FIGURE SUMMARY
Title

Loss of the novel Vcp (valosin containing protein) interactor Washc4 interferes with autophagy-mediated proteostasis in striated muscle and leads to myopathy in vivo

Authors
Kustermann, M., Manta, L., Paone, C., Kustermann, J., Lausser, L., Wiesner, C., Eichinger, L., Clemen, C.S., Schröder, R., Kestler, H.A., Sandri, M., Rottbauer, W., Just, S.
Source
Full text @ Autophagy

Phenotypic analysis of zebrafish after morpholino-mediated knockdown of vcp or ML240 treatment. (a to d) Lateral view of embryos at 60 hpf. Zebrafish embryos were either injected with 5 base pair (bp) mismatch vcp start morpholino (MO1-control; a) and vcp start MO (MO1-vcp; b) or treated with DMSO (c) and VCP-Inhibitor ML240 (VCP-I; d). scale bar: 500 μm (a, c); scale bar: 650 μm (b, d). (e) Statistical analysis of affected embryos after MO1/2-control or MO1/2-vcp injection and DMSO or ML240 treatment. N = 3/4 Experiments with in total n = 259 (MO1-control), n = 161 (MO2-control), n = 242 (MO1-vcp), n = 212 (MO2-vcp), n = 152 (DMSO), n = 148 (VCP-I); N two-sided Fisher exact tests combined with a modified Fisher method. Ratios of N experiments are displayed. MO1-vcp-injected embryos reveal reduced levels of Vcp by western blot analysis. Actb1/β-actin was used as loading control. RT-PCR after MO2-vcp injection shows specific effect on mRNA splicing. (f) Ventricular fractional shortening (FS) measurements of MO1-control and MO1-vcp-injected embryos at 48, 72 and 96 hpf, n = 18 to 30 (MO1-control), n = 19 to 32 (MO1-vcp). The individual measurements are shown (two-sided Wilcoxon rank-sum test). (g) Heart beat per minute at 48, 72 and 96 hpf of vcp morphants and control embryos. n = 30 (MO1-control), n = 29 or 30 (MO1-vcp). The individual measurements are shown (two-sided unpaired Wilcoxon rank-sum test. (h and i) False-colored superimposed overviews of spontaneous movement assay with MO1-control- (h) and MO1-vcp-injected (i) embryos at 24 hpf; red pictures = 0 s; green pictures = 10 s; yellow = merge. scale bar: 1 mm. (j) Quantification of spontaneous movement assay; N = 3 experiments with n = 30 (MO1-control), n = 29 or 30 (MO1-vcp) per experiment (N two-sided Fisher exact tests combined with modified Fisher method). The ratios of N experiments are shown. (k) Statistical analysis of touch-evoke flight response of control and vcp morphants at 72 hpf. n = 30, N = 3 (MO1-control), n = 29 or 30, N = 3 (MO1-vcp); data represent means.

Vcp deficiency results in a severe skeletal muscle myopathy. (a and b) Lateral view of control (a) and vcp morphants (b) showing birefringence at 48 hpf. scale bar: 450 μm. (c) Densitometric analysis of birefringence (n = 23/25). Individual samples are shown (two-sided Wilcoxon rank-sum test). (d and e) Skeletal muscle H&E-stained sagittal histological sections of MO1-control- (d) and MO1-vcp-injected embryos (e) at 72 hpf; scale bar: 100 μm. (f to g``) Transmission electron microscopy in control embryos (f to f``) and vcp morphants (g to g``) at 48 hpf. (f to f``) MO1-control-injected embryos show organized myofibrils and sarcomeric units with highly structured thick and thin filaments (f`) and mitochondria with many cristae (f``). (g to g``) VCP-deficient muscle cells present disorganized myofibrils with Z-disc alterations (arrow, g`), dysmorphic mitochondria (g``) and an accumulation of numerous vesicular bodies (double arrowheads, g); scale bar: 2.5 μm (f, g); scale bar: 1 μm (f` and f``, g` and g``).

Inactivation of Vcp leads to an accumulation of ubiquitinated proteins and Sqstm1. (a) Western blot analysis using an anti-K48-linkage specific ubiquitin antibody after MO1-control and MO1-vcp injection and DMSO or MG132 treatment (n = 4). (b) Vcp morphants show a significant increase of Sqstm1 by western blot analysis (n = 3); quantification of gray values. Actb1/β-actin was used as loading control. Data represent means ± SD, unpaired Student t test, *P value< 0.03 (c) Western blot analysis of Lc3 after MO1-control and MO1-vcp injection and DMSO or Ammonium chloride (NH4Cl) treatment (top) and quantification of gray values (bottom). NH4Cl treated control embryos reveal significant increase of Lc3-II levels, compared to DMSO treated embryos (n = 5). Actb1/β-actin was used as loading control. The individual samples are displayed (two-sided Wilcoxon rank-sum test).

VCP and its interactor WASHC4 colocalize in HEK293T cells and mouse FDB fibers. (a) Western blot analysis of a representative Washc4 pull-down experiment using anti-His (upper panel) and anti-GST (middle and lower panels) antibodies. Extracts of E. coli cells expressing recombinant His-tagged zebrafish (Danio rerio, Dr) Washc4 (His-DrWashc4) were incubated with either no beads (ctrl) or glutathione beads carrying GST (GST), GST–DrVcp wt or GST–DrVcpR155H. Coomassie stained SDS–PAGE gel of a Washc4 affinity isolation experiment (lower panel). ctrl = beads incubated with PBS. (b to b``) Transfection of HEK293T cells using a VCP-eGFP expression construct (b`) and staining with an anti-WASHC4 antibody (b) show cytoplasmic colocalization of both proteins. (c to c``) HEK293T cell immunostaining of CANX (c`) and WASHC4 (c) display colocalization at the endoplasmic reticulum. Scale bar = 5 µm. (d to d``) FDB muscle fiber of a male CD1 mouse transfected with WASHC4-eGFP (d) and VCP-mCherry (d`) expressing constructs demonstrated colocalization of both proteins. (e to e``) Close-ups of the squared area of the FDB muscle fiber of D``. (e```) Line-scan profiles demonstrated overlapping fluorescence intensities. Cell nuclei were counterstained with DAPI.

