Recently our research has focused on two neuromuscular diseases: autosomal recessive spinal muscular atrophy (SMA) due to deficiency of the protein SMN, and spinal and bulbar muscular atrophy (SBMA) due to polyglutamine expansion in the androgen receptor. We also collaborated in a mass spectrometry study of iPS cells and in a study of the mechanism of hereditary neuropathy associated with mutation in GARS. Specific research accomplishments include the following: (1) Polyglutamine expansion in androgen receptor (AR) is responsible for SBMA and leads to selective loss of lower motor neurons. Using SBMA as a model, we explored the relationship between protein structure/function and neurodegeneration in polyglutamine diseases. We show here that protein arginine methyltransferase 6 (PRMT6) is a specific co-activator of normal and mutant AR and that the interaction of PRMT6 with AR is significantly enhanced in the AR mutant. AR and PRMT6 interaction occurs through the PRMT6 steroid receptor interaction motif, LXXLL, and the AR activating function 2 surface. AR transactivation requires PRMT6 catalytic activity and involves methylation of arginine residues at Akt consensus site motifs, which is mutually exclusive with serine phosphorylation by Akt. The enhanced interaction of PRMT6 and mutant AR leads to neurodegeneration in cell and fly models of SBMA. These findings demonstrate a direct role of arginine methylation in polyglutamine disease pathogenesis. (2) The emergence of new methods for reprogramming of adult somatic cells into induced pluripotent stem cells (iPSC) led to the development of new approaches in drug discovery and regenerative medicine. Investigation of the molecular mechanisms underlying the self-renewal, expansion and differentiation of human iPSC (hiPSC) should lead to improvements in the manufacture of safe and reliable cell therapy products. The goal of our study was qualitative and quantitative proteomic characterizations of hiPSC by means of electrospray ionization (ESI)-MS(e) and MALDI-TOF/TOF mass spectrometry (MS). Proteomes of hiPSCs of different somatic origins: fibroblasts and peripheral blood CD34(+) cells, reprogrammed by the same technique, were compared with the original somatic cells and hESC. Quantitative proteomic comparison revealed approximately 220 proteins commonly up-regulated in all three pluripotent stem cell lines compared to the primary cells. Expression of 21 proteins previously reported as pluripotency markers was up-regulated in both hiPSCs (8 were confirmed by Western blot). A number of novel candidate marker proteins with the highest fold-change difference between hiPSCs/hESC and somatic cells discovered by MS were confirmed by Western blot. A panel of 22 candidate marker proteins of hiPSC was developed and expression of these proteins was confirmed in 8 additional hiPSC lines. (3) Charcot-Marie-Tooth (CMT) neuropathies are collectively the most common hereditary neurological condition and a major health burden for society. Dominant mutations in the gene GARS, encoding the ubiquitous enzyme, glycyl-tRNA synthetase (GlyRS), cause peripheral nerve degeneration and lead to CMT disease type 2D. This genetic disorder exemplifies a recurring motif in neurodegeneration, whereby mutations in essential, widely expressed genes have selective deleterious consequences for the nervous system. Using novel Drosophila models, we showed a potential solution to this phenomenon. Ubiquitous expression of mutant GlyRS leads to motor deficits, progressive neuromuscular junction (NMJ) denervation and pre-synaptic build-up of mutant GlyRS. Intriguingly, neuronal toxicity is, at least in part, non-cell autonomous, as expression of mutant GlyRS in mesoderm or muscle alone results in similar pathology. This mutant GlyRS toxic gain-of-function, which is WHEP domain-dependent, coincides with abnormal NMJ assembly, leading to synaptic degeneration, and, ultimately, reduced viability. Our findings suggest that mutant GlyRS gains access to ectopic sub-compartments of the motor neuron, providing a possible explanation for the selective neuropathology caused by mutations in a widely expressed gene. Ref: Grice SJ, Sleigh JN, Motley WW, Liu JL, Burgess RW, Talbot K, Cader MZ: Dominant, toxic gain-of-function mutations in gars lead to non-cell autonomous neuropathology. Hum Mol Genet 24:4397-4406, 2015
| Wang, Isabel X; Grunseich, Christopher; Fox, Jennifer et al. (2018) Human proteins that interact with RNA/DNA hybrids. Genome Res 28:1405-1414 |
| Manzano, Raquel; Sorarú, Gianni; Grunseich, Christopher et al. (2018) Beyond motor neurons: expanding the clinical spectrum in Kennedy's disease. J Neurol Neurosurg Psychiatry 89:808-812 |
| Pourshafie, Naemeh; Lee, Philip R; Chen, Ke-Lian et al. (2018) Systemic Delivery of MicroRNA Using Recombinant Adeno-associated Virus Serotype 9 to Treat Neuromuscular Diseases in Rodents. J Vis Exp : |
| Bennett, Craig L; Dastidar, Somasish G; Ling, Shuo-Chien et al. (2018) Senataxin mutations elicit motor neuron degeneration phenotypes and yield TDP-43 mislocalization in ALS4 mice and human patients. Acta Neuropathol 136:425-443 |
| Guber, Robert D; Schindler, Alice B; Budron, Maher S et al. (2018) Nucleocytoplasmic transport defect in a North American patient with ALS8. Ann Clin Transl Neurol 5:369-375 |
| Fernandopulle, Michael S; Prestil, Ryan; Grunseich, Christopher et al. (2018) Transcription Factor-Mediated Differentiation of Human iPSCs into Neurons. Curr Protoc Cell Biol 79:e51 |
| Grunseich, Christopher; Wang, Isabel X; Watts, Jason A et al. (2018) Senataxin Mutation Reveals How R-Loops Promote Transcription by Blocking DNA Methylation at Gene Promoters. Mol Cell 69:426-437.e7 |
| Shi, Yingxiao; Lin, Shaoyu; Staats, Kim A et al. (2018) Haploinsufficiency leads to neurodegeneration in C9ORF72 ALS/FTD human induced motor neurons. Nat Med 24:313-325 |
| Plehn, Jonathan F; Hasbani, Keren; Ernst, Inez et al. (2017) The Subclinical Cardiomyopathy of Friedreich's Ataxia in a Pediatric Population. J Card Fail : |
| Guber, Robert D; Takyar, Varun; Kokkinis, Angela et al. (2017) Nonalcoholic fatty liver disease in spinal and bulbar muscular atrophy. Neurology 89:2481-2490 |
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