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PubMed: 28083634

Title
Tau interactome mapping based identification of Otub1 as Tau deubiquitinase involved in accumulation of pathological Tau forms in vitro and in vivo.
Journal
Acta neuropathologica
Volume
133
Issue
None
Pages
731-749
Date
2017-05-01
Authors
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
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These results indicate that Otub1 regulates Tau degradation, dependent on its catalytic activity (Fig. 5c).

p(HGNC:OTUB1) regulates deg(p(HGNC:MAPT)) View Subject | View Object

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Evidence 51f4399004
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Interestingly, Otub1 expression is gradually downregulated with increasing Tau-seeded Tau aggregation (Fig. S4a), which could be considered as a protective mechanism.

a(HBP:"Tau aggregates") negativeCorrelation p(HGNC:OTUB1) View Subject | View Object

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Annotations
MeSH
Hippocampus

p(HGNC:OTUB1) negativeCorrelation a(HBP:"Tau aggregates") View Subject | View Object

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Annotations
MeSH
Hippocampus

Evidence 43a3c4f0b7
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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).

bp(GO:aging) increases p(MGI:Otub1) View Subject | View Object

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Annotations
MeSH
Hippocampus

Evidence c62136b67a
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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.

complex(p(HGNC:MAPT), p(HGNC:OTUB1)) increases p(HGNC:MAPT, pmod(Ph)) View Subject | View Object

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complex(p(HGNC:MAPT), p(HGNC:OTUB1)) increases a(HBP:"Tau aggregates") View Subject | View Object

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p(HGNC:OTUB1) increases complex(p(HGNC:MAPT), p(HGNC:OTUB1)) View Subject | View Object

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Evidence 3854bdb29b
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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

p(HGNC:OTUB1) decreases p(HGNC:MAPT, pmod(Ub)) View Subject | View Object

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p(HGNC:OTUB1) increases complex(p(HGNC:MAPT), p(HGNC:OTUB1)) View Subject | View Object

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p(HGNC:USP5) decreases p(HGNC:MAPT, pmod(Ub)) View Subject | View Object

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p(HGNC:USP5) increases complex(p(HGNC:MAPT), p(HGNC:USP5)) View Subject | View Object

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p(HGNC:USP9X) decreases p(HGNC:MAPT, pmod(Ub)) View Subject | View Object

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p(HGNC:USP9X) increases complex(p(HGNC:MAPT), p(HGNC:USP9X)) View Subject | View Object

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Evidence 189e37d2de
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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).

p(HGNC:OTUB1) increases a(HBP:"Tau aggregates") View Subject | View Object

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p(HGNC:USP5) causesNoChange a(HBP:"Tau aggregates") View Subject | View Object

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p(HGNC:USP9X) causesNoChange a(HBP:"Tau aggregates") View Subject | View Object

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Evidence 9a645e0613
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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).

p(HGNC:OTUB1) increases a(HBP:"Tau aggregates") View Subject | View Object

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Evidence f950b70c0e
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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.

p(HGNC:OTUB1) increases a(HBP:"Tau aggregates") View Subject | View Object

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p(HGNC:OTUB1) regulates p(HGNC:MAPT) View Subject | View Object

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Evidence cd6704e9db
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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.

p(HGNC:OTUB1) increases a(HBP:"Tau aggregates") View Subject | View Object

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MeSH
Neurons

Evidence e958e83ef0
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Otub1 also significantly increased total Tau protein level compared with GFP control (Fig. 3b).

p(HGNC:OTUB1) increases p(HGNC:MAPT) View Subject | View Object

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Evidence a64dbc480e
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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.

p(HGNC:OTUB1) decreases deg(p(HGNC:MAPT)) View Subject | View Object

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p(HGNC:OTUB1, var("p.Cys91Ala")) causesNoChange deg(p(HGNC:MAPT)) View Subject | View Object

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Evidence c2db685991
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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).

p(HGNC:OTUB1) decreases p(HGNC:MAPT, pmod(Ph, Ser, 202), pmod(Ph, Thr, 205)) View Subject | View Object

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Annotations
MeSH
Neurons

Evidence c9d135b72c
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Biochemical analysis confirmed a sharp increase of phosphorylated Tau at Ser202/Thr205 in Otub1-infected primary neurons (Fig. 3b).

