Studies of Hereditary Neurological Disease: Disease Mechanisms

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Recently our research has focused on two hereditary motor neuron diseases: spinal and bulbar muscular atrophy (SBMA) due to mutation in the androgen receptor (AR) and amyotrophic lateral sclerosis type 4 (ALS4) due to mutation in senataxin (SETX). Specific research accomplishments include the following: (1) In work started in our lab and continued in the labs of Dr. Craig Blackstone at NINDS and Dr. Chris Ross at Johns Hopkins, Dr. Xia Feng used CRISPR-Cas9 gene editing to engineer isogenic human induced pluripotent stem cell (hiPSC) models, consisting of isogenic AR knockout, control, and disease lines expressing mutant AR with distinct repeat lengths, as well as control and disease lines expressing wildtype and mutant AR, respectively. Adapting a small-molecule-directed approach, Dr. Feng differentiated the isogenic hiPSC models into an enriched population of motor neuron-like cells to uncover cell-type-specific mechanisms underlying SBMA and to distinguish gain- from loss-of-function properties of mutant AR in diseased motor neurons. Dr. Feng demonstrated that ligand-free mutant AR causes mitochondrial dysfunction in neurites of differentiated disease motor neurons due to toxic gain-of-function, and such cytotoxicity can be amplified upon ligand (androgen) treatment. She further showed that aberrant interaction between ligand-free, mitochondria-localized mutant AR and F-ATP synthase is associated with compromised mitochondrial respiration and other mitochondrial impairments. These findings counter the established notion that androgens are required for mutant AR-induced cytotoxicity in SBMA, reveal a mechanistic link between ligand-free mutant AR, F-ATP synthase, and mitochondrial dysfunction, and provide insights into motor neuron-specific therapeutic interventions for SBMA. (2) Synaptojanin 2 binding protein (SYNJ2BP) is an outer mitochondrial membrane protein with a cytosolic PDZ domain that functions as a cellular signaling hub. Few studies have evaluated its role in disease. We used iPSC-derived motor neurons and post-mortem tissue from patients with SBMA and ALS4, and showed that SYNJ2BP expression is increased in diseased motor neurons. Similarly, we showed that SYNJ2BP expression increases in iPSC-derived motor neurons undergoing stress. Using proteomic analysis, we found that elevated SYNJ2BP alters the cellular distribution of mitochondria and increases mitochondrial-ER membrane contact sites. Furthermore, decreasing SYNJ2BP levels improves mitochondrial oxidative function in the diseased motor neurons. Together, our observations offer new insight into the molecular pathology of motor neuron disease and the role of SYNJ2BP in mitochondrial dysfunction. (3) A three-stranded nucleic acid structure, the R-loop, is increasingly recognized for its role in gene regulation. Initially, R-loops were thought to be by-products of transcription; but recent findings of fewer R-loops in diseased cells made it clear that R-loops have functional roles in a variety of human cells. It is important to understand the roles of R-loops and how cells balance their abundance. A challenge in the field is the quantitation of R-loops since much of the work relies on the S9.6 monoclonal antibody, the specificity of which for RNA-DNA hybrids has been questioned. Here, we use dot-blots with the S9.6 antibody to quantify R-loops and show the sensitivity and specificity of this assay with RNase H, RNase T1, and RNase III that cleave RNA-DNA hybrids, single-stranded RNA, and double-stranded RNA, respectively. This method is highly reproducible and provides results within two days. This assay can be used in research and clinical settings to quantify R-loops and assess the effect of mutations in genes such as SETX on R-loop abundance.