Mechanism of DNA strand-break repair deficiency in Huntington's disease

 描述（由申请人提供）：亨廷顿病（HD）是一种无法治愈的常染色体显性神经退行性疾病，由亨廷顿（HTT）基因中CAG三联体重复扩增引起。突变HTT基因中扩增的CAG重复序列被翻译成多聚谷氨酰胺（polyQ）段，这对突变蛋白质赋予了有害的功能增益。结构改变的突变体（m）HTT与关键因子相互作用并使其失活;这些因子的功能丧失破坏HD中特定基因的转录、突触功能、钙稳态和线粒体动力学和能量学。此外，DNA损伤的积累和DNA损伤反应（DDR）共济失调毛细血管扩张突变（ATM）信号传导的异常激活也涉及HD病理机制。尽管这些发现定义了潜在的疾病机制，但mHTT诱导这些病理变化并驱动功能下降和神经元损失的早期引发过程仍然未知。这种知识差距是开发预防HD神经功能下降的靶向治疗策略的主要限速步骤;由于这种限制，这种退行性疾病仍然无法治疗，并且总是致命的。在重要的一步 我们进一步发现，正常HTT募集多核苷酸激酶3 ′磷酸酶（PNKP）（一种主要的DNA链断裂修复酶）、共济失调蛋白-3（ATXN 3）（一种去泛素化酶）和RNA聚合酶II（RNAPII），以形成转录依赖性多蛋白DNA修复复合物，该复合物可能参与活跃转录基因的修复。相比之下，mHTT破坏了这种修复复合物的功能完整性并消除了PNKP活性，导致DNA链断裂积累，从而刺激HD中DNA损伤反应信号传导的致病性激增。该项目的总体目标是表征mHTT损害PNKP和ATXN 3的酶活性，干扰活跃转录基因的修复，并有助于HD病理机制的分子相互作用，以便我们最终能够开发这种终末期疾病的预防性治疗。这些研究旨在为HD中的神经变性提供机制基础，并揭示DNA修复在其中的关键作用。目的1应证实mHTT改变转录偶联修复复合物的功能完整性，并灭活PNKP，以诱导HD中活跃转录基因的DNA链断裂。因此，这些研究应该提供重要的见解polyQ扩增如何干扰特定的DNA修复机制，以诱导核和mtDNA损伤的HD。目的2将提供关键的机制的见解mHTT如何触发促凋亡刺激，导致神经毒性的HD。目的3将测试PNKP过表达在突变细胞和HD转基因小鼠模型中减少DNA损伤、预防或减缓HD中DDR途径活化的有效性。这些研究应证实突变HTT介导的神经元DNA修复损伤有助于HD的神经毒性，并确定近端分子靶点，以开发基于机制的HD治疗策略。

 DESCRIPTION (provided by applicant): Huntington's disease (HD) is an incurable autosomal dominant neurodegenerative disease that is caused by an expansion of CAG triplet repeats in the huntingtin (HTT) gene. The expanded CAG repeats in the mutant HTT gene are translated into a polyglutamine (polyQ) tract that confers a deleterious gain of function on the mutant protein. The structurally altered mutant (m)HTT interacts with and inactivates critical factors; loss of function of these factors disrupts transcription of specific genes, synaptic function, calcium homeostasis and mitochondrial dynamics and energetics in HD. Additionally, accumulation of DNA damage and aberrant activation of DNA damage-response (DDR) ataxia telangiectasia-mutated (ATM) signaling is also implicated in the HD pathomechanism. Notwithstanding these discoveries defining the underlying disease mechanisms, the early instigating processes by which mHTT induces these pathological changes and drives functional decline and neuronal loss remain unknown. This knowledge gap is a major rate-limiting step in developing a targeted therapeutic strategy to prevent neurological decline in HD; due to this limitation this degenerative illness remains untreatable and invariably fatal. In an important step forward, we have discovered that the normal HTT recruits polynucleotide kinase 3' phosphatase (PNKP), a major DNA strand break repair enzyme; ataxin-3 (ATXN3), a deubiquitinating enzyme; and RNA polymerase II (RNAPII) to form a transcription-dependent multi- protein DNA repair complex likely to be involved in the repair of actively transcribing genes. In contrast, mHTT disrupts the functional integrity of this repair complex and abrogates PNKP activity, resulting in DNA strand break accumulation to stimulate a pathogenic surge of DNA damage-response signaling in HD. The overall objective of this project is to characterize the molecular interactions by which mHTT impairs the enzymatic activities of PNKP and ATXN3, perturbs repair of the actively transcribed genes, and contributes to HD pathomechanism so that we can ultimately develop a preventive therapy for this terminal disease. These studies are designed to provide a mechanistic basis for neurodegeneration in HD and uncover the critical role of DNA repair therein. Aim 1 should confirm that mHTT alters the functional integrity of the transcription coupled repair complex, and inactivates PNKP to induce DNA strand breaks in the actively transcribing genes in HD. These studies should thus provide important insights into how polyQ expansion interferes with a specific DNA repair machinery to induce nuclear and mtDNA damage in HD. Aim 2 will provide critical mechanistic insights into how mHTT triggers pro-apoptotic stimuli to cause neurotoxicity in HD. Aim 3 will test the effectiveness of PNKP overexpression in the mutant cells and in HD transgenic mouse model in decreasing DNA lesions, preventing or slowing down DDR pathway activation in HD. These studies should confirm that mutant HTT-mediated impairment of neuronal DNA repair contributes to neurotoxicity in HD, and identify proximal molecular targets to develop mechanism-based therapeutic strategies for HD.