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Anne-Cécile Durieux,
Stéphane Vassilopoulos,
Jeanne Lainé, Bodvaël Fraysse,
Laura Briñas,
Bernard Prudhon,
Josiane Castells,
Damien Freyssenet,
Gisèle Bonne,
Pascale Guicheney,
Marc Bitoun
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ABSTRACT: Dynamin 2 (Dnm2) is involved in endocytosis and intracellular membrane trafficking through its function in vesicle formation from distinct membrane compartments. Heterozygous (HTZ) mutations in the DNM2 gene cause dominant centronuclear myopathy or Charcot-Marie-Tooth neuropathy. We generated a knock-in Dnm2R465W mouse model expressing the most frequent human mutation and recently reported that HTZ mice progressively developed a myopathy. We investigated here the cause of neonatal lethality occurring in homozygous (HMZ) mice. We show that HMZ mice present at birth with a reduced body weight, hypoglycemia, increased liver glycogen content and hepatomegaly, in agreement with a defect in neonatal autophagy. In vitro studies performed in HMZ embryonic fibroblasts point out to a decrease in the autophagy flux prior to degradation at the autolysosome. We show that starved HMZ cells have a higher number of immature autophagy-related structures probably due to a defect of acidification. Our results highlight the role of Dnm2 in the cross talk between endosomal and autophagic pathways and evidence a new role of Dnm2-dependent membrane trafficking in autophagy which may be relevant in DNM2-related human diseases.
Traffic 02/2012; 13(6):869-79. · 4.92 Impact Factor
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Anne-Cécile Durieux,
Alban Vignaud,
Bernard Prudhon,
Mai Thao Viou,
Maud Beuvin,
Stéphane Vassilopoulos, Bodvaël Fraysse,
Arnaud Ferry,
Jeanne Lainé,
Norma B Romero,
Pascale Guicheney,
Marc Bitoun
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ABSTRACT: Autosomal dominant centronuclear myopathy (AD-CNM) is due to mutations in the gene encoding dynamin 2 (DNM2) involved in endocytosis and intracellular membrane trafficking. To understand the pathomechanisms resulting from a DNM2 mutation, we generated a knock-in mouse model expressing the most frequent AD-CNM mutation (KI-Dnm2(R465W)). Heterozygous (HTZ) mice developed a myopathy showing a specific spatial and temporal muscle involvement. In the primarily and prominently affected tibialis anterior muscle, impairment of the contractile properties was evidenced at weaning and was progressively associated with atrophy and histopathological abnormalities mainly affecting mitochondria and reticular network. Expression of genes involved in ubiquitin-proteosome and autophagy pathways was up-regulated during DNM2-induced atrophy. In isolated muscle fibers from wild-type and HTZ mice, Dnm2 localized in regions of intense membrane trafficking (I-band and perinuclear region), emphasizing the pathophysiological hypothesis in which DNM2-dependent trafficking would be altered. In addition, HTZ fibers showed an increased calcium concentration as well as an intracellular Dnm2 and dysferlin accumulation. A similar dysferlin retention, never reported so far in congenital myopathies, was also demonstrated in biopsies from DNM2-CNM patients and can be considered as a new marker to orientate direct genetic testing. Homozygous (HMZ) mice died during the first hours of life. Impairment of clathrin-mediated endocytosis, demonstrated in HMZ embryonic fibroblasts, could be the cause of lethality. Overall, this first mouse model of DNM2-related myopathy shows the crucial role of DNM2 in muscle homeostasis and will be a precious tool to study DNM2 functions in muscle, pathomechanisms of DNM2-CNM and developing therapeutic strategies.
