Cellular and Molecular Studies of SBMA Neuromuscular Disease

 描述(申请人提供)：脊髓和延髓肌肉萎缩症(SBMA)是一种进行性神经肌肉疾病，影响近端脊髓和延髓运动神经元和骨骼肌。到目前为止，还没有有效的疗法来治愈这种毁灭性的疾病。SBMA的产生源于雄激素受体(AR)基因中CAG三核苷酸重复序列的多态扩张，导致AR蛋白中谷氨酸的扩张。多聚谷氨酰胺的膨胀使突变的AR蛋白有毒，导致突变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理解细胞和分子。 SBMA的发病机制，希望这种洞察力将导致更好的治疗方法的发展。为了达到这一目标，我们首先确定了在体内可能调节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-like Kinase(NLK)是一种进化上保守的丝氨酸/苏氨酸激酶，是潜在调节SBMA疾病状态的关键因素。在这个方案中，我们将检验NLK可以通过调节突变AR的翻译后修饰或表达来调控SBMA发病的想法。为了研究这一想法，我们提出了以下三个具体目标。在目标1中，我们将以小鼠为模型系统，确定NLK是否能够调节SBMA相关的表型。我们将利用几种以神经肌肉病理和行为缺陷为特征的SBMA小鼠模型。具体地说，(1)我们将首先测试一个功能性NLK等位基因的结构性丢失(NLK减少50%)是否会抑制SBMA的神经肌肉表型。(2)我们还将利用NLK条件基因敲除小鼠，以组织特异性的方式评估NLK在SBMA中的作用。我们将重点关注运动神经元和骨骼肌，因为它们是已知的受SBMA影响的组织。在目标2中，我们将确定NLK调节SBMA疾病状态的分子机制。我们将确定NLK活性是如何调节SBMA的神经肌肉表型的。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，并将表征它们对SBMA毒性的贡献。在目标3中，我们将确定肽基-Pro异构酶Pin1在SBMA病理中的作用。我们将测试Pin1是否作用于NLK诱导的突变AR的下游，以调节SBMA的毒性。为了做到这一点，我们将利用细胞培养方法以及果蝇和老鼠的遗传学。我们相信，从这项应用中建议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.