La coordinación entre esfingolípidos y fosfoinosítidos es esencial para la diferenciación de las células tubulares renales

La diferenciación de las células epiteliales se produce por maduración de la membrana apical y desarrollo de cilio primario. Resultados previos de nuestro laboratorio demostraron que la hipertonicidad extracelular induce la diferenciación de las células MDCK, una línea celular derivada de túbulos colectores renales caninos, y que en este proceso son esenciales los esfingolípidos. Por otro lado, los fosfoinosítidos, otros lípidos bioactivos, son considerados determinantes de la polaridad apico - basal, dada su ubicación en dominios específicos de membrana en células epiteliales polarizadas: con PI(4,5)P2 localizado en el dominio apical y PI(3,4,5)P3 en el basolateral. Esta ubicación específica es la que determina el reclutamiento de complejos importantes de polaridad, como el Par3/Par6/aPKC.Fil: Pescio Lucila G.. Universidad de Buenos AiresFil: Romero, Daniela J.. Universidad de Buenos AiresFil: Santacreu, Bruno J.. Universidad de Buenos AiresFil: Francisco, María N..Fil: Favale, Nicolás O.. Universidad de Buenos AiresFil: Sterin-Speziale, Norma B.. Universidad de Buenos Aire

Human Skeletal myopathy myosin mutations disrupt myosin head sequestration

Myosin heavy chains encoded by MYH7 and MYH2 are abundant in human skeletal muscle, and important for muscle contraction. However, it is unclear how mutations in these genes disrupt myosin structure and function leading to skeletal muscle myopathies termed myosinopathies. Here, we used multiple approaches to analyse the effects of common MYH7 and MYH2 mutations in the light meromyosin region of myosin (LMM). Analyses of expressed and purified MYH7 and MYH2 LMM mutant proteins combined with in-silico modelling showed that myosin coiled-coil structure and packing of filaments in vitro are commonly disrupted. Using muscle biopsies from patients, and Mant-ATP chase protocols to estimate the proportion of myosin heads that were super-relaxed, together with X-ray diffraction measurements to estimate myosin head order we found that basal myosin ATP consumption was increased and the myosin super-relaxed state was decreased in vivo. In addition, myofibre mechanics experiments to investigate contractile function showed myofibre contractility was not affected. These findings indicate that the structural remodelling associated with LMM mutations induces a pathogenic state in which formation of shutdown heads is impaired, thus increasing myosin head ATP demand in the filaments, rather than affecting contractility. These key findings will help design future therapies for myosinopathies

Muscle histopathology in nebulin-related nemaline myopathy : ultrastrastructural findings correlated to disease severity and genotype

Peer reviewe

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

Mild clinical presentation in KLHL40-related nemaline myopathy (NEM 8).

Nemaline myopathies are clinically and genetically heterogeneous muscle diseases characterized by the presence of nemaline bodies (rods) in muscle fibers. Mutations in the KLHL40 (kelch-like family member 40) gene (NEM 8) are common cause of severe/lethal nemaline myopathy. We report an 8-year-old girl born to consanguineous Moroccan parents, who presented with hypotonia and poor sucking at birth, delayed motor development, and further mild difficulties in walking and fatigability. A muscle biopsy revealed the presence of nemaline bodies. KLHL40 gene Sanger sequencing disclosed a never before reported pathogenic homozygous mutation which resulted in absent KLHL40 protein expression in the muscle. This further expands the phenotypical spectrum of KLHL40 related nemaline myopathy

Missense mutations in small muscle protein X-linked (SMPX) cause distal myopathy with protein inclusions

