Autophagy as a new therapeutic target in Duchenne muscular dystrophy

Abstract

A resolutive therapy for Duchene muscular dystrophy, a severe degenerative disease of the skeletal muscle, is still lacking. Because autophagy has been shown to be crucial in clearing dysfunctional organelles and in preventing tissue damage, we investigated its pathogenic role and its suitability as a target for new therapeutic interventions in Duchenne muscular dystrophy (DMD). Here we demonstrate that autophagy is severely impaired in muscles from patients affected by DMD and mdx mice, a model of the disease, with accumulation of damaged organelles. The defect in autophagy was accompanied by persistent activation via phosphorylation of Akt, mammalian target of rapamycin (mTOR) and of the autophagy-inhibiting pathways dependent on them, including the translation-initiation factor 4E-binding protein 1 and the ribosomal protein S6, and downregulation of the autophagy-inducing genes LC3, Atg12, Gabarapl1 and Bnip3. The defective autophagy was rescued in mdx mice by long-term exposure to a low-protein diet. The treatment led to normalisation of Akt and mTOR signalling; it also reduced significantly muscle inflammation, fibrosis and myofibre damage, leading to recovery of muscle function. This study highlights novel pathogenic aspects of DMD and suggests autophagy as a new effective therapeutic target. The treatment we propose can be safely applied and immediately tested for efficacy in humans.

Figures

Figure 1

Autophagy is defective in mdx mice. (a) Muscle homogenates from mdx and WT mice (60 μg per lane) were immunoblotted with antibodies against LC3 and p62. LC3 lipidation (LC3 II) in TA and in diaphragm muscles of mdx mice is reduced when compared with that of WT mice. The mdx mice also show accumulation of p62. Densitometric quantification of the LC3 II/I ratio and of p62, normalised against actin, is also shown. (b) In muscle homogenates (60 μg per lane), phosphorylation levels of Akt, S6 and 4E-BP1 are enhanced in mdx mice when compared with those of WT. We used the same protein extracts as in panel a. Densitometric analyses show the ratio between phosphorylated and corresponding total protein. In both panels, western blots are representative, and quantifications correspond to 15 animals per group. The asterisks indicate statistical significance versus WT (*P<0.05 and **P<0.01). Error bars represent S.E.M.

Figure 2

Chloroquine treatment of WT and mdx mice. (a and b) The western blots show the levels of LC3 and p62 in TA (a) and diaphragm (b) muscles of mdx and WT mice treated (+) or not (−) with chloroquine diphosphate at 50 mg/kg body weigh daily for 7 days. The absence of choloroquine effects in mdx (but not WT) mice indicate that the defect in LC3 II expression is due to an actual deficit of autophagy and not the consequence of autophagic vesicle exhaustion secondary to hyperactivation of autophagy. Western blotting are representative, and quantifications correspond to 15 animals per group. Asterisks indicate statistical significance versus untreated WT (**P<0.01 and ***P<0.001). Error bars represent S.E.M.

Figure 3

Autophagy induction is impaired in mdx mice. (a and b) Western blots showing LC3 and p62 levels in TA (a) and diaphragm (b) muscles (60 μg per lane) of fed and 15 h-starved (15 h) WT and mdx mice, treated (+) or not (−) with chloroquine 50 mg/kg body weight daily for 7 days. Densitometric quantification of LC3 II/I ratio and of p62 levels, normalised against actin, shows induction of autophagy only in WT mice after 15 h of starvation. (c and d) Western blots, using the same protein extracts of panels a and b, reveal reduction of Akt and 4E-BP1 phosphorylation in diaphragm and TA muscles of 15 h-starved WT mice. The mdx muscles display no changes in the phosphorylation levels of both proteins. (e) Quantitative RT-PCR analysis of LC3β, Atg12, Gaparapl1 and Bnip3 mRNA levels in diaphragm and TA muscles of fed and 15 h-starved WT and mdx mice. The genes downstream of FoxO3 are upregulated in WT mice after starvation, but not in the mdx mice. Western blots are representative, and quantifications correspond to 15 animals per group. Asterisks indicate statistical significance versus WT-fed mice (*P<0.05, **P<0.01 and ***P<0.001); crosses indicate statistical significance versus WT-starved mice (+++P<0.001). Error bars represent S.E.M.

