IBMPFD mutant-expressing cells increase ubiquitinated proteins. A, characterization of tetracycline-inducible U20S cell lines expressing Myc-tagged p97/VCP, ATPase inactive p97/VCP-E578Qm, or IBMPFD mutant p97/VCP-R155H, R95G, or A232E. Cell lysates from uninduced (-) and 16 h tetracycline-induced (+) U20S cells were separated by SDS-PAGE and subjected to Western blot analysis to detect Myc-tagged p97/VCP (top panel) or actin (bottom panel). B, the same induced cells as above were immunoblotted with an antibody to ubiquitin. The lanes were taken from the same gel at the same exposure and realigned for presentation purposes. Note the increase in high molecular weight ubiquitinated species in IBMPFD mutant-expressing cells. C, ubiquitin immunostaining of control U20S cells or U20S cells expressing p97/VCP-WT, ATPase inactive p97/VCP-E578Q, or IBMPFD mutant p97/VCP-R155H or -A232E either untreated (left panels) or treated with proteasome inhibitor MG132 (right panels). Note that p97/VCP-WT-expressing cells or untransfected cells have small perinuclear aggresomes that are not present in IBMPFD mutant-expressing cells. D, MTT assays of U20S cells expressing p97/VCP-WT or IBMPFD mutant p97/VCP-R155H or -A232E following 16 h of application of 10-fold dilutions (original concentrations, 1 mm) as indicated of the proteasome inhibitors MG132 (mg, top panel) or lactacystin (lact, bottom panel). Units are the change of treated cells compared with untreated cells (arbitrarily set to 1) in each condition.

Aggresome formation is impaired in IBMPFD mutant-expressing cells and tissue. Electron micrographs of U20S cells co-expressing p97/VCP-WT (A) or p97/VCP-R155H (B-E) along with polyQ80-CFP for 48 h. A, p97/VCP-WT-expressing cells have large perinuclear inclusions with active autophagic degradation consistent with an aggresome. The inset shows a double membrane containing autophagic vacuole with electron dense debris. B and C, IBMPFD mutant-expressing cells have multiple small electron dense inclusions that are within membranous structures or non-membrane-associated within the cytoplasm. D and E, an IBMPFD mutant-expressing cell with multiple inclusions attached to microtubules. Similar results were also obtained with p97/VCP-A232E-expressing cells.

IBMPFD mutants are “trapped” on aggregated polyglutamine. A, FRAP analysis of the molecular interaction between polyQ80-CFP and p97/VCP-DsReds (WT, E305Q/E578Q, R155H, or A232E) co-localizing inclusions. The relative fluorescence intensity was determined for each time point and is represented as the percentage of the prebleaching relative fluorescence intensity value. FRAP analysis was typically performed on >2 cells/field and >3 fields/experimental condition. Each experiment was repeated three times with different transfectants for each experiment. B, representative FRAP analysis images of co-localized p97/VCP-DsRed/polyQ80-CFP inclusions before bleaching, immediately after photobleaching and after recovery. Inclusions within cells were photobleached in a small region of interest (outlined box) and monitored for recovery of fluorescence into the region of interest. C, U20S control and U20S cells stably expressing tetracycline-inducible p97/VCP-WT-Myc, dominant negative p97/VCP-E578Q-Myc or IBMPFD mutant p97/VCP-R155H-Myc, -A232E-Myc, and -L198W-Myc were transfected with polyQ80-CFP. 36 h later lysates were collected, and p97/VCP was immunoprecipitated with an anti-Myc. The immunoprecipitate was subsequently immunoblotted for associated polyQ80-CFP with anti-GFP. Similar amounts of p97/VCP were immunoprecipitated. Total lysates (T) and unbound (U) fractions were separated by SDS-PAGE and immunoblotted with anti-GFP antibody. All of the blots are representative of three independent experiments.

Model of proposed p97/VCP function. p97/VCP triages ubiquitinated proteins to the UPS or the aggresome via interactions with other ubiquitin-binding proteins (i.e. rad23) or HDAC6 respectively. IBMPFD mutant p97/VCP avidly binds to ubiquitinated and aggregated proteins failing to efficiently release them to the UPS or aggresome. This results in the accumulation of undegraded ubiquitinated and aggregated proteins.

