Inactivating the lipid kinase activity of PI3KC2β is sufficient to rescue myotubular myopathy in mice

Phosphoinositides (PI) are membrane lipids that regulate signal transduction and vesicular trafficking. X-linked centronuclear myopathy (XLCNM), also called myotubular myopathy, results from loss-of-function mutations in the Mtm1 gene, which encodes the myotubularin phosphatidylinositol 3-phosphate (PtdIns3P) lipid phosphatase. No therapy for this disease is currently available. Previous studies showed that loss of expression of the class II phosphoinositide 3-kinase (PI3K) PI3K-C2β protein improved the phenotypes of a XLCNM mouse model. PI3Ks are well known to have extensive scaffolding functions and the importance of the catalytic activity of this PI3K for rescue remains unclear. Here, using PI3K-C2β kinase-dead mice, we show that the selective inactivation of PI3K-C2β kinase activity is sufficient to fully prevent muscle atrophy and weakness, histopathology, and sarcomere and triad disorganization in Mtm1 knockout mice. This rescue correlates with normalization of PtdIns3P level and mTORC1 activity, a key regulator of protein synthesis and autophagy. Conversely, lack of PI3K-C2β kinase activity did not rescue the histopathology of the BIN1 autosomal centronuclear myopathy mouse model. Overall, these findings support the development of specific PI3K-C2β kinase inhibitors to cure myotubular myopathy

Expression of the neuropathy-associated MTMR2 gene rescues MTM1-associated myopathy

Myotubularins (MTMs) are active or dead phosphoinositides phosphatases defining a large protein family conserved through evolution and implicated in different neuromuscular diseases. Loss-of-function mutations in MTM1 cause the severe congenital myopathy called myotubular myopathy (or X-linked centronuclear myopathy) while mutations in the MTM1-related protein MTMR2 cause a recessive Charcot-Marie-Tooth peripheral neuropathy. Here we aimed to determine the functional specificity and redundancy of MTM1 and MTMR2, and to assess their abilities to compensate for a potential therapeutic strategy. Using molecular investigations and heterologous expression of human MTMs in yeast cells and in Mtm1 knockout mice, we characterized several naturally occurring MTMR2 isoforms with different activities. We identified the N-terminal domain as responsible for functional differences between MTM1 and MTMR2. An N-terminal extension observed in MTMR2 is absent in MTM1, and only the short MTMR2 isoform lacking this N-terminal extension behaved similarly to MTM1 in yeast and mice. Moreover, adeno-associated virus-mediated exogenous expression of several MTMR2 isoforms ameliorates the myopathic phenotype owing to MTM1 loss, with increased muscle force, reduced myofiber atrophy, and reduction of the intracellular disorganization hallmarks associated with myotubular myopathy. Noteworthy, the short MTMR2 isoform provided a better rescue when compared with the long MTMR2 isoform. In conclusion, these results point to the molecular basis for MTMs functional specificity. They also provide the proof-of-concept that expression of the neuropathy-associated MTMR2 gene improves the MTM1-associated myopathy, thus identifying MTMR2 as a novel therapeutic target for myotubular myopathy

Dissecting the autism-associated 16p11.2 locus identifies multiple drivers in neuroanatomical phenotypes and unveils a male-specific role for the major vault protein

Background Using mouse genetic studies and systematic assessments of brain neuroanatomical phenotypes, we set out to identify which of the 30 genes causes brain defects at the autism-associated 16p11.2 locus. Results We show that multiple genes mapping to this region interact to regulate brain anatomy, with female mice exhibiting far fewer brain neuroanatomical phenotypes. In male mice, among the 13 genes associated with neuroanatomical defects (Mvp, Ppp4c, Zg16, Taok2, Slx1b, Maz, Fam57b, Bola2, Tbx6, Qprt, Spn, Hirip3, and Doc2a), Mvp is the top driver implicated in phenotypes pertaining to brain, cortex, hippocampus, ventricles, and corpus callosum sizes. The major vault protein (MVP), the main component of the vault organelle, is a conserved protein found in eukaryotic cells, yet its function is not understood. Here, we find MVP expression highly specific to the limbic system and show that Mvp regulates neuronal morphology, postnatally and specifically in males. We also recapitulate a previously reported genetic interaction and show that Mvp+/−;Mapk3+/− mice exhibit behavioral deficits, notably decreased anxiety-like traits detected in the elevated plus maze and open field paradigms. Conclusions Our study highlights multiple gene drivers in neuroanatomical phenotypes, interacting with each other through complex relationships. It also provides the first evidence for the involvement of the major vault protein in the regulation of brain size and neuroanatomy, specifically in male mice

AAV-mediated intramuscular delivery of myotubularin corrects the myotubular myopathy phenotype in targeted murine muscle and suggests a function in plasma membrane homeostasis

