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Synthesis and Assessment of Novel Probes for Imaging Tau Pathology in Transgenic Mouse and Rat Models
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  • Lindsay McMurray
    Lindsay McMurray
    Molecular Imaging Chemistry Laboratory, Wolfson Brain Imaging Centre, University of Cambridge, Cambridge CB2 0QQ, United Kingdom
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  • Jennifer A. Macdonald
    Jennifer A. Macdonald
    MRC Laboratory of Molecular Biology, Cambridge CB2 0QH, United Kingdom
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  • Nisha Kuzhuppilly Ramakrishnan
    Nisha Kuzhuppilly Ramakrishnan
    Molecular Imaging Chemistry Laboratory, Wolfson Brain Imaging Centre, University of Cambridge, Cambridge CB2 0QQ, United Kingdom
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  • Yanyan Zhao
    Yanyan Zhao
    Molecular Imaging Chemistry Laboratory, Wolfson Brain Imaging Centre, University of Cambridge, Cambridge CB2 0QQ, United Kingdom
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  • David W. Williamson
    David W. Williamson
    Molecular Imaging Chemistry Laboratory, Wolfson Brain Imaging Centre, University of Cambridge, Cambridge CB2 0QQ, United Kingdom
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  • Ole Tietz
    Ole Tietz
    Molecular Imaging Chemistry Laboratory, Wolfson Brain Imaging Centre, University of Cambridge, Cambridge CB2 0QQ, United Kingdom
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  • Xiaoyun Zhou
    Xiaoyun Zhou
    Molecular Imaging Chemistry Laboratory, Wolfson Brain Imaging Centre, University of Cambridge, Cambridge CB2 0QQ, United Kingdom
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  • Steven Kealey
    Steven Kealey
    Molecular Imaging Chemistry Laboratory, Wolfson Brain Imaging Centre, University of Cambridge, Cambridge CB2 0QQ, United Kingdom
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  • Steven G. Fagan
    Steven G. Fagan
    Department of Clinical Neurosciences, University of Cambridge, Cambridge CB2 0QQ, United Kingdom
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  • Tomáš Smolek
    Tomáš Smolek
    Axon Neuroscience R&D Services SE, Bratislava, Slovak Republic 811 02
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  • Veronika Cubinkova
    Veronika Cubinkova
    Axon Neuroscience R&D Services SE, Bratislava, Slovak Republic 811 02
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  • Norbert Žilka
    Norbert Žilka
    Axon Neuroscience R&D Services SE, Bratislava, Slovak Republic 811 02
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  • Maria Grazia Spillantini
    Maria Grazia Spillantini
    Department of Clinical Neurosciences, University of Cambridge, Cambridge CB2 0QQ, United Kingdom
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  • Aviva M. Tolkovsky
    Aviva M. Tolkovsky
    Department of Clinical Neurosciences, University of Cambridge, Cambridge CB2 0QQ, United Kingdom
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  • Michel Goedert
    Michel Goedert
    MRC Laboratory of Molecular Biology, Cambridge CB2 0QH, United Kingdom
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  • Franklin I. Aigbirhio*
    Franklin I. Aigbirhio
    Molecular Imaging Chemistry Laboratory, Wolfson Brain Imaging Centre, University of Cambridge, Cambridge CB2 0QQ, United Kingdom
    Department of Clinical Neurosciences, University of Cambridge, Cambridge CB2 0QQ, United Kingdom
    *Email: [email protected]
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    Orcidhttp://orcid.org/0000-0001-9453-5257
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ACS Chemical Neuroscience

Cite this: ACS Chem. Neurosci. 2021, 12, 11, 1885–1893
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https://doi.org/10.1021/acschemneuro.0c00790
Published March 10, 2021

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CC-BY 4.0 .

Abstract

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Aggregated tau protein is a core pathology present in several neurodegenerative diseases. Therefore, the development and application of positron emission tomography (PET) imaging radiotracers that selectively bind to aggregated tau in fibril form is of importance in furthering the understanding of these disorders. While radiotracers used in human PET studies offer invaluable insight, radiotracers that are also capable of visualizing tau fibrils in animal models are important tools for translational research into these diseases. Herein, we report the synthesis and characterization of a novel library of compounds based on the phenyl/pyridinylbutadienylbenzothiazoles/benzothiazolium (PBB3) backbone developed for this application. From this library, we selected the compound LM229, which binds to recombinant tau fibrils with high affinity (Kd = 3.6 nM) and detects with high specificity (a) pathological 4R tau aggregates in living cultured neurons and mouse brain sections from transgenic human P301S tau mice, (b) truncated human 151-351 3R (SHR24) and 4R (SHR72) tau aggregates in transgenic rat brain sections, and (c) tau neurofibrillary tangles in brain sections from Alzheimer’s disease (3R/4R tau) and progressive supranuclear palsy (4R tau). With LM229 also shown to cross the blood–brain barrier in vivo and its effective radiolabeling with the radioisotope carbon-11, we have established a novel platform for PET translational studies using rodent transgenic tau models.

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Introduction

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Imaging protein aggregates, characteristic features of a variety of neurodegenerative diseases, (1) has become a powerful means of investigating their pathology. In particular, positron emission tomography (PET) is the method of choice to study their pathophysiology in vivo using imaging probes selective for the various aggregated protein forms. PET probes were initially developed for imaging β-amyloid (Aβ) aggregated protein, present as plaque deposits in Alzheimer’s disease (AD), (2) then probes selective for protein tau aggregates, which form intracellular neurofibrillary tangles (NFT) in AD. (3,4) In addition to AD, pathological tau aggregates are a constituent feature of several other neurodegenerative disorders, collectively termed tauopathies; (5−7) hence tau PET imaging is a key biomarker technology. Since the first reports of [18F]T807 ([18F]AV1451) (8) as a suitable PET radiotracer for selective imaging of NFTs, several radiotracers capable of binding to and visualizing tau have now been used for clinical PET studies, (4,9) including [11C]PBB3, (10) [18F]PI-2620, (11) and [18F]MK6240. (12)
While the final aim is to employ tau PET probes for human diagnostics and investigation of disease mechanisms, there is a need to underpin their application by studying experimental animal models of neurodegeneration. These provide a critical translational link between laboratory research from cellular and animal models to human disease and, conversely, allow human pathophysiological data to be used to design more appropriate experimental models and studies. In particular, transgenic animal models of dementia are important for understanding the involvement of Aβ and NFTs in disease, especially given their use in longitudinal and behavioral studies. (13) Therefore, the development of PET radiotracers that are capable of visualizing tau aggregates within these animal models is of great importance in advancing both the understanding of the disease and the development of new therapeutics alongside clinical studies.
While the present range of tau PET radiotracers has shown excellent affinities for tau fibrils, they are still limited in their ability to visualize tau NFTs in animal models as well as in humans. For example, [18F]T807 (AV1451) showed no specific binding to tau tangles in transgenic mice expressing mutant human P301L tau as assessed by in vitro (14) and microPET in vivo (15) imaging studies. Studies with P301S tau mice indicated a difference in retention of [18F]AV1451 in their brainstem of no more than 23% compared to control wild-type (WT) mice. (16) More successful have been the radiotracers [11C]PBB3, which has been shown to bind to tau pathology in PS19 tau transgenic mice (10) and [18F]THK5117, which bound to tau aggregates in two transgenic mice mouse models, P301S tau and GSK3-β X P301L tau (biGT). (17) Even though these can detect tau fibrils, higher specificity is needed to analyze subtle early changes in tau aggregation. Notably, imaging tau pathology in a rat model of tauopathy has yet to be reported. With the rat brain being approximately six times larger than the mouse brain, this would bring distinct advantages for research involving small animal PET imaging. For example, it would enable more accurate quantification of radioactivity concentration in brain regions and blood sampling for gold standard blood-based kinetic analysis.
Therefore, to improve the development of PET imaging probes for visualizing tau pathology in transgenic models of neurodegeneration, herein, we report the synthesis and characterization of a novel series of compounds based on the phenyl/pyridinylbutadienylbenzothiazoles/benzothiazolium (PBB3) structure (Scheme 1A), which has been shown to bind to tau aggregates in both mouse models and humans. (10) Binding affinities to heparin-induced synthetic recombinant tau fibrils were determined as well as binding to aggregated tau in a transgenic human P301S tau mouse model. (18) The most promising compound from this series, LM229, was then assessed for its binding to pathological tau in transgenic mouse and rat brain tissues in vitro and in vivo and in two human tauopathies. The successful radiolabeling of LM229 with the carbon-11 (t1/2 = 20 min) PET radioisotope is described.

Scheme 1

Scheme 1. (A) Development of Novel Compounds for Imaging Tau Aggregation in Animal Models of Tau and (B) Streamlined Convergent Synthetic Route for a Compound Library
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Results and Discussion

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Synthesis of a Compound Library

At the outset of our investigation, a library of unlabeled analogues based on the core structure of PBB3 (19) was synthesized. To rapidly access compounds of interest, we developed a streamlined convergent route centered on a Horner–Wadsworth–Emmons coupling of two key fragments: aldehyde C and phosphonate D (Scheme 1B). Synthesis of aldehyde C commenced with palladium(II)-catalyzed Heck reaction of a range of commercially available bromopyridines (A) with methyl acrylate. DIBAL reduction followed by immediate benzylic oxidation with manganese dioxide furnished the desired aldehyde C. The desired benzothiazoles (B) were either available from commercial sources or synthesized from simple starting materials. Deprotonation with lithium diisopropylamide (LDA) followed by addition of diethylchlorophosphate afforded the desired phosphonates D. Coupling of the aldehyde C and phosphonate D proceeded smoothly for all derivatives to furnish the desired library of PBB3 analogues.

Assessment of Binding Affinities

All compounds were assessed for their ability to bind to synthetic recombinant tau fibrils using a fluorescence quenching assay (20) (Table 1), for which we first established a Kd value for PBB3 of 1.4 nM, in good agreement with the previously reported value of 1.3 nM. (21) Binding affinities for some compounds (141, 164, 166, 169, 171, and 207) could not be assessed with this assay because of negligible changes in fluorescence intensity. While several compounds showed affinities below the micromolar range, LM3-229 (abbreviated LM229 or 229) showed a Kd of 3.8 nM, in the nanomolar range expected of a PET ligand, combined with having chemical moieties with the potential to be radiolabeled by either carbon-11 or fluorine-18 for development as a PET imaging probe. This compound was then selected for further characterization and development.
Table 1. Kd (nM) of Compound Binding to Heparin-Induced Synthetic Tau Fibrils Determined by Fluorimetry
nameR1R2R3XYKd (nM)
PBB3HOHNHMeC(H)N1.4
148HOHFC(H)N56
152HOHFNC(H)353
229FOHNHMeC(H)N3.6
225FOMeNHMeC(H)N1.1
171aOHHFC(H)N 
166aOHHFNC(H) 
169aHHFC(H)N 
164aHHFNC(H) 
236OCH2CH2FHNHMeC(H)N454
207aHFNHMeC(H)N 
141aFHNHMeC(H)N 
a

Due to negligible change of fluorescence intensity, binding affinities could not be determined.

Binding to Transgenic P301S Tau Mouse Brain Sections

We then screened all of the compounds for their binding to tau inclusions in brain sections from transgenic P301S tau mice, which express the shortest isoform (0N4R) of human 4-repeat (4R) tau, (18) using their autofluorescence properties to rapidly analyze their binding. Although some of the compounds recognized tau aggregates, they also showed significant fluorescence in WT brain sections, giving a high background and low specificity, therefore unlikely to be useful as PET radiotracers. However, compound LM229, showed intense, specific binding that correlated with hyperphosphorylated pathological tau (AT8 antibody, pS202/pS205) and β-sheet-rich filamentous tau (AT100 antibody, pT212/pS214/pT217 (18,22)), consistent with its high binding affinity to recombinant tau fibrils (Figure 1).

Figure 1

Figure 1. LM229 binding to mouse brain sections is specific to neurons with pathological tau aggregates. (Top) No specific binding to brainstem sections from WT animals but coincident LM229 binding (green) and (middle) AT8 or (bottom) AT100 antibody staining (red) in phenotypic P301S tau mice; AT100 binding correlates with tau fibrils. Scale bar = 20 μm.

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To further confirm that binding of LM229 was specific to aggregated tau rather than to soluble tau, binding to brain sections from P301S tau mice at 2 and 6 months of age was compared, based on the fact that a significant amount of tau aggregates is detected in older mice (>3 months). (18,23) Rare binding was detected in brain sections from 2 months old mice, but significant binding to large populations of neurons was evident at 6 months of age (Supporting Information, Figure S1).
We next looked at the ability of LM229 to cross the blood–brain barrier (BBB), a key property of any viable neuro-radiotracer. LM229 was injected intravenously (i.v.) (1 mg/kg weight), and after 1 h, WT and P301S tau mice were perfused-fixed and brain sections were imaged under a fluorescence microscope. Figure 2 shows binding of LM229 to some nerve cells from P301S tau transgenic mice, with little binding to the brains of non-transgenic control mice., confirming that the compound crosses the BBB. Moreover, when compared with PBB3 (Figure 3), and based on the compounds having similar extinction coefficients, LM229 showed comparably less non-specific binding and more specific binding to tau aggregates by fluorescence microscopy, suggesting that LM229 was highly suitable as a ligand for tau aggregate tracing within the transgenic mouse model. However, we await a comparison of PBB3 and LM229 by PET imaging to determine their relativity properties as in vivo imaging probes.

Figure 2

Figure 2. Fluorescence image of brain section from WT (A) and P301S tau mice (B) fixed 1 h after i.v. injection of LM229 showing entry into brain from periphery. Scale bar = 100 μm.

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Figure 3

Figure 3. P301S tau brain tissue from (E) ex vivo experiments with PBB3 (i.v. injection 1 h prior to injection) colocalized with AT8 showing binding of PBB3 with significant nonspecific binding; (F) ex vivo experiments with LM229 showing significant specific binding to tau aggregates as confirmed with colocalization with AT8.

