Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Ubiquitin-specific peptidase-19 links TDP-43 aggregation to ER stress

doi: 10.1073/pnas.2514355123

Figure Lengend Snippet: Identification of USP19 as a TDP-43-directed deubiquitinase. ( A ) Schematic of Duolink® PLA principle for detection of ubiquitin (Ub)-TDP-43 complexes. ( B ) Representative images of Ub-TDP-43 PLA puncta (red), TDP-43 (green), and DAPI (blue) in tetracycline (tet)-induced HeLa-GFP-TDP-43 cells transfected with Smartpool siRNAs targeting 20 different USPs. ( C ) Quantification of Ub-TDP-43 PLA puncta numbers per cell (one-way ANOVA, F(20, 42) = 2.711, P = 0.0032; post hoc Dunnett compared to control siRNA (Cont), * P = 0.0270; n = 3 independent experiments per USP with 4 to 9 images averaged per experiment). ( D ) Schematic of USP19-flag constructs: ER-localized USP19 (USP19-ER), cytosolic USP19 (USP19-Cyto), and USP19-ER lacking catalytic DUB activity (USP19-CS-ER). ( E ) Representative blots showing co-IP of Myc-TDP-43 with USP19-flag variants (flag pulldown) in HEK293T cells cotransfected ± USP19-flag variants or vector control ± Myc-TDP-43. ( F ) Representative images of USP19–TDP-43 PLA puncta (red: myc + flag PLA) and DAPI (blue) from HeLa cells transfected with Myc-TDP-43 and USP19-flag variants or vector control. One probe - lacks one secondary antibody probe. ( G ) Representative blots of polyubiquitin conjugates (Ub-K48 & Ub-K63) in TDP-43 pulldown (myc) from HEK293T cells transfected with ubiquitin-HA (Ub-HA) and myc-TDP-43 with USP19-flag variants or vector control.

Article Snippet: Primary antibody used: Ubiquitin (Cell signaling, #3936); Flag (ThermoFisher, MA5-48063), myc (Cell signaling, #2276s), USP19 (Abcam, #93159), TDP-43 (Proteintech, #67345), and Calnexin (Cell signaling, #2679).

Techniques: Ubiquitin Proteomics, Transfection, Control, Construct, Activity Assay, Co-Immunoprecipitation Assay, Plasmid Preparation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Ubiquitin-specific peptidase-19 links TDP-43 aggregation to ER stress

doi: 10.1073/pnas.2514355123

Figure Lengend Snippet: USP19 drives TDP-43 aggregation by increasing insoluble TDP-43 CTFs. ( A ) Representative blots of RIPA-soluble and RIPA-insoluble proteins from tet-induced HeLa-GFP-TDP-43 cell line transfected with control or USP19 siRNA. ( B – D ) Quantification of soluble (sol) and insoluble (insol) TDP-80 (GFP-TDP-43), TDP-43, and TDP-35 from RIPA-soluble and RIPA-insoluble fractions (one-sample t test, * P < 0.05, ** P < 0.01, n = 4 independent experiments with 2 to 3 replicates averaged per experiment). ( E ) Representative RIPA-soluble blots of HEK 293T cells transfected with TDP-35-myc and control siRNA or USP19 siRNA. ( F and G ) Quantification of TDP-35 and TDP-25 (one-sample t test, * P < 0.05; n = 4 independent experiments with 2 to 3 replicates averaged per experiment). ( H ) Representative blots of RIPA-soluble and RIPA-insoluble proteins from TAR4 +/+ primary neurons transduced with control or USP19 adenovirus. ( I – L ) Quantification of TDP-43 and TDP-35 from RIPA-soluble and RIPA-insoluble fractions (one-sample t test, * P < 0.05, ns: not significant; n = 2 independent experiments with two replicates averaged per experiment).

Article Snippet: Primary antibody used: Ubiquitin (Cell signaling, #3936); Flag (ThermoFisher, MA5-48063), myc (Cell signaling, #2276s), USP19 (Abcam, #93159), TDP-43 (Proteintech, #67345), and Calnexin (Cell signaling, #2679).

