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。亨廷顿蛋白基因编码一种350kD的蛋白质；导致疾病的突变是由超过36个连续的谷氨酸组成的氨基末端多谷氨酰胺重复序列的扩大。我们广泛的研究目标是了解HD发病的分子基础，从而达到靶向治疗的目的。由于HD是一种遗传性疾病，我们预计突变的等位基因将至少有一个单核苷酸多态性，从而为RNAi的基因沉默提供了一个靶点。因此，我们的核心观点是，RNA沉默有助于选择性地减少突变的Huntingtin的表达，减缓或阻断HD神经元的功能障碍和死亡。我们假设，选择性地敲除突变的Huntingtin可以恢复正常的神经元功能，但过度沉默通过干扰必要的信号事件来损害神经元功能。目的1在小鼠体内检测等位基因特异性沉默是否可以延缓或预防HD神经病和异常行为。目的2研究人死后HD脑内Huntingtin mRNA的转录后调控，寻找有利于其合成的分子过程。我们将使用一种新的策略来量化每个Huntingtin等位基因的mRNA，基于等位基因的SNP杂合性。了解AIMS 1和AIMS 2的知识对于将信使核糖核酸沉默应用于HD患者至关重要。目的3研究RNAi恢复HD细胞基本神经元信号活动的细胞机制以及野生型Huntingtin恢复正常神经元完整性的阈值。目的3结合等位基因RNAi和调节谷胱甘肽清除活性氧的半胱氨酸转运体的神经元信号。目的4通过评估先天免疫和炎症反应，检查给予等位基因特异性siRNA的安全性。这一建议满足了NINDS在翻译科学方面的目标：基因沉默疗法的翻译、早期状态疗法的开发以及确定神经系统功能的基础机制。创新包括针对RNAi的mRNA等位基因，使用只表达人类Huntingtin基因的HD小鼠模型，基于SNP杂合性的Huntingtin等位基因mRNA的定量测量，深度测序分析以确定3个非编码区Huntingtin mRNA调控，以及在基因组水平上消除Huntingtin等位基因的锌指核酸策略。这一建议应用基础生物学来理解HD的发病机制，并将这些知识转化为更好的治疗方法。 公共卫生相关性：亨廷顿病(HD)患者会出现认知缺陷、抑郁和运动异常。脑细胞过早死亡。我们将研究基因沉默如何减少导致HD的突变亨廷顿蛋白的产生，从而防止HD动物模型中的神经元和培养中的HD神经元的功能障碍和死亡。我们将研究引发基因沉默的分子在大脑中的安全性。这项建议通过研究沉默致病基因的基本机制来解决HD的治疗问题。

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. PUBLIC HEALTH RELEVANCE: Patients with Huntington's disease (HD) develop cognitive deficits, depression and movement abnormalities. Brain cells die early. We will examine how gene silencing can reduce production of the mutant huntingtin protein that causes HD, thereby preventing dysfunction and death in neurons in animal models of HD and in HD neurons in culture. We will study safety, in brain, of the molecules that initiate gene silencing. This proposal addresses treatment of HD through study of basic mechanisms of silencing the gene that causes the disease.