Signaling Mechanisms in Neuronal Degeneration

描述（由申请方提供）：亨廷顿病（HD）是最常见的常染色体显性遗传三核苷酸重复神经退行性疾病。亨廷顿蛋白基因编码350 kD的蛋白质;致病突变是氨基末端多聚谷氨酰胺重复序列超过36个连续谷氨酰胺的扩增。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HD是一种遗传性疾病，我们预期突变等位基因将与野生型至少有一个单核苷酸多态性不同，从而为RNAi的基因沉默提供了一个靶点。因此，我们的核心思想是，RNA沉默是有用的，选择性地减少突变亨廷顿蛋白的表达和减缓或阻止神经元功能障碍和死亡的HD。我们假设，选择性敲低突变亨廷顿蛋白恢复正常的神经元功能，但过度沉默损害神经元功能的干扰与必要的信号事件。目的1在小鼠体内检测等位基因特异性沉默是否可以延迟或预防HD神经病变和异常行为。目的2研究死后HD大鼠脑亨廷顿蛋白mRNA的转录后调控，寻找有利于突变亨廷顿蛋白mRNA合成的分子过程。我们将使用一种新的策略来量化每个亨廷顿等位基因mRNA，基于SNP杂合性的等位基因。目标1和2的知识对于将mRNA沉默应用于HD患者至关重要。目的3研究RNAi恢复HD细胞中必需的神经元信号活性的细胞机制以及野生型亨廷顿蛋白恢复正常神经元完整性的阈值。目的3结合了等位基因RNAi和半胱氨酸转运蛋白的神经元信号传导，该半胱氨酸转运蛋白调节谷胱甘肽对活性氧的清除。目的4通过评估先天免疫和炎症反应来检查施用等位基因特异性siRNA的安全性。该提案满足了NINDS在转化科学方面的目标：基因沉默疗法的翻译，早期治疗开发以及确定神经系统功能的基础机制。创新包括靶向RNAi的mRNA等位基因，使用仅表达人亨廷顿蛋白基因的HD小鼠模型，基于SNP杂合性定量测量亨廷顿蛋白等位基因mRNA，鉴定3个UTR亨廷顿蛋白mRNA调控的深度测序分析，以及在基因组水平消除亨廷顿蛋白等位基因的锌指核酸酶策略。该提案应用基础生物学来了解HD的发病机制，并将这些知识转化为更好的治疗方法。

DESCRIPTION (provided by applicant): Huntington's disease (HD) is the most prevalent autosomal dominant, trinucleotide repeat neurodegenerative disease. The huntingtin gene encodes a protein of 350 kD; the disease causing mutation is 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 HD is an inherited disease, we expected that the mutant allele will differ from wild-type by at least a single nucleotide polymorphism, thereby offering a target for gene silencing by RNAi. Thus demonstrated, our core idea is that RNA silencing is useful to selectively reduce mutant huntingtin expression and slow or block neuronal dysfunction and death in HD. We hypothesize that selective knockdown of mutant huntingtin restores normal neuronal function, but excessive silencing impairs neuronal function by interfering with essential signaling events. Aim 1 examines in vivo in mice whether allele specific silencing can delay or prevent HD neuropathy and abnormal behaviors. Aim 2 investigates in post-mortem HD brain post- transcriptional regulation of huntingtin mRNA, to find molecular processes that favor synthesis of mutant huntingtin mRNA. We will use a new strategy to quantify each huntingtin allelic mRNA, based on SNP heterozygosities of the alleles. Knowledge from aims 1 and 2 is essential in applying mRNA silencing to HD patients. Aim 3 studies cellular mechanisms by which RNAi restores essential neuronal signaling activity in HD cells and the threshold for wild type huntingtin to main normal neuronal integrity. Aim 3 combines allelic RNAi and neuronal signaling of the cysteine transporter that regulates glutathione clearance of reactive oxygen species. Aim 4 examines the safety of administration of allele specific siRNA, by evaluating innate immunity and inflammatory responses. This proposal satisfies NINDS goals in translational science: translation of gene silencing therapeutics, early-state therapy development, and identifying mechanisms that underlie nervous system function. Innovations include targeting mRNA alleles for RNAi, use of HD mouse models that express only human huntingtin genes, quantitative measurement of huntingtin allelic mRNA based on SNP heterozygosities, deep sequencing analysis to identify 3 UTR huntingtin mRNA regulation, and zinc finger nuclease strategy to eliminate huntingtin alleles at the genomic level. This proposal applies fundamental biology to understand pathogenesis of HD and translate this knowledge into better therapies.