Spinocerebellar Ataxia Type 2 Gene and Gene Product

描述（由申请人提供）：神经退行性疾病代表着不断增加的社会和经济负担，世卫组织估计表明，到2040年，它们将取代癌症成为第二大死亡原因。退行性共济失调是影响小脑浦肯野细胞（PCs）和其他中枢神经系统神经元的一种常见的神经退行性疾病。脊髓小脑性共济失调2型（SCA2）是一种常染色体显性形式的共济失调，由编码CAG重复扩增引起，属于聚谷氨酰胺（polyQ）疾病。SCA2 （ATXN2）基因的突变也可能导致或促成帕金森病和ALS的发展，这是两种常见的神经退行性疾病。没有症状性或神经保护剂被确定用于治疗人类共济失调。在之前的资助期内，我们开发了一种多学科方法，结合形态学、生化、生理学和行为学技术来表征几种SCA2小鼠模型。我们使用小脑切片准备显示，PC放电频率的下降与运动功能的下降密切相关。重要的是，显著的转录组和神经生理变化发生之前，任何树突或细胞损失在小脑明显。我们发现突变体ATXN2通过增强与肌醇-三磷酸受体（ITPR1）的相互作用诱导内质网异常钙释放。基于这些发现，提出了两个具体目标。在Aim 1中，我们将验证mGluR1-ITPR1轴在表达polyQ扩增ATXN2的SCA2小鼠小脑中过度活跃的假设。我们将使用小脑切片制备来评估mGluR1信号以及PC放电与细胞内钙的关系。我们还将通过与mGluR1单倍不足的小鼠系的遗传相互作用，在体内测试mGluR1- itpr1轴。在目标2中，我们将在症状前时间点使用转录组分析来识别参与SCA2发病机制的新基因。为了提高灵敏度和识别较少pc表达基因变化的能力，我们建立了小脑区域的激光捕获显微解剖（LCM）。转录组将通过GO和KEGG通路分析进行分析，特别强调mGluR1-ERK下游靶标。关键基因的变化将通过qPCR和ICC/ western blot分析进行验证。我们假设一些表达变化将是稳态的，但其他的将有助于发病机制。我们将通过在体内使用AAV转导使各自基因的表达正常化来区分这些替代方案。将根据多个SCA2小鼠模型中是否存在早期变化和是否存在变化来选择五个基因。小鼠将通过行为测试和小脑切片分析来评估。提出的实验解决了与SCAs相关的两个重要问题：细胞内钙水平对PC功能和存活的重要性，以及在多个SCA2啮齿动物模型中共享的新途径的鉴定。这些问题的答案将有助于确定治疗SCA2和其他退行性共济失调的新途径。

DESCRIPTION (provided by applicant): Neurodegenerative diseases represent an ever-increasing societal and economic burden with WHO estimates indicating that they will replace cancer as the 2nd leading cause of death by 2040. Degenerative ataxias are a common form of neurodegeneration affecting cerebellar Purkinje cells (PCs) and other neurons in the CNS. Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant form of ataxia caused by expansion of a coding CAG repeat and belongs to the group of polyglutamine (polyQ) diseases. Mutations in the SCA2 (ATXN2) gene can also cause or contribute to the development of Parkinson disease and ALS, two common forms of neurodegeneration. Neither symptomatic nor neuroprotective agents have been identified for the treatment of human ataxias. In the previous funding period we developed a multidisciplinary approach to characterize several SCA2 mouse models combining morphologic, biochemical, physiologic, and behavioral techniques. We used the cerebellar slice preparation to show that a decrease in PC firing frequency closely mirrored the decline in motor function. Importantly, significant transcriptome and neurophysiological changes occurred before any dendritic or cell loss was apparent in the cerebellum. We discovered that mutant ATXN2 induced abnormal calcium release from the ER via enhanced interaction with the inositol-triphosphate receptor (ITPR1). Based on these findings, two specific aims are proposed. In Aim 1, we will test the hypothesis that the mGluR1-ITPR1 axis is hyperactive in cerebella from SCA2 mice expressing polyQ expanded ATXN2. We will use the cerebellar slice preparation to evaluate mGluR1 signaling and the relationship between PC firing and intracellular calcium. We will also test the mGluR1-ITPR1 axis in vivo by genetic interaction with mGluR1 haploinsufficient mouse lines. In aim 2, we will use transcriptome profiling at presymptomatic time points to identify novel genes involved in SCA2 pathogenesis. To increase sensitivity and the ability to identify changes in less abundantly PC-expressed genes we have established laser-capture microdissection (LCM) of cerebellar regions. Transcriptomes will be analyzed by GO and KEGG pathway analysis with a particular emphasis on mGluR1-ERK downstream targets. Changes in key genes will be verified by qPCR and ICC/ western blot analysis. We hypothesize that some expression changes will be homeostatic, but others will contribute to pathogenesis. We will differentiate between these alternatives by normalizing expression of the respective gene using AAV- transduction in vivo. Five genes will be chosen based on presence of early changes and presence of changes in multiple SCA2 mouse models. Mice will be evaluated by behavioral testing as well as analysis in the cerebellar slice. The proposed experiments address two significant questions relating to SCAs: the importance of intracellular calcium levels on PC function and survival and the identification of novel pathways that are shared across multiple SCA2 rodent models. Answers to these questions will help in the identification of new avenues towards treatments of SCA2 and other degenerative ataxias.