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Neuronal interactome changes upon aggregation of Tau

ABSTRACT: To understand at molecular level how aggregation of Tau modulates its interactions with proteins we reveal key determinant for derailing Tau protein network. By performing quantitative AP-MS we define a new set of interactors which bind Tau upon fibril formation. These interactors contain disordered regions with a unique amino acidic footprint. Such regions are enriched in positively charged Arginines which can engage aberrant binding to Tau fibrils through their guanidinium group via pi-stacking. We also find that the Hsp90 chaperone stalls formation of Tau fibrils and reshapes their abnormal interactome.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Rattus Norvegicus (rat)

TISSUE(S): Brain

SUBMITTER: Riccardo Stucchi

LAB HEAD: Casper Hoogenraad

PROVIDER: PXD015432 | Pride | 2020-01-30

REPOSITORIES: Pride

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			Action	DRS
	EXP_HSP90MaxQuantproteinGroups.txt	Txt
	Figure2B.xlsx	Xlsx
	Figure2C.xlsx	Xlsx
	Figure2D.xlsx	Xlsx
	Figure6B.xlsx	Xlsx

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Publications

Arginine π-stacking drives binding to fibrils of the Alzheimer protein Tau.

Ferrari Luca L Stucchi Riccardo R Konstantoulea Katerina K van de Kamp Gerarda G Kos Renate R Geerts Willie J C WJC van Bezouwen Laura S LS Förster Friedrich G FG Altelaar Maarten M Hoogenraad Casper C CC Rüdiger Stefan G D SGD

Nature communications 20200129 1

Aggregation of the Tau protein into fibrils defines progression of neurodegenerative diseases, including Alzheimer's Disease. The molecular basis for potentially toxic reactions of Tau aggregates is poorly understood. Here we show that π-stacking by Arginine side-chains drives protein binding to Tau fibrils. We mapped an aggregation-dependent interaction pattern of Tau. Fibrils recruit specifically aberrant interactors characterised by intrinsically disordered regions of atypical sequence featur ...[more]

PMID: 31996674

Similar Datasets

Are Mouse Models a surrogate for human disease? A temporal, quantitative and molecular perspective.

Project description:It is unclear to what extent Tau molecular pathology in murine models reflect human Tauopathies. Nevertheless, mouse models that overexpress human mutant Tau (P301S and P301L) are widely used in studies of Tauopathies and Alzheimer’s Disease (AD). In this study, we perform an in-depth temporally and spatially resolved mass spectrometry-based proteomic analysis of P301S (hTau.P301S) and P301L (rTg(tauP301L)4510) mice as well as human patients with AD or frontotemporal dementia due to the P301L mutation, to identify differences and similarities between human AD, animal models and human P301L patients. Both mouse models and human P301L patients show progressive Tau accumulation driven by Tau phosphorylation during disease progression as also observed in early human AD. However, Tau ubiquitination and acetylation, important in human AD, are less or not represented in the mouse models or in P301L patients. Our analyses provide guidance regarding designing mechanistic studies and testing of Tau directed therapeutics.

2023-02-03 | PXD033965 | Pride

Identification of proteins that interact with extracellularly applied fibrillar Tau assemblies

Project description:The microtubule associated protein Tau (MAPT) expressed in neurons is involved in microtubules stabilization, cell morphogenesis and axonal transport. In pathological conditions, Tau assembles into high molecular weight assemblies leading to neuropathological Tau deposits, the hallmark of several neurodegenerative diseases collectively named “tauopathies”, including Alzheimer’s disease. These pathologic Tau assemblies are released by affected neuronal cells and taken up by naïve neighbor cells, propagate from one cell to another and amplify by seeding the aggregation of endogenous Tau. In order to identify plasma membrane proteins exposed extracellularly that interact with extracellularly applied fibrillar Tau assemblies, we exposed pure-neuronal cultures to fibrillar Tau for 10 min, pulled down the associated proteins, and identified them using a proteomic-based approach. Of the six Tau isoforms produced by alternative splicing of the MAPT gene and differing from each other by the presence or absence of one or two inserts in the N-terminal part (0N, 1N or 2N) of the protein and by the presence of either three or four repeated microtubule binding motifs in the protein C-terminal part (3R or 4R), we have expressed and purified 1N3R and 1N4R Tau isoforms and further assembled them into 1N3R and 1N4R Tau fibrils. Using pull-down of whole cell lysates and mass spectrometry, we have identified proteins interacting with extracellularly applied Tau fibrils. We have performed two different experiments with either 1N3R Tau fibrils or 1N4R Tau fibrils (condition 1 and condition 2 respectively). Each condition consists in six experimental replicates of cells exposed 10 min to Tau fibrils and of the non-treated cells used as controls.

2019-01-09 | PXD009294 | Pride

Targeted mass spectrometry of Tau preparations purified from Alzheimer’s Disease brains plus either the deubiquitylase USP21 or its activity-dead mutant C221A.

