Cellular and Molecular Studies of SBMA Neuromuscular Disease

 描述（由申请方提供）：脊髓延髓肌萎缩症（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。到目前为止，还没有有效的方法来治疗这种毁灭性的疾病。SBMA由雄激素受体（AR）基因中多态性CAG三核苷酸重复序列的扩增引起，导致AR蛋白中谷氨酰胺的扩增。多聚谷氨酰胺扩增使突变体AR蛋白具有毒性，导致突变体蛋白聚集体的形成和细胞死亡。这个项目的首要目标是更好地了解细胞和分子 SBMA的发病机制，希望这种见解将导致更好的治疗方法的发展。为了达到这一目标，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，是可能潜在地调节SBMA疾病状况的关键因子。在这个提议中，我们将测试的想法，NLK可以调节SBMA的发病机制，通过调节翻译后修饰或表达的突变体AR。为了研究这个想法，我们提出了以下三个具体目标。在目标1中，我们将确定NLK是否可以使用小鼠作为模型系统调节SBMA相关的表型。我们将利用几种SBMA小鼠模型，其特征在于神经肌肉病理学和行为缺陷。具体地，（1）我们将首先测试一个功能性Nlk等位基因的组成性缺失（50%NLK减少）是否抑制SBMA的神经肌肉表型。(2)我们还将通过使用Nlk条件性敲除小鼠以组织特异性方式评估NLK在SBMA中的作用。我们将集中在运动神经元和骨骼肌，因为它们是已知的SBMA影响的组织。在目标2中，我们将确定NLK调节SBMA疾病状况的分子机制。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，并表征其对SBMA毒性的贡献。在目标3中，我们将确定肽基脯氨酰异构酶Pin 1在SBMA病理中的作用。我们将测试Pin 1是否在NLK诱导的对突变体AR的作用的下游起作用以调节SBMA中的毒性。为此，我们将利用细胞培养方法和果蝇和小鼠遗传学。我们相信，从本申请中提出的研究中获得的知识将促进我们对SBMA潜在的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的治疗干预措施。

 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.