Signaling Mechanisms in Neuronal Degeneration

描述（申请人提供）：亨廷顿舞蹈病（HD）是最常见的常染色体显性、三核苷酸重复的神经退行性疾病。huntingtin （htt）基因编码一个350 kD的蛋白；致病突变导致氨基末端多谷氨酰胺重复扩增超过36个连续的谷氨酰胺。我们广泛的研究目标是了解HD发病机制的分子基础，以靶向治疗。由于亨廷顿氏病是一种遗传性疾病，我们预计突变等位基因与野生型至少有一个单核苷酸多态性（SNP）的差异。我们的核心思想是，RNA沉默可以用来选择性地减少突变htt的产生，从而减缓或阻断HD疾病中的神经元功能障碍和死亡。我们提出5个目标。目的1将鉴定htt mRNA中存在的所有snp，以鉴定可以设计snp选择性sirma的SNP9。Aim 2将开发snp选择性sirna，然后研究其在细胞模型中的有效性：在HeLa细胞、转染人htt的X-57永生化神经元细胞以及通过慢病毒转导表达人htt的初级纹状体和皮质神经元中进行荧光素酶报告基因检测。htt RNAi对HD发病的分子相关因素（htt片段积累、htt聚集和自噬）的影响将被测量。目的3将检验针对非snp选择性的htt mRNA的sirna的有效性。我们将研究htt sirna的脱靶效应。Aim 4将采取另一种策略来阻止。通过创造一种分子链，招募miRNA来靶向ririrna。Aim 5将成为亨廷顿舞蹈病小鼠模型中最活跃的、snp选择性的siRNA，通过递送结合的htt siRNA和慢病毒htt shRNA。HD分子相关因素，神经生理和行为将被测量。初步和已发表的研究支持这些目标，包括htt snp选择性高功能sirna的开发，sirna向初级神经元的传递，以及分子和整个动物终点的传递。这些目标有望提供HD治疗和对标记其发病机制的分子相关的见解。

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.