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Binding of LM229 to Cultured Neurons from P301S Tau Transgenic Mice

P301S tau mice with aggregated tau in the brain also express aggregated tau in DRG neurons. (24−26) Unlike neurons from the brain, which do not survive in culture when extracted beyond neonatal ages, DRG neurons with tau filaments can be cultured from adult mice when filamentous tau pathology is well-developed. To estimate the apparent affinity of LM229 to tau aggregates directly in living neurons, we imaged live neurons after 20 min incubation with various concentrations of LM229 (Figure 4A). Analysis of fluorescence intensity by nonlinear curve fitting to a Michaelis–Menten equation yielded an IC50 of 2.7 ± 0.7 μM (R2 = 0.97) (Figure 4B). A dose–response conducted after fixation and permeabilization of the neurons (Figure 4C) gave a similar affinity (IC50 = 1.7 ± 0.3 μM, R2 = 0.99, Figure 4B), showing that the plasma membrane does not form a barrier to LM229 entry/binding. LM229 binding was specific to neurons with aggregated tau, as detected with the AT100 antibody (18,22,23,26) (Figure 4D). Notably, inspection of the neurons 3 days after LM229 exposure and washes showed that the LM229 fluorescence was still retained albeit to a lower intensity. Although PBB3 also bound to DRG neurons with tau aggregates at 30–100 μM, it was not possible to derive an IC50 value because of its low affinity in this assay (Supporting Information, Figure S2). Thus, LM229 is highly cell permeable, and binding follows a single saturable binding algorithm. The shift from nanomolar to micromolar affinity between binding to heparin tau fibrils and tau aggregates in P301S tau neurons may reflect the different environment and assay conditions or the different conformations that tau aggregates assume in neurons compared to heparin–tau complexes. (27)

Figure 4

Figure 4. (A) Fluorescence images of LM229 binding to live DRG neurons (green). Phase contrast images show total live neurons. (B) Fluorescence intensity per neuron as a function of LM229 concentration (mean ± SD, 5–15 neurons per concentration, three independent cultures; black line, result of nonlinear curve fitting, green and blue dashed lines, 95% confidence intervals; live neurons: IC50 = 2.97 ± 0.73; R2 = 0.968; fixed neurons: IC50 = 1.67 ± 0.28; R2 = 0.990). (C) Dose–response of LM229 binding to fixed neurons. (D) Co-staining of LM229 (10 μM, green) and antiphospho-tau antibody AT100 (red). Blue in merged image shows cell nuclei in the culture.

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Binding to Tau Transgenic Rat Brain Tissue

Since future PET studies for development of these compounds would require kinetic modeling for their assessment in vivo, we tested their performance in tau transgenic rats. Rat brains and blood volumes are about 6- and 10-fold larger, respectively, than those of mice and thus enable higher resolution imaging and ample volume for repeated blood sampling. We used two transgenic rat lines expressing human-truncated tau comprising the proline-rich region and either three microtubule binding domains (3R tau151-391, SH24) (28) or four microtubule binding domains (4R tau151-391, SHR72). (29) Both LM229 (Figure 5A) and PBB3 (Figure 6A) bound with similar properties to pathological aggregated tau in brain sections from SHR24 rats, which develop tangle pathology in the cortex, as confirmed by colocalization with AT8 immunofluorescence (Figures 5B and 6B). Likewise, with similar properties, both LM229 (Figure 5C) and PBB3 (Figure 6C) bound to tau in brain sections from SHR72 rats, which develop tangles mainly in the brainstem, as confirmed by colocalization with AT8 immunofluorescence (Figures 5D and Figure 6D). However, compared to the SHR24 rat brain sections, there were less inclusions detected, which seems to indicate the compounds have preferential binding toward the three repeat forms of tau in these rat models. To our knowledge, this is the first time that tau PET imaging compounds have been shown to bind to pathological tau in a rat transgenic model. These results establish the basis to perform in vivo PET imaging studies, including further investigations into the selectivity of LM229 toward the three and four repeat forms of tau.

Figure 5

Figure 5. LM229 (green) binding to (A) 3R tau in cortical sections from SHR24 rat brains and (B) 4R tau in brainstem sections from SHR72 rats. (C, D) Colocalization with AT8 (red).

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Figure 6

Figure 6. Binding of PBB3 (green) to (A) 3R tau in the cortex of SHR24 brain tissue and (B) to 4R tau in the brainstem of SHR72 and (C, D) colocalization with staining AT8 (red).

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Binding to Human Tau Pathology

To further validate LM229 as a potential radiotracer for translational research, we investigated its binding to tau aggregates in human globus pallidus/putamen from progressive supranuclear palsy (PSP) subjects (Figure 7) and its selectivity for tau over β-amyloid pathology in AD frontal cortex sections (Figure 8). An intense signal was emitted by LM229 binding to PSP sections (Figure 6A), which was extensively colocalized with AT8 binding.

Figure 7

Figure 7. (A) Post-mortem PSP tissue stained with LM229; (B) Double staining of PSP tissue with LM229 and AT8 (red) to confirm colocalization of LM229 with tau aggregates.

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Figure 8

Figure 8. Post mortem AD tissue stained with LM229 and (A,C) and double staining with AT8 (B) and beta-amyloid antibodies (D), suggesting binding to tau rather than amyloid aggregates.

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In slices from frontal cortex of AD brains, LM229 gave rise to strong signal (Figure 8A), which colocalized with AT8 immunofluorescence (Figure 8B). In contrast, LM229 fluorescence intensity that colocalized with Aβ (Figure 8C) was lower (Figure 8D),suggesting selectivity for tau versus β-amyloid protein.

Radiosynthesis of [11C]LM229

Having characterized the in vitro properties of unlabeled LM229, we next synthesized a [11C]radiolabeled version for application as a PET imaging probe. An analogous route to that reported by Wang et al. (19) for the synthesis of [11C]PBB3 was developed. [11C]methyl iodide was trapped in a mixture of tert-butyldimethylsilyl (TBS)-protected E and potassium hydroxide in dimethylsulfoxide. The resulting mixture was heated to 125 °C for 5 min to access intermediate F. Addition of water, followed by heating to 60 °C for 2 min, enabled the deprotection of the TBS group to furnish [11C]LM229 (Scheme 2). It was found that a loading of potassium hydroxide higher than that reported was required to prevent formation of undesired side products. Due to the heterogeneity of the reaction, mixing of the suspension of E and KOH via vortex prior to the reaction also proved to be crucial in the formation of [11C]LM229. Purification of the crude reaction mixture by semipreparative HPLC yielded [11C]LM229 (Supporting Information, Figure S1), which was reformulated into ethanolic saline using a C18-light Sep-Pak cartridge to afford [11C]LM229 1.0–1.4 GBq at end-of-synthesis (10% DCY, 96.6% RCP, 250 GBq/μmol molar activity).

Scheme 2

Scheme 2. Two-Step Radiosynthesis of [11C]LM229
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PET Studies with [11C]LM229

We then performed preliminary in vivo PET studies with [11C]L229 in both WT and transgenic P310S tau mice. These studies confirmed blood–barrier brain penetration by the radiotracer with maximum brain uptake (%ID/g max; WT = 1.56, P301S = 2.38) within the first minute followed by washout within the 90 min scan (Figure 9). The brain exposure was higher in the P301S mouse (area under the curve, AUC = 60) compared to the wild-type mouse (AUC = 34). Further PET studies are required to fully characterize the in vivo pharmacokinetics of the radiotracer in both mice and rats.

Figure 9

Figure 9. Time–activity curves for [11C]LM229 from in vivo PET studies in wild-type and P301S mice.

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Conclusion

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In order to establish transgenic tau rodent models as a translational platform for PET imaging research in neurodegenerative disorders, we have developed a range of novel compounds based on the PBB3 structure. One of these compounds, LM229, is shown to have high binding affinity to recombinant tau fibrils. Further evaluation of LM229 showed it to bind to pathological tau inclusions in transgenic human P301S tau mouse brains, with selectivity for tau inclusions in PSP and AD, dementias with different fibrillar tau isoform compositions. In addition, LM229 and PBB3 PET compounds were both shown to bind to tau pathology in brain tissue from two transgenic tau rat models with either 3R and 4R tau inclusions with preferential binding toward the 3R tau inclusions. To our knowledge, this is the first time any tau PET imaging compounds have been shown to bind to tau inclusions in a transgenic rat model. Finally, we have established that LM229 can be used for in vivo translational PET research, by developing a method for radiolabeling with the carbon-11 radioisotope and confirmation of BBB entry in mice.

Methods

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General Methods and Materials

All reagents and solvents were used as supplied or purified using standard procedures as necessary. Analytical separation was conducted on an Ultimate 3000 HPLC system (Thermo Scientific). [11C]CO2 was produced via the 14N(p,α) nuclear reaction using GE PETtrace cyclotron applying 16.5 MeV protons onto 0.5% oxygen in nitrogen gas. [11C]Methyl iodide was synthesized on a GE Medical Systems MeI MicroLab. A F-4500 FL spectrophotometer (Chemical Engineering department, University of Cambridge) was used to do fluorescence scan of all of the compounds mixed with tau fibrils. Excitation wavelength was 365 nm, and emission was scanned from 400 to 600 nm. With parameters, setting excitation slit was 5.0 nm, emission slit was 20.0 nm, PMT voltage was 950 V, and response was 0.5 s.
All mouse studies were carried out in the Laboratory of Molecular Imaging or the Clifford Allbutt Building, University of Cambridge, and the MRC Laboratory of Molecular Biology. P301S tau transgenic mice (18) and WT mice were on a pure C57Bl/6 Jax background. The SHR24, SHR72, and SHR wild-type rat brain slices were developed and obtained from AXON Neuroscience. (28) PSP brain tissue was obtained from the Cambridge Brain Bank, with the PSP cases being part of the cohort created by Prof. James Rowe. AD brain tissue was supplied by the London Neurodegenerative Diseases Brain Bank and Brains for Dementia Research.

Preparation of tau fibrils

Monomeric tau (tau-441 human) lyophilized powder was obtained from Eurogentec. Tau monomers (100 μM) were prepared in 20 mM BES buffer (pH 7.4 with 25 mM NaCl and 2 mM dithiothreitol and incubated at 56 °C for 10 min. After being cooled to room temperature in a water bath, heparin (25 μM) and a protease inhibitor mix were added and incubated at 37 °C for 10 days.

In Vitro and Ex Vivo Fluorescence Microscopy

Mice were perfused with PBS followed by 4% PFA. Following 24 h in 4% PFA, the brains were transferred to 20% sucrose prior to being embedded in OCT for cryo-sectioning. Sections (14 μm) were incubated with 50 μM LM229 or PBB3 for 1 h, washed in PBS + 0.1% Triton X-100, and cover-slipped with Vectashield mounting medium. Sections were imaged using a Leica DM6000 microscope. Mouse monoclonal AT8 (MN1020) or AT100 (MN1060) were from ThermoFisher and used at 1:1000. Anti-mouse secondary antibody was conjugated to AlexaFluor647.

Ex Vivo Mice Studies

Mice were injected intravenously (i.v.) with 50 μM LM229 or PBB3 (DMSO) diluted in 50:50 ethanol/PBS. Animals were then perfused (4% PFA) 1 h post-i.v.

Rat Brain

Fresh frozen rat brain coronal sections (10 μm) were stored on slides at −80 °C until use. Sections were fixed in ice cold acetone for 10 min and air-dried and then rehydrated twice for 5 min with buffer (10 mM PBS with 0.1% Triton X-100) before application of 5% bovine serum albumin for 1 h and two more 5 min buffer washes. Primary monoclonal antibody AT8 (Thermo MN1020, 1:1000) was added overnight at 4 °C, followed by 1 h incubation with a mixture of secondary AlexaFluor555 goat anti-mouse antibody (1:1000) and 50 μM PBB3 or LM229 in 50:50 ethanol/buffer. Following two 5 min washes in buffer, the slides were air-dried and cover-slipped using Fluorsave before analysis. The green autofluorescence of PBB3 and LM229 was visualized using a 488 nm filter and fluorescence of AT8 using a 568 nm filter on a Leica DM6000 microscope.

DRG Neurons

Neurons were prepared from phenotypic end stage 5–7 month old P301S tau mice and cultured as described previously. (24) For live staining, neurons cultured for 2 days were washed in PBS and incubated with various concentrations of LM229 for 20 min at room temperature. After three washes in PBS, neurons were imaged on a Leica DMI 4000B microscope using a DFC3000 G camera and application suite 4.0.0.11706. The dead cell stain Propidium iodide (1 μg/mL) was added to ensure that only live cells were imaged. Phase contrast images were taken to show viable neurons. For fixed cell staining, neurons were fixed for 20 min at room temperature in 4% paraformaldehyde, washed in PBS, and permeabilized in PBS containing 0.3% Triton X-100. LM299 was added as described for live cells, and cells were probed with AT100 (1:1000, MN1060) followed by anti-mouse AlexaFluor568 secondary antibody. Cultures were co-stained with the nuclear dye DAPI (blue) to reveal total cells.

Radiosynthesis of [11C]LM229

TBS-protected precursor E (1.5 mg) in DMSO (150 μL) was added to a suspension of powdered KOH (15 mg) in DMSO (300 μL), and the suspension mixed via vortex for 1 min. [11C]Methyl iodide was trapped in the suspension and heated to 125 °C for 5 min. Water (200 μL) was then added and the mixture heated to 60 °C for 2 min. The mixture was diluted with water (0.8 mL), and the radioactive material was loaded into a preparative HPLC system for purification (42% acetonitrile/50 mM ammonium formate, 6 mL/min, Luna 5μ, 10.00 mm internal diameter × 250 mm (Phenomenex)). The fraction corresponding to [11C]LM229 was collected in water (100 mL) and reformulated into saline (6 mL) containing ethanol (400 μL) and 25% ascorbic acid (200 μL) using a C18-light sep-pak cartridge to afford [11C]LM229 (1.0–1.4 GBq, 10% DCY). Analytical HPLC (Luna 5μ, 4.60 mm internal diameter × 250 mm (Phenomenex), 50% acetonitrile/50 mM ammonium formate, 1 mL/min) confirmed the formulated product was radiochemically pure (>95%). The specific activity of [11C]LM229 was >200 GBq/μmol.

