Studies of Hereditary Neurological Disease: Disease Mechanisms

遗传性神经系统疾病的研究：疾病机制

基本信息

批准号：
10708607
负责人：
Kenneth Fischbeck
金额：
$ 39.11万
依托单位：
NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10708607
关键词：
Amyotrophic Lateral Sclerosis Androgen Receptor Androgens Animal Model Antibodies Antibody Specificity Autopsy Binding Proteins Biological Assay CRISPR/Cas technology Cell Culture Techniques Cells Clinical DNA Disease Dot Immunoblotting Double-Stranded RNA Engineering Equilibrium Gene Expression Regulation Genes Genetic Transcription Goals Human Hybrids Impairment Inherited Kennedy Syndrome Knock-out Length Ligands Link Lower Motor Neuron Disease Membrane Methods Mitochondria Mitochondrial Membrane Protein Modeling Monoclonal Antibodies Motor Neuron Disease Motor Neurons Mutation National Institute of Neurological Disorders and Stroke Neurites Neuromuscular Diseases Outer Mitochondrial Membrane Patients Population Property Proteomics RNA Reproducibility Research Respiration Respiratory distress Ribonuclease H Ribonuclease III Ribonucleases Role SYNJ1 gene Sensitivity and Specificity Signal Transduction Site Spinal Muscular Atrophy Stress Therapeutic Intervention Tissues Work cell type cytotoxicity gain of function improved induced pluripotent stem cell insight loss of function mitochondrial dysfunction molecular pathology motor neuron degeneration mutant nervous system disorder nucleic acid structure protein expression small molecule spinal and bulbar muscular atrophy stem cell model

项目摘要

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.

最近我们的研究集中在两种遗传性运动神经元疾病：由于雄激素受体（AR）突变引起的脊髓和延髓肌萎缩症（SBMA）和由于senataxin（SETX）突变引起的4型肌萎缩侧索硬化症（ALS4）。具体研究成果包括： (1) 在我们实验室开始并在 NINDS 的 Craig Blackstone 博士和约翰霍普金斯大学的 Chris Ross 博士的实验室继续进行的工作中，夏峰博士使用 CRISPR-Cas9 基因编辑来设计同基因人类诱导多能干细胞 (hiPSC) ）模型，由等基因 AR 敲除、对照和疾病系组成，表达具有不同重复长度的突变 AR，以及分别表达野生型和突变 AR 的对照和疾病系。冯博士采用小分子定向方法，将同基因 hiPSC 模型分化为丰富的运动神经元样细胞群，以揭示 SBMA 背后的细胞类型特异性机制，并区分运动神经元样细胞的功能增益和功能丧失特性。患病运动神经元中的突变 AR。冯博士证明，无配体的突变体AR会因毒性功能获得而导致分化疾病运动神经元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、线粒体定位的突变 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。这些发现反驳了既定观念，即突变体 AR 诱导的 SBMA 细胞毒性需要雄激素，揭示了无配体突变体 AR、F-ATP 合酶和线粒体功能障碍之间的机制联系，并为运动神经元特异性治疗干预提供了见解。 SBMA。 (2) Synaptojanin 2 结合蛋白 (SYNJ2BP) 是一种线粒体外膜蛋白，具有胞质 PDZ 结构域，可充当细胞信号传导中枢。很少有研究评估其在疾病中的作用。我们使用 iPSC 衍生的运动神经元和 SBMA 和 ALS4 患者的死后组织，结果表明患病运动神经元中 SYNJ2BP 表达增加。同样，我们发现在承受压力的 iPSC 来源的运动神经元中 SYNJ2BP 表达增加。通过蛋白质组学分析，我们发现升高的 SYNJ2BP 会改变线粒体的细胞分布并增加线粒体 - ER 膜接触位点。此外，降低 SYNJ2BP 水平可以改善患病运动神经元的线粒体氧化功能。总之，我们的观察结果为运动神经元疾病的分子病理学以及 SYNJ2BP 在线粒体功能障碍中的作用提供了新的见解。 (3) 三链核酸结构 R 环因其在基因调控中的作用而日益得到认可。最初，R 环被认为是转录的副产物；但最近发现患病细胞中 R 环较少，这清楚地表明 R 环在多种人类细胞中具有功能作用。了解 R 环的作用以及细胞如何平衡其丰度非常重要。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，并显示了使用 RNase H、RNase T1 和 RNase III 进行的检测的灵敏度和特异性，这些检测可切割 RNA-DNA 杂合体、单链 RNA 和分别为双链RNA。该方法具有高度重复性，并可在两天内提供结果。该测定可用于研究和临床环境中，以量化 R 环并评估 SETX 等基因突变对 R 环丰度的影响。