Aggresome formation is impaired in IBMPFD mutant-expressing cells and tissue. A, representative live cell images of cells co-expressing polyQ80-CFP (blue) with DsRed tagged p97/VCP-WT (WT) or IBMPFD mutants R155H (RH), A232E (AE), and R95G (RG). Note that although p97/VCP co-localizes with polyQ80-CFP, there is impaired inclusion formation in IBMPFD mutant-expressing cells. B, control U20S or U20S cells stably expressing tetracycline-inducible p97/VCP-WT, ATPase inactive p97/VCP-E578Q (EQ) or IBMPFD mutant p97/VCP R155H, A232E, and L198W (LW) were transfected with polyQ80-CFP for 48 h. The cells were fixed with 3% paraformaldyhyde and processed for fluorescence microscopic analysis of inclusion bodies. The cells were scored for the presence of ≤ 3 inclusions or >3 inclusions. ^*, p < 0.05 versus U20S control containing less than three aggregated proteins. C, representative low power fields from U20S cells stably expressing tetracycline-inducible p97/VCP-WT or IBMPFD mutant p97/VCP R155H and A232E transfected with polyQ80-CFP for 48 h. The filled arrows denote cells with ≤3 inclusions, and the open arrows denote cells with >3 inclusions. D, confocal micrograph of p97/VCP-dsRed and CD3δ-YFP co-expression. Note that overexpressed CD3δ-YFP forms a perinuclear inclusion in p97/VCP-WT-expressing cells and generates multiple smaller inclusions in IBMPFD mutant cells (R155H and A232E). E, control or U20S cells expressing p97/VCP-WT or IBMPFD mutant p97/VCP R155H or A232E along with polyQ80-CFP. Ubiquitinated proteins were identified via immunohistochemical analysis with FK2 antibody. Note that polyQ80-CFP inclusions are ubiquitin-positive. F, non-transgenic control, p97/VCP-WT or p97/VCP-R155H-expressing transgenic animals electroporated with a polyQ80-CFP expression construct into the TA muscle 7 days prior to analysis. Images are CFP autofluorescence from the TA muscles. Control and p97/VCP-WT skeletal muscle typically have a few large inclusion bodies/myofiber, although IBMPFD mutant R155H transgenic muscle has multiple small inclusion bodies occasionally surrounding a larger inclusion body. The brackets denote groups of inclusions within single myofibers.

Polyglutamine inclusions fail to co-localize and are less efficiently degraded by the autophagic machinery in IBMPFD mutant-expressing cells. U20S cells stably expressing tetracycline-inducible p97/VCP-WT or IBMPFD mutant p97/VCP R155H, A232E were co-transfected with polyQ80-CFP and expression vectors GFP-LC3 (A) and mcherry-ATG5 (B) or immunostained to visualize endogeneous p62 (C) and HDAC6 (D). Representative confocal images are shown. All of the images were taken at the same gain and laser power, allowing comparison in the intensity of fluorescence between samples. E, control or U20S cells stably expressing tetracycline-inducible p97/VCP-WT or IBMPFD mutant p97/VCP R155H, A232E, and L198W were transfected with polyQ80-CFP. 48 h later lysates were prepared in 1% SDS and filtered through a nitrocellulose membrane and blotted with anti-GFP antibody. Note the darker band in IBMPFD mutant-expressing lysates. Total polyQ80 protein levels were similar between cell lines (see C). F, U20S cells expressing a tetracycline-inducible polyQ80-CFP were transfected with p97/VCP-WT or IBMPFD mutant p97/VCP R155H, R95G, and A232E. Forty-eight hours after transfection, the cells were washed with PBS to remove tetracycline and incubated in medium without tetracycline. Aggregate-containing transfected cells were counted and plotted as the percentages of aggregates/total transfectants.

HDAC6 co-expression improves aggresome formation and rescue cell death in IBMPFD mutant-expressing cells. A, quantification of polyQ80-CFP-positive inclusion bodies (cells containing <3or ≥3 inclusions) in p97/VCP-WT or IBMPFD mutant-expressing (R155H, R95G, and A232E) cells with or without co-expression of HDAC6-FLAG. Note that all IBMPFD mutant cell lines have fewer cells with <3 inclusion than p97/VCP-WT. In addition, HDAC6 increases the number of cells with <3 inclusions in IBMPFD mutant-expressing cells. Non-HDAC6 transfected controls (<3 inclusions/cell) of each group versus HDAC6 transfected (<3 inclusions/cell) of each group have a p < 0.05 (^*). B, control U20S cells and U20S cells stably expressing tetracycline-inducible p97/VCP-WT or IBMPFD mutant p97/VCP-R155H, -R95G, and -A232E were transfected with polyQ80-CFP with or without co-expression of HDAC6-FLAG. Forty-eight hours after transfection, the cell number was measured via MTT assay. Note that the number of IBMPFD mutant p97/VCP expressing is decreased compared with p97-VCP-WT, and HDAC6 increases the number of cells when co-expressed with IBMPFD mutant p97/VCP. Cell death is given as a ratio of the nontransfected controls of each group. The results are means ± S.E. and representative of at least three independent experiments. ^*, p < 0.05 versus non-HDAC6 overexpressing controls of each group. C, representative confocal micrographs of cells expressing p97/VCP-Myc, polyQ80-CFP, and HDAC6-FLAG. Note the co-localization of HDAC6 with a perinuclear inclusion body in IBMPFD mutant-expressing cells.