Title: C9ORF72-related cellular pathology in skeletal muscle cells derived from ALS-patient induced pluripotent stem cells
Legend: (Top) Our workflow for in vitro modeling to study cellular pathology in skeletal myocytes derived from induced pluripotent stem (iPS) cells derived from familial ALS patients with C9ORF72 repeat expansion (C9-ALS). (Left bottom) Immunocytochemical images of titin-positive differentiated myocytes with sarcomere structure. (Middle bottom) Fluorescence in situ hybridization for GGGGCC-repeating RNA foci in C9-ALS myocytes. (Right bottom) Immunostaining for the dipeptide repeat protein poly-GR in the differentiated myocytes.
Citation: Lynch E, Semrad T, Belsito VS, FitzGibbons C, Reilly M, Hayakawa K, Suzuki M. C9ORF72-related cellular pathology in skeletal myocytes derived from ALS-patient induced pluripotent stem cells. Disease Models & Mechanisms. 2019 Aug;12(8): dmm039552. PubMed PMID: 31439573; PubMed Central PMCID: PMC6737948.
Abstract: Amyotrophic lateral sclerosis (ALS) is a late-onset neuromuscular disease with no cure and limited treatment options. Patients experience a gradual paralysis leading to death from respiratory complications on average only 2-5 years after diagnosis. There is increasing evidence that skeletal muscle is affected early in the disease process, yet the pathological processes occurring in the skeletal muscle of ALS patients are still mostly unknown. Specifically, the most common genetic cause of ALS, a hexanucleotide repeat expansion in the C9ORF72 gene, has yet to be fully characterized in the context of skeletal muscle. In this study, we used the protocol previously developed in our lab to differentiate skeletal myocytes from induced pluripotent stem cells (iPSCs) of C9ORF72 ALS (C9-ALS) patients in order to create an in vitro disease model of C9-ALS skeletal muscle pathology. Of the three C9ORF72mutation hallmarks, we did not see any evidence of haploinsufficiency, but we did detect RNA foci and dipeptide repeat (DPR) proteins. Additional abnormalities included changes in the expression of mitochondrial genes and a susceptibility to oxidative stress, indicating that mitochondrial dysfunction may be a critical feature of C9-ALS skeletal muscle pathology. Finally, the C9-ALS myocytes had increased expression and aggregation of TDP-43. Together, these data show that skeletal muscle cells experience pathological changes due to the C9ORF72 mutation. Our in vitro model could facilitate further study of cellular and molecular pathology in ALS skeletal muscle in order to discover new therapeutic targets against this devastating disease.
About the Lab: The Suzuki group has demonstrated the therapeutic benefits of ex vivo gene therapy (stem cell-based growth/trophic factor delivery) targeting the skeletal muscle to prevent degeneration of motor neurons and associated neuromuscular junctions during ALS. Although most ALS research has focused on mechanisms of motor neuron cell death, degeneration is also observed in skeletal muscle, particularly at the neuromuscular connection. Glial cell line-derived neurotrophic factor and vascular endothelial growth factor (VEGF) promote survival of motor neurons and their neuromuscular junctions in neuromuscular disorders, such as ALS. Most recently, the lab delivered a combination of GDNF and/or VEGF to muscles using hMSCs; the hMSCs survive and synthesize and release growth factors, which slow disease progression in familial ALS model rats.