Dystrophin glycoprotein complex dysfunction:a regulatory link between muscular dystrophy and cancer cachexia

SummaryCachexia contributes to nearly a third of all cancer deaths, yet the mechanisms underlying skeletal muscle wasting in this syndrome remain poorly defined. We report that tumor-induced alterations in the muscular dystrophy-associated dystrophin glycoprotein complex (DGC) represent a key early event in cachexia. Muscles from tumor-bearing mice exhibited membrane abnormalities accompanied by reduced levels of dystrophin and increased glycosylation on DGC proteins. Wasting was accentuated in tumor mdx mice lacking a DGC but spared in dystrophin transgenic mice that blocked induction of muscle E3 ubiquitin ligases. Furthermore, DGC deregulation correlated positively with cachexia in patients with gastrointestinal cancers. Based on these results, we propose that, similar to muscular dystrophy, DGC dysfunction plays a critical role in cancer-induced wasting

Impaired regeneration in LGMD2A supported by increased Pax7 positive satellite cell content and muscle specific microRNA dysregulation

Introduction—Recent in vitro studies suggest that CAPN3 deficiency leads initially to accelerated myofiber formation followed by depletion of satellite cells (SC). In normal muscle, upregulation of miR-1 and miR-206 facilitates transition from proliferating SCs to differentiating myogenic progenitors. Methods—We examined the histopathological stages, Pax7 SC content, and muscle specific microRNA expression in biopsy specimens from well-characterized LGMD 2A patients to gain insight into disease pathogenesis. Results—Three distinct stages of pathological changes were identified that represented the continuum of the dystrophic process from prominent inflammation with necrosis and regeneration to prominent fibrosis, which correlated with age and disease duration. Pax7-positive SCs were highest in fibrotic group and correlated with down-regulation of miR-1, miR-133a, and miR-206. Conclusions—These observations, and other published reports, are consistent with microRNA dysregulation leading to inability of Pax7-positive SCs to transit from proliferation to differentiation. This results in impaired regeneration and fibrosis.This work was supported by NIH NIAMS U54 AR050733-05, Jesse’s Journey, and the muscular Dystrophy Associatio

Persistent Expression of FLAG-tagged Micro dystrophin in Nonhuman Primates Following Intramuscular and Vascular Delivery

Animal models for Duchenne muscular dystrophy (DMD) have species limitations related to assessing function, immune response, and distribution of micro- or mini-dystrophins. Nonhuman primates (NHPs) provide the ideal model to optimize vector delivery across a vascular barrier and provide accurate dose estimates for widespread transduction. To address vascular delivery and dosing in rhesus macaques, we have generated a fusion construct that encodes an eight amino-acid FLAG epitope at the C-terminus of micro-dystrophin to facilitate translational studies targeting DMD. Intramuscular (IM) injection of AAV8.MCK.micro-dys.FLAG in the tibialis anterior (TA) of macaques demonstrated robust gene expression, with muscle transduction (50–79%) persisting for up to 5 months. Success by IM injection was followed by targeted vascular delivery studies using a fluoroscopy-guided catheter threaded through the femoral artery. Three months after gene transfer, >80% of muscle fibers showed gene expression in the targeted muscle. No cellular immune response to AAV8 capsid, micro-dystrophin, or the FLAG tag was detected by interferon-γ (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) at any time point with either route. In summary, an epitope-tagged micro-dystrophin cassette enhances the ability to evaluate site-specific localization and distribution of gene expression in the NHP in preparation for vascular delivery clinical trials

Gene Delivery to Spinal Motor Neurons

This study demonstrates the direct delivery of plasmid gene constructs into spinal motor neurons utilizing retrograde axoplasmic transport. The plasmid vectors contained the Lac Z gene under the control of both the Rous sarcoma virus (RSV) and Simian virus (SV)40 promoters. β-Galactosidase expression was observed in α and γ motor neurons by histochemical staining following direct injection into the sciatic nerve or gastrocnemius muscle. The presence of LacZ gene constructs was confirmed by the polymerase chain reaction (PCR). The ability to introduce gene constructs into motor neurons allows for the study of gene regulation and permits the development of gene therapy strategies for motor neuron diseases including the spinal muscular atrophies (SMA) and amyotrophic lateral sclerosis (ALS)

Mutation of FIG4 causes a rapidly progressive, asymmetric neuronal degeneration

Recessive Charcot-Marie-Tooth disease type-4J (CMT4J) and its animal model, the pale tremor mouse (plt), are caused by mutations of the FIG4 gene encoding a PI(3,5)P2 5-phosphatase. We describe the 9-year clinical course of CMT4J, including asymmetric, rapidly progressive paralysis, in two siblings. Sensory symptoms were absent despite reduced numbers of sensory axons. Thus, the phenotypic presentation of CMT4J clinically resembles motor neuron disease. Time-lapse imaging of fibroblasts from CMT4J patients demonstrates impaired trafficking of intracellular organelles because of obstruction by vacuoles. Further characterization of plt mice identified axonal degeneration in motor and sensory neurons, limited segmental demyelination, lack of TUNEL staining and lack of accumulation of ubiquitinated protein in vacuoles of motor and sensory neurons. This study represents the first documentation of the natural history of CMT4J. Physical obstruction of organelle trafficking by vacuoles is a potential novel cellular mechanism of neurodegeneration

Impaired regeneration in calpain-3 null muscle is associated with perturbations in mTORC1 signaling and defective mitochondrial biogenesis

Abstract Background Previous studies in patients with limb-girdle muscular dystrophy type 2A (LGMD2A) have suggested that calpain-3 (CAPN3) mutations result in aberrant regeneration in muscle. Methods To gain insight into pathogenesis of aberrant muscle regeneration in LGMD2A, we used a paradigm of cardiotoxin (CTX)-induced cycles of muscle necrosis and regeneration in the CAPN3-KO mice to simulate the early features of the dystrophic process in LGMD2A. The temporal evolution of the regeneration process was followed by assessing the oxidative state, size, and the number of metabolic fiber types at 4 and 12 weeks after last CTX injection. Muscles isolated at these time points were further investigated for the key regulators of the pathways involved in various cellular processes such as protein synthesis, cellular energy status, metabolism, and cell stress to include Akt/mTORC1 signaling, mitochondrial biogenesis, and AMPK signaling. TGF-β and microRNA (miR-1, miR-206, miR-133a) regulation were also assessed. Additional studies included in vitro assays for quantifying fusion index of myoblasts from CAPN3-KO mice and development of an in vivo gene therapy paradigm for restoration of impaired regeneration using the adeno-associated virus vector carrying CAPN3 gene in the muscle. Results At 4 and 12 weeks after last CTX injection, we found impaired regeneration in CAPN3-KO muscle characterized by excessive numbers of small lobulated fibers belonging to oxidative metabolic type (slow twitch) and increased connective tissue. TGF-β transcription levels in the regenerating CAPN3-KO muscles were significantly increased along with microRNA dysregulation compared to wild type (WT), and the attenuated radial growth of muscle fibers was accompanied by perturbed Akt/mTORC1 signaling, uncoupled from protein synthesis, through activation of AMPK pathway, thought to be triggered by energy shortage in the CAPN3-KO muscle. This was associated with failure to increase mitochondria content, PGC-1α, and ATP5D transcripts in the regenerating CAPN3-KO muscles compared to WT. In vitro studies showed defective myotube fusion in CAPN3-KO myoblast cultures. Replacement of CAPN3 by gene therapy in vivo increased the fiber size and decreased the number of small oxidative fibers. Conclusion Our findings provide insights into understanding of the impaired radial growth phase of regeneration in calpainopathy