PET Studies with [11C]LM229

These studies were regulated under the Animals (Scientific Procedures) Act 1986 Amendment Regulations 2012 following ethical review by the University of Cambridge Animal Welfare and Ethical Review Body (AWERB). The mice were anesthetized with 5% isoflurane, and general anesthesia was maintained using 1.5% isoflurane. The anesthetized mice were placed prone on the bed of a microPET Focus-220 scanner (Concorde Microsystems, Knoxville, TN). Body temperature was maintained at 37 °C using a heating blanket connected to a rectal thermistor probe. Blood oxygen saturation as well as heart rate and breathing rate were monitored and maintained within normal limits using a noninvasive mouseOX pulse-oximeter sensor (Starr Life Science Corp., Oakmont, PA) attached to the thigh. Before the injection of a tracer, a transmission scan was performed with a 68Ge point source for attenuation and scatter correction of 511 keV photons. [11C]LM229 was injected via the tail vein over 30 s, followed by a 15 s heparin–saline flush. Dynamic data were acquired in list mode for 90 min. Data were subsequently Fourier rebinned in following time frames: 12 × 5 s, 6 × 10 s, 3 × 20 s, 4 × 30 s, 5 × 60 s, 10 × 120 s, 12 × 5 min. Corrections were applied for randoms, dead time, normalization, attenuation, and decay. Fourier rebinning was used to compress the 4D sinograms to 3D before reconstruction with a 2D-filtered back projection with a Hann window cutoff at the Nyquist frequency. The image voxel size was 0.95 × 0.95 × 0.80 mm, with an array size of 128 × 128 × 95.
Three-dimensional volumes of interest (VOIs) for mice brain MRI template available on PMOD software (version 3.8; PMOD technologies, Zurich, Switzerland) were modified to obtain a whole brain VOI. Individual PET images were then co-registered with this MRI template, and the VOI transferred from MRI to PET. Whole-brain time–activity curves were obtained for each of the animals. The results were expressed as % injected dose per gram, assuming a specific gravity of 1 g·mL–1 for brain tissue.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acschemneuro.0c00790.

  • Synthetic and analytical data of compounds, immunohistochemistry, and binding affinity methods (PDF)

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Author Information

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  • Corresponding Author
    • Franklin I. Aigbirhio - Molecular Imaging Chemistry Laboratory, Wolfson Brain Imaging Centre, University of Cambridge, Cambridge CB2 0QQ, United Kingdom;  Department of Clinical Neurosciences, University of Cambridge, Cambridge CB2 0QQ, United Kingdom;  Orcidhttp://orcid.org/0000-0001-9453-5257;  Email: [email protected]
  • Authors
    • Lindsay McMurray - Molecular Imaging Chemistry Laboratory, Wolfson Brain Imaging Centre, University of Cambridge, Cambridge CB2 0QQ, United Kingdom
    • Jennifer A. Macdonald - MRC Laboratory of Molecular Biology, Cambridge CB2 0QH, United Kingdom
    • Nisha Kuzhuppilly Ramakrishnan - Molecular Imaging Chemistry Laboratory, Wolfson Brain Imaging Centre, University of Cambridge, Cambridge CB2 0QQ, United Kingdom
    • Yanyan Zhao - Molecular Imaging Chemistry Laboratory, Wolfson Brain Imaging Centre, University of Cambridge, Cambridge CB2 0QQ, United Kingdom
    • David W. Williamson - Molecular Imaging Chemistry Laboratory, Wolfson Brain Imaging Centre, University of Cambridge, Cambridge CB2 0QQ, United Kingdom
    • Ole Tietz - Molecular Imaging Chemistry Laboratory, Wolfson Brain Imaging Centre, University of Cambridge, Cambridge CB2 0QQ, United Kingdom
    • Xiaoyun Zhou - Molecular Imaging Chemistry Laboratory, Wolfson Brain Imaging Centre, University of Cambridge, Cambridge CB2 0QQ, United Kingdom
    • Steven Kealey - Molecular Imaging Chemistry Laboratory, Wolfson Brain Imaging Centre, University of Cambridge, Cambridge CB2 0QQ, United Kingdom
    • Steven G. Fagan - Department of Clinical Neurosciences, University of Cambridge, Cambridge CB2 0QQ, United Kingdom
    • Tomáš Smolek - Axon Neuroscience R&D Services SE, Bratislava, Slovak Republic 811 02
    • Veronika Cubinkova - Axon Neuroscience R&D Services SE, Bratislava, Slovak Republic 811 02
    • Norbert Žilka - Axon Neuroscience R&D Services SE, Bratislava, Slovak Republic 811 02
    • Maria Grazia Spillantini - Department of Clinical Neurosciences, University of Cambridge, Cambridge CB2 0QQ, United Kingdom
    • Aviva M. Tolkovsky - Department of Clinical Neurosciences, University of Cambridge, Cambridge CB2 0QQ, United Kingdom
    • Michel Goedert - MRC Laboratory of Molecular Biology, Cambridge CB2 0QH, United Kingdom
  • Author Contributions

    The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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Funding was provided by UK Medical Research Council grants (MR/K02308X/1 and a Proximity to Discovery award), a UK Engineering and Physical Science Council grant (EP/P008224/1) and EU/EFPIA/Innovative Medicines Initiative 2 Joint Undertaking (IMPRiND Grant No. 116060). We gratefully acknowledge the London Neurodegenerative Diseases Brain, The Cambridge Brain Bank, and Brains for Dementia Research for human AD and PSP tissue. PSP brain tissue was part of the cohort created by Professor James Rowe. The Cambridge Brain Bank is supported by the National Institute for Health Research (NIHR) Cambridge Biomedical Research Centre. Mass spectrometry data were acquired at the EPSRC UK National Mass Spectrometry Facility at Swansea University.

Abbreviations

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PET

positron emission tomography

Aβ

β-amyloid

AD

Alzheimer’s disease

PSP

progressive supranuclear palsy

PiB

Pittsburgh Compound B

NFT

neurofibrillary tangle

DTT

dithiothreitol

4R

four repeat tau

3R

three repeat tau

References

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This article references 29 other publications.

