VCP/p97 is essential for maturation of ubiquitin-containing autophagosomes and this function is impaired by mutations that cause IBMPFD

Abstract

VCP (VCP/p97) is a ubiquitously expressed member of the AAA(+)-ATPase family of chaperone-like proteins that regulates numerous cellular processes including chromatin decondensation, homotypic membrane fusion and ubiquitin-dependent protein degradation by the proteasome. Mutations in VCP cause a multisystem degenerative disease consisting of inclusion body myopathy, Paget disease of bone, and frontotemporal dementia (IBMPFD). Here we show that VCP is essential for autophagosome maturation. We generated cells stably expressing dual-tagged LC3 (mCherry-EGFP-LC3) which permit monitoring of autophagosome maturation. We determined that VCP deficiency by RNAi-mediated knockdown or overexpression of dominant-negative VCP results in significant accumulation of immature autophagic vesicles, some of which are abnormally large, acidified and exhibit cathepsin B activity. Furthermore, expression of disease-associated VCP mutants (R155H and A232E) also causes this autophagy defect. VCP was found to be essential to autophagosome maturation under basal conditions and in cells challenged by proteasome inhibition, but not in cells challenged by starvation, suggesting that VCP might be selectively required for autophagic degradation of ubiquitinated substrates. Indeed, a high percentage of the accumulated autophagic vesicles contain ubiquitin-positive contents, a feature that is not observed in autophagic vesicles that accumulate following starvation or treatment with Bafilomycin A. Finally, we show accumulation of numerous, large LAMP-1 and LAMP-2-positive vacuoles and accumulation of LC3-II in myoblasts derived from patients with IBMPFD. We conclude that VCP is essential for maturation of ubiquitin-containing autophagosomes and that defect in this function may contribute to IBMPFD pathogenesis.

Figure 1. Disease-associated VCP mutants do not cause a general impairment of ubiquitin-dependent proteasomal degradation

(A) Mel Juso cells stably expressing the ubiquitin fusion degradation (UFD) substrate UbG76V-YFP were transiently transfected with expression vectors encoding GFP-tagged wild type VCP (VCP-wt), VCP-R155H, VCP-A232E and dominant negative VCP (VCP-DN). The VCP-wt and disease-associated mutants did not cause accumulation of the UbG76V-YFP substrate in contrast to the VCP-DN mutant. As a control, untransfected UbG76V-YFP Mel Juso cells were treated for 6 hrs with 10 μM of the proteasome inhibitor MG132. Scale bars equal 50 μm. (B) Quantification of the YFP fluorescence in UbG76V-YFP Mel Juso cells expressing wt and mutant VCPs. Values were normalized for the fluorescence intensity of untransfected cells. VCP-DN caused a significant increase in UbG76V-YFP levels (**unpaired t test, P < 0.001). Error bars represent standard deviation (n=30–150 cells). (C) Scatter plots of the YFP and GFP levels in UbG76V-YFP Mel Juso cells expressing GFP-tagged wt and mutant VCPs. (D) Mel Juso cells stably expressing the ERAD substrate CD3δ-YFP were transiently transfected with expression vectors encoding GFP-tagged VCP-wt, VCP-R155H, VCP-A232E and VCP-DN. The VCP-wt and disease-associated mutants did not cause accumulation of the CD3δ-YFP substrate whereas the VCP-DN mutant caused a general increase in CD3δ-YFP levels. Scale bars equal 50 μm. Error bars represent standard deviation. (E) Quantification of the YFP fluorescent values in CD3δ-YFP Mel Juso cells expressing wt and mutant VCPs. VCP-DN caused a significant increase in CD3δ-YFP levels (**unpaired t test, p<0.001). Error bars represent standard deviation (n=30–150 cells). (F) Scatter plots of the YFP and GFP levels in CD3δ-YFP Mel Juso cells expressing GFP-tagged wt and mutant VCP.