Project description:To characterise the abundance of ubiquitylated Tau peptides in the presence of active or inactive variants (wt 196-565 vs. C221A) of the broad spectrum deubiquitylase USP21. Tau peptides are obtained from Tau fibrils purified from post mortem human brains diagnosed with Alzheimer’s. Their detection is facilitated by previous training of the mass spectrometer with in vitro ubiquitylated Tau. The same samples were used to evaluate the percentage of peptides modified with ubiquitin

2024-06-18 | PXD046645 | Pride

Combinatorial native MS and LC-MS/MS approach reveals high intrinsic phosphorylation of human Tau

Project description:Abnormal changes in the neuronal microtubule-associated protein Tau, such as high phosphorylation and aggregation, are considered hallmarks of cognitive deficits in Alzheimer disease. Abnormal phosphorylation is thought to take place before aggregation, and therefore it is often assumed that phosphorylation predisposes Tau towards aggregation. However, the nature and extent of phosphorylation has remained ill-defined. Tau protein contains up to 85 potential phosphorylation sites (80 Ser/Thr, and 5 Tyr P-sites), many of which can be phosphorylated by various kinases because the unfolded structure of Tau makes them accessible. However, limitations in methods (e.g. in mass spectrometry of phosphorylated peptides, or antibodies against phospho-epitopes) have led to conflicting results regarding the overall degree of phosphorylation of Tau in cells. Here we present results from a new approach, that is based on native mass spectrometry analysis of intact Tau expressed in a eukaryotic cell system (Sf9) which reveals Tau in different phosphorylation states. The extent of phosphorylation is remarkably heterogeneous with up to ~20 phosphates (Pi) per molecule and distributed over 51 sites (including all P-sites published so far and additional 18 P-sites). The medium phosphorylated fraction Pm showed overall occupancies centered at 8 Pi (± 5 Pi) with a bell-shaped distribution, the highly phosphorylated fraction Ph had 14 Pi (± 6 Pi). The distribution of sites was remarkably asymmetric (with 71% of all P-sites located in the C-terminal half of Tau). All phosphorylation sites were on Ser or Thr residues, but none on Tyr. Other known posttranslational modifications of Tau were near or below our detection limit (e.g. acetylation, ubiquitination).

2020-11-20 | PXD020985 | Pride

Quantitative PTM Maps of Human Pathologic Tau Identify Patient Heterogeneity and Define Critical Steps in Alzheimer’s Disease Progression - angular gyrus

Project description:To elucidate the role of Tau isoforms and PTM stoichiometry in Alzheimer’s disease (AD), we generated a high resolution quantitative proteomic map of 88 PTMs on multiple isoforms of Tau isolated from the post-mortem human tissue from 49 AD and 42 control subjects. While Tau PTM maps reveal heterogeneity across subjects, a subset of PTMs display high occupancy and patient frequency for AD suggesting importance in disease. Unsupervised analyses indicate that PTMs occur in an ordered manner leading to Tau aggregation. The processive addition and minimal set of PTMs associated with seeding activity was further defined by the analysis of size fractionated Tau. To summarize, critical features within the Tau protein for disease intervention at different stages of disease are identified, including enrichment of 0N and 4R isoforms, underrepresentation of the C-terminal, an increase in negative charge in the PRR and a decrease in positive charge in the MBD.

2020-12-04 | PXD020517 | Pride

Truncated Tau - Role of the Tau N-terminal region in microtubule stabilization revealed by new endogenous truncated forms

Project description:Tau is a microtubule-associated protein that ensures neuronal shape and function. Besides, Tau is a central player in Alzheimer’s disease (AD) and related Tauopathies where it is found aggregated in degenerating neurons. Mechanisms leading to Tau pathology and its progression are far from being elucidated. Among Tau species found in AD brains, several yet unidentified truncated Tau fragments are showed. A major step forward in the understanding of the role of Tau truncation would be to identify the precise cleavage sites of Tau species. This key step is mandatory to generate appropriate experimental tools in order to investigate the impact of each identified truncated-species on Tau function/dysfunction. Here, we achieved an optimized proteomics approach and succeed in identifying a number of new N-terminally truncated-Tau species from human brain. As N-terminal residues of these fragments are located broadly across Tau sequence, one could expect to have different effects on Tau. We initiated cell-based functional studies by analyzing biochemical characteristics of two N-terminally truncated Tau species starting at residues Met11 and Gln124 (accordingly to the longest Tau isoform) regarding Tau microtubule function. Our results surprisingly showed that the ability of Tau to bind and stabilize microtubules was greater when the first 123 residues are truncated, suggesting that Tau N-terminus would have a role in regulation of microtubule stabilization. Overall, future studies based on our new N-terminally truncated-Tau species will provide new knowledge on the role of truncation in Tau biology as well as in AD pathological process.

