Studies of Hereditary Neurological Disease: Disease Mechanisms

最近，我们的研究集中在两个遗传性运动神经元疾病：由于雄激素受体（AR）突变的脊髓和延髓肌萎缩症（SBMA）和由于senataxin（SETX）突变的肌萎缩侧索硬化症4型（ALS 4）。具体的研究成果包括： (1)在我们实验室开始并在NINDS的克雷格黑石博士和约翰霍普金斯的克里斯·罗斯博士的实验室继续进行的工作中，夏峰博士使用CRISPR-Cas9基因编辑来设计等基因人类诱导多能干细胞（hiPSC）模型，包括表达具有不同重复长度的突变体AR的等基因AR敲除、对照和疾病系，以及表达野生型和突变体AR的对照和疾病系，分别Feng博士采用小分子导向的方法，将等基因hiPSC模型分化为运动神经元样细胞的富集群体，以揭示SBMA背后的细胞类型特异性机制，并区分患病运动神经元中突变体AR的功能获得与丧失特性。Feng博士证明，由于毒性功能获得，无配体突变体AR导致分化疾病运动神经元的神经突中的线粒体功能障碍，并且这种细胞毒性可以在配体（雄激素）治疗后放大。她进一步表明，无配体的、位于线粒体的突变体AR和F-ATP合酶之间的异常相互作用与线粒体呼吸受损和其他线粒体损伤有关。这些发现反驳了SBMA中突变AR诱导的细胞毒性需要雄激素的既定观点，揭示了无配体突变AR，F-ATP合酶和线粒体功能障碍之间的机制联系，并为SBMA的运动神经元特异性治疗干预提供了见解。 (2)Synaptojanin 2 binding protein（SYNJ 2BP）是一种线粒体外膜蛋白，具有细胞溶质PDZ结构域，作为细胞信号传导枢纽。很少有研究评估它在疾病中的作用。我们使用了来自SBMA和ALS 4患者的iPSC衍生的运动神经元和死后组织，并表明SYNJ 2BP表达在患病的运动神经元中增加。类似地，我们发现SYNJ 2BP表达在经历应激的iPSC衍生的运动神经元中增加。使用蛋白质组学分析，我们发现升高的SYNJ 2BP改变了线粒体的细胞分布，并增加了线粒体-ER膜接触位点。此外，降低SYNJ 2BP水平改善了患病运动神经元中的线粒体氧化功能。总之，我们的观察为运动神经元疾病的分子病理学和SYNJ 2BP在线粒体功能障碍中的作用提供了新的见解。 (3)一个三链核酸结构，R环，越来越多地认识到其在基因调控中的作用。最初，R环被认为是转录的副产物;但最近发现患病细胞中的R环较少，这清楚地表明R环在各种人类细胞中具有功能性作用。了解R环的作用以及细胞如何平衡它们的丰度是很重要的。该领域的一个挑战是R环的定量，因为大部分工作依赖于S9.6单克隆抗体，其对RNA-DNA杂交体的特异性受到质疑。在这里，我们使用S9.6抗体进行斑点印迹来定量R环，并显示了该检测方法的灵敏度和特异性，其中RNase H、RNase T1和RNase III分别切割RNA-DNA杂交体、单链RNA和双链RNA。该方法重现性高，可在两天内提供结果。该检测可用于研究和临床环境中，以定量R环，并评估基因突变（如SETX）对R环丰度的影响。

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.