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    Declercq, Lieven; Celen, Sofie; Lecina, Joan; Ahamed, Muneer; Tousseyn, Thomas; Moechars, Diederik; Alcazar, Jesus; Ariza, Manuela; Fierens, Katleen; Bottelbergs, Astrid; Marien, Jonas; Vandenberghe, Rik; Andres, Ignacio Jose; Van Laere, Koen; Verbruggen, Alfons; Bormans, Guy
    Molecular Imaging (2016), 15 (Jan.-Dec.), 1-15CODEN: MIOMBP; ISSN:1536-0121. (Sage Publications)
    Early clin. results of two tau tracers, [18F]T808 and [18F]T807, have recently been reported. In the present study, the biodistribution, radiometabolite quantification, and competition-binding studies were performed in order to acquire comparative preclin. data as well as to establish the value of T808 and T807 as benchmark compds. for assessment of binding affinities of eight new/other tau tracers. Biodistribution studies in mice showed high brain uptake and fast washout. In vivo radiometabolite anal. using high-performance liq. chromatog. showed the presence of polar radiometabolites in plasma and brain. No specific binding of [18F]T808 was found in transgenic mice expressing mutant human P301L tau. In semiquant. autoradiog. studies on human Alzheimer disease slices, we obsd. more than 50% tau selective blocking of [18F]T808 in the presence of 1 μmol/L of the novel ligands. This study provides a straightforward comparison of the binding affinity and selectivity for tau of the reported radiolabeled tracers BF-158, BF-170, THK5105, lansoprazole, astemizole, and novel tau positron emission tomog. ligands against T807 and T808. Therefore, these data are helpful to identify structural requirements for selective interaction with tau and to compare the performance of new highly selective and specific radiolabeled tau tracers.
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  15. 15
    Xia, C. F., Arteaga, J., Chen, G., Gangadharmath, U., Gomez, L. F., Kasi, D., Lam, C., Liang, Q., Liu, C., Mocharla, V. P. (2013) [(18)F]T807, a novel tau positron emission tomography imaging agent for Alzheimer’s disease. Alzheimer's Dementia 9 (6), 666– 76,  DOI: 10.1016/j.jalz.2012.11.008
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    15
    (18)F]T807, a novel tau positron emission tomography imaging agent for Alzheimer's disease
    Xia Chun-Fang; Arteaga Janna; Chen Gang; Gangadharmath Umesh; Gomez Luis F; Kasi Dhanalakshmi; Lam Chung; Liang Qianwa; Liu Changhui; Mocharla Vani P; Mu Fanrong; Sinha Anjana; Su Helen; Szardenings A Katrin; Walsh Joseph C; Wang Eric; Yu Chul; Zhang Wei; Zhao Tieming; Kolb Hartmuth C
    Alzheimer's & dementia : the journal of the Alzheimer's Association (2013), 9 (6), 666-76 ISSN:.
    OBJECTIVE: We wished to develop a highly selective positron emission tomography (PET) imaging agent targeting PHF-tau in human Alzheimer's disease (AD) brains. METHODS: To screen potential tau binders, human AD brain sections were used as a source of native paired helical filament (PHF)-tau and Aβ rather than synthetic tau aggregates or Aβ fibrils generated in vitro to measure the affinity and selectivity of [(18)F]T807 to tau and Aβ. Brain uptake and biodistribution of [(18)F]T807 in mice were also tested. RESULTS: In vitro autoradiography results show that [(18)F]T807 exhibits strong binding to PHF-tau-positive human brain sections. A dissociation constant (Kd) of [(18)F]T807 (14.6 nM) was measured using brain sections from the frontal lobe of AD patients. A comparison of autoradiography and double immunohistochemical staining of PHF-tau and Aβ on adjacent sections demonstrated that [(18)F]T807 binding colocalized with immunoreactive PHF-tau pathology, but did not highlight Aβ plaques. In vivo studies in mice demonstrated that [(18)F]T807 was able to cross the blood-brain barrier and washed out quickly. CONCLUSIONS: [(18)F]T807 demonstrates high affinity and selectivity to PHF-tau as well as favorable in vivo properties, making this a promising candidate as an imaging agent for AD.
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  16. 16
    Brendel, M., Yousefi, B. H., Blume, T., Herz, M., Focke, C., Deussing, M., Peters, F., Lindner, S., von Ungern-Sternberg, B., Drzezga, A. (2018) Comparison of (18)F-T807 and (18)F-THK5117 PET in a Mouse Model of Tau Pathology. Front. Aging Neurosci. 10, 174,  DOI: 10.3389/fnagi.2018.00174
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    16
    Comparison of 18F-T807 and 18 F-THK5117 PET in a mouse model of tau pathology
    Brendel, Matthias; Yousefi, Behrooz H.; Blume, Tanja; Herz, Michael; Focke, Carola; Deussing, Maximilian; Peters, Finn; Lindner, Simon; von Ungern-Sternberg, Barbara; Drzezga, Alexander; Bartenstein, Peter; Haass, Christian; Okamura, Nobuyuki; Herms, Jochen; Yakushev, Igor; Rominger, Axel
    Frontiers in Aging Neuroscience (2018), 10 (), 174/1-174/9CODEN: FANRC5; ISSN:1663-4365. (Frontiers Media S.A.)
    Positron-emission-tomog. (PET) imaging of tau pathol. has facilitated development of anti-tau therapies. While members of the arylquinoline and pyridoindole families have been the most frequently used tau radioligands so far, analyses of their comparative performance in vivo are scantly documented. Here, we conducted a head-to-head PET comparison of the arylquinoline 18FT807 and the pyridoindole 18FTHK5117 PET in a mouse model of tau pathol. PET recordings were obtained in groups of (N = 5-7) P301S and wild-type (WT) mice at 6 and 9 mo of age. Vol.-of-interest based anal. (std.-uptake-value ratio, SUVR) was used to calc. effect sizes (Cohen's d) for each tracer and age. Statistical parametric mapping (SPM) was used to assess regional similarity (dice coeff.) of tracer binding alterations for the two tracers. Immunohistochem. staining of neurofibrillary tangles was performed for validation ex vivo. Significantly elevated 18F-T807 binding in the brainstem of P301S mice was already evident at 6 mo ( + 14%, p < 0.01, d = 1.64), and increased further at 9 mo ( + 23%, p < 0.001, d = 2.70). 18F-THK5117 indicated weaker increases and effect sizes at 6 mo ( + 5%, p < 0.05, d = 1.07) and 9 mo ( + 10%, p < 0.001, d = 1.49). Regional similarity of binding of the two tracers was high (71%) at 9 mo. 18F-T807 was more sensitive than 18F-THK5117 to tau pathol. in this model, although both tracers present certain obstacles, which need to be considered in the design of longitudinal preclin. tau imaging studies.
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    Brendel, M., Jaworska, A., Probst, F., Overhoff, F., Korzhova, V., Lindner, S., Carlsen, J., Bartenstein, P., Harada, R., Kudo, Y. (2016) Small-Animal PET Imaging of Tau Pathology with 18F-THK5117 in 2 Transgenic Mouse Models. J. Nucl. Med. 57 (5), 792– 8,  DOI: 10.2967/jnumed.115.163493
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    17
    Small-animal PET imaging of tau pathology with 18F-THK5117 in 2 transgenic mouse models
    Brendel, Matthias; Jaworska, Anna; Probst, Federico; Overhoff, Felix; Korzhova, Viktoria; Lindner, Simon; Carlsen, Janette; Bartenstein, Peter; Harada, Ryuichi; Kudo, Yukitsuka; Haass, Christian; Van Leuven, Fred; Okamura, Nobuyuki; Herms, Jochen; Rominger, Axel
    Journal of Nuclear Medicine (2016), 57 (5), 792-798CODEN: JNMEAQ; ISSN:1535-5667. (Society of Nuclear Medicine and Molecular Imaging)
    Abnormal accumulation of tau aggregates in the brain is one of the hallmarks of Alzheimer disease neuropathol. We visualized tau deposition in vivo with the previously developed 2-arylquinoline deriv. 18F-THK5117 using small-animal PET in conjunction with autoradiog. and immunohistochem. gold std. assessment in 2 transgenic mouse models expressing hyperphosphorylated tau. Small-animal PET recordings were obtained in groups of P301S (n = 11) and biGT mice (n = 16) of different ages, with age-matched wild-type (WT) serving as controls. After i.v. administration of 16 ± 2 MBq of 18F-THK5117, a dynamic 90-min emission recording was initiated for P301S mice and during 20-50 min after injection for biGT mice, followed by a 15-min transmission scan. After coregistration to the MRI atlas and scaling to the cerebellum, we performed vol.-of-interest-based anal. (SUV ratio [SUVR]) and statistical parametric mapping. Small-animal PET results were compared with autoradiog. ex vivo and in vitro and further validated with AT8 staining for neurofibrillary tangles. SUVRs calcd. from static recordings during the interval of 20-50 min after tracer injection correlated highly with ests. of binding potential based on the entire dynamic emission recordings (R = 0.85). SUVR increases were detected in the brain stem of aged P301S mice (+11%; P < 0.001) and in entorhinal/amygdaloidal areas (+15%; P < 0.001) of biGT mice when compared with WT, whereas aged WT mice did not show increased tracer uptake. Immunohistochem. tau loads correlated with small-animal PET SUVR for both P301S (R = 0.8; P < 0.001) and biGT (R = 0.7; P < 0.001) mice, and distribution patterns of AT8-pos. neurons matched voxelwise statistical parametric mapping anal. Saturable binding of the tracer was verified by autoradiog. blocking studies. In the first dedicated small-animal PET study in 2 different transgenic tauopathy mouse models using the tau tracer 18F-THK5117, the temporal and spatial progression could be visualized in good correlation with gold std. assessments of tau accumulation. The serial small-animal PET method could afford the means for preclin. testing of novel therapeutic approaches by accommodating interanimal variability at baseline, while detection thresholds in young animals have to be considered.
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    Allen, B., Ingram, E., Takao, M., Smith, M. J., Jakes, R., Virdee, K., Yoshida, H., Holzer, M., Craxton, M., Emson, P. C. (2002) Abundant tau filaments and nonapoptotic neurodegeneration in transgenic mice expressing human P301S tau protein. J. Neurosci. 22 (21), 9340– 51,  DOI: 10.1523/JNEUROSCI.22-21-09340.2002
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    18
    Abundant tau filaments and nonapoptotic neurodegeneration in transgenic mice expressing human P301S Tau protein
    Allen, Bridget; Ingram, Esther; Takao, Masaki; Smith, Michael J.; Jakes, Ross; Virdee, Kanwar; Yoshida, Hirotaka; Holzer, Max; Craxton, Molly; Emson, Piers C.; Atzori, Cristiana; Migheli, Antonio; Crowther, R. Anthony; Ghetti, Bernardino; Spillantini, Maria Grazia; Goedert, Michel
    Journal of Neuroscience (2002), 22 (21), 9340-9351CODEN: JNRSDS; ISSN:0270-6474. (Society for Neuroscience)
    The identification of mutations in the Tau gene in frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) has made it possible to express human tau protein with pathogenic mutations in transgenic animals. Here the authors report on the prodn. and characterization of a line of mice transgenic for the 383 aa isoform of human tau with the P301S mutation. At 5-6 mo of age, homozygous animals from this line developed a neurol. phenotype dominated by a severe paraparesis. According to light microscopy, many nerve cells in brain and spinal cord were strongly immunoreactive for hyperphosphorylated tau. According to electron microscopy, abundant filaments made of hyperphosphorylated tau protein were present. The majority of filaments resembled the half-twisted ribbons described previously in cases of FTDP-17, with a minority of filaments resembling the paired helical filaments of Alzheimer's disease. Sarkosyl-insol. tau from brains and spinal cords of transgenic mice ran as a hyperphosphorylated 64 kDa band, the same apparent mol. mass as that of the 383 aa tau isoform in the human tauopathies. Perchloric acid-sol. tau was also phosphorylated at many sites, with the notable exception of serine 214. In the spinal cord, neurodegeneration was present, as indicated by a 49% redn. in the no. of motor neurons. No evidence for apoptosis was obtained, despite the extensive colocalization of hyperphosphorylated tau protein with activated MAP kinase family members. The latter may be involved in the hyperphosphorylation of tau.
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    Wang, M., Gao, M., Xu, Z., and Zheng, Q. H. (2015) Synthesis of a PET tau tracer [(11)C]PBB3 for imaging of Alzheimer’s disease. Bioorg. Med. Chem. Lett. 25 (20), 4587– 92,  DOI: 10.1016/j.bmcl.2015.08.053
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    19
    Synthesis of a PET tau tracer [11C]PBB3 for imaging of Alzheimer's disease
    Wang, Min; Gao, Mingzhang; Xu, Zhidong; Zheng, Qi-Huang
    Bioorganic & Medicinal Chemistry Letters (2015), 25 (20), 4587-4592CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)
    The authentic std. PBB3 and its precursor N-desmethyl-PBB3 as well as TBS-protected N-desmethyl-PBB3 precursor for radiolabeling were synthesized from 5-bromo-2-nitropyridine, acrolein di-Et acetal, 6-methoxy-2-methylbenzothiazole, and di-Et chlorophosphate with overall chem. yield 1% in six steps, 2% in five steps, and 1% in six steps, resp. [11C]PBB3 was prepd. from either desmethyl-PBB3 or TBS-protected desmethyl-PBB3 with [11C]CH3OTf through N-[11C]methylation and isolated by HPLC combined with SPE in 20-25% and 15-20% radiochem. yield, resp., based on [11C]CO2 and decay cor. to end of bombardment (EOB). The radiochem. purity was >99%, and the specific activity at EOB was 370-1110 GBq/μmol with a total synthesis time of ∼40-min from EOB.
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    Bragina, M. E., Stergiopulos, N., and Fraga-Silva, R. A. (2017) Fluorescence-Based Binding Assay for Screening Ligands of Angiotensin Receptors. Methods Mol. Biol. 1614, 165– 174,  DOI: 10.1007/978-1-4939-7030-8_13
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    20
    Fluorescence-based binding assay for screening ligands of angiotensin receptors
    Bragina, Maiia E.; Stergiopulos, Nikolaos; Fraga-Silva, Rodrigo A.
    Methods in Molecular Biology (New York, NY, United States) (2017), 1614 (Renin-Angiotensin-Aldosterone System), 165-174CODEN: MMBIED; ISSN:1940-6029. (Springer)
    A review. Binding assay is a common technique used to characterize ability of a ligand to interact with a specific biol. target. A no. of parameters, such as binding affinity, receptor d., and assocn./dissocn. rate consts., can be measured by means of this technique. In most cases, implementation of the binding assay requires specific infrastructure for labeling and detecting the ligand, which impedes realization of this technique in a std. lab. Here we describe a simple fluorescence-based binding assay for angiotensin peptides and receptors, which does not require complex equipment and can be used for initial screening of the novel ligands or mutational studies.
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    Ono, M., Sahara, N., Kumata, K., Ji, B., Ni, R., Koga, S., Dickson, D. W., Trojanowski, J. Q., Lee, V. M., Yoshida, M. (2017) Distinct binding of PET ligands PBB3 and AV-1451 to tau fibril strains in neurodegenerative tauopathies. Brain 140 (3), 764– 780,  DOI: 10.1093/brain/aww339
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    21
    Distinct binding of PET ligands PBB3 and AV-1451 to tau fibril strains in neurodegenerative tauopathies
    Ono Maiko; Sahara Naruhiko; Kumata Katsushi; Ji Bin; Suhara Tetsuya; Zhang Ming-Rong; Higuchi Makoto; Ono Maiko; Ni Ruiqing; Nordberg Agneta; Koga Shunsuke; Dickson Dennis W; Trojanowski John Q; Lee Virginia M-Y; Yoshida Mari; Hozumi Isao; Yoshiyama Yasumasa; van Swieten John C
    Brain : a journal of neurology (2017), 140 (3), 764-780 ISSN:.
    Diverse neurodegenerative disorders are characterized by deposition of tau fibrils composed of conformers (i.e. strains) unique to each illness. The development of tau imaging agents has enabled visualization of tau lesions in tauopathy patients, but the modes of their binding to different tau strains remain elusive. Here we compared binding of tau positron emission tomography ligands, PBB3 and AV-1451, by fluorescence, autoradiography and homogenate binding assays with homologous and heterologous blockades using tauopathy brain samples. Fluorescence microscopy demonstrated intense labelling of non-ghost and ghost tangles with PBB3 and AV-1451, while dystrophic neurites were more clearly detected by PBB3 in brains of Alzheimer's disease and diffuse neurofibrillary tangles with calcification, characterized by accumulation of all six tau isoforms. Correspondingly, partially distinct distributions of autoradiographic labelling of Alzheimer's disease slices with 11C-PBB3 and 18F-AV-1451 were noted. Neuronal and glial tau lesions comprised of 4-repeat isoforms in brains of progressive supranuclear palsy, corticobasal degeneration and familial tauopathy due to N279K tau mutation and 3-repeat isoforms in brains of Pick's disease and familial tauopathy due to G272V tau mutation were sensitively detected by PBB3 fluorescence in contrast to very weak AV-1451 signals. This was in line with moderate 11C-PBB3 versus faint 18F-AV-1451 autoradiographic labelling of these tissues. Radioligand binding to brain homogenates revealed multiple binding components with differential affinities for 11C-PBB3 and 18F-AV-1451, and higher availability of binding sites on progressive supranuclear palsy tau deposits for 11C-PBB3 than 18F-AV-1451. Our data indicate distinct selectivity of PBB3 compared to AV-1451 for diverse tau fibril strains. This highlights the more robust ability of PBB3 to capture wide-range tau pathologies.
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    Delobel, P., Lavenir, I., Fraser, G., Ingram, E., Holzer, M., Ghetti, B., Spillantini, M. G., Crowther, R. A., and Goedert, M. (2008) Analysis of tau phosphorylation and truncation in a mouse model of human tauopathy. Am. J. Pathol. 172 (1), 123– 31,  DOI: 10.2353/ajpath.2008.070627
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    22
    Analysis of Tau phosphorylation and truncation in a mouse model of human tauopathy
    Delobel, Patrice; Lavenir, Isabelle; Fraser, Graham; Ingram, Esther; Holzer, Max; Ghetti, Bernardino; Spillantini, Maria Grazia; Crowther, R. Anthony; Goedert, Michel
    American Journal of Pathology (2008), 172 (1), 123-131CODEN: AJPAA4; ISSN:0002-9440. (American Society for Investigative Pathology)
    Recent evidence has suggested that truncation of tau protein at the caspase cleavage site D421 precedes hyperphosphorylation and may be necessary for the assembly of tau into filaments in Alzheimer's disease and other tauopathies. Here we have investigated the time course of the appearance of phosphorylated and truncated tau in the brain and spinal cord of mice transgenic for mutant human P301S tau protein. This mouse line recapitulates the essential mol. and cellular features of the human tauopathies, including tau hyperphosphorylation, tau filament formation, and neurodegeneration. Sol. tau was strongly phosphorylated at 1 to 6 mo of age. Low levels of phosphorylated, sarkosyl-insol. tau were detected at 2 mo, with a steady increase up to 6 mo of age. Tau truncated at D421 was detected at low levels in Tris-sol. and detergent-sol. tau at 3 to 6 mo of age. By immunoblotting, it was not detected in sarkosyl-insol. tau. However, by immunoelectron microscopy, a small percentage of tau in filaments from brain and spinal cord of transgenic mice was truncated at D421. Similar findings were obtained using dispersed filaments from Alzheimer's disease and FTDP-17 brains. The late appearance and low abundance of tau ending at D421 indicate that it is unlikely that truncation at this site is necessary for the assembly of tau into filaments.
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    Macdonald, J. A., Bronner, I. F., Drynan, L., Fan, J., Curry, A., Fraser, G., Lavenir, I., and Goedert, M. (2019) Assembly of transgenic human P301S Tau is necessary for neurodegeneration in murine spinal cord. Acta Neuropathol Commun. 7 (1), 44,  DOI: 10.1186/s40478-019-0695-5
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    23
    Assembly of transgenic human P301S Tau is necessary for neurodegeneration in murine spinal cord
    Macdonald Jennifer A; Drynan Lesley; Fan Juan; Lavenir Isabelle; Goedert Michel; Bronner Iraad F; Curry Annabelle; Fraser Graham
    Acta neuropathologica communications (2019), 7 (1), 44 ISSN:.
    A pathological pathway leading from soluble monomeric to insoluble filamentous Tau is characteristic of many human neurodegenerative diseases, which also exhibit dysfunction and death of brain cells. However, it is unknown how the assembly of Tau into filaments relates to cell loss. To study this, we first used a mouse line transgenic for full-length human mutant P301S Tau to investigate the temporal relationship between Tau assembly into filaments, assessed using anti-Tau antibody AT100, and motor neuron numbers, in the lumbar spinal cord. AT100 immunoreactivity preceded nerve cell loss. Murine Tau did not contribute significantly to either Tau aggregation or neurodegeneration. To further study the relevance of filament formation for neurodegeneration, we deleted hexapeptides (275)VQIINK(280) and (306)VQIVYK(311), either singly or in combination, from human 0N4R Tau with the P301S mutation. These hexapeptides are essential for the assembly of Tau into filaments. Homozygous mice transgenic for P301S Tau with the hexapeptide deletions, which expressed Tau at a similar level to the heterozygous line transgenic for P301S Tau, had a normal lifespan, unlike mice from the P301S Tau line. The latter had significant levels of sarkosyl-insoluble Tau in brain and spinal cord, and exhibited neurodegeneration. Mice transgenic for P301S Tau with the hexapeptide deletions failed to show significant levels of sarkosyl-insoluble Tau or neurodegeneration. Recombinant P301S Tau with the hexapeptide deletions failed to form β-sheet structure and filaments following incubation with heparin. Taken together, we conclude that β-sheet assembly of human P301S Tau is necessary for neurodegeneration in transgenic mice.
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    Mellone, M., Kestoras, D., Andrews, M. R., Dassie, E., Crowther, R. A., Stokin, G. B., Tinsley, J., Horne, G., Goedert, M., Tolkovsky, A. M. (2013) Tau pathology is present in vivo and develops in vitro in sensory neurons from human P301S tau transgenic mice: a system for screening drugs against tauopathies. J. Neurosci. 33 (46), 18175– 89,  DOI: 10.1523/JNEUROSCI.4933-12.2013
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    24
    Tau pathology is present in vivo and develops in vitro in sensory neurons from human P301S tau transgenic mice: a system for screening drugs against tauopathies
    Mellone, Manuela; Kestoras, Dimitra; Andrews, Melissa R.; Dassie, Elisa; Crowther, R. Anthony; Stokin, Gorazd B.; Tinsley, Jon; Horne, Graeme; Goedert, Michel; Tolkovsky, Aviva M.; Spillantini, Maria Grazia
    Journal of Neuroscience (2013), 33 (46), 18175-18189CODEN: JNRSDS; ISSN:0270-6474. (Society for Neuroscience)
    Intracellular tau aggregates are the neuropathol. hallmark of several neurodegenerative diseases, including Alzheimer's disease, progressive supranuclear palsy, and cases of frontotemporal dementia, but the link between these aggregates and neurodegeneration remains unclear. Neuronal models recapitulating the main features of tau pathol. are necessary to investigate the mol. mechanisms of tau malfunction, but current models show little and inconsistent spontaneous tau aggregation. We show that dorsal root ganglion (DRG) neurons in transgenic mice expressing human P301S tau (P301S-htau) develop tau pathol. similar to that found in brain and spinal cord and a significant redn. in mechanosensation occurs before detectable fibrillar tau formation. DRG neuronal cultures established from adult P301S-htau mice at different ages retained the pattern of aberrant tau found in vivo. Moreover, htau became progressively hyperphosphorylated over 2 mo in vitro beginning with nonsymptomatic neurons, while hyperphosphorylated P301S-htau-pos. neurons from 5-mo-old mice cultured for 2 mo died preferentially. P301S-htau-pos. neurons grew aberrant axons, including spheroids, typically found in human tauopathies. Neurons cultured at advanced stages of tau pathol. showed a 60% decrease in the fraction of moving mitochondria. SEG28019, a novel O-GlcNAcase inhibitor, reduced steady-state pSer396/pSer404 phosphorylation over 7 wk in a significant proportion of DRG neurons showing for the first time the possible beneficial effect of prolonged dosing of O-GlcNAcase inhibitor in vitro. Our system is unique in that fibrillar tau forms without external manipulation and provides an important new tool for understanding the mechanisms of tau dysfunction and for screening of compds. for treatment of tauopathies.
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    Brelstaff, J., Spillantini, M. G., and Tolkovsky, A. M. (2015) pFTAA: a high affinity oligothiophene probe that detects filamentous tau in vivo and in cultured neurons. Neural Regener. Res. 10 (11), 1746– 7,  DOI: 10.4103/1673-5374.165298
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    25
    pFTAA: a high affinity oligothiophene probe that detects filamentous tau in vivo and in cultured neurons
    Brelstaff, Jack; Spillantini, Maria Grazia; Tolkovsky, Aviva M.
    Neural Regeneration Research (2015), 10 (11), 1746-1747CODEN: NRREBM; ISSN:1673-5374. (Publishing House of Neural Regeneration Research)
    Tauopathies describe a group of neurodegenerative diseases in which the protein tau, encoded by the gene MAPT, is aberrantly misfolded, leading to tau aggregation, neural dysfunction, and cell death. In Alzheimer's disease (AD), tau forms the characteristic intracellular neurofibrillary tangles (NFTs), which are thought to be the major cause of neurodegeneration. The use of pFTAA has solved a major problem in trying to decipher mechanisms of cell death induced by filamentous forms of tau, as we can now follow living neurons to their death. We also noted previously that DRG neurons with abnormal forms of tau regenerate abnormally, with splayed growth cones, spheroids (a typical feature of human tauopathies), and thick short axons, after they are severed prior to culture. Given that we can identify pFTAA+ve neurons and axons, we can now approach the question whether tau filaments, or sol. abnormal tau, are responsible for inducing these aberrant retarded growth patterns. This will be esp. interesting in studying the regeneration of DRG neurons in vivo, and ultimately CNS neuronal axons and dendrites during synapse formation.
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    Brelstaff, J., Ossola, B., Neher, J. J., Klingstedt, T., Nilsson, K. P., Goedert, M., Spillantini, M. G., and Tolkovsky, A. M. (2015) The fluorescent pentameric oligothiophene pFTAA identifies filamentous tau in live neurons cultured from adult P301S tau mice. Front. Neurosci. 9, 184,  DOI: 10.3389/fnins.2015.00184
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    26
    The fluorescent pentameric oligothiophene pFTAA identifies filamentous tau in live neurons cultured from adult P301S tau mice
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  • Abstract