Using deep phenotyping and high-throughput sequencing, we have identified a novel type of distal myopathy caused by mutations in the Small muscle protein X-linked (SMPX) gene. Four different missense mutations were identified in ten patients from nine families in five different countries, suggesting that this disease could be prevalent in other populations as well. Haplotype analysis of patients with similar ancestry revealed two different founder mutations in Southern Europe and France, indicating that the prevalence in these populations may be higher. In our study all patients presented with highly similar clinical features: adult-onset, usually distal more than proximal limb muscle weakness, slowly progressing over decades with preserved walking. Lower limb muscle imaging showed a characteristic pattern of muscle involvement and fatty degeneration. Histopathological and electron microscopic analysis of patient muscle biopsies revealed myopathic findings with rimmed vacuoles and the presence of sarcoplasmic inclusions, some with amyloid-like characteristics. In silico predictions and subsequent cell culture studies showed that the missense mutations increase aggregation propensity of the SMPX protein. In cell culture studies, overexpressed SMPX localized to stress granules and slowed down their clearance.Peer reviewe

Myoglobinopathy is an adult-onset autosomal dominant myopathy with characteristic sarcoplasmic inclusions

Myoglobin, encoded by MB, is a small cytoplasmic globular hemoprotein highly expressed in cardiac myocytes and oxidative skeletal myofibers. Myoglobin binds O-2, facilitates its intracellular transport and serves as a controller of nitric oxide and reactive oxygen species. Here, we identify a recurrent c.292C>T ( p.His98Tyr) substitution in MB in fourteen members of six European families suffering from an autosomal dominant progressive myopathy with highly characteristic sarcoplasmic inclusions in skeletal and cardiac muscle. Myoglobinopathy manifests in adulthood with proximal and axial weakness that progresses to involve distal muscles and causes respiratory and cardiac failure. Biochemical characterization reveals that the mutant myoglobin has altered O-2 binding, exhibits a faster heme dissociation rate and has a lower reduction potential compared to wild-type myoglobin. Preliminary studies show that mutant myoglobin may result in elevated superoxide levels at the cellular level. These data define a recognizable muscle disease associated with MB mutation.Peer reviewe

Class-modeling analysis reveals T-cell homeostasis disturbances involved in loss of immune control in elite controllers

Despite long-lasting HIV replication control, a significant proportion of elite controller (EC) patients may experience CD4 T-cell loss. Discovering perturbations in immunological parameters could help our understanding of the mechanisms that may be operating in those patients experiencing loss of immunological control. Methods A case–control study was performed to evaluate if alterations in different T-cell homeostatic parameters can predict CD4 T-cell loss in ECs by comparing data from EC patients showing significant CD4 decline (cases) and EC patients showing stable CD4 counts (controls). The partial least-squares–class modeling (PLS-CM) statistical methodology was employed to discriminate between the two groups of patients, and as a predictive model. Results Herein, we show that among T-cell homeostatic alterations, lower levels of naïve and recent thymic emigrant subsets of CD8 cells and higher levels of effector and senescent subsets of CD8 cells as well as higher levels of exhaustion of CD4 cells, measured prior to CD4 T-cell loss, predict the loss of immunological control. Conclusions These data indicate that the parameters of T-cell homeostasis may identify those EC patients with a higher proclivity to CD4 T-cell loss. Our results may open new avenues for understanding the mechanisms underlying immunological progression despite HIV replication control, and eventually, for finding a functional cure through immune-based clinical trials.projects RD12/0017/0031, RD16/0025/ 0013, and SAF2015-66193-R as part of the Health Research and Development Strategy, State Plan for Scientific and Technical Research and Innovation (2008– 2011 and 2013–2016) and cofinanced by the Institute of Health Carlos III (ISCIII), Sub-Directorate General for Research Assessment and Promotion and European Regional Development Fund. NR is a Miguel Servet investigator from the ISCIII (CP14/00198), Madrid, Spain. C Restrepo was funded by project RD12/0017/ 0031 and is currently funded by project RD16/0025/0013. M García is a predoctoral student co-funded by grant CP14/00198 and an Intramural Research Scholarship from Instituto de Investigación Sanitaria-Fundación Jiménez Díaz (IIS-FJD)