Cellular and Molecular Studies of SBMA Neuromuscular Disease

SBMA 神经肌肉疾病的细胞和分子研究

• 批准号: 9325082
• 负责人: Janghoo Lim
• 金额: $ 36.42万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2020-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9325082
• 关键词: Affect; Alleles; Alpha Cell; Androgen Receptor; Animal Model; Behavioral; Biochemical; Biochemistry; Biological Assay; Biological Models; Biology; CAG repeat; Cell Culture System; Cell Culture Techniques; Cell Death; Data; Development; Disease; Drosophila genus; Functional disorder; Genetic; Glutamine; Goals; Grant; Immunofluorescence Immunologic; Intervention; Knockout Mice; Knowledge; Lead; Light; Mediating; Modeling; Molecular; Molecular Biology; Motor Neurons; Mus; Muscle Cells; Neurodegenerative Disorders; Neuromuscular Diseases; Pathogenesis; Pathogenicity; Pathologic; Pathology; Pathway interactions; Peptidylprolyl Isomerase; Phenotype; Phosphorylation; Phosphotransferases; Pin1 protein; Post-Translational Protein Processing; Protein-Serine-Threonine Kinases; Proteins; Quality Control; Receptor Gene; Research; Role; Signal Transduction; Skeletal Muscle; Spinal; Spinal Cord; Staining method; Stains; System; Testing; Therapeutic; Tissues; Toxic effect; Treatment Efficacy; Western Blotting; base; effective therapy; genetic analysis; improved; in vivo; insight; mouse model; mutant; nemo-like kinase; neurodegenerative phenotype; neuromuscular; novel therapeutic intervention; novel therapeutics; polyglutamine; protein aggregate; protein degradation; public health relevance; receptor; recombinase-mediated cassette exchange; spinal and bulbar muscular atrophy; therapeutic target

 DESCRIPTION (provided by applicant): Spinal and Bulbar Muscular Atrophy (SBMA) is a progressive neuromuscular disease affecting the proximal spinal and bulbar motor neurons and the skeletal muscles. To date, there is no effective cure for this devastating disease. SBMA arises from the expansion of a polymorphic CAG trinucleotide repeat in the Androgen Receptor (AR) gene, resulting in an expansion of glutamines in the AR protein. Polyglutamine expansion renders the mutant AR protein toxic, resulting in the formation of mutant protein aggregates and cell death. The overarching goal of this project is to better understand the cellular and molecular mechanisms underlying the pathogenesis of SBMA with the hope that such insight will lead to the development of better therapeutics. In order to reach this goal, we began by identifying factors that could potentially modulate the SBMA disease condition in vivo. The preliminary data presented in this application clearly show that Nemo-Like Kinase (NLK), an evolutionarily conserved serine/threonine kinase, is a key factor that could potentially modulate the SBMA disease condition. In this proposal, we will test the idea that NLK can modulate SBMA pathogenesis via regulating the posttranslational modification or expression of the mutant AR. To investigate this idea, we propose the following three specific aims. In Aim 1, we will determine whether NLK can modulate SBMA-related phenotypes using mouse as a model system. We will utilize several SBMA mouse models that are characterized by both neuromuscular pathology and behavioral deficits. Specifically, (1) we will first test whether constitutive loss of one functional Nlk allele (50% NLK reduction) suppresses the neuromuscular phenotypes of SBMA. (2) We will also evaluate the role of NLK in SBMA in a tissue-specific manner by using Nlk conditional knockout mice. We will focus on motor neurons and skeletal muscle, as they are known SBMA- affected tissues. In Aim 2, we will identify the molecular mechanism by which NLK regulates the SBMA disease condition. We will determine how NLK kinase activity is responsible for mediating the neuromuscular phenotypes of SBMA. The role of NLK in (1) AR posttranslational modification and (2) AR protein degradation/clearance will be determined and their contribution to SBMA toxicity will be characterized. In Aim 3, we will determine the role of the peptidyl-prolyl isomerase Pin1 in SBMA pathology. We will test whether Pin1 acts downstream of the NLK-induced effects on mutant AR to modulate toxicity in SBMA. To do this, we will utilize cell culture approaches and Drosophila and mouse genetics. We believe that the knowledge gained from the studies proposed in this application will advance our basic understanding of the cellular and molecular mechanisms underlying SBMA and will suggest new therapeutic interventions aimed at reducing the burden of SBMA and other neuromuscular diseases.

 描述（由申请人提供）：脊髓和延髓肌萎缩症（SBMA）是一种影响近端脊髓和延髓运动神经元以及骨骼肌的进行性神经肌肉疾病。迄今为止，这种毁灭性疾病还没有有效的治疗方法。 SBMA 源自雄激素受体 (AR) 基因中多态性 CAG 三核苷酸重复的扩展，导致 AR 蛋白中谷氨酰胺的扩展。聚谷氨酰胺扩增使突变 AR 蛋白有毒，导致突变蛋白聚集体的形成和细胞死亡。该项目的总体目标是更好地了解细胞和分子 SBMA 发病机制的潜在机制，希望这种见解能够导致更好的治疗方法的开发。为了实现这一目标，我们首先确定可能在体内调节 SBMA 疾病状况的因素。本申请中提供的初步数据清楚地表明，Nemo 样激酶 (NLK)（一种进化上保守的丝氨酸/苏氨酸激酶）是可能调节 SBMA 疾病状况的关键因素。在本提案中，我们将测试 NLK 可以通过调节突变 AR 的翻译后修饰或表达来调节 SBMA 发病机制的想法。为了研究这个想法，我们提出以下三个具体目标。在目标 1 中，我们将使用小鼠作为模型系统来确定 NLK 是否可以调节 SBMA 相关表型。我们将利用几种以神经肌肉病理学和行为缺陷为特征的 SBMA 小鼠模型。具体来说，（1）我们将首先测试一个功能性 Nlk 等位基因的组成性缺失（NLK 减少 50%）是否会抑制 SBMA 的神经肌肉表型。 (2)我们还将通过使用Nlk条件敲除小鼠以组织特异性方式评估NLK在SBMA中的作用。我们将重点关注运动神经元和骨骼肌，因为它们是已知的 SBMA 受影响的组织。在目标 2 中，我们将确定 NLK 调节 SBMA 疾病的分子机制。我们将确定 NLK 激酶活性如何负责介导 SBMA 的神经肌肉表型。将确定 NLK 在 (1) AR 翻译后修饰和 (2) AR 蛋白降解/清除中的作用，并表征它们对 SBMA 毒性的贡献。在目标 3 中，我们将确定肽基脯氨酰异构酶 Pin1 在 SBMA 病理学中的作用。我们将测试 Pin1 是否在 NLK 诱导的突变 AR 下游发挥作用，以调节 SBMA 的毒性。为此，我们将利用细胞培养方法以及果蝇和小鼠遗传学。我们相信，从本申请中提出的研究中获得的知识将增进我们对 SBMA 基础细胞和分子机制的基本理解，并将提出旨在减轻 SBMA 和其他神经肌肉疾病负担的新治疗干预措施。