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    Scheme 1

    Scheme 1. (A) Development of Novel Compounds for Imaging Tau Aggregation in Animal Models of Tau and (B) Streamlined Convergent Synthetic Route for a Compound Library
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    Figure 1

    Figure 1. LM229 binding to mouse brain sections is specific to neurons with pathological tau aggregates. (Top) No specific binding to brainstem sections from WT animals but coincident LM229 binding (green) and (middle) AT8 or (bottom) AT100 antibody staining (red) in phenotypic P301S tau mice; AT100 binding correlates with tau fibrils. Scale bar = 20 μm.

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    Figure 2

    Figure 2. Fluorescence image of brain section from WT (A) and P301S tau mice (B) fixed 1 h after i.v. injection of LM229 showing entry into brain from periphery. Scale bar = 100 μm.

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    Figure 3

    Figure 3. P301S tau brain tissue from (E) ex vivo experiments with PBB3 (i.v. injection 1 h prior to injection) colocalized with AT8 showing binding of PBB3 with significant nonspecific binding; (F) ex vivo experiments with LM229 showing significant specific binding to tau aggregates as confirmed with colocalization with AT8.

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    Figure 4

    Figure 4. (A) Fluorescence images of LM229 binding to live DRG neurons (green). Phase contrast images show total live neurons. (B) Fluorescence intensity per neuron as a function of LM229 concentration (mean ± SD, 5–15 neurons per concentration, three independent cultures; black line, result of nonlinear curve fitting, green and blue dashed lines, 95% confidence intervals; live neurons: IC50 = 2.97 ± 0.73; R2 = 0.968; fixed neurons: IC50 = 1.67 ± 0.28; R2 = 0.990). (C) Dose–response of LM229 binding to fixed neurons. (D) Co-staining of LM229 (10 μM, green) and antiphospho-tau antibody AT100 (red). Blue in merged image shows cell nuclei in the culture.

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    Figure 5

    Figure 5. LM229 (green) binding to (A) 3R tau in cortical sections from SHR24 rat brains and (B) 4R tau in brainstem sections from SHR72 rats. (C, D) Colocalization with AT8 (red).

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    Figure 6

    Figure 6. Binding of PBB3 (green) to (A) 3R tau in the cortex of SHR24 brain tissue and (B) to 4R tau in the brainstem of SHR72 and (C, D) colocalization with staining AT8 (red).

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    Figure 7

    Figure 7. (A) Post-mortem PSP tissue stained with LM229; (B) Double staining of PSP tissue with LM229 and AT8 (red) to confirm colocalization of LM229 with tau aggregates.

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    Figure 8

    Figure 8. Post mortem AD tissue stained with LM229 and (A,C) and double staining with AT8 (B) and beta-amyloid antibodies (D), suggesting binding to tau rather than amyloid aggregates.

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    Scheme 2

    Scheme 2. Two-Step Radiosynthesis of [11C]LM229
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    Figure 9

    Figure 9. Time–activity curves for [11C]LM229 from in vivo PET studies in wild-type and P301S mice.

