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

基本信息

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
项目类别：
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

项目摘要

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）：6型脊髓脑性共济失调（SCA6）是一种主要的，不可治疗的神经退行性疾病，其特征是进行性共济失调和Purkinje细胞变性与基因中CAG重复膨胀相关的Purkinje细胞变性。我们最近的证据表明，该疾病归因于在第二个CACNA1A基因产物A1ACT中表达PolyQ重复膨胀的表达，通常是小脑皮质发育至关重要的转录因子（TF），并且通过这种转录因子（TF），并且通过这种转录因子（TF），并且通过这种转录因子（TF），并且通过这种转录因子（TF）。编码区域内隐秘的细胞内部核糖体入口位点（IRE）的作用。 A1ACT TF中的SCA6大小的PolyQ膨胀中断其细胞和分子功能，导致细胞死亡在体外，共济失调和皮质变薄。该项目的长期目标是通过表征A1ACTSCA6在体内确认CACNA1A IRES在体内的作用并制定抑制IRES化合物作为潜在疗法的潜在IRES的筛查策略来改变基因表达，从而了解SCA6的TE发病机理。具体来说，我们提出要问：1。在转录因子（A1ACTSCA6）中，Sca6 PolyQ扩展会改变基因结合模式和Purkinje细胞基因的表达模式吗？我们将利用染色质免疫沉淀（CHIP），然后使用下一代测序（CHIP-SEQ）进行详细分析A1ACTWT和A1ACTSCA6的详细结合曲线，并在PC12细胞中表达的A1ACTSCA6和在PC12细胞中表达的RNA和分离rNA从表达A1ACTWT或A1ACTWT或A1ACTSCA6的Purkinje细胞中的RNA分离出RNA，以促进A1ATCWT或A1ACTWT或A1ACTWT或A1ACTSCA 6与转录活性结合并确定基因表达模式的等位基因特异性变化。 2。cacna1a ires和启动子的表达是否会重现A1ACT的正常和病理功能？我们将生成新的A1ACT转基因小鼠的新系，该小鼠仅通过cap独立翻译产生A1ACTWT或A1ACTSCA6蛋白，并使用Tet-Owd-off表达系统驱动条件表达并使用CACNA1A1A-TTA转基因来产生内源性表达模式。 3。我们可以鉴定有选择性抑制A1ACC翻译的IRES指导分子，而不是A1A亚基表达？我们将使用双酸酸酯酶双散发性报告基因来确定IRES功能所涉及的最佳RNA序列，识别EMSA的ITAF，并预测CACNA1A IRES的二级结构。我们将进行高吞吐量筛选分析，以识别干扰A1ACT IRES功能的化合物。