Contribution of transcriptional mutagenesis of oxidative DNA lesions to generatingnew mutant alpha-synuclein species and aggregation toward the pathogenesis of Parkinson'sdisease

氧化DNA损伤的转录突变对产生新的突变α-突触核蛋白种类和聚集对帕金森病发病机制的贡献

基本信息

批准号：
10405538
负责人：
YOON-SEONG KIM
金额：
$ 39.25万
依托单位：
RBHS-ROBERT WOOD JOHNSON MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-05-15 至 2024-04-30
项目状态：
已结题

项目摘要

PROJECT SUMMARY The most frequent DNA lesion caused by oxidative stress is 8-oxo-7,8-dihydroguanine (8-oxodG) and it is often associated with neurodegenerative diseases including PD and aging processes. In terminally differentiated cells like neurons, 8-oxodG DNA lesions in the transcribed strand of an active gene could be bypassed by RNA polymerase II, and generate erroneous proteins through a process called transcriptional mutagenesis. Studies have reported selective increase of 8-oxodG in the substantia nigra dopaminergic neurons of PD brain tissue. Decreased activity of the 8-oxodG-specific repair enzyme, 8-oxoguanine-DNA glycosylase (OGG1), was also documented in PD and aging conditions. Coding region of human SNCA contains 43 potential sites for transcriptional mutagenesis. We recently found that oxidative stress or Ogg1 knockdown increase transcriptional mutagenesis of α-SYN, leading to protein ag- gregation. Moreover using a novel technique, RNase H2-dependent PCR, we were able to identify various TM- generated α-SYN mutants including S42Y and A53E from human PD brain samples. We have also found S42Y- positive Lewy bodies from postmortem brain samples of PD and dementia with Lewy bodies (DLB) using highly specific anti-S42Y antibody. Together, our preliminary results strongly suggest that transcriptional mutagenesis contributes to generation of novel pathogenic species of α-SYN in 8-oxodG accumulation conditions such as Parkinson's disease and other synucleinopathy. Currently, there are major gaps in knowledge regarding the mechanism by which these mutant species may affect α-SYN pathology and if α-SYN aggregates in LBs contain mutant proteins produced by transcriptional mutagenesis. Our central hypothesis is that 8-oxodG-mediated transcriptional mutagenesis event leads to the generation of novel mutant variants of α-SYN which causes nucleation-dependent aggregation and toxicity as seen in PD. The objective here is to identify oxidative stress-derived TM mutant species of α-SYN and investigate their contribution to α-SYN aggregation and the pathogenesis of PD. The following three specific aims will be pursued: In Aim 1, levels of 8-oxodG and the entire profile of TM- derived mutant variants of α-SYN mRNA in human postmortem brain samples of PD and control will be meas- ured. In Aim 2, the role of TM-generated α-SYN mutants in nucleation-dependent aggregation process will be investigated and α-SYN TM mutant proteins will be detected in human postmortem brain samples. In Aim 3, the collective effect of TM-generated mutants on α-SYN aggregation, toxicity, and neuron-to-neuron transmission will be assessed. Successful completion of the project will create a paradigm shift in our understanding of the molecular mech- anisms underlying oxidative stress-mediated α-SYN pathology in PD. Knowledge of TM events in α-SYN might be equally important to understand other molecules, such as Aβ and tau in other neurodegenerative conditions.

项目概要氧化应激引起的最常见的 DNA 损伤是 8-oxo-7,8-二氢鸟嘌呤 (8-oxodG)，它是通常与神经退行性疾病（包括 PD 和终末分化过程）相关。在神经元等细胞中，活性基因转录链中的 8-oxodG DNA 损伤可以被 RNA 绕过聚合酶 II，并通过称为转录诱变的过程产生错误的蛋白质。报道了PD脑组织黑质多巴胺能神经元中8-oxodG的选择性增加。 8-oxodG 特异性修复酶 8-oxoguanine-DNA 糖基化酶 (OGG1) 的活性也降低记录在 PD 和老化条件中。我们最近发现人类 SNCA 的编码区包含 43 个潜在的转录突变位点。氧化应激或 Ogg1 敲低会增加 α-SYN 的转录突变，导致蛋白质 ag- 此外，使用一种新技术，RNase H2 依赖性 PCR，我们能够识别各种 TM-。我们还从人类 PD 大脑样本中产生了 α-SYN 突变体，包括 S42Y 和 A53E。 PD 和痴呆症的死后脑样本呈路易体阳性，路易体 (DLB) 使用高度总之，我们的初步结果强烈表明转录突变。有助于在 8-oxodG 积累条件下产生新型 α-SYN 致病菌，例如帕金森病和其他突触核蛋白病。目前，关于这些突变物种可能的机制的知识还存在重大差距。影响 α-SYN 病理学，并且 LB 中的 α-SYN 聚集体是否含有转录产生的突变蛋白我们的中心假设是 8-oxodG 介导的转录诱变事件导致产生新的 α-SYN 突变体，导致成核依赖性聚集和毒性此处的目的是鉴定氧化应激衍生的 α-SYN TM 突变种并进行研究。它们对 α-SYN 聚集和 PD 发病机制的贡献。将追求以下三个具体目标：在目标 1 中，8-oxodG 的水平和 TM- 的整体概况 PD 和对照的人类死后脑样本中衍生的 α-SYN mRNA 突变体将被测量在目标 2 中，TM 生成的 α-SYN 突变体在成核依赖性聚集过程中的作用将被确定。在目标 3 中，将在人类死后大脑样本中检测到 α-SYN TM 突变蛋白。 TM 生成的突变体对 α-SYN 聚集、毒性和神经元间传递的集体效应将被评估。该项目的成功完成将使我们对分子机制的理解发生范式转变。 PD 中氧化应激介导的 α-SYN 病理学的本质是 α-SYN 中 TM 事件的知识。了解其他分子（例如其他神经退行性疾病中的 Aβ 和 tau）同样重要。