The current study characterizes a cohort of limb-girdle muscular dystrophy (LGMD) in the United States using whole exome sequencing. Fifty-five families affected by LGMD were recruited using an institutionally-approved protocol. Exome sequencing was performed on probands and selected parental samples. Pathogenic mutations and co-segregation patterns were confirmed by Sanger sequencing. Twenty-two families (40%) had novel and previously reported pathogenic mutations, primarily in LGMD genes, but also in genes for Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy, congenital myopathy, myofibrillar myopathy, inclusion body myopathy, and Pompe disease. One family was diagnosed via clinical testing. Dominant mutations were identified in COL6A1, COL6A3, FLNC, LMNA, RYR1, SMCHD1, and VCP, recessive mutations in ANO5, CAPN3, GAA, LAMA2, SGCA, and SGCG, and X-linked mutations in DMD. A previously reported variant in DMD was confirmed to be benign. Exome sequencing is a powerful diagnostic tool for LGMD. Despite careful phenotypic screening, pathogenic mutations were found in other muscle disease genes, largely accounting for the increased sensitivity of exome sequencing. Our experience suggests that broad sequencing panels are useful for these analyses due to the phenotypic overlap of many neuromuscular conditions. The confirmation of a benign DMD variant illustrates the potential of exome sequencing to help determine pathogenicity.

Abstract. Impaired lipid metabolism and inflammatory pathways have individually been implicated in the development of insulin resistance in skeletal muscle; however, little evidence is available to date linking the two in this context. In this study, we explored a potential molecular mechanism underlying insulin resistance in myoblasts mediated by the crosstalk between lipid accumulation and inflammatory pathways. We examined the influence of perilipin 2 (PLIN2), one of the most highly expressed lipid droplet-associated proteins in skeletal muscle, on glucose uptake and on the nucleotide-binding domain, leucine-rich repeat containing protein 3 (NLRP3) inflammasome in vitro. PLIN2 overexpression in C2C12 cells led to an increased expression of NLRP3, caspase-1 and interleukin (IL)-1β, along with an impaired insulin-induced glucose uptake. This defect was remedied by the RNAi-mediated knockdown of NLRP3 expression. We also found that insulin receptor substrate-1 (IRS-1), a component of insulin signaling, was negatively regulated by NLRP3 and IL-1β, and that IL-1β inhibited insulin-induced glucose uptake in myoblasts. These results suggest that PLIN2 inhibits insulin-induced glucose uptake by activating NLRP3, caspase-1 and IL-1β, leading to a decreased IRS-1 expression. This study provides in vitro evidence supporting an association between lipid metabolism and inflammatory pathways in the pathogenesis of insulin resistance in skeletal muscle, and suggests potential therapeutic targets that warrant further investigation.

Animal studies using chronic social defeat stress (CSDS) in mice showed that brain-derived neurotrophic factor (BDNF) signaling in the mesolimbic dopamine (DA) circuit is important for the development of social aversion. However, the downstream molecular targets after BDNF release from ventral tegmental area (VTA) DA terminals are unknown. Here, we show that depressive-like behaviors induced by CSDS are mediated in part by Gadd45b downstream of BDNF signaling in the nucleus accumbens (NAc). We show that Gadd45b mRNA levels are increased in susceptible but not resilient mice. Intra-NAc infusion of BDNF or optical stimulation of VTA DA terminals in NAc enhanced Gadd45b expression levels in the NAc. Importantly, Gadd45b downregulation reversed social avoidance in susceptible mice. Together, these data suggest that Gadd45b in NAc contributes to susceptibility to social stress. In addition, we investigated the function of Gadd45b in demethylating CpG islands of representative gene targets, which have been associated with a depressive phenotype in humans and animal models. We found that Gadd45b downregulation changes DNA methylation levels in a phenotype-, gene-, and locus-specific fashion. Together, these results highlight the contribution of Gadd45b and changes in DNA methylation in mediating the effects of social stress in the mesolimbic DA circuit.

