DNA Double Strand Break Repair Deficiency and Neurodegeneration

脊髓小脑性共济失调3型(SCA3)，又名Machado-Joseph病，是最常见的显性遗传性疾病 全球共济失调。它是Aaxin 3编码区CAG(谷氨酰胺)重复扩增的结果，a 含有聚谷氨酰胺(14-41个重复)的蛋白质。在研究SCA3的作用机制时，我们发现 野生型Aaxin 3刺激，而突变型则取消多核苷酸激酶3‘- 磷酸酶(PNKP)是一种重要的DNA修复蛋白。这导致了DNA双链的积累 SCA3患者和小鼠大脑的损伤。DNA损伤反应通路的持续激活 随之而来的细胞凋亡可能是SCA3的原因之一。我们最近的研究表明，PNKP发挥着 通过经典的非同源末端连接(C-NHEJ)在DNA双链断裂修复中起关键作用。我们有 证明PNKP介导的C-NHEJ修复途径是无错误的，具有同源的新生RNA 提供模板以修复转录基因中双链断裂部位的缺失序列。 与野生型相比，SCA3小鼠脑内DNA链断裂分析显示明显更多的链断裂 在转录的基因中，而非非转录的基因中。总的来说，这些数据表明差异基因组 神经细胞发生区域特异性的链断裂修复。我们的实验室发现，除了ATXN3，还有两个RNA- 结合/剪接蛋白(NONO和SFPQ)和糖酵解的关键变构调节因子参与了这一过程 路径。我们首次展示了Aaxin 3和糖酵解调节蛋白在经典非典型肺炎中的作用。 同源末端连接介导DNA双链断裂修复。值得注意的是，所有这些因素形成了一种生理上的 与RNA聚合酶II和其他经典的非同源末端连接蛋白形成复合体。我们还发现， NONO和SFPQ显著刺激PNKP的末端加工活性。我们推测这些与RNA的结合 蛋白质促进RNA-DNA杂交体的形成，从而依赖于RNA的DNA聚合酶可以 有效地使用RNA作为模板来恢复丢失的信息。然而，糖酵解诱导剂没有。 影响PNKP的活性，但诱导剂催化的代谢产物显著刺激PNKP的活性。此外， 代谢产物甚至可以恢复SCA3患者脑核提取液中PNKP的活性，提示 这种天然代谢物对SCA3有很好的治疗潜力。因此，这个项目将检验这些假设 因此，修复PNKP介导的C-NHEJ修复通路对于维持心肌细胞的完整性至关重要。 转录后的基因组，从而将神经细胞从突变的有害影响中拯救出来 ATXN3。理解转录基因无错误双链断裂修复的机制基础 在神经细胞中通过经典的非同源末端连接途径，以及一种天然的 代谢产物在这样的途径中，将极大地推进我们的知识，并应加速发展 SCA3和其他多Q疾病的新治疗方式。

Spinocerebellar ataxia type 3 (SCA3), aka Machado-Joseph Disease is the most common dominantly inherited ataxia worldwide. It is the result of a CAG (glutamine) repeat expansion in the coding region of Ataxin 3, a polyglutamine (14-41 repeats)-containing protein. While investigating the mechanism of SCA3, we have found that wild type Ataxin 3 stimulates, while the mutant form abrogates the activity of polynucleotide kinase 3’- phosphatase (PNKP), an essential DNA repair protein. This resulted in the accumulation of DNA double-strand breaks in the brains of SCA3 patients and mice. Constant activation of the DNA damage-response pathway with consequent cellular apoptosis is a plausible cause of SCA3. Our recent studies have revealed that PNKP plays a critical role in DNA double strand break repair via classical non-homologous end joining (C-NHEJ). We have demonstrated that PNKP-mediated C-NHEJ repair pathway is error-free, with homologous nascent RNA providing the template to restore the missing sequence at the double strand break site in transcribed genes. DNA strand break analysis in the SCA3 mouse brain versus wild type showed significantly more strand breaks in the transcribed but, not the non-transcribed genes. Collectively, these data indicate that differential genomic region-specific strand break repair occurs in neuronal cells. Our lab found that, in addition to ATXN3, two RNA- binding/splicing proteins (NONO and SFPQ) and a key allosteric regulator of glycolysis are involved in this pathway. We are the first to show the roles of Ataxin 3 and the glycolysis-regulatory protein in classical non- homologous end joining mediated DNA double strand break repair. Notably, all these factors form a physiological complex with RNA polymerase II and other classical non-homologous end joining proteins. We also found that NONO and SFPQ significantly stimulated PNKP’s end-processing activity. We postulate that these RNA-binding proteins facilitate the formation of the RNA-DNA hybrid so that the RNA-dependent DNA polymerase can effectively use the RNA as a template to restore the missing information. However, the glycolysis inducer did not affect PNKP’s activity, but the inducer-catalyzed metabolite significantly stimulated PNKP’s activity. Furthermore, the metabolite can even restore the activity of PNKP in SCA3 patients’ brain nuclear extract, suggesting the promising therapeutic potential of this natural metabolite for SCA3. Hence, this project will test the hypotheses that: restoration of the PNKP-mediated C-NHEJ repair pathway is crucial for maintaining the integrity of the transcribed genome, and thereby rescuing neuronal cells from the deleterious effects of mutant ATXN3. Understanding the mechanistic basis of error-free double strand break repair of the transcribed genes in neuronal cells via classical non-homologous end joining pathway, and the modulatory effect of a natural metabolite in such a pathway, will significantly advance our knowledge and should accelerate the development of new treatment modalities for SCA3 and other polyQ diseases.