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(申请人提供)：脊髓小脑性共济失调6型(SCA6)是一种以进行性共济失调和浦肯野细胞变性为特征的主要遗传性、不可治疗的神经退行性疾病，与CACNA1A基因中CAG重复扩增有关。我们最近的证据表明，这种疾病是由于第二个CACNA1A基因产物a1ACT中的PolyQ重复扩增所致，该基因产物通常是小脑皮质发育的关键转录因子(Tf)，而这种转录因子是通过 编码区内隐蔽的细胞内部核糖体进入位点(IRES)的作用。在a1ACT TF中，SCA6大小的多聚Q的扩张干扰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a1ACTSCA6是如何改变基因表达的、通过确认CACNA1AIRES在体内的作用以及通过开发潜在的IRES抑制化合物作为潜在的治疗方法来了解SCA6的发病机制。具体地说，我们建议问：1.转录因子(A1ACTSCA6)中的a1ACT中的SCA6 PolyQ扩展是否改变了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)和下一代测序(CHIP-SEQ)详细分析在PC12细胞中表达的a1ACTWT和a1ACTSCA6的结合图谱，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，将a1ACTWT-DNA结合与转录活性相关联，并确定基因表达模式中等位基因特异性的变化。2.CACNA1AIRES和启动子表达的a1ACT是否具有a1ACT的正常和病理功能？我们将建立新的a1ACT转基因小鼠系，通过CAP非依赖的翻译只产生a1ACTWT或a1ACTSCA6蛋白，并使用TET-OFF表达系统来驱动条件表达，并使用CACNA1A-TTA转基因来产生内源性表达模式。3.我们能否识别IRES导向的分子选择性地抑制A1ACT翻译，而不是A1A亚单位的表达？我们将使用我们的双荧光素酶双顺反子报告基因来确定IRES功能涉及的最佳RNA序列，通过EMSA鉴定ITAFs，并预测CACNA1AIRES的二级结构。我们将进行高通量筛选试验，以确定干扰IRES功能的化合物。