p(HGNC:OTUB1) decreases p(HGNC:MAPT, pmod(Ph, Ser, 202), pmod(Ph, Thr, 205)) View Subject | View Object

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Annotations
MeSH
Neurons

Evidence 8aa279b8c5
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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).

p(HGNC:OTUB1) increases p(HGNC:MAPT, pmod(Ph, Ser, 202), pmod(Ph, Thr, 205)) View Subject | View Object

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p(HGNC:OTUB1, frag("?")) increases p(HGNC:MAPT, pmod(Ph, Ser, 202), pmod(Ph, Thr, 205)) View Subject | View Object

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p(HGNC:OTUB1, var("p.Cys91Ala")) causesNoChange p(HGNC:MAPT, pmod(Ph, Ser, 202), pmod(Ph, Thr, 205)) View Subject | View Object

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Evidence 83c023a180
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This revealed a significant increase of oligomeric Tau forms in AAV– Otub1-infected neurons compared with AAV–GFP-infected neurons (Fig. 4c).

p(HGNC:OTUB1) increases a(HBP:"Tau oligomers") View Subject | View Object

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Annotations
MeSH
Neurons

Evidence a5880eff65
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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.

p(HGNC:OTUB1) increases a(HBP:"Tau oligomers") View Subject | View Object

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Evidence 02e34d6876
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Otub1 displayed strong preference for disassembling Lys48-linked polyubiquitin chains, while leaving Lys63-linked polyubiquitin chains unaffected (Fig. 5a).

p(HGNC:OTUB1) decreases p(HGNC:MAPT, pmod(UbK48)) View Subject | View Object

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p(HGNC:OTUB1) causesNoChange p(HGNC:MAPT, pmod(UbK63)) View Subject | View Object

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Evidence 295a2c699d
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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.

p(HGNC:OTUB1, frag("?")) decreases p(HGNC:MAPT, pmod(UbK48)) View Subject | View Object

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Evidence b34e84dea0
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Analysis of a catalytically dead mutant with a C91A mutation revealed no effect on Lys48-linked polyubiquitination of Tau

p(HGNC:OTUB1, var("p.Cys91Ala")) causesNoChange p(HGNC:MAPT, pmod(UbK48)) View Subject | View Object

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Evidence 78075cbcac
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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.

p(MGI:Otub1) increases p(MGI:Mapt, pmod(Ph, Ser, 202), pmod(Ph, Thr, 205)) View Subject | View Object

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Evidence 05d9e1e62a
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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.

p(MGI:Otub1) increases p(MGI:Mapt, pmod(Ph, Ser, 202), pmod(Ph, Thr, 205)) View Subject | View Object

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Evidence 0bc05c580e
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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).

p(MGI:Otub1) increases p(MGI:Mapt, pmod(Ph, Ser, 202), pmod(Ph, Thr, 205)) View Subject | View Object

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p(MGI:Otub1, frag("?")) increases p(MGI:Mapt, pmod(Ph, Ser, 202), pmod(Ph, Thr, 205)) View Subject | View Object

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p(MGI:Otub1, var("p.Cys91Ala")) causesNoChange p(MGI:Mapt, pmod(Ph, Ser, 202), pmod(Ph, Thr, 205)) View Subject | View Object

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Evidence 429f70549b
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This revealed a significant increase of monomeric Tau and oligomeric Tau in AAV– Otub1-injected mice compared with AAV–GFP-injected mice (Fig. 7c).

p(MGI:Otub1) increases p(MGI:Mapt) View Subject | View Object

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p(MGI:Otub1) increases a(HBP:"Tau oligomers") View Subject | View Object

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Evidence ec083d17b9
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This revealed significantly increased oligomeric Tau in AAV– Otub1-injected mice compared with AAV–GFP-injected control cases (Fig. 7d).

p(MGI:Otub1) increases a(HBP:"Tau oligomers") View Subject | View Object

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Evidence 26381c5ed7
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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).

p(MGI:Otub1) increases a(HBP:"Tau oligomers") View Subject | View Object

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p(MGI:Otub1, var("p.Cys91Ala")) causesNoChange a(HBP:"Tau oligomers") View Subject | View Object

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