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      There is no corresponding record for this reference.
    9. 9
      Leuzy, A., Chiotis, K., Lemoine, L., Gillberg, P. G., Almkvist, O., Rodriguez-Vieitez, E., and Nordberg, A. (2019) Tau PET imaging in neurodegenerative tauopathies-still a challenge. Mol. Psychiatry 24 (8), 1112– 1134,  DOI: 10.1038/s41380-018-0342-8
      9
      Tau PET imaging in neurodegenerative tauopathies-still a challenge
      Leuzy, Antoine; Chiotis, Konstantinos; Lemoine, Laetitia; Gillberg, Per-Goeran; Almkvist, Ove; Rodriguez-Vieitez, Elena; Nordberg, Agneta
      Molecular Psychiatry (2019), 24 (8), 1112-1134CODEN: MOPSFQ; ISSN:1359-4184. (Nature Research)
      A review. The accumulation of pathol. misfolded tau is a feature common to a collective of neurodegenerative disorders known as tauopathies, of which Alzheimer's disease (AD) is the most common. Related tauopathies include progressive supranuclear palsy (PSP), corticobasal syndrome (CBS), Down's syndrome (DS), Parkinson's disease (PD), and dementia with Lewy bodies (DLB). Investigation of the role of tau pathol. in the onset and progression of these disorders is now possible due the recent advent of tau-specific ligands for use with positron emission tomog. (PET), including first- (e.g., [18F]THK5317, [18F]THK5351, [18F]AV1451, and [11C]PBB3) and second-generation compds. [namely [18F]MK-6240, [18F]RO-948 (previously referred to as [18F]RO69558948), [18F]PI-2620, [18F]GTP1, [18F]PM-PBB3, and [18F]JNJ64349311 ([18F]JNJ311) and its deriv. [18F]JNJ-067)]. In this review we describe and discuss findings from in vitro and in vivo studies using both initial and new tau ligands, including their relation to biomarkers for amyloid-β and neurodegeneration, and cognitive findings. Lastly, methodol. considerations for the quantification of in vivo ligand binding are addressed, along with potential future applications of tau PET, including therapeutic trials.
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    10. 10
      Maruyama, M., Shimada, H., Suhara, T., Shinotoh, H., Ji, B., Maeda, J., Zhang, M. R., Trojanowski, J. Q., Lee, V. M., Ono, M. (2013) Imaging of tau pathology in a tauopathy mouse model and in Alzheimer patients compared to normal controls. Neuron 79 (6), 1094– 108,  DOI: 10.1016/j.neuron.2013.07.037
      10
      Imaging of Tau Pathology in a Tauopathy Mouse Model and in Alzheimer Patients Compared to Normal Controls
      Maruyama, Masahiro; Shimada, Hitoshi; Suhara, Tetsuya; Shinotoh, Hitoshi; Ji, Bin; Maeda, Jun; Zhang, Ming-Rong; Trojanowski, John Q.; Lee, Virginia M.-Y.; Ono, Maiko; Masamoto, Kazuto; Takano, Harumasa; Sahara, Naruhiko; Iwata, Nobuhisa; Okamura, Nobuyuki; Furumoto, Shozo; Kudo, Yukitsuka; Chang, Qing; Saido, Takaomi C.; Takashima, Akihiko; Lewis, Jada; Jang, Ming-Kuei; Aoki, Ichio; Ito, Hiroshi; Higuchi, Makoto
      Neuron (2013), 79 (6), 1094-1108CODEN: NERNET; ISSN:0896-6273. (Cell Press)
      Accumulation of intracellular tau fibrils has been the focus of research on the mechanisms of neurodegeneration in Alzheimer's disease (AD) and related tauopathies. Here, we have developed a class of tau ligands, phenyl/pyridinyl-butadienyl-benzothiazoles/benzothiazoliums (PBBs), for visualizing diverse tau inclusions in brains of living patients with AD or non-AD tauopathies and animal models of these disorders. In vivo optical and positron emission tomog. (PET) imaging of a transgenic mouse model demonstrated sensitive detection of tau inclusions by PBBs. A pyridinated PBB, [11C]PBB3, was next applied in a clin. PET study, and its robust signal in the AD hippocampus wherein tau pathol. is enriched contrasted strikingly with that of a senile plaque radioligand, [11C]Pittsburgh Compd.-B ([11C]PIB). [11C]PBB3-PET data were also consistent with the spreading of tau pathol. with AD progression. Furthermore, increased [11C]PBB3 signals were found in a corticobasal syndrome patient neg. for [11C]PIB-PET.
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    11. 11
      Mueller, A., Bullich, S., Barret, O., Madonia, J., Berndt, M., Papin, C., Perrotin, A., Koglin, N., Kroth, H., Pfeifer, A. (2020) Tau PET imaging with (18)F-PI-2620 in patients with Alzheimer’s disease and healthy controls: a first-in-human study. J. Nucl. Med. 61, 911,  DOI: 10.2967/jnumed.119.236224
      11
      Tau PET imaging with 18F-PI-2620 in patients with Alzheimer disease and healthy controls: a first-in-humans study
      Mueller, Andre; Bullich, Santiago; Barret, Olivier; Madonia, Jennifer; Berndt, Mathias; Papin, Caroline; Perrotin, Audrey; Koglin, Norman; Kroth, Heiko; Pfeifer, Andrea; Tamagnan, Gilles; Seibyl, John P.; Marek, Kenneth; De Santi, Susan; Dinkelborg, Ludger M.; Stephens, Andrew W.
      Journal of Nuclear Medicine (2020), 61 (6), 911-919CODEN: JNMEAQ; ISSN:1535-5667. (Society of Nuclear Medicine and Molecular Imaging)
      The purpose of this first-in-humans study was to evaluate the ability of 18F-PI-2620 to detect tau pathol. in AD patients using PET imaging, as well as to assess the safety and tolerability of this new tau PET tracer. Methods: Participants with a clin. diagnosis of probable AD and healthy controls (HCs) underwent dynamic 18F-PI-2620 PET imaging for 180 min. 18F-PI-2620 binding was assessed visually and quant. using distribution vol. ratios (DVR) estd. from noninvasive tracer kinetics and SUV ratio (SUVR) measured at different time points after injection, with the cerebellar cortex as the ref. region. Results:18F-PI-2620 showed peak brain uptake around 5 min after injection and fast washout from nontarget regions. Very low background signal was obsd. in HCs. 18F-PI-2620 administration was safe and well tolerated. SUVR time-activity curves in most regions and subjects achieved a secular equil. after 40 min after injection. A strong correlation (R2 > 0.93) was found between noninvasive DVR and SUVR for all imaging windows starting at more than 30 min after injection. Similar effect sizes between AD and HC groups were obtained across the different imaging windows. 18F-PI-2620 uptake in neocortical regions significantly correlated with the degree of cognitive impairment. Conclusion: Initial clin. data obtained in AD and HC subjects demonstrated a high image quality and excellent signal-to-noise ratio of 18F-PI-2620 PET for imaging tau deposition in AD subjects.
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    12. 12
      Hostetler, E. D., Walji, A. M., Zeng, Z., Miller, P., Bennacef, I., Salinas, C., Connolly, B., Gantert, L., Haley, H., Holahan, M. (2016) Preclinical Characterization of 18F-MK-6240, a Promising PET Tracer for In Vivo Quantification of Human Neurofibrillary Tangles. J. Nucl. Med. 57 (10), 1599– 1606,  DOI: 10.2967/jnumed.115.171678
      12
      Preclinical characterization of 18F-MK-6240, a promising PET tracer for in vivo quantification of human neurofibrillary tangles
      Hostetler, Eric D.; Walji, Abbas M.; Zeng, Zhizhen; Miller, Patricia; Bennacef, Idriss; Salinas, Cristian; Connolly, Brett; Gantert, Liza; Haley, Hyking; Holahan, Marie; Purcell, Mona; Riffel, Kerry; Lohith, Talakad G.; Coleman, Paul; Soriano, Aileen; Ogawa, Aimie; Xu, Serena; Zhang, Xiaoping; Joshi, Elizabeth; Della Rocca, Joseph; Hesk, David; Schenk, David J.; Evelhoch, Jeffrey L.
      Journal of Nuclear Medicine (2016), 57 (10), 1599-1606CODEN: JNMEAQ; ISSN:1535-5667. (Society of Nuclear Medicine and Molecular Imaging)
      A PET tracer is desired to help guide the discovery and development of disease-modifying therapeutics for neurodegenerative diseases characterized by neurofibrillary tangles (NFTs), the predominant tau pathol. in Alzheimer disease (AD). We describe the preclin. characterization of the NFT PET tracer 18F-MK-6240. Methods: In vitro binding studies were conducted with 3H-MK-6240 in tissue slices and homogenates from cognitively normal and AD human brain donors to evaluate tracer affinity and selectivity for NFTs. Immunohistochem. for phosphorylated tau was performed on human brain slices for comparison with 3H-MK-6240 binding patterns on adjacent brain slices. PET studies were performed with 18F-MK-6240 in monkeys to evaluate tracer kinetics and distribution in the brain. 18F-MK-6240 monkey PET studies were conducted after dosing with unlabeled MK-6240 to evaluate tracer binding selectivity in vivo. Results: The 3H-MK-6240 binding pattern was consistent with the distribution of phosphorylated tau in human AD brain slices. 3H-MK-6240 bound with high affinity to human AD brain cortex homogenates contg. abundant NFTs but bound poorly to amyloid plaque-rich, NFT-poor AD brain homogenates. 3H-MK-6240 showed no displaceable binding in the subcortical regions of human AD brain slices and in the hippocampus/entorhinal cortex of non-AD human brain homogenates. In monkey PET studies, 18F-MK-6240 displayed rapid and homogeneous distribution in the brain. The 18F-MK-6240 vol. of distribution stabilized rapidly, indicating favorable tracer kinetics. No displaceable binding was obsd. in self-block studies in rhesus monkeys, which do not natively express NFTs. Moderate defluorination was obsd. as skull uptake. Conclusion: 18F-MK-6240 is a promising PET tracer for the in vivo quantification of NFTs in AD patients.
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    13. 13
      Götz, J., Bodea, L. G., and Goedert, M. (2018) Rodent models for Alzheimer disease. Nat. Rev. Neurosci. 19 (10), 583– 598,  DOI: 10.1038/s41583-018-0054-8
      13
      Rodent models for Alzheimer disease
      Gotz, Jurgen; Bodea, Liviu-Gabriel; Goedert, Michel
      Nature Reviews Neuroscience (2018), 19 (10), 583-598CODEN: NRNAAN; ISSN:1471-003X. (Nature Research)
      A review. Animal models are indispensable tools for Alzheimer disease (AD) research. Over the course of more than two decades, an increasing no. of complementary rodent models has been generated. These models have facilitated testing hypotheses about the etiol. and progression of AD, dissecting the assocd. pathomechanisms and validating therapeutic interventions, thereby providing guidance for the design of human clin. trials. However, the lack of success in translating rodent data into therapeutic outcomes may challenge the validity of the current models. This Review critically evaluates the genetic and non-genetic strategies used in AD modeling, discussing their strengths and limitations, as well as new opportunities for the development of better models for the disease.
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    14. 14
      Declercq, L., Celen, S., Lecina, J., Ahamed, M., Tousseyn, T., Moechars, D., Alcazar, J., Ariza, M., Fierens, K., and Bottelbergs, A. (2016) Comparison of New Tau PET-Tracer Candidates With [18F]T808 and [18F]T807. Mol. Imaging 15, 1– 15,  DOI: 10.1177/1536012115624920
      14
      Comparison of new tau PET-tracer candidates with [18F]T808 and [18F]T807
      Declercq, Lieven; Celen, Sofie; Lecina, Joan; Ahamed, Muneer; Tousseyn, Thomas; Moechars, Diederik; Alcazar, Jesus; Ariza, Manuela; Fierens, Katleen; Bottelbergs, Astrid; Marien, Jonas; Vandenberghe, Rik; Andres, Ignacio Jose; Van Laere, Koen; Verbruggen, Alfons; Bormans, Guy
      Molecular Imaging (2016), 15 (Jan.-Dec.), 1-15CODEN: MIOMBP; ISSN:1536-0121. (Sage Publications)
      Early clin. results of two tau tracers, [18F]T808 and [18F]T807, have recently been reported. In the present study, the biodistribution, radiometabolite quantification, and competition-binding studies were performed in order to acquire comparative preclin. data as well as to establish the value of T808 and T807 as benchmark compds. for assessment of binding affinities of eight new/other tau tracers. Biodistribution studies in mice showed high brain uptake and fast washout. In vivo radiometabolite anal. using high-performance liq. chromatog. showed the presence of polar radiometabolites in plasma and brain. No specific binding of [18F]T808 was found in transgenic mice expressing mutant human P301L tau. In semiquant. autoradiog. studies on human Alzheimer disease slices, we obsd. more than 50% tau selective blocking of [18F]T808 in the presence of 1 μmol/L of the novel ligands. This study provides a straightforward comparison of the binding affinity and selectivity for tau of the reported radiolabeled tracers BF-158, BF-170, THK5105, lansoprazole, astemizole, and novel tau positron emission tomog. ligands against T807 and T808. Therefore, these data are helpful to identify structural requirements for selective interaction with tau and to compare the performance of new highly selective and specific radiolabeled tau tracers.
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    15. 15
      Xia, C. F., Arteaga, J., Chen, G., Gangadharmath, U., Gomez, L. F., Kasi, D., Lam, C., Liang, Q., Liu, C., Mocharla, V. P. (2013) [(18)F]T807, a novel tau positron emission tomography imaging agent for Alzheimer’s disease. Alzheimer's Dementia 9 (6), 666– 76,  DOI: 10.1016/j.jalz.2012.11.008
      15
      (18)F]T807, a novel tau positron emission tomography imaging agent for Alzheimer's disease
      Xia Chun-Fang; Arteaga Janna; Chen Gang; Gangadharmath Umesh; Gomez Luis F; Kasi Dhanalakshmi; Lam Chung; Liang Qianwa; Liu Changhui; Mocharla Vani P; Mu Fanrong; Sinha Anjana; Su Helen; Szardenings A Katrin; Walsh Joseph C; Wang Eric; Yu Chul; Zhang Wei; Zhao Tieming; Kolb Hartmuth C
      Alzheimer's & dementia : the journal of the Alzheimer's Association (2013), 9 (6), 666-76 ISSN:.
      OBJECTIVE: We wished to develop a highly selective positron emission tomography (PET) imaging agent targeting PHF-tau in human Alzheimer's disease (AD) brains. METHODS: To screen potential tau binders, human AD brain sections were used as a source of native paired helical filament (PHF)-tau and Aβ rather than synthetic tau aggregates or Aβ fibrils generated in vitro to measure the affinity and selectivity of [(18)F]T807 to tau and Aβ. Brain uptake and biodistribution of [(18)F]T807 in mice were also tested. RESULTS: In vitro autoradiography results show that [(18)F]T807 exhibits strong binding to PHF-tau-positive human brain sections. A dissociation constant (Kd) of [(18)F]T807 (14.6 nM) was measured using brain sections from the frontal lobe of AD patients. A comparison of autoradiography and double immunohistochemical staining of PHF-tau and Aβ on adjacent sections demonstrated that [(18)F]T807 binding colocalized with immunoreactive PHF-tau pathology, but did not highlight Aβ plaques. In vivo studies in mice demonstrated that [(18)F]T807 was able to cross the blood-brain barrier and washed out quickly. CONCLUSIONS: [(18)F]T807 demonstrates high affinity and selectivity to PHF-tau as well as favorable in vivo properties, making this a promising candidate as an imaging agent for AD.
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    16. 16
      Brendel, M., Yousefi, B. H., Blume, T., Herz, M., Focke, C., Deussing, M., Peters, F., Lindner, S., von Ungern-Sternberg, B., Drzezga, A. (2018) Comparison of (18)F-T807 and (18)F-THK5117 PET in a Mouse Model of Tau Pathology. Front. Aging Neurosci. 10, 174,  DOI: 10.3389/fnagi.2018.00174
      16
      Comparison of 18F-T807 and 18 F-THK5117 PET in a mouse model of tau pathology
      Brendel, Matthias; Yousefi, Behrooz H.; Blume, Tanja; Herz, Michael; Focke, Carola; Deussing, Maximilian; Peters, Finn; Lindner, Simon; von Ungern-Sternberg, Barbara; Drzezga, Alexander; Bartenstein, Peter; Haass, Christian; Okamura, Nobuyuki; Herms, Jochen; Yakushev, Igor; Rominger, Axel