Figure 4

Autophagy induction in WT and mdx mice. (a) electron micrographs of TA muscles from WT mice. No defects in the architecture of the contractile apparatus or in the mitochondrial pool are observed in WT-fed mice (scale bars 1 μm, 200 nm in the inset). (b) The 15 h-starved TA muscles of WT mice (b and insets) show alteration of mitochondrial pool and an increase of autophagosomes, identified as double-membrane structures associated with degenerating organelles (inset) (scale bars 1 μm, 200 nm in the insets). (c) Electron micrographs of TA muscles from fed mdx mice. No autophagosomes are detectable, and the defect in autophagy is accompanied by expansion of endoplasmic reticulum cisternae expansion (scale bar 1 μm). (d) The mdx-starved TA muscles present an increased level of altered organelles, in particular of mitochondria in the subsarcolemmal pool, that are not related with lytic organelles (d and inset) (scale bars 1 μm, 200 nm in the inset). Images are representative of reproducible results in 10 animals per group

Figure 5

Prolonged autophagy induction by LPD ameliorates dystrophy in mdx mice. WT and mdx mice were fed with LPD or SD for 3 months. (a and b) The representative western blots of homogenates (60 μg per lane) from TA (a) and diaphragm (b) muscles show that LPD enhances LC3 II and p62 expression, whereas reducing phosphorylation of Akt and 4E-BP1 both in WT and mdx mice. (c) Quantitative RT-PCR analysis of LC3β, Atg12, Gaparapl1 and Bnip3 mRNA levels in TA fed with SD or LPD. (d) In vivo analysis of muscle force was measured as the amount of whole body pulling force, calculated as the force relative to body weight in WT and mdx. WBT10 represents the media of 10 FPTs, measured as grams of tension, divided by body weight. WBT5 represents the media of top-five FPTs divided by body weight. LPD improved muscle force in mdx mice. (e) TA and diaphragm muscles were stained with H&E; centronucleated and necrotic myofibres were counted. LPD reduces necrotic myofibres and increases centronucleated myofibres. Scale bar 110 μm. (f) Distribution of CSA values of 700 myofibres of TA muscle of WT and mdx treated with LPD or SD. LPD normalises the CSA distribution in mdx. Images and western blots in the various panels are representative, and quantifications correspond to 15 animals per group. Asterisks indicate statistical significance versus WT (*P<0.05, **P<0.01 and ***P<0.001); crosses indicate statistical significance versus mdx normally fed (+P<0.05; ++P<0.01 and +++P<0.001). Error bars represent S.E.M.

Figure 6

Prolonged autophagy induction by LPD ameliorates dystrophy in mdx mice. WT and mdx mice were fed with LPD or SD for 3 months. (a) TA and diaphragm muscles representative sections, stained with the Masson's Trichrome procedure reveal reduced accumulation of collagen and fibrosis in LPD-treated mdx versus SD-treated mdx. Scale bar 110 μm. The graph shows the quantification carried out in five sections per animal. (b) Fibre membrane damage, evaluated by assessing EBD uptake, quantified by the Image 1.63 (Scion Corporation) software as % of Evans blue-positive cells. The representative images show that LPD reduce EBD uptake in mdx mice. Scale bar 110 μm. The graph shows the quantification carried out in five sections per animal. (c) Quantification of TUNEL-positive nuclei in TA muscles of WT and mdx mice; LPD reduces the number of TUNEL-positive nuclei compared with mdx. Quantification (number of TUNEL-positive nuclei normalised per mm2) was carried out with the Image 1.63 (Scion Corporation) software. Images in the various panels are representative, and quantifications correspond to 10 animals per group. Asterisks indicate statistical significance versus WT (*P<0.05, **P<0.01 and ***P<0.001). Error bars represent S.E.M.

Figure 7

Autophagy is defective in human DMD patients. (a) Analysis of the LC3 ratio and p62 levels in human muscle biopsies from four control subjects and five DMD patients. p62 expression is normalised on GAPDH. (b) Western blots for pAKT and p4E-BP1 in protein lysates of human biopsies using the same protein extract of panel a. DMD patients show higher Akt and 4E-BP1 phosphorylation compared with controls. Western blots are representative, and quantifications correspond to results in biopsies from five DMD patients and four controls. Asterisks indicate statistical significance versus controls (*P<0.05 and **P<0.01). Error bars represent S.E.M.