mTOR dysfunction contributes to vacuolar pathology and weakness in valosin-containing protein associated inclusion body myopathy

Abstract

Autophagy is dysfunctional in many degenerative diseases including myopathies. Mutations in valosin-containing protein (VCP) cause inclusion body myopathy (IBM) associated with Paget's disease of the bone, fronto-temporal dementia and amyotrophic lateral sclerosis (IBMPFD/ALS). VCP is necessary for protein degradation via the proteasome and lysosome. IBMPFD/ALS mutations in VCP disrupt autophagosome and endosome maturation resulting in vacuolation, weakness and muscle atrophy. To understand the regulation of autophagy in VCP-IBM muscle, we examined the AKT/FOXO3 and mammalian target of rapamycin (mTOR) pathways. Basal Akt and FOXO3 phosphorylation was normal. In contrast, the phosphorylation of mTOR targets was decreased. Consistent with this, global protein translation was diminished and autophagosome biogenesis was increased in VCP-IBM muscle. Further mTORC1 inhibition with rapamycin hastened weakness, atrophy and vacuolation in VCP-IBM mice. This was accompanied by the accumulation of autophagic substrates such as p62, LC3II and ubiquitinated proteins. The decrease in mTOR signaling was partially rescued by insulin and to a lesser extent by amino acid (AA) stimulation in VCP-IBM muscle. Cells expressing catalytically inactive VCP or treated with a VCP inhibitor also failed to activate mTOR upon nutrient stimulation. Expression of a constitutively active Rheb enhanced mTOR activity and increased the fiber size in VCP-IBM mouse skeletal muscle. These studies suggest that VCP mutations may disrupt mTOR signaling and contribute to IBMPFD/ALS disease pathogenesis. Treatment of some autophagic disorders with mTOR inhibitors such as rapamycin may worsen disease.

Figure 1.

mTOR activity is diminished in VCP-IBM mouse muscle. (A) Immunoblot analysis of phosphorylated Akt^Ser473 (pAkt), total Akt, phosphorylated FOXO3^Ser253 (pFOXO3), phosphorylated S6^Ser240/244 (pS6), total S6, VCP or actin in the TA muscle of control, VCP-WT or one of the two independent VCP-R155H expressing mouse lines (VCP-RH9 or VCP-RH12). (B) Quantitative PCR analysis of Atrogin-1 and Murf-1 transcript levels in TA of control, VCP-WT or VCP-RH12 mice. (C) Immunoblot of beclin-1, BNIP3 or actin in the TA of control, VCP-WT or VCP-RH12 mice. (D) Immunoblot of phosphorylated p70S6 kinase-1^Thr389 (pp70s6k), total p70S6 kinase-1 (p70s6k), phosphorylated 4EBP1^Thr37/46 (p4EBP1), total 4EBP1, pS6, S6 and actin in the TA muscle of control, VCP-WT, VCP-RH or control mice treated with 10 mg/kg i.p. rapamycin for 12 h. (E) Quantitation of the densitometric levels of the ratio of pp70S6K/p70S6K (n = 6 mice/condition); p4EBP1/4EBP1 (n = 8 mice/condition) and pS6/S6 (n = 4 mice/condition) in the skeletal muscle of VCP-WT and VCP-RH mice. **P < 0.001. All blots are representative of at least two independent experiments.

Figure 2.

Protein translation is decreased and autophagosome biogenesis is increased in VCP-IBM mouse muscle. (A) Immunoblot analysis for incorporated puromycin and actin in TA of control, VCP-WT, VCP-RH or control mice treated with 10 mg/kg i.p. rapamycin for 12 h prior to the administration of i.p. puromycin. (B) Immunoblot for phosphorylated ULK1^Ser757 (pULK1), total ULK1 and GAPDH from TA of control, VCP-WT, VCP-RH9 or RH12 mice. (C) Quantitation of the densitometric levels of the ratio of pULK1/GAPDH (n = 4 mice/condition) of control, VCP-WT, VCP-RH9 and VCP-RH12 mice. *P < 0.01 and **P < 0.001. (D) Immunoblot for LC3 and actin in TA of vehicle treated or 0.4 mg/kg i.p. colchicine for 24 h prior to harvesting skeletal muscle of control, VCP-WT and VCP-RH mice. (E) Quantitation of the densitometric levels of LC3II to actin ratio in TA of vehicle-treated (−) or 24 h of 0.4 mg/kg i.p. colchicine treatment (+) in VCP-WT and VCP-RH mice. **P < 0.001 and n = 6 mice/condition.

Figure 3.