      Frontiers in Aging Neuroscience (2018), 10 (), 174/1-174/9CODEN: FANRC5; ISSN:1663-4365. (Frontiers Media S.A.)
      Positron-emission-tomog. (PET) imaging of tau pathol. has facilitated development of anti-tau therapies. While members of the arylquinoline and pyridoindole families have been the most frequently used tau radioligands so far, analyses of their comparative performance in vivo are scantly documented. Here, we conducted a head-to-head PET comparison of the arylquinoline 18FT807 and the pyridoindole 18FTHK5117 PET in a mouse model of tau pathol. PET recordings were obtained in groups of (N = 5-7) P301S and wild-type (WT) mice at 6 and 9 mo of age. Vol.-of-interest based anal. (std.-uptake-value ratio, SUVR) was used to calc. effect sizes (Cohen's d) for each tracer and age. Statistical parametric mapping (SPM) was used to assess regional similarity (dice coeff.) of tracer binding alterations for the two tracers. Immunohistochem. staining of neurofibrillary tangles was performed for validation ex vivo. Significantly elevated 18F-T807 binding in the brainstem of P301S mice was already evident at 6 mo ( + 14%, p < 0.01, d = 1.64), and increased further at 9 mo ( + 23%, p < 0.001, d = 2.70). 18F-THK5117 indicated weaker increases and effect sizes at 6 mo ( + 5%, p < 0.05, d = 1.07) and 9 mo ( + 10%, p < 0.001, d = 1.49). Regional similarity of binding of the two tracers was high (71%) at 9 mo. 18F-T807 was more sensitive than 18F-THK5117 to tau pathol. in this model, although both tracers present certain obstacles, which need to be considered in the design of longitudinal preclin. tau imaging studies.
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    17. 17
      Brendel, M., Jaworska, A., Probst, F., Overhoff, F., Korzhova, V., Lindner, S., Carlsen, J., Bartenstein, P., Harada, R., Kudo, Y. (2016) Small-Animal PET Imaging of Tau Pathology with 18F-THK5117 in 2 Transgenic Mouse Models. J. Nucl. Med. 57 (5), 792– 8,  DOI: 10.2967/jnumed.115.163493
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      Small-animal PET imaging of tau pathology with 18F-THK5117 in 2 transgenic mouse models
      Brendel, Matthias; Jaworska, Anna; Probst, Federico; Overhoff, Felix; Korzhova, Viktoria; Lindner, Simon; Carlsen, Janette; Bartenstein, Peter; Harada, Ryuichi; Kudo, Yukitsuka; Haass, Christian; Van Leuven, Fred; Okamura, Nobuyuki; Herms, Jochen; Rominger, Axel
      Journal of Nuclear Medicine (2016), 57 (5), 792-798CODEN: JNMEAQ; ISSN:1535-5667. (Society of Nuclear Medicine and Molecular Imaging)
      Abnormal accumulation of tau aggregates in the brain is one of the hallmarks of Alzheimer disease neuropathol. We visualized tau deposition in vivo with the previously developed 2-arylquinoline deriv. 18F-THK5117 using small-animal PET in conjunction with autoradiog. and immunohistochem. gold std. assessment in 2 transgenic mouse models expressing hyperphosphorylated tau. Small-animal PET recordings were obtained in groups of P301S (n = 11) and biGT mice (n = 16) of different ages, with age-matched wild-type (WT) serving as controls. After i.v. administration of 16 ± 2 MBq of 18F-THK5117, a dynamic 90-min emission recording was initiated for P301S mice and during 20-50 min after injection for biGT mice, followed by a 15-min transmission scan. After coregistration to the MRI atlas and scaling to the cerebellum, we performed vol.-of-interest-based anal. (SUV ratio [SUVR]) and statistical parametric mapping. Small-animal PET results were compared with autoradiog. ex vivo and in vitro and further validated with AT8 staining for neurofibrillary tangles. SUVRs calcd. from static recordings during the interval of 20-50 min after tracer injection correlated highly with ests. of binding potential based on the entire dynamic emission recordings (R = 0.85). SUVR increases were detected in the brain stem of aged P301S mice (+11%; P < 0.001) and in entorhinal/amygdaloidal areas (+15%; P < 0.001) of biGT mice when compared with WT, whereas aged WT mice did not show increased tracer uptake. Immunohistochem. tau loads correlated with small-animal PET SUVR for both P301S (R = 0.8; P < 0.001) and biGT (R = 0.7; P < 0.001) mice, and distribution patterns of AT8-pos. neurons matched voxelwise statistical parametric mapping anal. Saturable binding of the tracer was verified by autoradiog. blocking studies. In the first dedicated small-animal PET study in 2 different transgenic tauopathy mouse models using the tau tracer 18F-THK5117, the temporal and spatial progression could be visualized in good correlation with gold std. assessments of tau accumulation. The serial small-animal PET method could afford the means for preclin. testing of novel therapeutic approaches by accommodating interanimal variability at baseline, while detection thresholds in young animals have to be considered.
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    18. 18
      Allen, B., Ingram, E., Takao, M., Smith, M. J., Jakes, R., Virdee, K., Yoshida, H., Holzer, M., Craxton, M., Emson, P. C. (2002) Abundant tau filaments and nonapoptotic neurodegeneration in transgenic mice expressing human P301S tau protein. J. Neurosci. 22 (21), 9340– 51,  DOI: 10.1523/JNEUROSCI.22-21-09340.2002
      18
      Abundant tau filaments and nonapoptotic neurodegeneration in transgenic mice expressing human P301S Tau protein
      Allen, Bridget; Ingram, Esther; Takao, Masaki; Smith, Michael J.; Jakes, Ross; Virdee, Kanwar; Yoshida, Hirotaka; Holzer, Max; Craxton, Molly; Emson, Piers C.; Atzori, Cristiana; Migheli, Antonio; Crowther, R. Anthony; Ghetti, Bernardino; Spillantini, Maria Grazia; Goedert, Michel
      Journal of Neuroscience (2002), 22 (21), 9340-9351CODEN: JNRSDS; ISSN:0270-6474. (Society for Neuroscience)
      The identification of mutations in the Tau gene in frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) has made it possible to express human tau protein with pathogenic mutations in transgenic animals. Here the authors report on the prodn. and characterization of a line of mice transgenic for the 383 aa isoform of human tau with the P301S mutation. At 5-6 mo of age, homozygous animals from this line developed a neurol. phenotype dominated by a severe paraparesis. According to light microscopy, many nerve cells in brain and spinal cord were strongly immunoreactive for hyperphosphorylated tau. According to electron microscopy, abundant filaments made of hyperphosphorylated tau protein were present. The majority of filaments resembled the half-twisted ribbons described previously in cases of FTDP-17, with a minority of filaments resembling the paired helical filaments of Alzheimer's disease. Sarkosyl-insol. tau from brains and spinal cords of transgenic mice ran as a hyperphosphorylated 64 kDa band, the same apparent mol. mass as that of the 383 aa tau isoform in the human tauopathies. Perchloric acid-sol. tau was also phosphorylated at many sites, with the notable exception of serine 214. In the spinal cord, neurodegeneration was present, as indicated by a 49% redn. in the no. of motor neurons. No evidence for apoptosis was obtained, despite the extensive colocalization of hyperphosphorylated tau protein with activated MAP kinase family members. The latter may be involved in the hyperphosphorylation of tau.
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    19. 19
      Wang, M., Gao, M., Xu, Z., and Zheng, Q. H. (2015) Synthesis of a PET tau tracer [(11)C]PBB3 for imaging of Alzheimer’s disease. Bioorg. Med. Chem. Lett. 25 (20), 4587– 92,  DOI: 10.1016/j.bmcl.2015.08.053
      19
      Synthesis of a PET tau tracer [11C]PBB3 for imaging of Alzheimer's disease
      Wang, Min; Gao, Mingzhang; Xu, Zhidong; Zheng, Qi-Huang
      Bioorganic & Medicinal Chemistry Letters (2015), 25 (20), 4587-4592CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)
      The authentic std. PBB3 and its precursor N-desmethyl-PBB3 as well as TBS-protected N-desmethyl-PBB3 precursor for radiolabeling were synthesized from 5-bromo-2-nitropyridine, acrolein di-Et acetal, 6-methoxy-2-methylbenzothiazole, and di-Et chlorophosphate with overall chem. yield 1% in six steps, 2% in five steps, and 1% in six steps, resp. [11C]PBB3 was prepd. from either desmethyl-PBB3 or TBS-protected desmethyl-PBB3 with [11C]CH3OTf through N-[11C]methylation and isolated by HPLC combined with SPE in 20-25% and 15-20% radiochem. yield, resp., based on [11C]CO2 and decay cor. to end of bombardment (EOB). The radiochem. purity was >99%, and the specific activity at EOB was 370-1110 GBq/μmol with a total synthesis time of ∼40-min from EOB.
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    20. 20
      Bragina, M. E., Stergiopulos, N., and Fraga-Silva, R. A. (2017) Fluorescence-Based Binding Assay for Screening Ligands of Angiotensin Receptors. Methods Mol. Biol. 1614, 165– 174,  DOI: 10.1007/978-1-4939-7030-8_13
      20
      Fluorescence-based binding assay for screening ligands of angiotensin receptors
      Bragina, Maiia E.; Stergiopulos, Nikolaos; Fraga-Silva, Rodrigo A.
      Methods in Molecular Biology (New York, NY, United States) (2017), 1614 (Renin-Angiotensin-Aldosterone System), 165-174CODEN: MMBIED; ISSN:1940-6029. (Springer)
      A review. Binding assay is a common technique used to characterize ability of a ligand to interact with a specific biol. target. A no. of parameters, such as binding affinity, receptor d., and assocn./dissocn. rate consts., can be measured by means of this technique. In most cases, implementation of the binding assay requires specific infrastructure for labeling and detecting the ligand, which impedes realization of this technique in a std. lab. Here we describe a simple fluorescence-based binding assay for angiotensin peptides and receptors, which does not require complex equipment and can be used for initial screening of the novel ligands or mutational studies.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXht12iurnO&md5=c4acdfaefdc451eaab4d9107f13aec21
    21. 21
      Ono, M., Sahara, N., Kumata, K., Ji, B., Ni, R., Koga, S., Dickson, D. W., Trojanowski, J. Q., Lee, V. M., Yoshida, M. (2017) Distinct binding of PET ligands PBB3 and AV-1451 to tau fibril strains in neurodegenerative tauopathies. Brain 140 (3), 764– 780,  DOI: 10.1093/brain/aww339
      21
      Distinct binding of PET ligands PBB3 and AV-1451 to tau fibril strains in neurodegenerative tauopathies
      Ono Maiko; Sahara Naruhiko; Kumata Katsushi; Ji Bin; Suhara Tetsuya; Zhang Ming-Rong; Higuchi Makoto; Ono Maiko; Ni Ruiqing; Nordberg Agneta; Koga Shunsuke; Dickson Dennis W; Trojanowski John Q; Lee Virginia M-Y; Yoshida Mari; Hozumi Isao; Yoshiyama Yasumasa; van Swieten John C
      Brain : a journal of neurology (2017), 140 (3), 764-780 ISSN:.
      Diverse neurodegenerative disorders are characterized by deposition of tau fibrils composed of conformers (i.e. strains) unique to each illness. The development of tau imaging agents has enabled visualization of tau lesions in tauopathy patients, but the modes of their binding to different tau strains remain elusive. Here we compared binding of tau positron emission tomography ligands, PBB3 and AV-1451, by fluorescence, autoradiography and homogenate binding assays with homologous and heterologous blockades using tauopathy brain samples. Fluorescence microscopy demonstrated intense labelling of non-ghost and ghost tangles with PBB3 and AV-1451, while dystrophic neurites were more clearly detected by PBB3 in brains of Alzheimer's disease and diffuse neurofibrillary tangles with calcification, characterized by accumulation of all six tau isoforms. Correspondingly, partially distinct distributions of autoradiographic labelling of Alzheimer's disease slices with 11C-PBB3 and 18F-AV-1451 were noted. Neuronal and glial tau lesions comprised of 4-repeat isoforms in brains of progressive supranuclear palsy, corticobasal degeneration and familial tauopathy due to N279K tau mutation and 3-repeat isoforms in brains of Pick's disease and familial tauopathy due to G272V tau mutation were sensitively detected by PBB3 fluorescence in contrast to very weak AV-1451 signals. This was in line with moderate 11C-PBB3 versus faint 18F-AV-1451 autoradiographic labelling of these tissues. Radioligand binding to brain homogenates revealed multiple binding components with differential affinities for 11C-PBB3 and 18F-AV-1451, and higher availability of binding sites on progressive supranuclear palsy tau deposits for 11C-PBB3 than 18F-AV-1451. Our data indicate distinct selectivity of PBB3 compared to AV-1451 for diverse tau fibril strains. This highlights the more robust ability of PBB3 to capture wide-range tau pathologies.
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    22. 22
      Delobel, P., Lavenir, I., Fraser, G., Ingram, E., Holzer, M., Ghetti, B., Spillantini, M. G., Crowther, R. A., and Goedert, M. (2008) Analysis of tau phosphorylation and truncation in a mouse model of human tauopathy. Am. J. Pathol. 172 (1), 123– 31,  DOI: 10.2353/ajpath.2008.070627
      22
      Analysis of Tau phosphorylation and truncation in a mouse model of human tauopathy
      Delobel, Patrice; Lavenir, Isabelle; Fraser, Graham; Ingram, Esther; Holzer, Max; Ghetti, Bernardino; Spillantini, Maria Grazia; Crowther, R. Anthony; Goedert, Michel
      American Journal of Pathology (2008), 172 (1), 123-131CODEN: AJPAA4; ISSN:0002-9440. (American Society for Investigative Pathology)
      Recent evidence has suggested that truncation of tau protein at the caspase cleavage site D421 precedes hyperphosphorylation and may be necessary for the assembly of tau into filaments in Alzheimer's disease and other tauopathies. Here we have investigated the time course of the appearance of phosphorylated and truncated tau in the brain and spinal cord of mice transgenic for mutant human P301S tau protein. This mouse line recapitulates the essential mol. and cellular features of the human tauopathies, including tau hyperphosphorylation, tau filament formation, and neurodegeneration. Sol. tau was strongly phosphorylated at 1 to 6 mo of age. Low levels of phosphorylated, sarkosyl-insol. tau were detected at 2 mo, with a steady increase up to 6 mo of age. Tau truncated at D421 was detected at low levels in Tris-sol. and detergent-sol. tau at 3 to 6 mo of age. By immunoblotting, it was not detected in sarkosyl-insol. tau. However, by immunoelectron microscopy, a small percentage of tau in filaments from brain and spinal cord of transgenic mice was truncated at D421. Similar findings were obtained using dispersed filaments from Alzheimer's disease and FTDP-17 brains. The late appearance and low abundance of tau ending at D421 indicate that it is unlikely that truncation at this site is necessary for the assembly of tau into filaments.
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    23. 23
      Macdonald, J. A., Bronner, I. F., Drynan, L., Fan, J., Curry, A., Fraser, G., Lavenir, I., and Goedert, M. (2019) Assembly of transgenic human P301S Tau is necessary for neurodegeneration in murine spinal cord. Acta Neuropathol Commun. 7 (1), 44,  DOI: 10.1186/s40478-019-0695-5
      23
      Assembly of transgenic human P301S Tau is necessary for neurodegeneration in murine spinal cord
