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

双顺反子钙通道基因在神经发育和神经元中的新作用

• 批准号: 8696017
• 负责人: Christopher Manuel Gomez
• 金额: $ 44.05万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-02-15 至 2019-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8696017
• 关键词: Affect; Alleles; Ataxia; Base Sequence; Binding; Binding Proteins; Biological Assay; CAG repeat; Calcium Channel; Cell Culture Techniques; Cell Death; Cells; Cerebellar cortex structure; Cistrons; Code; Consensus; DNA Binding; Development; Disease; EMSA; Elderly; Gene Expression; Gene Expression Profile; Gene Targeting; Genes; Genetic; Goals; Health; Human; In Vitro; Incubated; Inherited; Ion Channel; Length; Luciferases; Mediating; Messenger RNA; Modeling; Molecular; Molecular Profiling; Motor; Mus; Mutation; Nerve Degeneration; Neurites; Neurodegenerative Disorders; Neurons; PC12 Cells; Pathogenesis; Pattern; Phenotype; Preventive; Proteins; Purkinje Cells; RNA; RNA Sequences; Regulator Genes; Reporter; Role; Site; Structure; Structure of molecular layer of cerebellar cortex; System; Testing; Tetanus Helper Peptide; Therapeutic Agents; Toxic effect; Transgenes; Transgenic Mice; Transgenic Model; Translations; Type 1 Spinocerebellar Ataxia; Type 6 Spinocerebellar Ataxia; Ursidae Family; Variant; base; chromatin immunoprecipitation; drug development; gain of function; high throughput screening; in vivo; mutant; neurodevelopment; next generation sequencing; novel; polyglutamine; promoter; public health relevance; screening; small molecule; transcription factor; transcriptome sequencing; voltage; young adult

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, a1ACT, 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 a1ACT 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 (a1ACTSCA6) 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 a1ACTWT and a1ACTSCA6 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 a1ACT? We will generate new lines of a1ACT transgenic mice that will only yield a1ACTWT 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 a1ACT 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 a1ACT IRES function.

描述（由申请人提供）：脊髓小脑共济失调 6 型（SCA6）是一种显性遗传的、无法治疗的神经退行性疾病，其特征是进行性共济失调和浦肯野细胞变性，与 CACNA1A 基因中的 CAG 重复扩增相关。我们最近的证据表明，该疾病可归因于第二个 CACNA1A 基因产物 a1ACT 内的 PolyQ 重复扩增表达，该产物通常作为对小脑皮质发育至关重要的转录因子 (TF)，并且是通过 编码区内隐秘的细胞内部核糖体进入位点（IRES）的作用。 a1ACT TF 中 SCA6 大小的 PolyQ 扩增会中断其细胞和分子功能，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 a1ACTSCA6 如何改变基因表达、确认 CACNA1A IRES 在体内的作用以及开发筛选策略来筛选潜在的 IRES 抑制化合物作为潜在疗法，从而了解 SCA6 的发病机制。具体来说，我们建议问：1.转录因子（a1ACTSCA6）中a1ACT的SCA6 polyQ扩展是否会改变基因结合模式和浦肯野细胞基因的表达模式？我们将利用染色质免疫沉淀 (ChIP) 和新一代测序 (ChIP-seq) 来详细分析 PC12 细胞中表达的 a1ACTWT 和 a1ACTSCA6 的结合谱，并从表达 a1ACTWT 或 a1ACTSCA6 的小鼠浦肯野细胞中分离 RNA，通过 RNA-seq 生成全局基因表达谱，以关联 a1ACTWT-DNA 与转录活性结合并识别基因表达模式的等位基因特异性变化。 2. CACNA1A IRES 和启动子表达的 a1ACT 是否再现了 a1ACT 的正常和病理功能？我们将产生新的 a1ACT 转基因小鼠品系，它们仅通过 CAP 独立翻译产生 a1ACTWT 或 a1ACTSCA6 蛋白，并使用 tet-off 表达系统驱动条件表达，并使用 CACNA1A-tTA 转基因产生内源表达模式。 3. 我们能否鉴定出选择性抑制 a1ACT 翻译但不抑制 a1A 亚基表达的 IRES 定向分子？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 识别 ITAF 并预测 CACNA1A IRES 的二级结构。我们将进行高通量筛选测定，以鉴定干扰 a1ACT IRES 功能的化合物。