Rapamycin treatment exacerbates weakness and muscle pathology in VCP-IBM mice. Five-month-old control, VCP-WT or VCP-RH mice were treated with vehicle or every other day dosing of 10 mg/kg i.p. rapamycin for 21 days. Graphs represent change in grip strength (A) or rotarod performance (B) from day 1. Results are obtained from four mice per group and three independent experiments for grip strength analysis and four mice per group and two independent experiments for rotarod performance. *P < 0.01. (C) Serum creatine phosphokinase (CPK) levels from mice VCP-WT or VCP-RH mice treated with vehicle or every other day 10 mg/kg i.p. rapamycin. (D) Congo red staining or LC3 immunofluorescence of TA muscle from 5-month-old VCP-RH mice treated with vehicle or every other day rapamycin for 21 days. Open arrows denote vacuolated fibers and closed arrows denote angular and atrophic fibers. Bar is 100 microns. (E) Quantitation of the percentage of vacuolated fibers and (F) percentage of fibers having a CSA of <200 µm from TA muscle of 5-month-old VCP-WT or VCP-RH mice treated with vehicle or every other day rapamycin (+) for 21 days. **P < 0.001.

Figure 4.

Autophagic substrates accumulate in VCP-IBM mice following chronic rapamycin treatment. (A) Immunoblot analysis for ubiquitinated proteins (FK2), BNIP3, p62, LC3 and GAPDH in TA from control, VCP-WT or VCP-RH mice treated with vehicle or every other day dosing of 10 mg/kg i.p. rapamycin for 21 days. (B) Quantitation of the densitometric levels of the ratio of p62/GAPDH or LC3II/GAPDH (n = 4 mice/condition) from the skeletal muscle of VCP-RH12 mice treated with vehicle or rapamycin for 21 days. (C) Immunohistochemistry for p62 (red) from VCP-WT or VCP-RH mice treated with vehicle or every other day dosing of 10 mg/kg i.p. rapamycin for 21 days. Arrows denote p62-positive vacuolar structures. Rightmost image is an enlargement of box in the middle image. Bar is 50 microns.

Figure 5.

VCP-IBM mice have a diminished mTOR response. (A) VCP-WT or VCP-RH12 (RH) mice were treated with saline or 0.75 units/kg i.p. insulin 5 min prior to sacrifice. TA muscle was subjected to immunoblot for pAKT, AKT, pS6 or S6. (B) Levels of pAMPK, AMPK or pACC, ACC were measured in VCP-WT and VCP-RH12 mice. (C) Untreated VCP-WT or VCP-RH mice were starved for 16 h or starved and then treated with 360 µm/100 g l-leucine 1 h prior to sacrifice. TA muscle was immunoblotted for pS6 and S6. Blots are representative of at least two animals from three separate experiments. (D) Immunoblot of pp70S6K, p70S6K, p4EBP1, 4EBP1 and actin from VCP-WT or VCP-RH mice starved for 16 h and then treated with i.p. l-leucine 1 h prior to sacrifice.

Figure 6.

VCP activity is required for mTOR activation. (A) Immunoblot analysis of pS6, S6, 4EBP1 or actin from U20S cells treated for 18 h with DMSO, rapamycin or DBeQ. 4EBP1 immunoblots have multiple bands. The higher molecular weight bands represent phosphorylated 4EBP1 (double arrows). Rapamycin treatment and 10 µm DBeQ decrease the levels of pS6 and lead to a shift in 4EBP1 to lower molecular weight non-phosphorylated bands consistent with reduced mTOR activity. (B) Immunoblot for pS6, S6 or actin from serum-starved U20S cells. Some cells were stimulated with 10% FBS for 1 h or with drug plus serum for 1 h prior to harvesting cells. (C) Immunoblot for pS6, S6, actin and Flag-tagged caRheb from U20S cells stably expressing VCP-WT, VCP-E578Q or VCP-R155H and transfected with a plasmid expressing GFP or caRheb-Flag. The cells were starved of serum for 18 h and then restimulated with 10% FBS for 1 h. Some cells were also treated with 10 µm DBeQ at the time of serum stimulation. Samples were isolated and blotted under the same conditions. Gels are rearranged from the same experiment for presentation purposes.

Figure 7.

Rheb expression increases the myofiber size and activates mTOR in VCP-IBM mice. (A) Graph of the mean CSA of untransfected, caRheb-Flag or GFP transfected TA myofibers 7 days post-electroporation from 5-month-old control, VCP-WT or VCP-RH mice. **P < 0.001. Error bars denote standard error. (B) Immunofluorescence with anti-Flag (red) and anti-collagen IV (green) of TA myofibers 7 days post-electroporation from 5-month-old control, VCP-WT or VCP-RH mice image. Stars denote transfected fibers. Bar is 50 microns. (C) Histogram of the percent of myofibers with the indicated CSA for control, VCP-WT or VCP-RH mice electroporated with GFP or caRheb expressing plasmids. (D) Immunoblot for pp70S6k, p70SK1-HA, pS6 or S6 of VCP-WT or VCP-RH TA muscle following 7-day post-electroporation with GFP or caRheb-Flag with p70S6K1-HA expression vector. *denotes phosphorylated p70S6K1-HA.

Figure 8.

Model of mTOR function in IBMPFD/ALS. (A) Mutant VCP expression impairs autophagosome maturation leading to vacuolation and protein inclusions. (B) Diminished mTOR function leading to reduced protein synthesis and increased autophagosome biogenesis.