Figure 2. RNAi knock down of VCP impairs autophagosome maturation

(A) mCherry-EGFP-LC3b stable MEFs transfected with non-targeting, Rab7 or VCP siRNA. Scale bars equal 10 μm. (B) Quantification of autophagosomes and autophagolysosomes under basal conditions in cells transfected with non-targeting or VCP-targeting siRNA. * indicates p<0.01; n.s. indicates non-significant difference, error bars indicate standard errors. (C). Immunoblot against VCP, p62 and tubulin in non-targeting and VCP-targeting siRNA in MEFs. p62 and tubulin were observed simultaneously using a Li-Cor Odyssey system. (D) Quantification of p62 normalized against tubulin in cells transfected with non-targeting, VCP-targeting or Rab7-targeting siRNA. * indicates p<0.01; ** p<0.001; error bars indicate standard deviation from triplicates.

Figure 3. Defective autophagosome maturation in cells expressing disease-associated VCP mutants

(A) mCherry-EGFP-LC3b stable MEFs transfected with the empty vector or expressing VCP-wt, VCP-DN, VCP-R155H or VCP-A232E. Arrows indicate large LC3-positive vesicles. Scale bars equal 10 μm. (B) Quantification of autophagosomes (yellow) and autophagolysosomes (red) per cell under basal conditions. Cells were transfected with the empty vector, or with VCP-wt, VCP-DN, VCP-R155H or VCP-A232E. Empty vector transfected cells were also treated with bafilomycin (ev +Baf). * indicates p<0.05; ** p<0.01; *** p<0.001. Error bars indicate standard errors. (C) Size distribution of autophagic vesicles (AV) in cells transfected with wt or mutant forms of VCP. Size is estimated in μm. ** indicates p<0.01. (D) Quantification of the percent of cells containing “extra-large” vesicles (i.e. over 1.5 μm diameter) in cells transfected with empty vector, wt-VCP or mutant forms of VCP. Experiment was performed twice in triplicate. (E) Mutant forms of VCP lead to accumulation of p62 in HEK293T cells. Western blot against p62/rabbit and tubulin/mouse using a Li-Cor Odyssey system. (F) Quantification of p62 Western-Blot. p62 quantification was normalized to tubulin quantity. Error bars indicates standard deviation of three independent replicates. ** indicates p<0.01.

Figure 4. The large LC3-positive vesicles that accumulate in cells expressing mutant VCP are acidified and exhibit cathepsin B activity

Cells were co-transfected with GFP-LC3 and either wild type or mutant forms of VCP, and then labeled with acidotrophic dye LysoSensor Blue and Cathepsin B substrate MR-(RR)2. As a control, cells expressing VCP-wt were also treated with Bafilomycin A. Arrows indicate large GFP-LC3-positive vesicles that are labeled with LysoSensor Blue and MR(RR)2. Scale bars equal 10 μm. The control for efficiency of co-transfection is shown in Suppl. Fig. 4.

Figure 5. The large LC3-positive vesicles that accumulate in mutant VCP-expressing cells contain ubiquitin-positive material

(A) Cells transfected with LC3-GFP and immunostained with anti-ubiquitin (red) were observed under basal conditions (upper panel), or after Bafilomycin A treatment (middle panel) or starvation (lower panel). (B) Cells were co-transfected with indicated plasmid and GFP-LC3 and then immunostained with anti-ubiquitin (red). Scale bars equal 10 μm. Arrows indicate LC3-positive vesicles containing ubiquitin-positive material. (C) Graph representing the percentage ubiquitin-containing autophagosomes in cells expressing wild type or mutant VCP. Errors bars represent standard deviations of three independent replicates. ** means p<0,005; ***, p<0,001. See Suppl. Fig. 4 for analysis of co-transfection efficiency and Suppl. Fig. 5 for magnification indicating ubiquitin staining is lumenal.

Figure 6. IBMPFD patient myoblasts show accumulation of large LAMP-positive vesicles and LC3-II protein

(A) Immunostaining for VCP and LAMP-1 in myoblasts derived from normal control or an IBMPFD patient. Cells were observed with a 63× objective. Please see Suppl. Fig. 6 for cell lines from additional patients and additional markers. (B) Western blot analysis of LC3 in myoblast lysates from 2 control samples and samples from 2 IBMPFD patients.