Muscle inactivation of mTOR causes metabolic and dystrophin defects leading to severe myopathy

Mammalian target of rapamycin (mTOR) is a key regulator of cell growth that associates with raptor and rictor to form the mTOR complex 1 (mTORC1) and mTORC2, respectively. Raptor is required for oxidative muscle integrity, whereas rictor is dispensable. In this study, we show that muscle-specific inactivation of mTOR leads to severe myopathy, resulting in premature death. mTOR-deficient muscles display metabolic changes similar to those observed in muscles lacking raptor, including impaired oxidative metabolism, altered mitochondrial regulation, and glycogen accumulation associated with protein kinase B/Akt hyperactivation. In addition, mTOR-deficient muscles exhibit increased basal glucose uptake, whereas whole body glucose homeostasis is essentially maintained. Importantly, loss of mTOR exacerbates the myopathic features in both slow oxidative and fast glycolytic muscles. Moreover, mTOR but not raptor and rictor deficiency leads to reduced muscle dystrophin content. We provide evidence that mTOR controls dystrophin transcription in a cell-autonomous, rapamycin-resistant, and kinase-independent manner. Collectively, our results demonstrate that mTOR acts mainly via mTORC1, whereas regulation of dystrophin is raptor and rictor independent

Muscle inactivation of mTOR causes metabolic and dystrophin defects leading to severe myopathy

mTor, acting mainly via mTORC1, controls dystrophin transcription in a raptor- and rictor-independent mechanism

Identification of a new gene family specifically expressed in chicken embryonic stem cells and early embryo.

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A transgenic mouse expressing CHMP2B intron5 mutant in neurons develops histological and behavioural features of amyotrophic lateral sclerosis and frontotemporal dementia

International audienceMutations in the charged multivesicular body protein 2B (CHMP2B) are associated with frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), and with a mixed ALS-FTD syndrome. To model this syndrome, we generated a transgenic mouse line expressing the human CHMP2Bintron5 mutant in a neuron-specific manner. These mice developed a dose-dependent disease phenotype. A longitudinal study revealed progressive gait abnormalities, reduced muscle strength and decreased motor coordination. CHMP2Bintron5 mice died due to generalized paralysis. When paralyzed, signs of denervation were present as attested by altered electromyographic profiles, by decreased number of fully innervated neuromuscular junctions, by reduction in size of motor endplates and by a decrease of sciatic nerve axons area. However, spinal motor neurons cell bodies were preserved until death. In addition to the motor dysfunctions, CHMP2Bintron5 mice progressively developed FTD-relevant behavioural modifications such as disinhibition, stereotypies, decrease in social interactions, compulsivity and change in dietary preferences. Furthermore, neurons in the affected spinal cord and brain regions showed accumulation of p62-positive cytoplasmic inclusions associated or not with ubiquitin and CHMP2Bintron5 As observed in FTD3 patients, these inclusions were negative for TDP-43 and FUS. Moreover, astrogliosis and microgliosis developed with age. Altogether, these data indicate that the neuronal expression of human CHMP2Bintron5 in areas involved in motor and cognitive functions induces progressive motor alterations associated with dementia symptoms and with histopathological hallmarks reminiscent of both ALS and FTD

mTOR inactivation in myocardium from infant mice rapidly leads to dilated cardiomyopathy due to translation defects and p53/JNK-mediated apoptosis.

Mechanistic target of rapamycin (mTOR) is a central regulator of cell growth, proliferation, survival and metabolism, as part of mTOR complex 1 (mTORC1) and mTORC2. While partial inhibition of mTORC1 using rapamycin was shown to be cardioprotective, genetic studies in mouse models revealed that mTOR is essential for embryonic heart development and cardiac function in adults. However, the physiological role of mTOR during postnatal cardiac maturation is not fully elucidated. We have therefore generated a mouse model in which cardiac mTOR was inactivated at an early postnatal stage. Mutant mTORcmKO mice rapidly developed a dilated cardiomyopathy associated with cardiomyocyte growth defects, apoptosis and fibrosis, and died during their third week. Here, we show that reduced cardiomyocyte growth results from impaired protein translation efficiency through both 4E-BP1-dependent and -independent mechanisms. In addition, infant mTORcmKO hearts displayed markedly increased apoptosis linked to stretch-induced ANKRD1 (Ankyrin repeat-domain containing protein 1) up-regulation, JNK kinase activation and p53 accumulation. Pharmacological inhibition of p53 with pifithrin-α attenuated caspase-3 activation. Cardiomyocyte death did not result from activation of the MST1/Hippo pro-apoptotic pathway as reported in adult rictor/mTORC2 KO hearts. As well, mTORcmKO hearts showed a strong downregulation of myoglobin content, thereby leading to a hypoxic environment. Nevertheless, they lacked a HIF1α-mediated adaptive response, as mTOR is required for hypoxia-induced HIF-1α activation. Altogether, our results demonstrate that mTOR is critically required for cardiomyocyte growth, viability and oxygen supply in early postnatal myocardium and provide insight into the molecular mechanisms involved in apoptosis of mTOR-depleted cardiomyocyte

0452 : MTOR inactivation during early postnatal development of mice myocardium leads to severe dilated cardiomyopathy due to altered translational efficiency and hypoxia-induced apoptosis

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The questions you should ask yourself for efficient CRISPR/Cas9 genome editing

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Genome editing in primary cells and in vivo using viral-derived Nanoblades loaded with Cas9-sgRNA ribonucleoproteins

A current challenge in genome editing is delivering Cas9 and sgRNA into target cells. Here the authors engineer a delivery system based on murine leukemia virus-like particles loaded with Cas9-sgRNA ribonucleoproteins to induce efficient genome editing in both cell culture and in vivo in mouse

Severe congenital myasthenic syndromes caused by agrin mutations affecting secretion by motoneurons

International audienceAbstract Congenital myasthenic syndromes (CMS) are predominantly characterized by muscle weakness and fatigability and can be caused by a variety of mutations in genes required for neuromuscular junction formation and maintenance. Among them, AGRN encodes agrin, an essential synaptic protein secreted by motoneurons. We have identified severe CMS patients with uncharacterized p.R1671Q, p.R1698P and p.L1664P mutations in the LG2 domain of agrin . Overexpression in primary motoneurons cultures in vitro and in chick spinal motoneurons in vivo revealed that the mutations modified agrin trafficking, leading to its accumulation in the soma and/or in the axon. Expression of mutant agrins in cultured cells demonstrated accumulation of agrin in the endoplasmic reticulum associated with induction of unfolded protein response (UPR) and impaired secretion in the culture medium. Interestingly, evaluation of the specific activity of individual agrins on AChR cluster formation indicated that when secreted, mutant agrins retained a normal capacity to trigger the formation of AChR clusters. To confirm agrin accumulation and secretion defect, iPS cells were derived from a patient and differentiated into motoneurons. Patient iPS-derived motoneurons accumulated mutant agrin in the soma and increased XBP1 mRNA splicing, suggesting UPR activation. Moreover, co-cultures of patient iPS-derived motoneurons with myotubes confirmed the deficit in agrin secretion and revealed a reduction in motoneuron survival. Altogether, we report the first mutations in AGRN gene that specifically affect agrin secretion by motoneurons. Interestingly, the three patients carrying these mutations were initially suspected of spinal muscular atrophy (SMA). Therefore, in the presence of patients with a clinical presentation of SMA but without mutation in the SMN1 gene, it can be worth to look for mutations in AGRN