368 (1611), 20120027 ISSN:.Determining the organization of key molecules on the surface of live cells in two dimensions and how this changes during biological processes, such as signalling, is a major challenge in cell biology and requires methods with nanoscale spatial resolution and high temporal resolution. Here, we review biophysical tools, based on scanning ion conductance microscopy and single-molecule fluorescence and the combination of both of these methods, which have recently been developed to address these issues. We then give examples of how these methods have been be applied to provide new insights into cell membrane organization and function, and discuss some of the issues that will need to be addressed to further exploit these methods in the future.
- 23Zhu, C.; Shi, W.; Daleke, D. L.; Baker, L. A. Monitoring Dynamic Spiculation in Red Blood Cells with Scanning Ion Conductance Microscopy. Analyst 2018, 143 (5), 1087– 1093, DOI: 10.1039/C7AN01986F23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXntVKntg%253D%253D&md5=e6ee856e020daccea918e6afdf89f1f3Monitoring dynamic spiculation in red blood cells with scanning ion conductance microscopyZhu, Cheng; Shi, Wenqing; Daleke, David L.; Baker, Lane A.Analyst (Cambridge, United Kingdom) (2018), 143 (5), 1087-1093CODEN: ANALAO; ISSN:0003-2654. (Royal Society of Chemistry)Phospholipids are crit. structural components of the membrane of human erythrocytes and their asym. transbilayer distribution is essential for normal cell functions. Phospholipid asymmetry is maintained by transporters that shuttle phospholipids between the inner leaflet and the outer leaflet of the membrane bilayer. When an exogenous, short acyl chain, phosphatidylcholine (PC) or phosphatidylserine (PS) is incorporated into erythrocytes, a discocyte-to-echinocyte shape change is induced. PC treated cells remain echinocytic, while PS treated cells return to discocytes, and eventually stomatocytes, due to the action of an inwardly directed transporter. These morphol. changes have been well studied by light microscopy and SEM in the past few decades. However, most of this research is based on the glutaraldehyde fixed cells, which limits the dynamic study in discrete time points instead of continuous single cell measurements. Scanning ion conductance microscopy (SICM) is a scanning probe technique which is ideal for live cell imaging due to high resoln., in situ and non-contact scanning. To better understand these phospholipid-induced morphol. changes, SICM was used to scan the morphol. change of human erythrocytes after the incorporation of exogenous dilauroylphosphatidylserine (DLPS) and the results revealed single cell dynamic morphol. changes and the movement of spicules on the membrane surface.
- 24Rubfiaro, A. S.; Tsegay, P. S.; Lai, Y.; Cabello, E.; Shaver, M.; Hutcheson, J.; Liu, Y.; He, J. Scanning Ion Conductance Microscopy Study Reveals the Disruption of the Integrity of the Human Cell Membrane Structure by Oxidative DNA Damage. ACS Appl. Bio Mater. 2021, 4 (2), 1632– 1639, DOI: 10.1021/acsabm.0c0146124https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhtVOgt7s%253D&md5=80d72aa6c13b7d77bb1dd2db492b4b1aScanning Ion Conductance Microscopy Study Reveals the Disruption of the Integrity of the Human Cell Membrane Structure by Oxidative DNA DamageRubfiaro, Alberto S.; Tsegay, Pawlos S.; Lai, Yanhao; Cabello, Emmanuel; Shaver, Mohammad; Hutcheson, Joshua; Liu, Yuan; He, JinACS Applied Bio Materials (2021), 4 (2), 1632-1639CODEN: AABMCB; ISSN:2576-6422. (American Chemical Society)Oxidative stress can damage organs, tissues, and cells through reactive oxygen species (ROS) by oxidizing DNA, proteins, and lipids, thereby resulting in diseases. However, the underlying mol. mechanisms remain to be elucidated. Employing scanning ion conductance microscopy (SICM), the authors explored the early responses of human embryonic kidney (HEK293H) cells to oxidative DNA damage induced by potassium chromate (K2CrO4). The short term (1-2 h) exposure to a low concn. (10μM) of K2CrO4 damaged the lipid membrane of HEK293H cells, resulting in structural defects and depolarization of the cell membrane and reducing cellular secretion activity shortly after the treatment. Further the K2CrO4 treatment decreased the expression of the cytoskeleton protein, β-actin, by inducing oxidative DNA damage in the exon 4 of the β-actin gene. These results suggest that K2CrO4 caused oxidative DNA damage in cytoskeleton genes such as β-actin and reduced their expression, thereby disrupting the organization of the cytoskeleton beneath the cell membrane and inducing cell membrane damages. The authors' study provides direct evidence that oxidative DNA damage disrupted human cell membrane integrity by deregulating cytoskeleton gene expression.
