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VCP activator reverses nuclear proteostasis defects and enhances TDP-43 aggregate clearance in multisystem proteinopathy models
Jessica M. Phan, … , Carolyn N. Mann, Edward B. Lee
Jessica M. Phan, … , Carolyn N. Mann, Edward B. Lee
Published May 24, 2024
Citation Information: J Clin Invest. 2024;134(14):e169039. https://doi.org/10.1172/JCI169039.
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Research Article Neuroscience

VCP activator reverses nuclear proteostasis defects and enhances TDP-43 aggregate clearance in multisystem proteinopathy models

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Abstract

Pathogenic variants in valosin-containing protein (VCP) cause multisystem proteinopathy (MSP), a disease characterized by multiple clinical phenotypes including inclusion body myopathy, Paget’s disease of the bone, and frontotemporal dementia (FTD). How such diverse phenotypes are driven by pathogenic VCP variants is not known. We found that these diseases exhibit a common pathologic feature: ubiquitinated intranuclear inclusions affecting myocytes, osteoclasts, and neurons. Moreover, knock-in cell lines harboring MSP variants show a reduction in nuclear VCP. Given that MSP is associated with neuronal intranuclear inclusions comprised of TDP-43 protein, we developed a cellular model whereby proteostatic stress results in the formation of insoluble intranuclear TDP-43 aggregates. Consistent with a loss of nuclear VCP function, cells harboring MSP variants or cells treated with VCP inhibitor exhibited decreased clearance of insoluble intranuclear TDP-43 aggregates. Moreover, we identified 4 compounds that activate VCP primarily by increasing D2 ATPase activity, where pharmacologic VCP activation appears to enhance clearance of insoluble intranuclear TDP-43 aggregate. Our findings suggest that VCP function is important for nuclear protein homeostasis, that impaired nuclear proteostasis may contribute to MSP, and that VCP activation may be a potential therapeutic by virtue of enhancing the clearance of intranuclear protein aggregates.

Authors

Jessica M. Phan, Benjamin C. Creekmore, Aivi T. Nguyen, Darya D. Bershadskaya, Nabil F. Darwich, Carolyn N. Mann, Edward B. Lee

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Figure 3

TDP-4FL anisosomes become larger and more insoluble due to pathogenic A232E and R155H VCP variants.

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TDP-4FL anisosomes become larger and more insoluble due to pathogenic A2...
(A and B) Analysis of immunofluorescence images to determine the percentage of cells expressing (A) TDP-4FL or (B) eGFP as a percentage of total cell counts after 24 hours of transient TDP-4FL or eGFP transfection in WT HeLa cells or knock-in HeLa cells harboring A232E and R155H VCP variants. (Results are expressed as mean percentage values from each experiment and overall mean ± SEM. TDP-4FL, n = 6,390 cells counted across 3 cell lines over 3 independent experiments; 1-way ANOVA, **P < 0.01; Dunnett’s multiple comparison post hoc, *P < 0.05, **P < 0.01. eGFP, n = 2,135 cells counted across 3 cell lines over 3 independent experiments; 1-way ANOVA, P = 0.459) (C) Quantification of TDP-43 mRNA levels by qPCR (n = 4, results expressed as mean ± SEM; 1 sample t test: *P < 0.05). (D) Representative confocal images of immunofluorescence for TDP-4FL with no MG132 treatment or MG132 for 6 hours. Nuclear structures were categorized as either anisosomes or aggregates based on morphology. Scale bar: 10 μm. (E) Analysis of immunofluorescence images to determine the proportion of anisosome versus aggregate morphology in WT, A232E, or R155H cells treated with MG132 (n = 7,058 cells counted across 3 cell lines over 3 independent experiments; results are expressed as mean proportion values from each experiment and overall β ± SE, LME for A232E cells: time P < 0.0001, genotype P = 0.708, time × genotype **P < 0.01; LME for R155H cells: time P < 0.0001, genotype P = 0. 734, time × genotype *P < 0.05). (F) Immunoblots of soluble and insoluble TDP-FL protein (mouse anti-myc antibody) in WT, A232E, and R155H cells treated with MG132. GAPDH shown as a loading control. (G and H) Quantification of immunoblots for (G) soluble and (H) insoluble TDP-FL protein (normalized to soluble + insoluble TDP-FL protein levels within genotype) in WT, A232E, and R155H cells treated with MG132. (n = 3, results expressed as mean ± SEM; 2-way ANOVA for soluble protein: time *P < 0.05, genotype *P < 0.05, time × genotype P = 0.3613; for insoluble protein: time *P < 0.05, genotype *P < 0.05, time × genotype P = 0.3613; Dunnett’s post hoc analysis: *P < 0.05).

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