2015-05-15 | PXD001353 | Pride

Specific post-translational modifications of the soluble tau protein distinguish between Alzheimer’s disease, 4R-, and 3R-tauopathies

Project description:The tau protein aggregates in several neurodegenerative disorders, referred to as tauopathies. The type of tau isoforms (4R/3R) observed in brain aggregates is used to classify tauopathies. However, distinguishing tauopathies in cerebrospinal fluid remains challenging. Here, we demonstrated that the difference in tau isoforms between tauopathies is only observed in aggregates, not in soluble brain extracts. We therefore used untargeted mass spectrometry to characterize all post-translational modifications of both the aggregated and the soluble tau protein obtained from human brain tissue of patients with Alzheimer’s disease, cortico-basal degeneration, Pick’s disease, and fronto-temporal lobe degeneration. We found specific soluble signatures predictive of each tauopathy and its peculiar type of aggregated tau isoforms. These findings provide potential targets for developing cerebrospinal fluid assays able to distinguish between tauopathies in vivo. The comparison between soluble and aggregated tau features indicates potential mechanisms of tau aggregation, providing novel therapeutic leads for these diseases

2023-06-22 | PXD038901 | Pride

The AdipoR2 protein recruits an ER-localized fatty acid elongation complex to promote membrane homeostasis

Project description:The human AdipoR2 protein and its C. elegans homolog PAQR-2 are multi-pass plasma membrane proteins that protect cells against membrane rigidification. We seek to better understand the mechanisms by which these proteins can promote the synthesis and incorporation of membrane-fluidizing unsaturated fatty acids into phospholipids. To this end, we performed immunoprecipitations of tagged AdipoR2 and PAQR-2 expressed in HEK293 cells or whole C. elegans, respectively, and identified co-immunoprecipitated proteins using mass spectroscopy. Among the conserved interaction partners, we identified many proteins likely important for the protein life cycles (e.g. ribosome, proteasome, vesicle trafficking components). Importantly, several proteins important for fatty acid synthesis, elongation and incorporation into phospholipids were also identified as evolutionarily conserved AdipoR2/PAQR-2 interaction partners. We experimentally verified several of these interactions, and propose that AdipoR2 and PAQR-2 can recruit an ER-bound membrane complex to promote the production and incorporation of unsaturated fatty acids into phospholipids.

2023-07-20 | PXD031395 | Pride

Cancer-specific association between Tau (MAPT) and cellular pathways, clinical outcome, and drug response

Project description:Tau (MAPT) is a microtubule-associated protein causing frequent neurodegenerative diseases or inherited frontotemporal lobar degenerations. Emerging evidence for non-canonical functions of Tau in DNA protection and P53 regulation suggests its involvement in cancer. Indeed, Tau expression correlates with cancer-specific survival or response to microtubule therapeutics. These data may imply common molecular pathways involved in the pathogenesis of neurodegenerative disorders and cancer. To bring new evidence that Tau represents a key protein in cancer, we present an in silico pan-cancer analysis of MAPT transcriptomic profile in over 11000 clinical samples and over 1300 pre-clinical samples provided by the TCGA and the DEPMAP datasets respectively. We completed this analysis by exploring a possible interplay of MAPT with wild-type or mutated P53. Then, we calculated the impact of MAPT expression on clinical outcome and drug response. Overall, the results support a relevant role of the MAPT gene in several cancer types, although the contribution of Tau to cancer appears to very much depend on the cellular context.

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A pilot study for deciphering post-translational modifications and proteoforms of tau protein by capillary electrophoresis-mass spectrometry

Project description:Abnormal accumulation of tau proteins is one pathological hallmark of Alzheimer’s disease (AD). Many tau protein post-translational modifications (PTMs) are associated with the development of AD, such as phosphorylation, acetylation, and methylation. Therefore, a complete picture of PTM landscape of tau is critical for understanding the molecular mechanisms of AD progression. Here, we offered a pilot study of combining two complementary analytical techniques, capillary zone electrophoresis (CZE)-tandem mass spectrometry (MS/MS) and reversed-phase liquid chromatography (RPLC)-MS/MS, for bottom-up proteomics of tau-0N3R. We identified 53 phosphorylation sites of tau-0N3R in total, which is about 30% higher than that from RPLC-MS/MS alone. CZE-MS/MS provided more PTM sites (i.e., phosphorylation) and modified peptides of tau-0N3R than RPLC-MS/MS, and its predicted electrophoretic mobility helped improve the confidence of the identified modified peptides. We developed a highly efficient capillary isoelectric focusing (cIEF)-MS technique to offer a bird’s-eye view of tau-0N3R proteoforms, with 11 putative tau-0N3R proteoforms carrying up to ten phosphorylation sites and lower pI values from more phosphorylated proteoforms detected. Interestingly, under a native-like cIEF-MS condition, we observed three putative tau-0N3R dimers carrying phosphate groups. The findings demonstrate that CE-MS is a valuable analytical technique for the characterization of tau PTMs, proteoforms, and even oligomerization.

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