- 25Parres-Gold, J.; Chieng, A.; Wong Su, S.; Wang, Y. Real-Time Characterization of Cell Membrane Disruption by α-Synuclein Oligomers in Live SH-SY5Y Neuroblastoma Cells. ACS Chem. Neurosci. 2020, 11 (17), 2528– 2534, DOI: 10.1021/acschemneuro.0c0030925https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsFaqtrrJ&md5=ce8faca3d9088e072cee2c942983f1a7Real-Time Characterization of Cell Membrane Disruption by α-Synuclein Oligomers in Live SH-SY5Y Neuroblastoma CellsParres-Gold, Jacob; Chieng, Andy; Wong Su, Stephanie; Wang, YixianACS Chemical Neuroscience (2020), 11 (17), 2528-2534CODEN: ACNCDM; ISSN:1948-7193. (American Chemical Society)Aggregation of the natively unfolded protein α-synuclein (α-Syn) has been widely correlated to the neuronal death assocd. with Parkinson's disease. Mutations and protein overaccumulation can promote the aggregation of α-Syn into oligomers and fibrils. Recent work has suggested that α-Syn oligomers can permeabilize the neuronal membrane, promoting calcium influx and cell death. However, the mechanism of this permeabilization is still uncertain and has yet to be characterized in live cells. This work uses scanning ion conductance microscopy (SICM) to image, in real time and without using chem. probes, the topogs. of live SH-SY5Y neuroblastoma cells after exposure to α-Syn oligomers. Substantial morphol. changes were obsd., with micrometer-scale hills and troughs obsd. at lower α-Syn concns. (1.00μM) and large, transient pores obsd. at higher α-Syn concns. (6.0μM). These findings suggest that α-Syn oligomers may permeabilize the neuronal membrane by destabilizing the lipid bilayer and opening transient pores.
- 26Bridi, J. C.; Hirth, F. Mechanisms of α-Synuclein Induced Synaptopathy in Parkinson’s Disease. Front. Neurosci. 2018, 12, 80, DOI: 10.3389/fnins.2018.0008026https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1MroslGhtg%253D%253D&md5=c6ea0fd85d1ead1c07397d2b29afd4f9Mechanisms of α-Synuclein Induced Synaptopathy in Parkinson's DiseaseBridi Jessika C; Hirth FrankFrontiers in neuroscience (2018), 12 (), 80 ISSN:1662-4548.Parkinson's disease (PD) is characterized by intracellular inclusions of aggregated and misfolded α-Synuclein (α-Syn), and the loss of dopaminergic (DA) neurons in the brain. The resulting motor abnormalities mark the progression of PD, while non-motor symptoms can already be identified during early, prodromal stages of disease. Recent studies provide evidence that during this early prodromal phase, synaptic and axonal abnormalities occur before the degenerative loss of neuronal cell bodies. These early phenotypes can be attributed to synaptic accumulation of toxic α-Syn. Under physiological conditions, α-Syn functions in its native conformation as a soluble monomer. However, PD patient brains are characterized by intracellular inclusions of insoluble fibrils. Yet, oligomers and protofibrils of α-Syn have been identified to be the most toxic species, with their accumulation at presynaptic terminals affecting several steps of neurotransmitter release. First, high levels of α-Syn alter the size of synaptic vesicle pools and impair their trafficking. Second, α-Syn overexpression can either misregulate or redistribute proteins of the presynaptic SNARE complex. This leads to deficient tethering, docking, priming and fusion of synaptic vesicles at the active zone (AZ). Third, α-Syn inclusions are found within the presynaptic AZ, accompanied by a decrease in AZ protein levels. Furthermore, α-Syn overexpression reduces the endocytic retrieval of synaptic vesicle membranes during vesicle recycling. These presynaptic alterations mediated by accumulation of α-Syn, together impair neurotransmitter exocytosis and neuronal communication. Although α-Syn is expressed throughout the brain and enriched at presynaptic terminals, DA neurons are the most vulnerable in PD, likely because α-Syn directly regulates dopamine levels. Indeed, evidence suggests that α-Syn is a negative modulator of dopamine by inhibiting enzymes responsible for its synthesis. In addition, α-Syn is able to interact with and reduce the activity of VMAT2 and DAT. The resulting dysregulation of dopamine levels directly contributes to the formation of toxic α-Syn oligomers. Together these data suggest a vicious cycle of accumulating α-Syn and deregulated dopamine that triggers synaptic dysfunction and impaired neuronal communication, ultimately causing synaptopathy and progressive neurodegeneration in Parkinson's disease.