Human Molecular Genetics 12/2010; 19(24):4820-36. · 7.64 Impact Factor
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ABSTRACT: Muscular dystrophies are often associated with significant cardiac disease that can be the prominent feature associated with gene mutations in sarcoglycan. Cardiac cell death is a main feature of cardiomyopathy in sarcoglycan deficiency and may arise as a cardiomyocyte intrinsic process that remains unclear. Deficiency of delta-sarcoglycan (delta-SG) induces disruption of the dystrophin-associated glycoprotein complex, a known cause of membrane instability that may explain cardiomyocytes cytosolic Ca2+ increase. In this study we assessed the hypothesis that cytosolic Ca2+ increase triggers cardiomyocyte death through mitochondrial Ca2+ overload and dysfunction in the delta-SG-deficient CHF147 hamster. We showed that virtually all isolated CHF147 ventricular myocytes exhibited elevated cytosolic and mitochondrial Ca2+ levels by the use of the Fura-2 and Rhod-2 fluorescent probes. Observation of living cells with Mito-Tracker red lead to the conclusion that approximately 15% of isolated CHF147 cardiomyocytes had disorganized mitochondria. Transmission electron microscope imaging showed mitochondrial swelling associated with crest and membrane disruption. Analysis of the mitochondrial permeability transition pore (MPTP) activity using calcein revealed that mitochondria of CHF147 ventricular cells were twofold leakier than wild types, whereas reactive oxygen species production was unchanged. Bax, Bcl-2, and LC3 expression analysis by Western blot indicated that the intrinsic apoptosis and the cell death associated to autophagy pathways were not significantly activated in CHF147 hearts. Our results lead to conclusion that cardiomyocytes death in delta-SG-deficient animals is an intrinsic phenomenon, likely related to Ca2+-induced necrosis. In this process Ca2+ overload-induced MPTP activation and mitochondrial disorganization may have an important role.
AJP Cell Physiology 09/2010; 299(3):C706-13. · 3.54 Impact Factor
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ABSTRACT: Mutations of the selenoprotein N gene (SEPN1) cause SEPN1-related myopathy (SEPN1-RM), a novel early-onset muscle disorder formerly divided into four different nosological categories. Selenoprotein N (SelN) is the only selenoprotein involved in a genetic disease; its function being unknown, no treatment is available for this potentially lethal disorder. Our objective was to clarify the role of SelN and the pathophysiology of SEPN1-RM to identify therapeutic targets.
We established and analyzed an ex vivo model of SelN deficiency using fibroblast and myoblast primary cultures from patients with null SEPN1 mutations. DCFH assay, OxyBlot, Western blot, Fura-2, and cell survival studies were performed to measure intracellular oxidant activity, oxidative stress markers, calcium handling, and response to exogenous treatments.
SelN-depleted cells showed oxidative/nitrosative stress manifested by increased intracellular oxidant activity (reactive oxygen species and nitric oxide) and/or excessive oxidation of proteins, including the contractile proteins actin and myosin heavy chain II in myotubes. SelN-devoid myotubes showed also Ca(2+) homeostasis abnormalities suggesting dysfunction of the redox-sensor Ca(2+) channel ryanodine receptor type 1. Furthermore, absence of SelN was associated with abnormal susceptibility to H(2)O(2)-induced oxidative stress, demonstrated by increased cell death. This cell phenotype was restored by pretreatment with the antioxidant N-acetylcysteine.
SelN plays a key role in redox homeostasis and human cell protection against oxidative stress. Oxidative/nitrosative stress is a primary pathogenic mechanism in SEPN1-RM, which can be effectively targeted ex vivo by antioxidants. These findings pave the way to SEPN1-RM treatment, which would represent a first specific pharmacological treatment for a congenital myopathy.
Annals of Neurology 04/2009; 65(6):677-86. · 11.09 Impact Factor
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ABSTRACT: Using fura-2 and the manganese quenching technique, we show here that sarcolemmal permeability to cations (SP-Ca) of slow-twitch muscles is greater than that of fast-twitch ones. This appears to be related to a higher expression and/or activity of stretch-activated channels, whereas leak channel activities are similar. During hindlimb suspension (HU), we found highly correlated decreases in SPCa and resting calcium of soleus muscle toward values of extensor digitorum longus (EDL) muscle. This was significant as soon as 3 days of suspension, contrary to soleus muscle caffeine sensitivity and responsiveness that were not modified after this HU period. After 14 days of HU, SP-Ca, resting calcium, and caffeine response of soleus muscle became similar to that normally observed in EDL muscle. These results demonstrate that the correlated decreases in SP-Ca and resting calcium precede most functional changes due to HU. Given the known shortening of HU soleus muscle, we proposed that this could induce a decrease of SP-Ca and a consequent reduction of resting calcium. According to the crucial role of resting cytosolic free calcium in the maintenance and the adaptation of muscle phenotype, our results suggest that slow-to-fast transition of HU soleus muscle is calcium dependent.
The FASEB Journal 11/2003; 17(13):1916-8. · 5.71 Impact Factor
