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 have also supported work by Dr. Ami Mankodi's lab on myofibrillar myopathy caused by mutations in ZASP, and collaborated in studies of Charcot-Marie-Tooth disease (CMT), hereditary spastic paraplegia, ALS4, and ataxia with oculomotor apraxia type 2. Specific research accomplishments include the following: (1) SMA is one of the most common severe hereditary diseases of infancy and early childhood in North America, Europe, and Asia. SMA is usually caused by deletions of the SMN1 gene. A closely related gene, SMN2, modifies the disease severity. SMA carriers have only 1 copy of SMN1 and are relatively common (1 in 30-50) in populations of European and Asian descent. SMN copy numbers and SMA carrier frequencies have not been reliably estimated in Malians and other sub-Saharan Africans. We used a quantitative polymerase chain reaction assay to determine SMN1 and SMN2 copy numbers in 628 Malians, 120 Nigerians, and 120 Kenyans. We also explored possible mechanisms for SMN1 and SMN2 copy number differences in Malians, and investigated their effects on SMN mRNA and protein levels. The SMA carrier frequency in Malians is 1 in 209, lower than in Eurasians. Malians and other sub-Saharan Africans are more likely to have ≥3 copies of SMN1 than Eurasians, and more likely to lack SMN2 than Europeans. There was no evidence of gene conversion, gene locus duplication, or natural selection from malaria resistance to account for the higher SMN1 copy numbers in Malians. High SMN1 copy numbers were not associated with increased SMN mRNA or protein levels in human cell lines. SMA carrier frequencies are much lower in sub-Saharan Africans than in Eurasians. This finding is important to consider in SMA genetic counseling in individuals with black African ancestry. (2) While SMA is characterized by motor neuron degeneration, it is unclear whether and how much SMN protein deficiency in muscle contributes to the pathophysiology of the disease. There is increasing evidence from patients and SMA model organisms that SMN deficiency causes intrinsic muscle defects. Here we investigated the role of SMN in muscle development using muscle cell lines and primary myoblasts. Formation of multinucleate myotubes by SMN-deficient muscle cells is inhibited at a stage preceding plasma membrane fusion. We found increased expression and reduced induction of key muscle development factors, such as MyoD and myogenin, with differentiation of SMN-deficient cells. In addition, SMN-deficient muscle cells had impaired cell migration and altered organization of focal adhesions and the actin cytoskeleton. Partially restoring SMN inhibited the premature expression of muscle differentiation markers, corrected the cytoskeletal abnormalities and improved myoblast fusion. These findings are consistent with a role for SMN in myotube formation through effects on muscle differentiation and cell motility. (3) Although in SBMA degeneration occurs in the spinal cord and muscle, the exact mechanism is not clear. Induced pluripotent stem cells from spinal and bulbar muscular atrophy patients provide a useful model for understanding the disease mechanism and designing effective therapy. Stem cells were generated from six patients and compared to control lines from three healthy individuals. Motor neurons from four patients were differentiated from stem cells and characterized to understand disease-relevant phenotypes. Stem cells created from patient fibroblasts express less androgen receptor than control cells, but show androgen-dependent stabilization and nuclear translocation. The expanded repeat in several stem cell clones was unstable, with either expansion or contraction. Patient stem cell clones produced a similar number of motor neurons compared to controls, with or without androgen treatment. The stem cell-derived motor neurons had immunoreactivity for HB9, Isl1, ChAT, and SMI-32, and those with the largest repeat expansions were found to have increased acetylated α-tubulin and reduced HDAC6. Reduced HDAC6 was also found in motor neuron cultures from two other patients with shorter repeats. Evaluation of stably transfected mouse cells and SBMA spinal cord showed similar changes in acetylated α-tubulin and HDAC6. Perinuclear lysosomal enrichment, an HDAC6 dependent process, was disrupted in motor neurons from two patients with the longest repeats. SBMA stem cells present new insights into the disease, and the observations of reduced androgen receptor levels, repeat instability, and reduced HDAC6 provide avenues for further investigation of the disease mechanism and development of effective therapy. (4) The core of skeletal muscle Z-discs consists of actin filaments from adjacent sarcomeres that are cross-linked by α-actinin homodimers. ZASP interacts with α-actinin, myotilin, and other Z-disc proteins via the PDZ domain. However, these interactions are not sufficient to maintain the Z-disc structure. We showed that ZASP directly interacts with skeletal actin filaments. The actin-binding domain is between the modular PDZ and LIM domains. This ZASP region is alternatively spliced so that each isoform has unique actin-binding domains. All ZASP isoforms contain the exon 6-encoded ZASP-like motif that is mutated in zaspopathy, a myofibrillar myopathy (MFM), whereas the exon 8-11 junction-encoded peptide is exclusive to the postnatal long ZASP isoform (ZASP-LΔex10). MFM is characterized by disruption of skeletal muscle Z-discs and accumulation of myofibrillar degradation products. Wild-type and mutant ZASP interact with α-actin, α-actinin, and myotilin. Expression of mutant, but not wild-type, ZASP leads to Z-disc disruption and F-actin accumulation in mouse skeletal muscle, as in MFM. Mutations in the actin-binding domain of ZASP-LΔex10, but not other isoforms, cause disruption of the actin cytoskeleton in muscle cells. These isoform-specific mutation effects highlight the essential role of the ZASP-LΔex10 isoform in F-actin organization. Our results show that MFM-associated ZASP mutations in the actin-binding domain have deleterious effects on the core structure of the Z-discs in skeletal muscle. (5) Mutations in six tRNA synthetases specifically affect the peripheral nerves and cause CMT. The CMT-causing mutations in tyrosyl- and glycyl-tRNA synthetases (YARS and GARS, respectively) alter the activity of the proteins in a range of ways (some mutations do not impact charging function, while others abrogate it), making a loss of function in tRNA charging unlikely to be the cause of disease pathology. It is currently unknown which cellular mechanisms are triggered by the mutant enzymes and how this leads to neurodegeneration. Here, by expressing two pathogenic mutations (G240R, P234KY) in Drosophila, we generated a model for GARS-associated neuropathy. We observed compromised viability, and behavioral, electrophysiological and morphological impairment in flies expressing the cytoplasmic isoform of mutant GARS. Their features recapitulated several hallmarks of CMT pathophysiology and were similar to the phenotypes identified in our previously described Drosophila model of YARS-associated neuropathy. Furthermore, CG8316 and CG15599 - genes identified in a retinal degeneration screen to modify mutant YARS, also modified the mutant GARS phenotypes. Our study presents genetic evidence for common mutant-specific interactions between two CMT-associated aminoacyl-tRNA synthetases, lending support for a shared mechanism responsible for the synthetase-induced peripheral neuropathies.
| 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 |
| 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 |
| 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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