- 27Winner, B.; Jappelli, R.; Maji, S. K.; Desplats, P. A.; Boyer, L.; Aigner, S.; Hetzer, C.; Loher, T.; Vilar, M.; Campioni, S.; Tzitzilonis, C.; Soragni, A.; Jessberger, S.; Mira, H.; Consiglio, A.; Pham, E.; Masliah, E.; Gage, F. H.; Riek, R. In Vivo Demonstration That α-Synuclein Oligomers Are Toxic. Proc. Natl. Acad. Sci. U. S. A. 2011, 108 (10), 4194– 4199, DOI: 10.1073/pnas.110097610827https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXjsF2nu78%253D&md5=7af57c2b6e3726b17c5cb2e035f92ff5In vivo demonstration that α-synuclein oligomers are toxicWinner, Beate; Jappelli, Roberto; Maji, Samir K.; Desplats, Paula A.; Boyer, Leah; Aigner, Stefan; Hetzer, Claudia; Loher, Thomas; Vilar, Marial; Campioni, Silvia; Tzitzilonis, Christos; Soragni, Alice; Jessberger, Sebastian; Mira, Helena; Consiglio, Antonella; Pham, Emiley; Masliah, Eliezer; Gage, Fred H.; Riek, RolandProceedings of the National Academy of Sciences of the United States of America (2011), 108 (10), 4194-4199, S4194/1-S4194/12CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The aggregation of proteins into oligomers and amyloid fibrils is characteristic of several neurodegenerative diseases, including Parkinson disease (PD). In PD, the process of aggregation of α-synuclein (α-syn) from monomers, via oligomeric intermediates, into amyloid fibrils is considered the disease-causative toxic mechanism. We developed α-syn mutants that promote oligomer or fibril formation and tested the toxicity of these mutants by using a rat lentivirus system to investigate loss of dopaminergic neurons in the substantia nigra. The most severe dopaminergic loss in the substantia nigra is obsd. in animals with the α-syn variants that form oligomers (i.e., E57K and E35K), whereas the α-syn variants that form fibrils very quickly are less toxic. We show that α-syn oligomers are toxic in vivo and that α-syn oligomers might interact with and potentially disrupt membranes.
- 28Bigi, A.; Ermini, E.; Chen, S. W.; Cascella, R.; Cecchi, C. Exploring the Release of Toxic Oligomers from α-Synuclein Fibrils with Antibodies and STED Microscopy. Life 2021, 11 (5), 431, DOI: 10.3390/life1105043128https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXis1Cis7bK&md5=95009700ddba46421a6db0da96886037Exploring the release of toxic oligomers from α-synuclein fibrils with antibodies and STED microscopyBigi, Alessandra; Ermini, Emilio; Chen, Serene W.; Cascella, Roberta; Cecchi, CristinaLife (Basel, Switzerland) (2021), 11 (5), 431CODEN: LBSIB7; ISSN:2075-1729. (MDPI AG)α-Synuclein (αS) is an intrinsically disordered and highly dynamic protein involved in dopamine release at presynaptic terminals. The abnormal aggregation of αS as mature fibrils into intraneuronal inclusion bodies is directly linked to Parkinson's disease. Increasing exptl. evidence suggests that sol. oligomers formed early during the aggregation process are the most cytotoxic forms of αS. This study investigated the uptake by neuronal cells of pathol. relevant αS oligomers and fibrils exploiting a range of conformation-sensitive antibodies, and the super-resoln. stimulated emission depletion (STED) microscopy. We found that prefibrillar oligomers promptly penetrate neuronal membranes, thus resulting in cell dysfunction. By contrast, fibril docking to the phospholipid bilayer is accompanied by αS conformational changes with a progressive release of A11-reactive oligomers, which can enter into the neurons and trigger cell impairment. Our data provide important evidence on the role of αS fibrils as a source of harmful oligomers, which resemble the intermediate conformers formed de novo during aggregation, underling the dynamic and reversible nature of protein aggregates responsible for α-synucleinopathies.