Myotubular myopathy (XLMTM, OMIM 310400) is a severe congenital muscular disease due to mutations in the myotubularin gene (MTM1) and characterized by the presence of small myofibers with frequent occurrence of central nuclei. Myotubularin is a ubiquitously expressed phosphoinositide phosphatase with a muscle-specific role in man and mouse that is poorly understood. No specific treatment exists to date for patients with myotubular myopathy. We have constructed an adeno-associated virus (AAV) vector expressing myotubularin in order to test its therapeutic potential in a XLMTM mouse model. We show that a single intramuscular injection of this vector in symptomatic Mtm1-deficient mice ameliorates the pathological phenotype in the targeted muscle. Myotubularin replacement in mice largely corrects nuclei and mitochondria positioning in myofibers and leads to a strong increase in muscle volume and recovery of the contractile force. In addition, we used this AAV vector to overexpress myotubularin in wild-type skeletal muscle and get insight into its localization and function. We show that a substantial proportion of myotubularin associates with the sarcolemma and I band, including triads. Myotubularin overexpression in muscle induces the accumulation of packed membrane saccules and presence of vacuoles that contain markers of sarcolemma and T-tubules, suggesting that myotubularin is involved in plasma membrane homeostasis of myofibers. This study provides a proof-of-principle that local delivery of an AAV vector expressing myotubularin can improve the motor capacities of XLMTM muscle and represents a novel approach to study myotubularin function in skeletal muscle

Models of <i>KPTN</i>-related disorder implicate mTOR signalling in cognitive and overgrowth phenotypes

KPTN-related disorder is an autosomal recessive disorder associated with germline variants in KPTN (previously known as kaptin), a component of the mTOR regulatory complex KICSTOR. To gain further insights into the pathogenesis of KPTN-related disorder, we analysed mouse knockout and human stem cell KPTN loss-of-function models. Kptn -/- mice display many of the key KPTN-related disorder phenotypes, including brain overgrowth, behavioural abnormalities, and cognitive deficits. By assessment of affected individuals, we have identified widespread cognitive deficits (n = 6) and postnatal onset of brain overgrowth (n = 19). By analysing head size data from their parents (n = 24), we have identified a previously unrecognized KPTN dosage-sensitivity, resulting in increased head circumference in heterozygous carriers of pathogenic KPTN variants. Molecular and structural analysis of Kptn-/- mice revealed pathological changes, including differences in brain size, shape and cell numbers primarily due to abnormal postnatal brain development. Both the mouse and differentiated induced pluripotent stem cell models of the disorder display transcriptional and biochemical evidence for altered mTOR pathway signalling, supporting the role of KPTN in regulating mTORC1. By treatment in our KPTN mouse model, we found that the increased mTOR signalling downstream of KPTN is rapamycin sensitive, highlighting possible therapeutic avenues with currently available mTOR inhibitors. These findings place KPTN-related disorder in the broader group of mTORC1-related disorders affecting brain structure, cognitive function and network integrity.</p

An integrated diagnosis strategy for congenital myopathies

Congenital myopathies are severe muscle disorders affecting adults as well as children in all populations. The diagnosis of congenital myopathies is constrained by strong clinical and genetic heterogeneity. Moreover, the majority of patients present with unspecific histological features, precluding purposive molecular diagnosis and demonstrating the need for an alternative and more efficient diagnostic approach. We used exome sequencing complemented by histological and ultrastructural analysis of muscle biopsies to identify the causative mutations in eight patients with clinically different skeletal muscle pathologies, ranging from a fatal neonatal myopathy to a mild and slowly progressive myopathy with adult onset. We identified RYR1 (ryanodine receptor) mutations in six patients and NEB (nebulin) mutations in two patients. We found novel missense and nonsense mutations, unraveled small insertions/deletions and confirmed their impact on splicing and mRNA/protein stability. Histological and ultrastructural findings of the muscle biopsies of the patients validated the exome sequencing results. We provide the evidence that an integrated strategy combining exome sequencing with clinical and histopathological investigations overcomes the limitations of the individual approaches to allow a fast and efficient diagnosis, accelerating the patient's access to a better healthcare and disease management. This is of particular interest for the diagnosis of congenital myopathies, which involve very large genes like RYR1 and NEB as well as genetic and phenotypic heterogeneity

Azimuthal anisotropy of charged jet production in root s(NN)=2.76 TeV Pb-Pb collisions

We present measurements of the azimuthal dependence of charged jet production in central and semi-central root s(NN) = 2.76 TeV Pb-Pb collisions with respect to the second harmonic event plane, quantified as nu(ch)(2) (jet). Jet finding is performed employing the anti-k(T) algorithm with a resolution parameter R = 0.2 using charged tracks from the ALICE tracking system. The contribution of the azimuthal anisotropy of the underlying event is taken into account event-by-event. The remaining (statistical) region-to-region fluctuations are removed on an ensemble basis by unfolding the jet spectra for different event plane orientations independently. Significant non-zero nu(ch)(2) (jet) is observed in semi-central collisions (30-50% centrality) for 20 <p(T)(ch) (jet) <90 GeV/c. The azimuthal dependence of the charged jet production is similar to the dependence observed for jets comprising both charged and neutral fragments, and compatible with measurements of the nu(2) of single charged particles at high p(T). Good agreement between the data and predictions from JEWEL, an event generator simulating parton shower evolution in the presence of a dense QCD medium, is found in semi-central collisions. (C) 2015 CERN for the benefit of the ALICE Collaboration. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).Peer reviewe

Forward-central two-particle correlations in p-Pb collisions at root s(NN)=5.02 TeV

Two-particle angular correlations between trigger particles in the forward pseudorapidity range (2.5 2GeV/c. (C) 2015 CERN for the benefit of the ALICE Collaboration. Published by Elsevier B. V.Peer reviewe

Event-shape engineering for inclusive spectra and elliptic flow in Pb-Pb collisions at root(NN)-N-S=2.76 TeV

Peer reviewe