Techniques: Transfection, Control, Transduction

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Ubiquitin-specific peptidase-19 links TDP-43 aggregation to ER stress

doi: 10.1073/pnas.2514355123

Figure Lengend Snippet: USP19-ER isoform preferentially deubiquitinates and increases insoluble TDP-CTF through phase separation. ( A ) Representative blots of RIPA-soluble and RIPA-insoluble proteins from tet-induced GFP-TDP-43 cells transfected with vector control or USP19 variants. ( B – G ) Quantification of TDP-80 (GFP-TDP-43), TDP-43, and TDP-35 from RIPA-soluble (sol) and RIPA-insoluble (insol) fractions (Insol TDP-35: one-way ANOVA, F(3, 8) = 6.108, P = 0.0183; post hoc Dunnett, ** P < 0.01; ns: not significant; n = 3 independent experiments with 1 to 2 replicates averaged per experiment). ( H ) Representative blots of polyubiquitin conjugates (Ub-K48 & Ub-K63) in myc-TDP-35 pulldown (myc) from HEK293T cells transfected with ubiquitin-HA (Ub-HA) and myc-TDP-35 with USP19-flag variants or vector control. ( I ) Schematic model blue light-induced phase separation of Cry2olig-TDP-LCD-mCherry. ( J ) Representative images of Cry2olig-TDP-LCD-mCherry T(red) before and after blue light stimulation (488 nm, 1 s) in HeLa cells transfected with Cry2olig-TDP-LCD and USP19 variants (green) or vector control. ( K ) Quantification of Cry2olig-TDP-LCD puncta numbers normalized to cell area (two-way ANOVA, F(3, 39) = 21.33, P < 0.0001; post hoc Dunnett compared to control, **** P < 0.0001, ns: not significant; n = 2 independent experiments with 2 to 3 replicates averaged per experiment).

Article Snippet: Primary antibody used: Ubiquitin (Cell signaling, #3936); Flag (ThermoFisher, MA5-48063), myc (Cell signaling, #2276s), USP19 (Abcam, #93159), TDP-43 (Proteintech, #67345), and Calnexin (Cell signaling, #2679).

Techniques: Transfection, Plasmid Preparation, Control, Ubiquitin Proteomics

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Ubiquitin-specific peptidase-19 links TDP-43 aggregation to ER stress

doi: 10.1073/pnas.2514355123

Figure Lengend Snippet: USP19 promotes cytoplasmic mislocalization of TDP-43 and enhances ER stress. ( A ) Representative images of TDP-43-tomato (red), USP19 (green), and DAPI (blue) in HeLa cells transfected with TDP-43 and control siRNA or USP19 siRNA ± proteasome inhibition with MG132 treatment (10 μM, 4 h). ( B ) Quantification of TDP-43-tomato in the cytoplasm vs. nucleus with MG132 treatment (one-sample t test, * P < 0.05; n = 3 independent experiments with 10 to 32 cells/condition averaged per experiment). ( C ) Representative images of TDP-43-tomato (red), USP19 (green), and DAPI (blue) in HeLa cells transfected with TDP-43 and USP19 variants or GFP control. ( D ) Quantification of TDP-43-tomato in the cytoplasm vs. nucleus (one-way ANOVA, F(2, 6) = 22.73, P = 0.0016; post hoc Dunnett, ** P < 0.005, n = 3 independent experiments with 13 to 33 cells/condition averaged per experiment). ( E ) Representative images of endogenous USP19–TDP-43 PLA puncta (green) and DAPI (blue) in HeLa cells transfected with control or USP19 siRNA. Negative controls with only 1 primary antibody or 1 secondary antibody probe ( Bottom ). ( F ) Representative images of endogenous USP19–TDP-43 PLA puncta (red) and DAPI (blue) in HeLa cells treated ± ER stressor tunicamycin (2 μM) for 24 h. Negative controls with only 1 primary antibody or 1 secondary antibody probe ( Bottom ). ( G ) Quantification of USP19–TDP-43 PLA intensity ± tunicamycin treatment (2 μM, 24 h) (one-sample t test, *** P < 0.001, n = 3 independent experiments with 29 to 36 cells/condition averaged per experiment). ( H ) Schematic of unfolded protein response (UPR) triggered by ER stressors, such as misfolded proteins, illustrating the activation of IRE1α-XBP1, PERK-ATF4, and ATF6 pathways, leading to CHOP induction. ( I ) Representative blots of ER stress-induced UPR markers from tet-induced HeLa-GFP-TDP-43 cells transfected with control or USP19 siRNA and treated ± tunicamycin (2 μM; 0, 8, 24 h). ( J – M ) Quantification of CHOP, cleaved ATF6 (cl-ATF6), ATF4, and IRE1α (two-way ANOVA; CHOP: F(1, 18) = 24.49, P = 0.0001; cl-ATF6: F(1, 18) = 41.93, P < 0.0001; ATF-4: F(1, 18) = 4.343, P = 0.0517; IRE1α: F(1, 18) = 2.80, P = 0.1571; post hoc Sidak, * P < 0.05, ** P < 0.005, *** P < 0.001, ns: not significant; n = 4 independent experiments with 2 to 3 replicates averaged per experiment).

