Signaling Mechanisms in Neuronal Degeneration

描述(申请人提供)：亨廷顿病(HD)是最常见的常染色体显性、三核苷酸重复重复的神经退行性疾病。亨廷顿蛋白(HTT)基因编码一种350kD的蛋白质；这种致病突变导致超过36个连续谷氨酸的氨基末端多谷氨酰胺重复序列的扩张。我们广泛的研究目标是了解HD发病的分子基础，并进行靶向治疗。由于亨廷顿病是一种遗传性疾病，我们预计突变等位基因将与野生型至少有一个单核苷酸多态(SNP)。我们的核心想法是，RNA沉默可以被用来选择性地减少突变的HTT的产生，从而减缓或阻止HD疾病中的神经元功能障碍和死亡。我们提出了五个目标。目标1将确定HTT mRNA中存在的所有SNPs，以确定哪些SNP9可以设计为SNP选择性siRMA。目的研究SNP选择性siRNAs在HeLa细胞、转人HTT的X-57永生化神经细胞以及慢病毒转导表达人HTT的原代纹状体和皮质神经元中的作用。将测量HTT RNAi对HD发病的分子相关因素(HTT片段聚集、HTT聚集和自噬)的影响。目标3将检测针对非SNP选择性的HTT mRNA的siRNAs的有效性。我们将研究HTT siRNAs的脱靶效应。Aim 4将寻求一种替代战略来阻止。HTT mRNA，通过创建一种分子纽带，招募miRNA来靶向rriRNA。目的5将通过传递结合的HTT siRNA和慢病毒HTT shRNA，在亨廷顿病小鼠模型中获得最活跃的、SNP选择性的siRNA。HD分子相关性、神经生理学和行为将被测量。初步和已发表的研究支持每个目标，包括开发HTT SNP选择性高功能siRNAs，向初级神经元递送siRNAs，以及分子和整个动物终点。这些目标有望提供HD治疗，并深入了解其发病机制的分子相关性。

DESCRIPTION (provided by applicant): Huntington's disease (HD) is the most prevalent autosomal dominant, trinucleotide repeat neurodegenerative disease. The huntingtin (htt) gene encodes a protein of 350 kD; the disease-causing mutation causes an expansion of an amino-terminal polyglutamine repeat of more than 36 successive glutamines. Our broad research goal is to understand the molecular basis of HD pathogenesis, to target therapy. Because Huntington's disease is an inherited disease, we expect that the mutant allele will differ from wild-type by at least a single nucleotide polymorphism (SNP). Our core idea is that RNA silencing could be used) to selectively reduce mutant htt production and thereby slow or block neuronal dysfunction and death in HD disease. We propose 5 Aims. Aim 1 will identify all the SNPs present in the htt mRNA, to identify those SNP9 to which SNP-selective siRMAs can be designed. Aim 2 will develop SNP-selective siRNAs and then study their efficacy in cell models: luciferase reporter assays in HeLa cells, X-57 immortalized neuronal cells transfected with human htt, and primary striatal and cortical neurons expressing human htt through lentiviral transduction. Effects of htt RNAi on molecular correlates of HD pathogenesis (htt fragment accumulation, htt aggregation and autophagy) will be measured. Aim 3 will examine the effectiveness of siRNAs directed against htt mRNA that are not SNPselective. Off-target effects of htt siRNAs will be studied. Aim 4 will pursue an alternative strategy to block. htt mRNA, by creating a molecular tether that recruits miRNA to target rriRNAs. Aim 5 will iesi the most active, SNP-selective siRNA in mouse models of Huntington's disease, through delivery of conjugated htt siRNAs and lentivirus htt shRNA. HD molecular correlates, neurophysiology and behavior will be measured. Preliminary and published studies support each of the aims, including development of htt SNP-selective hyper functional siRNAs, delivery of siRNAs to primary neurons, and molecular and whole animal endpoints. These aims are expected to provide HD therapy and insights into molecular correlates that mark its pathogenesis.