Studies of Hereditary Neurological Disease: Disease Mechanisms

遗传性神经系统疾病的研究：疾病机制

• 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
• 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

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上：脊柱和鳞茎肌肉萎缩（SBMA），这是由于雄激素受体（AR）和肌萎缩性侧面硬化症4型（ALS4）引起的突变引起的，这是由于鼻毒素突变引起的（SETX）。具体的研究成就包括以下内容： (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突变体AR。 Feng博士调整了小型分子指导的方法，将等源性HIPSC模型区分为富集的运动神经元样细胞群，以发现SBMA的细胞类型特异性机制，以区分有害运动神经元中突变AR的功能性能丧失。冯博士证明，无配体突变体AR会导致由于功能毒性获得的分化疾病运动神经元神经突中的线粒体功能障碍，并且在配体（雄激素）处理后可以扩增这种细胞毒性。她进一步表明，无配体，线粒体 - 定位的突变体AR和F-ATP合酶之间的异常相互作用与线粒体呼吸和其他线粒体损伤的损害有关。这些发现对抗了既定的观念，即SBMA突变AR诱导的细胞毒性所必需的雄激素揭示了无配体突变抗AR，F-ATP合酶和线粒体功能障碍之间的机械联系，并为SBMA的运动神经元特异性治疗疗法提供了洞察力。 （2）Synaptojanin 2结合蛋白（Synj2bp）是一种外部线粒体膜蛋白，具有胞质PDZ结构域，可作为细胞信号枢纽。很少有研究评估其在疾病中的作用。我们使用了SBMA和ALS4患者的IPSC衍生运动神经元和验尸后组织，并表明患病运动神经元中Synj2BP的表达增加。同样，我们表明SYNJ2BP表达在受压力的IPSC衍生运动神经元中增加。使用蛋白质组学分析，我们发现Synj2bp升高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，降低的Synj2bp水平可改善患病运动神经元中的线粒体氧化功能。总之，我们的观察结果对运动神经元疾病的分子病理和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-loop）因其在基因调节中的作用而越来越认可。最初，R环被认为是转录的副产品。但是最近在患病细胞中较少的R环的发现清楚地表明，R环在多种人类细胞中具有功能性。重要的是要了解R环的作用以及细胞如何平衡其丰度。该领域的一个挑战是对R环的定量，因为许多工作依赖于S9.6单克隆抗体，该抗体对RNA DNA杂交的特异性受到质疑。在这里，我们使用与S9.6抗体的点印象来量化R环，并用RNase H，RNase T1和RNase III的敏感性和特异性分别裂解RNA杂种，单链RNA，单链RNA和双链RNA。该方法高度可重现，并在两天内提供结果。该测定可以用于研究和临床环境中，以量化R环并评估SETX等基因中突变对R环丰度的影响。