Article Snippet: Primary antibody used: Ubiquitin (Cell signaling, #3936); Flag (ThermoFisher, MA5-48063), myc (Cell signaling, #2276s), USP19 (Abcam, #93159), TDP-43 (Proteintech, #67345), and Calnexin (Cell signaling, #2679).

Techniques: Transfection, Control, Inhibition, Activation Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Ubiquitin-specific peptidase-19 links TDP-43 aggregation to ER stress

doi: 10.1073/pnas.2514355123

Figure Lengend Snippet: Elevated levels of USP19 in FTLD-TDP brains colocalize with pTDP-43 pathology. ( A ) Representative blots of RIPA-soluble USP19 and tubulin from the frontal cortex of FTLD-TDP and nondementia control cases. ( B ) Quantification of USP19 levels (unpaired Student’s t test, *** P = 0.0002, n = 5 FTLD-TDP and n = 11 nondementia controls). ( C 1 and C 2) Representative double-stained immunohistochemical (IHC) images of phospho-TDP-43 (pS403/pS404, green) and USP19 (red) in 4 FTLD-TDP cases and three controls, showing extensive colocalization of USP19 with pTDP-43 pathology (merged) in FTLD-TDP. Secondary antibody only negative controls (C2, Right ).

Article Snippet: Primary antibody used: Ubiquitin (Cell signaling, #3936); Flag (ThermoFisher, MA5-48063), myc (Cell signaling, #2276s), USP19 (Abcam, #93159), TDP-43 (Proteintech, #67345), and Calnexin (Cell signaling, #2679).

Techniques: Control, Staining, Immunohistochemical staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Ubiquitin-specific peptidase-19 links TDP-43 aggregation to ER stress

doi: 10.1073/pnas.2514355123

Figure Lengend Snippet: Usp19 reduction mitigates TDP-43 pathology, lowers ER stress, and reverses LTP and motor deficits. ( A ) Representative IHC images of cytoplasmic pTDP-43 pathology (pS409/pS410, green) and DAPI (blue) in the cortex of 10-mo-old TAR4 +/− and TAR4 +/− ; usp19 +/− mice. ( B ) Quantification of pTDP-43 pathology (pS409/pS410) in the cortex (unpaired Student’s t test, ** P = 0.003, n = 4 mice/genotype with 4 to 10 images averaged per mouse). ( C ) Immunoblots of human TDP-43 and TDP-35 in cytoplasmic (Cyto) and nuclear (Nucl) fractions of TAR4 +/− and TAR4 +/− ; usp19 +/− mice cortex. ( D – F ) Quantification of cytoplasmic and nuclear TDP-43 and TDP-35 (unpaired Student’s t test, * P < 0.05, ** P < 0.01, ns: not significant; n = 3 to 4 mice/genotype). ( G ) Representative IHC images of GFAP (green) and DAPI (blue) in the hippocampus of 10-mo-old WT, TAR4 +/− and TAR4 +/− ; usp19 +/− mice. ( H ) Quantification of GFAP intensity in the hippocampus (one-way ANOVA, F(2, 12) = 5.565, P = 0.0195; post hoc Dunnett, * P < 0.05; n = 4 to 6 mice/genotype with 3 to 6 images averaged per mouse). ( I ) Representative IHC images of CHOP (red) and DAPI (blue) in the cortex of 10-mo-old WT, TAR4 +/− and TAR4 +/− ; usp19 +/− mice. ( J ) Quantification of CHOP intensity in the cortex (one-way ANOVA, F(2, 9) = 6.678, P = 0.0167; post hoc Dunnett, * P < 0.05, n = 4 mice/genotype with 8 to 34 images averaged per mouse). ( K ) Quantification of LTP induced by theta burst stimulation from 9-mo-old mouse brain slices (two-way ANOVA, F(2, 9198) = 870.6, P < 0.0001; post hoc Dunnett, **** P < 0.0001; n = 29 to 51 slices from 5 to 7 mice/genotype). ( L ) Quantification of latency to fall (sec) in rotarod test from 9 to 10-mo-old mice (one-way ANOVA, F(2, 51) = 18.13, P < 0.0001; post hoc Dunnett, ** P < 0.005, **** P < 0.0001, n = 14 to 20 mice/genotype).

Article Snippet: Primary antibody used: Ubiquitin (Cell signaling, #3936); Flag (ThermoFisher, MA5-48063), myc (Cell signaling, #2276s), USP19 (Abcam, #93159), TDP-43 (Proteintech, #67345), and Calnexin (Cell signaling, #2679).

Techniques: Western Blot