A novel role for a bicistronic calcium channel gene in neurodevelopment and neuro

描述（由申请人提供）：脊髓小脑共济失调6型（SCA 6）是一种显性遗传、不可治疗的神经退行性疾病，其特征为进行性共济失调和浦肯野细胞变性，与基因CACNA 1A中的CAG重复扩增相关。我们最近的证据表明，这种疾病是由于第二个CACNA 1A基因产物α1ACT中polyQ重复扩增的表达，α1ACT通常作为小脑皮质发育的关键转录因子（TF），并通过 在编码区内的隐蔽的细胞内部核糖体进入位点（IRES）的作用。α1ACT TF中SCA 6大小的polyQ扩增中断了其细胞和分子功能，导致体外细胞死亡、共济失调和皮质变薄。该项目的长期目标是通过表征a1 ACTSCA 6如何改变基因表达，通过确认CACNA 1A IRES在体内的作用以及通过开发潜在IRES抑制化合物作为潜在疗法的筛选策略来了解SCA 6的发病机制。具体而言，我们提出以下问题：1。转录因子α 1ACT中的SCA 6 polyQ扩增（α 1ACTSCA 6）是否会改变浦肯野细胞基因的基因结合模式和表达模式？我们将利用染色质免疫沉淀（ChIP），然后进行下一代测序。（ChIP-seq）来详细分析在PC 12细胞中表达的α1ACTWT和α 1ACTSCA 6的结合谱，并从表达α 1ACTWT或α 1ACTSCA 6的小鼠的浦肯野细胞中分离RNA，以通过RNA-seq生成全局基因表达谱，从而将α 1ACTWT-DNA结合与转录活性相关联，并鉴定等位基因-基因表达模式的特定变化。2. CACNA 1A IRES及其启动子表达的α 1ACT是否能再现α1ACT的正常和病理功能？我们将产生新的a1 ACT转基因小鼠品系，其仅通过CAP非依赖性翻译产生α1ACTWT或a1 ACTSCA 6蛋白，并使用tet-off表达系统驱动条件表达，并使用CACNA 1A-tTA转基因产生内源性表达模式。3.我们能否鉴定出选择性抑制α1ACT翻译但不抑制α 1A亚基表达的IRES导向分子？我们将使用我们的双荧光素酶双顺反子报告基因来确定参与IRES功能的最佳RNA序列，通过EMSA鉴定ITAF并预测CACNA 1A IRES的二级结构。我们将进行高通量筛选试验，以鉴定干扰α1ACT IRES功能的化合物。

DESCRIPTION (provided by applicant): Spinocerebellar ataxia type 6 (SCA6) is a dominantly-inherited, untreatable neurodegenerative disease characterized by progressive ataxia and Purkinje cell degeneration associated with CAG repeat expansions in the gene, CACNA1A. Our recent evidence suggests that the disease is attributable to expression of a polyQ repeat expansion within a second CACNA1A gene product, α1ACT, that normally serves as a transcription factor (TF) critical for cerebellar cortical development, and that arises through the action of a cryptic cellular internal ribosomal entry site (IRES) within the coding region. SCA6-sized polyQ expansions in the α1ACT TF interrupt its cellular and molecular function, cause cell death in vitro, ataxia and cortical thinning. The long-term goal of this project is to understand te pathogenesis of SCA6 by characterizing how a1ACTSCA6 alters gene expression, by confirming the role of the CACNA1A IRES in vivo and by developing a screening strategy for potential IRES-inhibiting compounds as potential therapies. Specifically we propose to ask: 1. Does the SCA6 polyQ expansion in a1ACT in the transcription factor (α1ACTSCA6) changes the gene binding patterns and the expression patterns of Purkinje cell genes? We will utilize chromatin immunoprecipitation (ChIP) followed by next-generation sequencing (ChIP-seq) to analyze in detail binding profiles of α1ACTWT and α1ACTSCA6 expressed in PC12 cells and isolate RNA from Purkinje cells of mice expressing a1ACTWT or a1ACTSCA6 to generate global gene expression profiles by RNA-seq to correlate a1ACTWT-DNA binding with transcriptional activity and to identify allele-specific changes in gene expression patterns. 2. Does a1ACT expression by CACNA1A IRES and promoter reproduce the normal and pathological functions of α1ACT? We will generate new lines of a1ACT transgenic mice that will only yield α1ACTWT or a1ACTSCA6 protein by CAP-independent translation, and use the tet-off expression system to drive conditional expression and use a CACNA1A-tTA transgene to generate an endogenous pattern of expression. 3. Can we identify IRES-directed molecules that selectively suppress α1ACT translation, but not a1A subunit expression? We will use our dual-luciferase bicistronic reporter to determine the optimal RNA sequences involved in IRES function, identify the ITAFs by EMSA and predict the secondary structure of CACNA1A IRES. We will carry out a high throughput screening assay to identify compounds that interfere with α1ACT IRES function.