Phenotypic analysis washc4 morpholino-mediated knockdown in zebrafish embryos. (a to d) Lateral view of embryos at 48-hpf injected with washc4 5 bp mismatch start or splice morpholino (MO1/2-control; a, c) or washc4 start or splice MO (MO1/2-washc4; b, d). MO1/2- washc4 injection leads to a myopathic phenotype (b, d). scale bar: 450 μm. (e) Quantification of myopathic embryos after MO1/2-control or MO1/2- washc4 injection. N = 3/4 Experiments with in total n = 290 (MO1-control), n = 238 (MO2-control) n = 226 (MO1- washc4), n = 259 (MO2- washc4). The data were analyzed via N two-sided Fisher exact tests combined with a modified Fisher combination. The ratios of N experiments are shown. Western blot analysis shows reduction of Washc4 protein level in MO1-washc4-injected embryos. Actb1/β-actin was used as loading control. MO2-washc4 injection effects on mRNA splicing shown by RT-PCR. (f) Ventricular fractional shortening (FS) measurements at 48, 72 and 96 hpf of control and washc4 morphants. n = 30 (MO1-control), n = 30 (MO1-washc4); The individual measurements are shown (two-sided Wilcoxon rank-sum test). (g) Heart beat per minute of MO1-control- or MO1-washc4-injected embryos at 48, 72 and 96 hpf. n = 30 (MO1-control), n = 30 (MO1- washc4). The individual measurements are shown (two-sided Wilcoxon rank-sum test). (h, i) Spontaneous movement assay shown by false-colored superimposed pictures of control embryos (h) or Washc4 morphants (i) at 24 hpf; red pictures = 0 s; green pictures = 10 s; yellow = merge. scale bar: 1 mm. (j) Statistical analysis of spontaneous movement assay; N = 3 experiments with in total n = 56 (MO1-control), n = 60 (MO1-washc4) were performed (N two-sided Fisher exact tests combined with a modified Fisher method). The ratios of N experiments are shown. (k) Quantification of touch-evoke flight response assay of MO1-control- or MO1-washc4-injected embryos at 60 hpf. n = 40, N = 3 (MO1-control), n = 40, N = 3 (MO1-washc4); data represent means.

Inactivation of Washc4 in zebrafish embryos results in skeletal muscle ultrastructural alterations. (a and b) Lateral view of MO1-control- (a) and MO1-washc4-injected (b) embryos showing birefringence at 72 hpf. scale bar: 400 μm. (c) Statistical analysis of birefringence signal. n = 30 (MO1-control), n = 30 (MO1-washc4); two-sided Wilcoxon rank-sum tests. (d and e) H&E-stained sagittal histological sections of skeletal muscle of control embryos (d) and Washc4 morphants (e) at 72 hpf; scale bar = 100 μm. (f to g```) Transmission electron microscopy in MO1-control (f to f``) and MO1-washc4 (g to g```) skeletal muscle at 72 hpf. (f to f``) Control muscle cells exhibit highly organized myofibrils (f``) and normal mitochondria, next to the sarcomeric Z-discs (f`). (g to g``) Washc4 morphant muscle cells show less myofibrils, dysmorphic mitochondria (g``) and an accumulation of various vesicular structures; AV = autophagic vesicle, M = mitochondria, MeB = membranous bodies; scale bar: 2 μm (f, g); scale bar = 1 μm (f` and f``, g` to g```). (j and k) Immunostaining using an anti-acetylated tubulin (acTub) antibody of MO1-control- (j) or MO1-washc4-injected (k) embryos at 72 hpf. scale bar: 200 μm.

Loss of Washc4 leads to an upregulation of ER stress markers and to an impaired autophagy. (a) Western blot analysis of ubiquitinated proteins after MO1-control and MO1-washc4 injection. (b) qRT-PCR of ER stress markers (hspa5, atf4 and ppp1r15a) of control embryos or washc4 morphants at 3 dpf. The individual measurements are shown (log10). The 3 markers were analyzed via Wilcoxon rank-sum tests. A combined P value (comb. p) was determined via a modified Fisher method. slc25a5 was used as housekeeping gene. (c) Western blot analysis using anti-Sqstm1 antibody after MO1-control and MO1-washc4 injection (upper panel); quantification of gray values (lower panel; n = 6). (d) Representative western blotting of Lc3 of control embryos or washc4 morphants (upper panel); quantification of gray values (lower panel; n = 6). Individual measurements are shown (Wilcoxon rank-sum test). (e) MO1-control-or MO1-washc4-injected embryos treated with DMSO or ammonium chloride (NH4Cl). Control embryos show a significant increase of Lc3-II levels after NH4Cl treatment in comparison to DMSO controls. Ammonium chloride treatment of Washc4 morphants did not significantly enhance the Lc3-II level in comparison to DMSO treated embryos (n = 6). Actb1/β-actin was used as loading control. The individual measurements are shown (Wilcoxon rank-sum test).

Acknowledgments
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