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Summary: Mitochondria-endoplasmic reticulum (ER) contact sites (MERCs) emerged to play critical roles in numerous cellular processes, and their dysregulation has been associated to neurodegenerative disorders. Mutations in the SPG4 gene coding for spastin are among the main causes of hereditary spastic paraplegia (HSP). Spastin binds and severs microtubules, and the long isoform of this protein, namely M1, spans the outer leaflet of ER membrane where it interacts with other ER-HSP proteins. Here, we showed that overexpressed M1 spastin localizes in ER-mitochondria intersections and that endogenous spastin accumulates in MERCs. We demonstrated in different cellular models that downregulation of spastin enhances the number of MERCs, alters mitochondrial morphology, and impairs ER and mitochondrial calcium homeostasis. These effects are associated with reduced mitochondrial membrane potential, oxygen species levels, and oxidative metabolism. These findings extend our knowledge on the role of spastin in the ER and suggest MERCs deregulation as potential causes of SPG4-HSP disease.

Abstract Nemaline myopathy (NM) is a muscle disorder with broad clinical and genetic heterogeneity. The clinical presentation of affected individuals ranges from severe perinatal muscle weakness to milder childhood-onset forms, and the disease course and prognosis depends on the gene and mutation type. To date, 14 causative genes have been identified, and ACTA1 accounts for more than half of the severe NM cases. ACTA1 encodes α-actin, one of the principal components of the contractile units in skeletal muscle. We established a homogenous cohort of ten unreported families with severe NM, and we provide clinical, genetic, histological, and ultrastructural data. The patients manifested antenatal or neonatal muscle weakness requiring permanent respiratory assistance, and most deceased within the first months of life. DNA sequencing identified known or novel ACTA1 mutations in all. Morphological analyses of the muscle biopsy specimens showed characteristic features of NM histopathology including cytoplasmic and intranuclear rods, cytoplasmic bodies, and major myofibrillar disorganization. We also detected structural anomalies of the perinuclear space, emphasizing a physiological contribution of skeletal muscle α-actin to nuclear shape. In-depth investigations of the nuclei confirmed an abnormal localization of lamin A/C, Nesprin-1, and Nesprin-2, forming the main constituents of the nuclear lamina and the LINC complex and ensuring nuclear envelope integrity. To validate the relevance of our findings, we examined muscle samples from three previously reported ACTA1 cases, and we identified the same set of structural aberrations. Moreover, we measured an increased expression of cardiac α-actin in the muscle samples from the patients with longer lifespan, indicating a potential compensatory effect. Overall, this study expands the genetic and morphological spectrum of severe ACTA1-related nemaline myopathy, improves molecular diagnosis, highlights the enlargement of the perinuclear space as an ultrastructural hallmark, and indicates a potential genotype/phenotype correlation.

Drugs targeting myostatin reverse muscle wasting in animal models, but have limited efficacy in patients. The authors show that the myostatin pathway is downregulated in patients, possibly explaining the poor outcome of anti-myostatin approaches, and that it can be reactivated by correcting disease-causing mutations in mice.

Myotubular myopathy (MTM) is a devastating pediatric neuromuscular disorder of phosphoinositide (PIP) metabolism resulting from mutations of the PIP phosphatase MTM1 for which there are no treatments. We have previously shown phosphatidylinositol-3-phosphate (PI3P) accumulation in animal models of MTM. Here, we tested the hypothesis that lowering PI3P levels may prevent or reverse the MTM disease process. To test this, we targeted class II and III PI3 kinases (PI3Ks) in an MTM1-deficient mouse model. Muscle-specific ablation of Pik3c2b, but not Pik3c3, resulted in complete prevention of the MTM phenotype, and postsymptomatic targeting promoted a striking rescue of disease. We confirmed this genetic interaction in zebrafish, and additionally showed that certain PI3K inhibitors prevented development of the zebrafish mtm phenotype. Finally, the PI3K inhibitor wortmannin improved motor function and prolonged lifespan of the Mtm1-deficient mice. In all, we have identified Pik3c2b as a genetic modifier of Mtm1 mutation and demonstrated that PIK3C2B inhibition is a potential treatment strategy for MTM. In addition, we set the groundwork for similar reciprocal inhibition approaches for treating other PIP metabolic disorders and highlight the importance of modifier gene pathways as therapeutic targets., Introduction Phosphoinositides (PIPs) are low-abundance phospholipids responsible for the regulation of myriad cellular processes, including membrane trafficking and intracellular signaling cascades (1, 2). The distribution and amount of PIPs are [...]