- 29Volpicelli-Daley, L. A.; Luk, K. C.; Lee, V. M.-Y. Addition of Exogenous α-Synuclein Preformed Fibrils to Primary Neuronal Cultures to Seed Recruitment of Endogenous α-Synuclein to Lewy Body and Lewy Neurite-like Aggregates. Nat. Protoc. 2014, 9 (9), 2135– 2146, DOI: 10.1038/nprot.2014.14329https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhtlGntb3K&md5=1273f2338024b4321357a3dfa8420ffcAddition of exogenous α-synuclein preformed fibrils to primary neuronal cultures to seed recruitment of endogenous α-synuclein to Lewy body and Lewy neurite-like aggregatesVolpicelli-Daley, Laura A.; Luk, Kelvin C.; Lee, Virginia M-Y.Nature Protocols (2014), 9 (9), 2135-2146CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)This protocol describes a primary neuronal model of formation of α-synuclein (α-syn) aggregates that recapitulate features of the Lewy bodies and Lewy neurites found in Parkinson's disease brains and other synucleinopathies. This model allows investigation of aggregate formation, their impact on neuron function, and development of therapeutics. Addn. of preformed fibrils (PFFs) synthesized from recombinant α-syn to neurons seeds the recruitment of endogenous α-syn into aggregates characterized by detergent insoly. and hyperphosphorylation. Aggregate formation follows a lag phase of 2-3 d, followed by formation in axons by days 4-7, spread to somatodendritic compartments by days 7-10 and neuron death ∼14 d after PFF addn. Here we provide methods and highlight the crucial steps for PFF formation, PFF addn. to cultured hippocampal neurons and confirmation of aggregate formation. Neurons derived from various brain regions from nontransgenic and genetically engineered mice and rats can be used, allowing interrogation of the effect of specific genes on aggregate formation.
- 30Shimozawa, A.; Fujita, Y.; Kondo, H.; Takimoto, Y.; Terada, M.; Sanagi, M.; Hisanaga, S.; Hasegawa, M. Effect of L-DOPA/Benserazide on Propagation of Pathological α-Synuclein. Front. Neurosci. 2019, 13, 595, DOI: 10.3389/fnins.2019.0059530https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3Mzis1GqtA%253D%253D&md5=8478f3d4503c417cc5a618f840deb2d3Effect of L-DOPA/Benserazide on Propagation of Pathological α-SynucleinShimozawa Aki; Fujita Yuuki; Kondo Hiromi; Terada Makoto; Hasegawa Masato; Shimozawa Aki; Fujita Yuuki; Hisanaga Shin-Ichi; Takimoto Yu; Sanagi MasanaoFrontiers in neuroscience (2019), 13 (), 595 ISSN:1662-4548.Parkinson's disease (PD) and related disorders are characterized by filamentous or fibrous structures consisting of abnormal α-synuclein in the brains of patients, and the distributions and spread of these pathologies are closely correlated with disease progression. L-DOPA (a dopamine precursor) is the most effective therapy for PD, but it remains unclear whether the drug has any effect on the formation and propagation of pathogenic abnormal α-synuclein in vivo. Here, we tested whether or not L-DOPA influences the prion-like spread of α-synuclein pathologies in a wild-type (WT) mouse model of α-synuclein propagation. To quantitative the pathological α-synuclein in mice, we prepared brain sections stained with an anti-phosphoSer129 (PS129) antibody after pretreatments with autoclaving and formic acid, and carefully analyzed positive aggregates on multiple sections covering the areas of interest using a microscope. Notably, a significant reduction in the accumulation of phosphorylated α-synuclein was detected in substantia nigra of L-DOPA/benserazide (a dopamine decarboxylase inhibitor)-treated mice, compared with control mice. These results suggest that L-DOPA may slow the progression of PD in vivo by suppressing the aggregation of α-synuclein in dopaminergic neurons and the cell-to-cell propagation of abnormal α-synuclein. This is the first report describing the suppressing effect of L-DOPA/benserazide on the propagation of pathological α-synuclein. The experimental protocols and detection methods in this study are expected to be useful for evaluation of drug candidates or new therapies targeting the propagation of α-synuclein.
- 31Conway, K. A.; Rochet, J.-C.; Bieganski, R. M.; Lansbury, P. T. Kinetic Stabilization of the α-Synuclein Protofibril by a Dopamine-α-Synuclein Adduct. Science 2001, 294 (5545), 1346– 1349, DOI: 10.1126/science.106352231https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXotlKmurk%253D&md5=ff9705bd276af2c78db588b7beb321cbKinetic stabilization of the α-synuclein protofibril by a dopamine-α-synuclein adductConway, Kelly A.; Rachet, Jean-Christophe; Bieganski, Robert M.; Lansbury, Peter T., Jr.Science (Washington, DC, United States) (2001), 294 (5545), 1346-1349CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)The substantia nigra in Parkinson's disease (PD) is depleted of dopaminergic neurons and contains fibrillar Lewy bodies comprising primarily α-synuclein. We screened a library t6 identify drug-like mols. to probe the relation between neurodegeneration and α-synuclein fibrilization. All but one of 15 fibril inhibitors were catecholamines related to dopamine. The inhibitory activity of dopamine depended on its oxidative ligation to α-synuclein and was selective for the protofibril-to-fibril conversion, causing accumulation of the α-synuclein protofibril. Adduct formation provides an explanation for the dopaminergic selectivity of α-synuclein-assocd. neurotoxicity in PD and has implications for current and future PD therapeutic and diagnostic strategies.
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