Bone marrow (BM) transplantation (BMT) represents a curative treatment for various hematological disorders. Prior to BMT, a large amount of the relevant anticancer drug needed to be administered to eliminate cancer cells. However, during this pre-BMT cytotoxic conditioning regimen, hematopoietic stem cells in the BM and thymic epithelial cells were also destroyed. The T cell receptor (TcR) recognizes diverse pathogen, tumor and environmental antigens, and confers immunological memory and self-tolerance. delayed thymus reconstitution following pre-BMT cytotoxic conditioning impedes de novo thymopoiesis and limits T cell-mediated immunity. Several cytokines, such as RANK ligand, interleukin (IL)-7, IL-22 and stem cell factor, were recently reported to improve thymopoiesis and immune function following BMT. In the present study, it was found that the co-transplantation of tonsil-derived mesenchymal stromal cells (T-MScs) with BM-derived cells (BMcs) accelerated the recovery of involuted thymuses in mice following partial pre-BMT conditioning with busulfan-cyclophosphamide treatment, possibly by inducing FMS-like tyrosine kinase 3 ligand (FLT3L) and fibroblast growth factor 7 (FGF7) production in T-MScs. The co-transplantation of T-MScs with BMcs also replenished the cd3 + cell population by inhibiting thymocyte apoptosis following pre-BMT cytotoxic conditioning. Furthermore, T-MSc co-transplantation improved the recovery of the TcR repertoire and led to increased thymus-generated T cell diversity.

Type 2 diabetes mellitus (T2DM) is a prevalent chronic metabolic disorder accompanied by high blood glucose, insulin resistance, and relative insulin deficiency. Endoplasmic reticulum (ER) stress induced by high glucose and free fatty acids has been suggested as one of the main causes of β-cell dysfunction and death in T2DM. Stem cell-derived insulin-secreting cells were recently suggested as a novel therapy for diabetes. In the present study, we demonstrate the therapeutic potential of tonsil-derived mesenchymal stem cells (TMSCs) to treat high-fat diet (HFD)-induced T2DM. To explore whether TMSC administration can alleviate T2DM, TMSCs were intraperitoneally injected in HFD-induced T2DM mice once every 2 weeks. TMSC injection markedly improved glucose tolerance and glucose-stimulated insulin secretion and prevented HFD-induced pancreatic β-cell hypertrophy and cell death. In addition, TMSC injection relieved the ER-stress response and preserved gene expression related to glucose sensing and insulin secretion. Moreover, administration of TMSC-derived conditioned medium induced similar therapeutic outcomes, suggesting paracrine effects. Finally, proteomic analysis revealed high secretion of insulin-like growth factor-binding protein 5 by TMSCs, and its expression was critical for the protective effects of TMSCs against HFD-induced glucose intolerance and ER-stress response in pancreatic islets. TMSC administration can alleviate HFD-induced-T2DM via preserving pancreatic islets and their function. These results provide novel evidence of TMSCs as an ER-stress modulator that may be a novel, alternative cell therapy for T2DM.

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Reactive oxygen species (ROS) generated by neutrophils provide a frontline defence against invading pathogens. We investigated the supportive effect of tonsil-derived mesenchymal stem cells (TMSCs) on ROS generation from neutrophils using promyelocytic HL-60 cells. Methods: Differentiated HL-60 (dHL-60) cells were cocultured with TMSCs isolated from 25 independent donors, and ROS generation in dHL-60 cells was measured using luminescence. RNA sequencing and real-time PCR were performed to identify the candidate genes of TMSCs involved in augmenting the oxidative burst of dHL-60 cells. Transcriptome analysis of TMSCs derived from 25 independent donors revealed high levels of procollagen C-endopeptidase enhancer 2 (PCOLCE2) in TMSCs, which were highly effective in potentiating ROS generation in dHL-60 cells. In addition, PCOLCE2 knockdown in TMSCs abrogated TMSC-induced enhancement of ROS production in dHL-60 cells, indicating that TMSCs increased the oxidative burst in dHL-60 cells via PCOLCE2. Furthermore, the direct addition of recombinant PCOLCE2 protein increased ROS production in dHL-60 cells. These results suggest that PCOLCE2 secreted by TMSCs may be used as a therapeutic candidate to enhance host defences by increasing neutrophil oxidative bursts. PCOLCE2 levels in TMSCs could be used as a marker to select TMSCs exhibiting high efficacy for enhancing neutrophil oxidative bursts.