GSK3β phosphorylation catalyzes the aggregation of Tau into Alzheimer’s disease-like amyloid strain

Chakraborty, Pijush; Ibáñez de Opakua, Alain; Purslow, Jeffrey A.; Fromm, Simon A.; Chatterjee, Debdeep; Zachrdla, Milan; Puri, Sambhavi; Wolozin, Benjamin; Zweckstetter, Markus

doi:10.1101/2023.12.19.572292

Cited by 4 publications

(1 citation statement)

References 53 publications

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“…Comparison of the phospho-Tau antibodies that detected recombinant GSK3beta, DYRK1A and CAMKIIA-phosphorylated Tau with the target sites that each respective kinase has been reported to phosphorylate in recombinant Tau. Data for GSK3beta target sites in recombinant Tau was compiled from [ 30 , 63 , 121 , 152 , 154 ], data for DYRK1A target sites in recombinant Tau was compiled from [ 91 , 124 ], and data for CAMKIIA target sites in recombinant Tau was compiled from [ 30 , 89 , 152 , 154 , 167 ]. As Tau phosphorylation by various kinases is highly interdependent, with some phosphorylation events by one kinase priming further phosphorylation events by a different kinase, only reports that tested phosphorylation of recombinant Tau with the respective kinase individually were considered, given that the recombinant pTau proteins used in this study were phosphorylated with only one kinase at a time.…”

Section: Additional Filesmentioning

confidence: 99%

Identification of high-performing antibodies for the reliable detection of Tau proteoforms by Western blotting and immunohistochemistry

Ellis,

Lekka,

Holden

et al. 2024

Acta Neuropathol

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Antibodies are essential research tools whose performance directly impacts research conclusions and reproducibility. Owing to its central role in Alzheimer’s disease and other dementias, hundreds of distinct antibody clones have been developed against the microtubule-associated protein Tau and its multiple proteoforms. Despite this breadth of offer, limited understanding of their performance and poor antibody selectivity have hindered research progress. Here, we validate a large panel of Tau antibodies by Western blot (79 reagents) and immunohistochemistry (35 reagents). We address the reagents’ ability to detect the target proteoform, selectivity, the impact of protein phosphorylation on antibody binding and performance in human brain samples. While most antibodies detected Tau at high levels, many failed to detect it at lower, endogenous levels. By WB, non-selective binding to other proteins affected over half of the antibodies tested, with several cross-reacting with the related MAP2 protein, whereas the “oligomeric Tau” T22 antibody reacted with monomeric Tau by WB, thus calling into question its specificity to Tau oligomers. Despite the presumption that “total” Tau antibodies are agnostic to post-translational modifications, we found that phosphorylation partially inhibits binding for many such antibodies, including the popular Tau-5 clone. We further combine high-sensitivity reagents, mass-spectrometry proteomics and cDNA sequencing to demonstrate that presumptive Tau “knockout” human cells continue to express residual protein arising through exon skipping, providing evidence of previously unappreciated gene plasticity. Finally, probing of human brain samples with a large panel of antibodies revealed the presence of C-term-truncated versions of all main Tau brain isoforms in both control and tauopathy donors. Ultimately, we identify a validated panel of Tau antibodies that can be employed in Western blotting and/or immunohistochemistry to reliably detect even low levels of Tau expression with high selectivity. This work represents an extensive resource that will enable the re-interpretation of published data, improve reproducibility in Tau research, and overall accelerate scientific progress.

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Section: Additional Filesmentioning

confidence: 99%

Identification of high-performing antibodies for the reliable detection of Tau proteoforms by Western blotting and immunohistochemistry

Ellis,

Lekka,

Holden

et al. 2024

Acta Neuropathol

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Synaptic sabotage: How Tau and α-Synuclein undermine synaptic health

Uytterhoeven,

Verstreken,

Nachman

2024

Journal of Cell Biology

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Synaptic dysfunction is one of the earliest cellular defects observed in Alzheimer’s disease (AD) and Parkinson’s disease (PD), occurring before widespread protein aggregation, neuronal loss, and cognitive decline. While the field has focused on the aggregation of Tau and α-Synuclein (α-Syn), emerging evidence suggests that these proteins may drive presynaptic pathology even before their aggregation. Therefore, understanding the mechanisms by which Tau and α-Syn affect presynaptic terminals offers an opportunity for developing innovative therapeutics aimed at preserving synapses and potentially halting neurodegeneration. This review focuses on the molecular defects that converge on presynaptic dysfunction caused by Tau and α-Syn. Both proteins have physiological roles in synapses. However, during disease, they acquire abnormal functions due to aberrant interactions and mislocalization. We provide an overview of current research on different essential presynaptic pathways influenced by Tau and α-Syn. Finally, we highlight promising therapeutic targets aimed at maintaining synaptic function in both tauopathies and synucleinopathies.

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Chaperone-mediated heterotypic phase separation regulates liquid-to-solid phase transitions into amyloid fibrils

Rai,

Khanna,

Sarbahi

et al. 2024

Preprint

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Biomolecular condensates formed via the phase separation of proteins and nucleic acids are thought to regulate a myriad of cellular processes with exquisite spatiotemporal precision. However, such highly dynamic, viscoelastic, mesoscopic, intracellular membraneless bodies can undergo aberrant liquid-to-solid transitions into a range of amyloid-like species. The formation of such pathological assemblies necessitates their clearance by the cellular protein quality control machinery comprising molecular chaperones. Nonetheless, the mechanism underlying the chaperone-mediated regulation of protein homeostasis within biomolecular condensates remains elusive. Here, we present a unique case demonstrating that a heat shock protein 40 (Hsp40), Ydj1, promotes the heterotypic phase separation of intrinsically disordered tau via intermolecular electrostatic and hydrophobic interactions. Through a diverse array of tools involving high-resolution fluorescence imaging, single-droplet steady-state and picosecond time-resolved fluorescence anisotropy, and single-molecule FRET (Förster resonance energy transfer), we elucidate the diverse structural conformations of tau present within phase-separated heterotypic condensates that are otherwise predisposed to aggregation. Our vibrational Raman spectroscopy and electron microscopy data show that the presence of Ydj1 in tau-Ydj1 condensates abolishes the formation of amyloid fibrils, unlike tau-only droplets. By sequentially deleting segments, we identify amyloidogenic hexapeptide motifs located in the hydrophobic microtubule-binding region of tau that foster contacts with the peptide-binding regions of Ydj1, promoting the formation of tau-Ydj1 binary condensates. Additionally, we show that the underlying network of interactions governing these condensates can be further tuned by RNA. Our results underscore an intriguing interplay of molecular drivers that govern chaperone-associated phase separation, with broader implications for the chaperoning of a wide range of intrinsically disordered proteins involved in physiology and disease.

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GSK3β phosphorylation catalyzes the aggregation of Tau into Alzheimer’s disease-like amyloid strain

Cited by 4 publications

References 53 publications

Identification of high-performing antibodies for the reliable detection of Tau proteoforms by Western blotting and immunohistochemistry

Identification of high-performing antibodies for the reliable detection of Tau proteoforms by Western blotting and immunohistochemistry

Synaptic sabotage: How Tau and α-Synuclein undermine synaptic health

Chaperone-mediated heterotypic phase separation regulates liquid-to-solid phase transitions into amyloid fibrils

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