Valosin containing protein associated inclusion body myopathy: abnormal vacuolization, autophagy and cell fusion in myoblasts

Abstract

Inclusion body myopathy associated with Paget's disease and frontotemporal dementia (IBMPFD) is caused by mutations in the valosin containing protein (VCP) gene. The disease is associated with progressive proximal muscle weakness, inclusions and vacuoles in muscle fibers, malfunction in the bone remodeling process resulting in Paget's disease, and premature frontotemporal dementia. VCP is involved in several cellular processes related to the endoplasmic reticulum associated degradation of proteins. To understand the pathological mechanisms underlying the myopathy in IBMPFD, we have studied the cellular consequences of VCP mutations in human primary myoblasts. Our results revealed that patients' myoblasts accumulate large vacuoles. Lysosomal membrane proteins Lamp1 and Lamp2 show increased molecular weights in patients' myoblasts due to differential N-glycosylation. Additionally, mutant myoblasts show increased autophagy when cultured in the absence of nutrients, as well as defective cell fusion and increased apoptosis. Our results elucidate that VCP mutations result in disturbances in several cellular processes, which will help us in the understanding of the pathological mechanisms resulting in muscle weakness and other features of VCP associated disease.

Figures

Figure 1. Vacuolization of IBMPFD myoblasts

A–D) Human myoblasts were fixed, stained with a VCP-specific antibody, and viewed with an immunofluorescence microscope. Nuclei were stained with DAPI. Some of the vacuoles are indicated by arrows (C, D). A = wild-type A, B = wild-type B, C = R155H, D = R155S. Magnification 630x. E–F) Electron micrographs of the human myoblasts. E = wild-type A, F = R155H. Some of the vacuoles are indicated by arrows (F). Magnification 900x. Scale bar = 2µm.

Figure 2. Differential N-glycosylation of Lamp1 and Lamp2 proteins in mutant myoblasts

Proteins were harvested from cultured myoblasts and analyzed by Western blotting using a Lamp1-specific antibody (upper panels) or a Lamp2-specific antibody (lower panel). Panels on the right show the results after PNGaseF treatments, and panels on the left show the results without treatments. Genotypes of the cell lines are indicated above. The brackets on the left indicate the glycosylated forms of Lamp1 and Lamp2 (110–130 kDa), and molecular weights of non-glycosylated Lamp1 and Lamp2 are shown on the right.

Figure 3. Down-regulation of troponin C, myogenin and M-cadherin in mutant myoblasts

Human myoblasts were induced with equine serum for 7 days. A) Proteins were harvested from cultured myoblasts and analyzed by Western blotting using a troponin C-specific antibody (upper panel), a myogenin-specific antibody (middle panel) or an actin-specific antibody (lower panel). The arrows on the left indicate the troponin C (TropoC), myogenin (Myog), and actin bands. Cell lines are indicated above. B) Proteins were harvested from cultures myoblasts and analyzed by Western blotting using an M-cadherin-specific antibody (upper panel) or an actin-specific antibody (lower panel). Genotypes of the cell lines are indicated above, and the arrows on the right indicate the M-cadherin (M-Cad), and actin bands.

Figure 4. Increased apoptosis in mutant myoblasts

A) Caspase-3 activity was measured using a CaspACE Assay System. Results were obtained by a spectrophotometer and are presented as relative values to wild-type A in the Y-axis. Cell lines are indicated in the X-axis. B–E) Nuclear DNA degradation of wild-type (B = wild-type A, C = wild-type B) and mutant (D = R155H, E = R155S) cells was analyzed by a TUNEL-system kit. Results were analyzed with an immunofluorescence microscope. Nuclei were stained with DAPI. Apoptotic cell nuclei are shown by arrows. Note that panel E has lower cell density than other panels. Magnification 100x.

Figure 5. Increased autophagy in starved mutant myoblasts

Proteins were harvested from starved, cultured myoblasts and analyzed by Western blotting using an LC3-specific antibody (upper panel) or an actin-specific antibody (lower panel). Cytosolic (LC3-I) and autophagic (LC3-II) forms of LC3, as well as actin bands are indicated on the left. Cell lines are indicated above.

Figure 6. Potential pathogenesis of the IBMPFD muscle

Lamp-proteins (short black lines) of the mutant cells are differentially glycosylated (small white balls in the lumen) and ubiquitinated (small black balls in the cytosol) in the lysosomes (L). These lysosomes are able to fuse with vacuoles (V) but are unable to degrade the vacuolar contents resulting in the accumulation of enlarged autophagosomes (A). The accumulation of undegraded material causes increased apoptosis and defective myotube formation, which eventually lead to progressive muscle weakness observed in IBMPFD patients. Nucleus is shown in grey.