      Macdonald Jennifer A; Drynan Lesley; Fan Juan; Lavenir Isabelle; Goedert Michel; Bronner Iraad F; Curry Annabelle; Fraser Graham
      Acta neuropathologica communications (2019), 7 (1), 44 ISSN:.
      A pathological pathway leading from soluble monomeric to insoluble filamentous Tau is characteristic of many human neurodegenerative diseases, which also exhibit dysfunction and death of brain cells. However, it is unknown how the assembly of Tau into filaments relates to cell loss. To study this, we first used a mouse line transgenic for full-length human mutant P301S Tau to investigate the temporal relationship between Tau assembly into filaments, assessed using anti-Tau antibody AT100, and motor neuron numbers, in the lumbar spinal cord. AT100 immunoreactivity preceded nerve cell loss. Murine Tau did not contribute significantly to either Tau aggregation or neurodegeneration. To further study the relevance of filament formation for neurodegeneration, we deleted hexapeptides (275)VQIINK(280) and (306)VQIVYK(311), either singly or in combination, from human 0N4R Tau with the P301S mutation. These hexapeptides are essential for the assembly of Tau into filaments. Homozygous mice transgenic for P301S Tau with the hexapeptide deletions, which expressed Tau at a similar level to the heterozygous line transgenic for P301S Tau, had a normal lifespan, unlike mice from the P301S Tau line. The latter had significant levels of sarkosyl-insoluble Tau in brain and spinal cord, and exhibited neurodegeneration. Mice transgenic for P301S Tau with the hexapeptide deletions failed to show significant levels of sarkosyl-insoluble Tau or neurodegeneration. Recombinant P301S Tau with the hexapeptide deletions failed to form β-sheet structure and filaments following incubation with heparin. Taken together, we conclude that β-sheet assembly of human P301S Tau is necessary for neurodegeneration in transgenic mice.
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    24. 24
      Mellone, M., Kestoras, D., Andrews, M. R., Dassie, E., Crowther, R. A., Stokin, G. B., Tinsley, J., Horne, G., Goedert, M., Tolkovsky, A. M. (2013) Tau pathology is present in vivo and develops in vitro in sensory neurons from human P301S tau transgenic mice: a system for screening drugs against tauopathies. J. Neurosci. 33 (46), 18175– 89,  DOI: 10.1523/JNEUROSCI.4933-12.2013
      24
      Tau pathology is present in vivo and develops in vitro in sensory neurons from human P301S tau transgenic mice: a system for screening drugs against tauopathies
      Mellone, Manuela; Kestoras, Dimitra; Andrews, Melissa R.; Dassie, Elisa; Crowther, R. Anthony; Stokin, Gorazd B.; Tinsley, Jon; Horne, Graeme; Goedert, Michel; Tolkovsky, Aviva M.; Spillantini, Maria Grazia
      Journal of Neuroscience (2013), 33 (46), 18175-18189CODEN: JNRSDS; ISSN:0270-6474. (Society for Neuroscience)
      Intracellular tau aggregates are the neuropathol. hallmark of several neurodegenerative diseases, including Alzheimer's disease, progressive supranuclear palsy, and cases of frontotemporal dementia, but the link between these aggregates and neurodegeneration remains unclear. Neuronal models recapitulating the main features of tau pathol. are necessary to investigate the mol. mechanisms of tau malfunction, but current models show little and inconsistent spontaneous tau aggregation. We show that dorsal root ganglion (DRG) neurons in transgenic mice expressing human P301S tau (P301S-htau) develop tau pathol. similar to that found in brain and spinal cord and a significant redn. in mechanosensation occurs before detectable fibrillar tau formation. DRG neuronal cultures established from adult P301S-htau mice at different ages retained the pattern of aberrant tau found in vivo. Moreover, htau became progressively hyperphosphorylated over 2 mo in vitro beginning with nonsymptomatic neurons, while hyperphosphorylated P301S-htau-pos. neurons from 5-mo-old mice cultured for 2 mo died preferentially. P301S-htau-pos. neurons grew aberrant axons, including spheroids, typically found in human tauopathies. Neurons cultured at advanced stages of tau pathol. showed a 60% decrease in the fraction of moving mitochondria. SEG28019, a novel O-GlcNAcase inhibitor, reduced steady-state pSer396/pSer404 phosphorylation over 7 wk in a significant proportion of DRG neurons showing for the first time the possible beneficial effect of prolonged dosing of O-GlcNAcase inhibitor in vitro. Our system is unique in that fibrillar tau forms without external manipulation and provides an important new tool for understanding the mechanisms of tau dysfunction and for screening of compds. for treatment of tauopathies.
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    25. 25
      Brelstaff, J., Spillantini, M. G., and Tolkovsky, A. M. (2015) pFTAA: a high affinity oligothiophene probe that detects filamentous tau in vivo and in cultured neurons. Neural Regener. Res. 10 (11), 1746– 7,  DOI: 10.4103/1673-5374.165298
      25
      pFTAA: a high affinity oligothiophene probe that detects filamentous tau in vivo and in cultured neurons
      Brelstaff, Jack; Spillantini, Maria Grazia; Tolkovsky, Aviva M.
      Neural Regeneration Research (2015), 10 (11), 1746-1747CODEN: NRREBM; ISSN:1673-5374. (Publishing House of Neural Regeneration Research)
      Tauopathies describe a group of neurodegenerative diseases in which the protein tau, encoded by the gene MAPT, is aberrantly misfolded, leading to tau aggregation, neural dysfunction, and cell death. In Alzheimer's disease (AD), tau forms the characteristic intracellular neurofibrillary tangles (NFTs), which are thought to be the major cause of neurodegeneration. The use of pFTAA has solved a major problem in trying to decipher mechanisms of cell death induced by filamentous forms of tau, as we can now follow living neurons to their death. We also noted previously that DRG neurons with abnormal forms of tau regenerate abnormally, with splayed growth cones, spheroids (a typical feature of human tauopathies), and thick short axons, after they are severed prior to culture. Given that we can identify pFTAA+ve neurons and axons, we can now approach the question whether tau filaments, or sol. abnormal tau, are responsible for inducing these aberrant retarded growth patterns. This will be esp. interesting in studying the regeneration of DRG neurons in vivo, and ultimately CNS neuronal axons and dendrites during synapse formation.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhs1Kqu7vK&md5=15693d14f2ed191208460ce793b2fd72
    26. 26
      Brelstaff, J., Ossola, B., Neher, J. J., Klingstedt, T., Nilsson, K. P., Goedert, M., Spillantini, M. G., and Tolkovsky, A. M. (2015) The fluorescent pentameric oligothiophene pFTAA identifies filamentous tau in live neurons cultured from adult P301S tau mice. Front. Neurosci. 9, 184,  DOI: 10.3389/fnins.2015.00184
      26
      The fluorescent pentameric oligothiophene pFTAA identifies filamentous tau in live neurons cultured from adult P301S tau mice
      Brelstaff Jack; Ossola Bernardino; Spillantini Maria Grazia; Tolkovsky Aviva M; Neher Jonas J; Klingstedt Therese; Goedert Michel; Nilsson K Peter R
      Frontiers in neuroscience (2015), 9 (), 184 ISSN:1662-4548.
      Identification of fluorescent dyes that label the filamentous protein aggregates characteristic of neurodegenerative disease, such as β-amyloid and tau in Alzheimer's disease, in a live cell culture system has previously been a major hurdle. Here we show that pentameric formyl thiophene acetic acid (pFTAA) fulfills this function in living neurons cultured from adult P301S tau transgenic mice. Injection of pFTAA into 5-month-old P301S tau mice detected cortical and DRG neurons immunoreactive for AT100, an antibody that identifies solely filamentous tau, or MC1, an antibody that identifies a conformational change in tau that is commensurate with neurofibrillary tangle formation in Alzheimer's disease brains. In fixed cultures of dorsal root ganglion (DRG) neurons, pFTAA binding, which also identified AT100 or MC1+ve neurons, followed a single, saturable binding curve with a half saturation constant of 0.14 μM, the first reported measurement of a binding affinity of a beta-sheet reactive dye to primary neurons harboring filamentous tau. Treatment with formic acid, which solubilizes filamentous tau, extracted pFTAA, and prevented the re-binding of pFTAA and MC1 without perturbing expression of soluble tau, detected using an anti-human tau (HT7) antibody. In live cultures, pFTAA only identified DRG neurons that, after fixation, were AT100/MC1+ve, confirming that these forms of tau pre-exist in live neurons. The utility of pFTAA to discriminate between living neurons containing filamentous tau from other neurons is demonstrated by showing that more pFTAA+ve neurons die than pFTAA-ve neurons over 25 days. Since pFTAA identifies fibrillar tau and other misfolded proteins in living neurons in culture and in animal models of several neurodegenerative diseases, as well as in human brains, it will have considerable application in sorting out disease mechanisms and in identifying disease-modifying drugs that will ultimately help establish the mechanisms of neurodegeneration in human neurodegenerative diseases.
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    27. 27
      Zhang, W., Falcon, B., Murzin, A. G., Fan, J., Crowther, R. A., Goedert, M., and Scheres, S. H. (2019) Heparin-induced tau filaments are polymorphic and differ from those in Alzheimer’s and Pick’s diseases. eLife 8, e43584,  DOI: 10.7554/eLife.43584
      27
      Heparin-induced tau filaments are polymorphic and differ from those in Alzheimer's and Pick's diseases
      Zhang, Wenjuan; Falcon, Benjamin; Murzin, Alexey G.; Fan, Juan; Crowther, R. Anthony; Goedert, Michel; Scheres, Sjors H. W.
      eLife (2019), 8 (), 43584CODEN: ELIFA8; ISSN:2050-084X. (eLife Sciences Publications Ltd.)
      Assembly of microtubule-assocd. protein tau into filamentous inclusions underlies a range of neurodegenerative diseases. Tau filaments adopt different conformations in Alzheimer's and Pick's diseases. Here, we used cryo- and immuno- electron microscopy to characterize filaments that were assembled from recombinant full-length human tau with four (2N4R) or three (2N3R) microtubule-binding repeats in the presence of heparin. 2N4R tau assembles into multiple types of filaments, and the structures of three types reveal similar 'kinked hairpin' folds, in which the second and third repeats pack against each other. 2N3R tau filaments are structurally homogeneous, and adopt a dimeric core, where the third repeats of two tau mols. pack in a parallel manner. The heparin-induced tau filaments differ from those of Alzheimer's or Pick's disease, which have larger cores with different repeat compns. Our results illustrate the structural versatility of amyloid filaments, and raise questions about the relevance of in vitro assembly.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXisVegtLjP&md5=5f3224e5ce2a209e04dc76fd091bfc60
    28. 28
      Filipcik, P., Zilka, N., Bugos, O., Kucerak, J., Koson, P., Novak, P., and Novak, M. (2012) First transgenic rat model developing progressive cortical neurofibrillary tangles. Neurobiol. Aging 33 (7), 1448– 56,  DOI: 10.1016/j.neurobiolaging.2010.10.015
      28
      First transgenic rat model developing progressive cortical neurofibrillary tangles
      Filipcik Peter; Zilka Norbert; Bugos Ondrej; Kucerak Juraj; Koson Peter; Novak Petr; Novak Michal
      Neurobiology of aging (2012), 33 (7), 1448-56 ISSN:.
      Neurofibrillary degeneration induced by misfolded protein tau is considered to be one of the key pathological hallmarks of Alzheimer's disease (AD). In the present study, we have introduced a novel transgenic rat model expressing a human truncated tau that encompasses 3 microtubule binding domains (3R) and a proline-rich region (3R tau151-391). The transgenic rats developed progressive age-dependent neurofibrillary degeneration in the cortical brain areas. Neurofibrillary tangles (NFTs) satisfied several key histological criteria used to identify neurofibrillary degeneration in human Alzheimer's disease including argyrophilia, Congo red birefringence, and Thioflavin S reactivity. Neurofibrillary tangles were also identified with antibodies used to detect pathologic tau in the human brain, including DC11, recognizing an abnormal tau conformation and antibodies that are specific for hyperphosphorylated forms of tau protein. Moreover, neurofibrillary degeneration was characterized by extensive formation of sarkosyl insoluble tau protein complexes consisting of rat endogenous and truncated tau species. Interestingly, the transgenic rats did not show neuronal loss either in the cortex or in the hippocampus. We suggest that novel transgenic rat model for human tauopathy represents a valuable tool in preclinical drug discovery targeting neurofibrillary degeneration of Alzheimer's type.
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    29. 29
      Koson, P., Zilka, N., Kovac, A., Kovacech, B., Korenova, M., Filipcik, P., and Novak, M. (2008) Truncated tau expression levels determine life span of a rat model of tauopathy without causing neuronal loss or correlating with terminal neurofibrillary tangle load. Eur. J. Neurosci 28 (2), 239– 46,  DOI: 10.1111/j.1460-9568.2008.06329.x
      29
      Truncated tau expression levels determine life span of a rat model of tauopathy without causing neuronal loss or correlating with terminal neurofibrillary tangle load
      Koson Peter; Zilka Norbert; Kovac Andrej; Kovacech Branislav; Korenova Miroslava; Filipcik Peter; Novak Michal
      The European journal of neuroscience (2008), 28 (2), 239-46 ISSN:.
      We have previously demonstrated in a transgenic rat model of tauopathy that human misfolded truncated tau derived from Alzheimer's disease suffices to drive neurofibrillary degeneration in vivo. We employed this model to investigate the impact of truncated tau expression levels on life span, neuronal loss and the final load of neurofibrillary tangles (NFTs) in transgenic rats. Two independent transgenic lines (SHR72, SHR318), that display different expression levels of truncated tau, were utilized in this study. We found that transgene expression levels in the brain of SHR72 rats were 44% higher than in SHR318 rats and that truncated tau protein levels determined the survival rate of transgenic rats. The line with higher expression levels of truncated tau (SHR72) showed decreased median survival (222.5 days) when compared with the line with lower expression (SHR318; 294.5 days). Interestingly, NFT loads (total NFT/total neurons) were very similar in terminal stages of disease in both transgenic lines (SHR72 - 10.9%; SHR318 - 11.6%), despite significantly different expression levels of truncated tau. Moreover, mean neuron numbers in the hippocampus (CA1-3) and brain stem (gigantocellular reticular nucleus) in the two transgenic rat strains in the terminal stages of disease were similar, and did not differ significantly from those observed in age-matched non-transgenic controls. These findings suggest that the expression levels of misfolded truncated tau determine the life span in a transgenic rat model of tauopathy without causing neuronal loss or correlating with terminal NFT load.
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