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

• 批准号: 10252937
• 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
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-05-15 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10252937
• 关键词: 8-Oxoguanine DNA Glycosylase; Adenine; Affect; Age; Aging; Alzheimer' s Disease; Amyloid beta-Protein; Antibodies; Autopsy; Biological Assay; Brain; Brain Diseases; Bypass; Cell Culture System; Cell Differentiation process; Cells; Code; Cytosine; DNA; DNA Damage; DNA Sequence Alteration; DNA lesion; Dementia with Lewy Bodies; Diagnostic; Event; Exhibits; Gene Expression Profile; Generations; Genes; Genetic Transcription; Genomic DNA; Guanine; Human; In Vitro; Knock-out; Knockout Mice; Knowledge; Lead; Lewy Bodies; Lipids; Measures; Mediating; Messenger RNA; Microfluidics; Molecular; Multiple System Atrophy; Mutagenesis; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Nucleic Acids; OGG1 gene; Oxidative Stress; Parkinson Disease; Parkinson' s Dementia; Pathogenesis; Pathogenicity; Pathologic; Pathology; Patients; Population; Process; Proteins; Public Health; RNA; RNA Polymerase II; RNA Sequences; Recombinant Proteins; Reporting; Research; Research Proposals; Ribonucleases; Role; Sampling; Seeds; Site; Stains; Substantia nigra structure; Techniques; Testing; Therapeutic; Toxic effect; United States; Variant; aged; alpha synuclein; base; brain tissue; cell type; cohort; combat; cross reactivity; deep sequencing; disorder control; dopaminergic neuron; in vivo; innovation; knock-down; macromolecule; mind control; mutant; neurotoxicity; next generation; novel; novel therapeutic intervention; oxidation; oxidative DNA damage; repair enzyme; synucleinopathy; tau Proteins; tool; transmission process

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.

项目总结 氧化应激引起的最常见的DNA损伤是8-氧代-7，8-二氢鸟嘌呤(8-oxodG)，它是 通常与包括帕金森病和衰老过程在内的神经退行性疾病有关。在终端分化中 像神经元一样的细胞，活性基因转录链中的8-oxodG DNA损伤可以被RNA绕过 聚合酶II，并通过一种称为转录突变的过程产生错误的蛋白质。研究 报道了帕金森病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糖基酶(OGG1)的活性也降低 记录在PD和老化条件下。 人类SNCA的编码区包含43个潜在的转录突变位点。我们最近发现 氧化应激或Ogg1基因敲除会增加α-SYN的转录突变，导致蛋白质Ag。 会众。此外，使用一种新的技术，RNaseH2依赖的PCR，我们能够识别各种TM- 从帕金森病患者的脑标本中产生了包括S42Y和A53E在内的α-SYN突变体。我们还发现了S42Y- 从帕金森病和路易体痴呆死后脑标本中提取的路易小体阳性 特异性抗S42Y抗体。综上所述，我们的初步结果强烈表明，转录突变 在8-oxodG积累条件下有助于产生新的α-SYN致病物种，如 帕金森氏病和其他联体核素病。 目前，关于这些突变物种可能产生的机制，人们的认识存在很大差距。 对α-SYN病理的影响以及LBS中的α-SYN聚集体是否含有由转录产生的突变蛋白 诱变。我们的中心假设是8-oxodG介导的转录突变事件导致 α-SYN新突变体的产生，可引起核依赖聚集和AS毒性 在警局看到的。本研究的目的是鉴定α-SYN因氧化应激而产生的TM突变种，并研究 它们在α-SYN聚集和PD发病机制中的作用。 将追求以下三个具体目标：在目标1中，8-oxodG水平和TM的整个概况- 帕金森病患者和对照组死后脑标本中α-SYN mRNA的衍生突变体将被检测。 精致的。在目标2中，TM产生的α-SYN突变体在依赖于成核的聚集过程中的作用将是 研究和α-SYN TM突变蛋白将在人死后大脑样本中检测到。在目标3中， TM突变体对α-SYN聚集、毒性和神经元-神经元传递的集体影响 将会被评估。 该项目的成功完成将使我们对分子机械的理解发生范式转变-- 帕金森病患者氧化应激介导的α-SYN病理基础异常。了解α-SYN中的TM事件可能 同样重要的是了解其他分子，如Aβ和tau在其他神经退行性疾病中的作用。