Mechanisms of motor neuron toxicity in Kennedy disease

描述（由申请人提供）：与蛋白质聚集相关的神经退行性疾病包括9种由CAG/谷氨酰胺束（polyQ）扩增引起的无法治疗的疾病。这些疾病之一，脊髓延髓肌萎缩症（SBMA），其特征在于下运动神经元变性，是由雄激素受体（AR）基因突变引起的。该突变蛋白经历了依赖于细胞核转位、解折叠和寡聚化，这些步骤对毒性和男性进行性近端肢体和延髓肌无力的发展至关重要。虽然20年前就发现了致病突变，但SBMA患者可用的治疗方法在很大程度上仍然是支持性的。此外，降解突变蛋白的细胞途径仍然不完全确定，这种知识的缺乏阻碍了疾病修饰疗法的发展。本申请的目的是确定基于Hsp 90的分子伴侣机制在控制polyQ AR降解的蛋白质质量控制决策中的作用。我们的中心假设是Hsp 70和Hsp 90在polyQ AR的分类中具有基本上相反的作用，因为Hsp 70促进底物泛素化，而Hsp 90抑制泛素化。这一假设源于我们的初步数据，表明与Hsp 90的结合稳定了polyQ AR，而突变蛋白的解折叠导致Hsp 70依赖性E3连接酶的泛素化。在这里，我们将使用遗传学和药理学工具来定义变构激活热休克蛋白70依赖性泛素化的后果。此外，由于我们的数据指向骨骼肌和运动神经元对疾病表型的贡献，我们将使用遗传方法来确定每个部位的毒性必须达到何种程度才能达到有益的治疗效果。拟议工作的基本原理是，建立调节polyQ AR降解的机制将确定可用于开发新疗法的靶点。将使用遗传和生物化学方法来确定Hsp 70的变构激活剂促进polyQ AR清除的程度（目的1），以鉴定SBMA小鼠中polyQ AR毒性的关键部位（目的2），并建立 新型小分子热休克蛋白70变构激活剂在SBMA小鼠中的作用（目的3）。这些研究通过定义限制SBMA毒性的途径，同时提供支持新治疗方法的概念验证数据，预计将产生显著的积极影响。由于热休克蛋白70还调节质量控制决策，管理其他突变蛋白，导致神经变性的营业额，我们预计，这里定义的方法将告知治疗策略，将广泛适用。 公共卫生相关性：拟议研究与公共卫生的相关性在于，它们将把Hsp 70的变构激活定义为促进polyQ AR降解的策略，polyQ AR是导致SBMA的突变蛋白。这项工作将表征遗传和药理学Hsp 70激活剂，期望它们将改善模型系统中的SBMA表型，并加快疾病修饰疗法的进展。

DESCRIPTION (provided by applicant): Neurodegenerative disorders associated with protein aggregation include nine untreatable diseases caused by CAG/glutamine tract (polyQ) expansions. One of these disorders, spinobulbar muscular atrophy (SBMA), is characterized by degeneration of lower motor neurons and is caused by a mutation in the androgen receptor (AR) gene. The mutant protein undergoes hormone-dependent nuclear translocation, unfolding and oligomerization, steps that are critical to toxicity and to the development of progressive proximal limb and bulbar muscle weakness in men. Although the disease causing mutation was identified two decades ago, treatments available to SBMA patients remain largely supportive. Furthermore, the cellular pathways that degrade the mutant protein remain incompletely defined, and this lack of knowledge hinders the development of disease-modifying therapies. The objective of this application is to define the role of the Hsp90-based chaperone machinery in the protein quality control decisions that govern polyQ AR degradation. Our central hypothesis is that Hsp70 and Hsp90 have essentially opposing roles in the triage of the polyQ AR, in that Hsp70 promotes substrate ubiquitination whereas Hsp90 inhibits ubiquitination. This hypothesis springs from our preliminary data showing that association with Hsp90 stabilizes the polyQ AR, while unfolding of the mutant protein leads to ubiquitination by Hsp70-dependent E3 ligases. Here we will use genetic and pharmacological tools to define the consequences of allosterically activating Hsp70-dependent ubiquitination. Additionally, as our data point to contributions from both skeletal muscle and motor neurons to the disease phenotype, we will use genetic approaches to determine the extent to which toxicity at each site must be targeted to achieve beneficial therapeutic effects. The rationale of the proposed work is that establishing the mechanisms that regulate polyQ AR degradation will identify targets that can be exploited by the development of new therapies. Genetic and biochemical approaches will be used to determine the extent to which allosteric activators of Hsp70 promote clearance of the polyQ AR (Aim 1), to identify critical sties of polyQ AR toxicity in SBMA mice (Aim 2), and to establish the effects of novel, small molecule allosteric activators of Hsp70 in SBMA mice (Aim 3). These studies are expected to have a significant positive impact by defining pathways that limit SBMA toxicity while providing proof-of-concept data supporting a new therapeutic approach. As Hsp70 also regulates quality control decisions governing the turnover of other mutant proteins that cause neurodegeneration, we expect that the approaches defined here will inform therapeutic strategies that will be broadly applicable. PUBLIC HEALTH RELEVANCE: The relevance of the proposed studies to public health is that they will define allosteric activation of Hsp70 as a strategy to promote degradation of the polyQ AR, the mutant protein that causes SBMA. This work will characterize both genetic and pharmacological Hsp70 activators, with the expectation that they will ameliorate the SBMA phenotype in model systems and speed the advance toward disease-modifying therapies.