PubMed: 28083634

Tau interactome mapping based identification of Otub1 as Tau deubiquitinase involved in accumulation of pathological Tau forms in vitro and in vivo.
Acta neuropathologica
Drewes G | Wang P | Dewachter I | Bantscheff M | Buist A | Faelth-Savitski M | Joberty G | Kienlen-Campard P | Moechars D | Octave JN | Pierrot N | Stancu IC | Vanoosthuyse A | Vasconcelos B

Evidence 097dd8575d

These results indicate that Otub1 regulates Tau degradation, dependent on its catalytic activity (Fig. 5c).

Evidence 51f4399004

Interestingly, Otub1 expression is gradually downregulated with increasing Tau-seeded Tau aggregation (Fig. S4a), which could be considered as a protective mechanism.

Evidence 43a3c4f0b7

Furthermore, analysis of hippocampal gene expression in aging mice, using publicly available data (NCBI database GDS2082) [73], indicates a significant increase in Otub1 expression with aging (Fig. S4b).

Evidence c62136b67a

Since Otub1 interacts with Tau and promotes phosphorylated and aggregated Tau levels in primary neurons, we hypothesized that Otub1 could act as a Tau deubiquitinase, interfering with pathological Tau degradation and hence Tau aggregation.

Evidence 3854bdb29b

We focused particularly on Tau deubiquitination and identified three major candidate deubiquitinating enzymes, including Otub1, USP5, and USP9x, with Otub1 as the strongest Tau interactor according to statistical analysis

Evidence 189e37d2de

Expression of the candidate deubiquitinases Otub1, USP5, and USP9x in this assay demonstrated significantly increased Tau aggregation following expression of Otub1 (Fig. 2a; Fig. S3), not observed following expression of USP5 or USP9x (Fig. S3).

Evidence 9a645e0613

Endogenous Otub1 depletion significantly inhibited Tau aggregation, with the level of inhibition correlating with the level of knockdown efficiency,while use of a control siRNA did not affect levels of Otub1 nor aggregation efficiency (Fig. 2b).

Evidence f950b70c0e

Taken together, our data indicate that Otub1, a deubiquitinating enzyme, is a novel positive regulator of Tau aggregation and Tau stability in vitro, in a nonneuronal cell line.

Evidence cd6704e9db

Otub1 expression strikingly enhanced detergent-resistant AT8-positive Tau accumulation compared with GFP-infected neurons (Fig. 4b), indicating that Otub1 increased Tau-seeded Tau aggregation in primary neurons, corroborating the results obtained in a nonneuronal cell line.

Evidence e958e83ef0

Otub1 also significantly increased total Tau protein level compared with GFP control (Fig. 3b).

Evidence a64dbc480e

We found that Tau degradation was significantly impaired in primary neurons infected with Otub1 WT, but not with catalytically dead mutant C91A, compared with GFP control.

Evidence c2db685991

Immunocytological analysis with AT8 antibody against phosphorylated Tau (Ser202/Thr205) demonstrated that Otub1 expression drastically increased phospho-Tau (p-Tau Ser202/Thr205) in primary neurons compared with GFP expression (Fig. 3a).

Evidence c9d135b72c

Biochemical analysis confirmed a sharp increase of phosphorylated Tau at Ser202/Thr205 in Otub1-infected primary neurons (Fig. 3b).

Evidence 8aa279b8c5

AAV-driven expression of Otub1-C91A did not increase AT8-positive Tau, in contrast to expression of wild-type Otub1 and the N-terminally truncated form of Otub1. This was demonstrated by immunofluorescence staining (Fig. 6a) and confirmed by biochemical analysis (Fig. 6b).

Evidence 83c023a180

This revealed a significant increase of oligomeric Tau forms in AAV– Otub1-infected neurons compared with AAV–GFP-infected neurons (Fig. 4c).

Evidence a5880eff65

The induction of oligomeric Tau forms was further confirmed using dot-blot analysis with T22 antibody (Fig. 4d), revealing a significant increase in oligomeric Tau following expression of Otub1.

Evidence 02e34d6876

Otub1 displayed strong preference for disassembling Lys48-linked polyubiquitin chains, while leaving Lys63-linked polyubiquitin chains unaffected (Fig. 5a).

Evidence 295a2c699d

Conversely, AAV-driven expression of N-terminally truncated Otub1 significantly decreased Lys48-linked polyubiquitin chains of Tau (Fig. 5b), demonstrating that the N-terminal domain of Otub1, which is crucial for its noncanonical role, is not involved in regulation of Tau ubiquitination.

Evidence b34e84dea0

Analysis of a catalytically dead mutant with a C91A mutation revealed no effect on Lys48-linked polyubiquitination of Tau

Evidence 78075cbcac

Immunostaining with AT8 antibody revealed abundant AT8-positive neurons in AAV–Otub1-injected mice, absent in AAV–GFP-infected mice (Fig. 7a), at 2 months postinjection.

Evidence 05d9e1e62a

Biochemical analysis further confirmed increased AT8-positive Tau by Otub1 expression (Fig. 7b), with AT8 phosphorylated Tau more robustly increased than total Tau, as reflected by the AT8/total Tau ratio.

Evidence 0bc05c580e

Immunostaining and biochemical analysis, 2 months postinjection, unambiguously revealed that the catalytically dead mutant C91A did not affect AT8-stained Tau in vivo, while N-terminal truncated mutant of Otub1 increased AT8-positive Tau in vivo, similarly as wild-type Otub1 (Fig. 8).

Evidence 429f70549b

This revealed a significant increase of monomeric Tau and oligomeric Tau in AAV– Otub1-injected mice compared with AAV–GFP-injected mice (Fig. 7c).

Evidence ec083d17b9

This revealed significantly increased oligomeric Tau in AAV– Otub1-injected mice compared with AAV–GFP-injected control cases (Fig. 7d).

Evidence 26381c5ed7

This revealed significantly increased oligomeric Tau forms, following expression of wild-type Otub1, but not following expression of GFP or of the catalytically inactive form of Otub1 (C91A) (Fig. 8c).


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