Spinal muscular atrophy (SMA) is an autosomal recessive disease of variable severity caused by mutations in the SMN1 gene. Deficiency of the ubiquitous SMN function results in spinal cord α-motor neuron degeneration and proximal muscle weakness. Gene replacement therapy with recombinant adeno-associated viral (AAV) vectors showed therapeutic efficacy in several animal models of SMA. Here, we report a study aimed at analyzing the efficacy and biodistribution of a serotype-9, self-complementary AAV vector expressing a codon-optimized human SMN1 coding sequence (coSMN1) under the control of the constitutive phosphoglycerate kinase (PGK) promoter in neonatal SMNΔ7 mice, a severe animal model of the disease. We administered the scAAV9-coSMN1 vector in the intracerebroventricular (ICV) space in a dose-escalating mode, and analyzed survival, vector biodistribution and SMN protein expression in the spinal cord and peripheral tissues. All treated mice showed a significant, dose-dependent rescue of lifespan and growth with a median survival of 346 days. Additional administration of vector by an intravenous route (ICV+IV) did not improve survival, and vector biodistribution analysis 90 days postinjection indicated that diffusion from the cerebrospinal fluid to the periphery was sufficient to rescue the SMA phenotype. These results support the preclinical development of SMN1 gene therapy by CSF vector delivery.

SUMMARY Myotubular myopathy (MTM) is a severe congenital muscle disease characterized by profound weakness, early respiratory failure and premature lethality. MTM is defined by muscle biopsy findings that include centralized nuclei and disorganization of perinuclear organelles. No treatments currently exist for MTM. We hypothesized that aberrant neuromuscular junction (NMJ) transmission is an important and potentially treatable aspect of the disease pathogenesis. We tested this hypothesis in two murine models of MTM. In both models we uncovered evidence of a disorder of NMJ transmission: fatigable weakness, improved strength with neostigmine, and electrodecrement with repetitive nerve stimulation. Histopathological analysis revealed abnormalities in the organization, appearance and size of individual NMJs, abnormalities that correlated with changes in acetylcholine receptor gene expression and subcellular localization. We additionally determined the ability of pyridostigmine, an acetylcholinesterase inhibitor, to ameliorate aspects of the behavioral phenotype related to NMJ dysfunction. Pyridostigmine treatment resulted in significant improvement in fatigable weakness and treadmill endurance. In all, these results describe a newly identified pathological abnormality in MTM, and uncover a potential disease-modifying therapy for this devastating disorder.

In mammalian skeletal muscle, the propagation of surface membrane depolarization into the interior of the muscle fibre along the transverse (T) tubular network is essential for the synchronized release of calcium from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyRs) in response to the conformational change in the voltage‐sensor dihydropyridine receptors. Deficiency in 3‐phosphoinositide phosphatase myotubularin (MTM1) has been reported to disrupt T‐tubules, resulting in impaired SR calcium release. Here confocal calcium transients recorded in muscle fibres of MTM1‐deficient mice were compared with the results from a model where propagation of the depolarization along the T‐tubules was modelled mathematically with disruptions in the network assumed to modify the access and transmembrane resistance as well as the capacitance. If, in simulations, T‐tubules were assumed to be partially or completely inaccessible to the depolarization and RyRs at these points to be prime for calcium‐induced calcium release, all the features of measured SR calcium release could be reproduced. We conclude that the inappropriate propagation of the depolarization into the fibre interior is the initial critical cause of severely impaired SR calcium release in MTM1 deficiency, while the Ca2+‐triggered opening of RyRs provides an alleviating support to the diseased process. Key points: Myotubular myopathy is a fatal disease due to genetic deficiency in the phosphoinositide phosphatase MTM1.Although the causes are known and corresponding gene therapy strategies are being developed, there is no mechanistic understanding of the disease‐associated muscle function failure.Resolving this issue is of primary interest not only for a fundamental understanding of how MTM1 is critical for healthy muscle function, but also for establishing the related cellular mechanisms most primarily or stringently affected by the disease, which are thus of potential interest as therapy targets.The mathematical modelling approach used in the present work proves that the disease‐associated alteration of the plasma membrane invagination network is sufficient to explain the dysfunctions of excitation–contraction coupling, providing the first integrated quantitative framework that explains the associated contraction failure. [ABSTRACT FROM AUTHOR]