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: 10203277
• 负责人: YOON-SEONG KIM
• 金额: $ 36.14万
• 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/10203277
• 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; Diagnostic; Event; Exhibits; Generations; Genes; Genetic Transcription; Genomic DNA; Guanine; Human; In Vitro; Knock-out; Knockout Mice; Knowledge; Lead; Lewy Bodies; Lewy Body Dementia; 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.

项目摘要 由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），并且它是 通常与包括PD和衰老过程的神经退行性疾病相关。在终末分化 在神经元等细胞中，RNA可以绕过活性基因转录链中的8-oxodG DNA损伤， 聚合酶II，并通过称为转录诱变的过程产生错误的蛋白质。研究 已经报道了PD脑组织的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（OGG 1）的活性降低， 记录在PD和老化条件中。 人SNCA编码区含有43个潜在的转录突变位点。我们最近发现 氧化应激或Ogg 1敲低增加α-SYN转录突变，导致蛋白ag- 种族隔离。此外，使用一种新的技术，RNA酶H2依赖的PCR，我们能够识别各种TM- 从人PD脑样品中产生包括S42 Y和A53 E的α-SYN突变体。我们还发现了S42 Y- 使用高密度聚乙烯凝胶，从PD和路易体痴呆（DLB）的尸检脑样品中获得阳性路易体， 特异性抗S42 Y抗体。总之，我们的初步结果有力地表明，转录诱变 有助于在8-oxodG积累条件下产生α-SYN的新致病性物种，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 影响α-SYN病理，如果LB中的α-SYN聚集体含有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了 产生α-SYN的新突变变体，其引起成核依赖性聚集和毒性， 在PD中看到本研究的目的是鉴定α-SYN的氧化应激衍生的TM突变体种类，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的水平和TM-1的整个概况。 在PD和对照的人死后脑样本中，将测量α-SYN mRNA的衍生突变变体。 ured。在目标2中，TM产生的α-SYN突变体在成核依赖性聚集过程中的作用将是 研究和α-SYN TM突变蛋白将检测人类死后脑样品。在目标3中， TM产生的突变体对α-SYN聚集、毒性和神经元-神经元传递的集体效应 将被评估。 该项目的成功完成将在我们对分子机制的理解中创造一个范式转变- PD中氧化应激介导的α-SYN病理学的潜在差异。α-SYN中TM事件的知识可能 同样重要的是要了解其他分子，如Aβ和tau在其他神经退行性疾病。