DNA repair and alpha-synucleinopathy in Lewy body disorders

路易体疾病中的 DNA 修复和 α-突触核蛋白病

• 批准号: 9978230
• 负责人: Rehana Khan Leak
• 金额: $ 40.83万
• 依托单位: DUQUESNE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-15 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9978230
• 关键词: Address; Affect; Animal Model; Anterior; Anxiety; Area; Astrocytes; Autopsy; Base Excision Repairs; Behavioral; Biological Assay; Brain; Brain region; Cell Count; Cell Nucleus; Cell model; Cells; Cleaved cell; Cognitive; Complex; Cysteine; DNA; DNA Binding; DNA Binding Domain; DNA Damage; DNA Repair; DNA Repair Gene; DNA Repair Inhibition; DNA Sequence Alteration; DNA ligase I; Data; Deposition; Disease; Dyes; Emotions; Endonuclease I; Ensure; Environmental Impact; Etiology; Event; Experimental Models; Exposure to; FOS gene; Female; Funding; Gender; Genes; Genome; Genome Stability; Genomics; Grant; Harvest; High Prevalence; Hippocampus (Brain); Histologic; Histopathology; Human; Immunoblotting; Impairment; Individual; Influentials; Infusion procedures; Interneurons; JUN gene; Lead; Lewy Body Disease; Lewy body pathology; Life; Light; Link; Maintenance; Measures; Mediating; Memory; Modeling; Mosaicism; Mus; Mutant Strains Mice; Mutation; Nature; Neurodegenerative Disorders; Neurologic; Neurons; Olfactory Pathways; Outcome; Oxidation-Reduction; Oxidative Stress; Oxides; Parkinson Disease; Parkinsonian Disorders; Pathologic; Pathology; Patients; Pattern; Pharmacology; Phenotype; Plant Roots; Polymerase; Proteins; Quality Control; Rattus; Reporting; Resistance; Risk; Role; Seeds; Sex Differences; Site; Smell Perception; Specificity; Stroke; Subfamily lentivirinae; System; Testing; Transgenic Organisms; Travel; Ubiquitin; Vertebral column; Woman; Work; XRCC1 gene; alpha synuclein; anxiety-like behavior; brain health; brain tissue; design; endonuclease; entorhinal cortex; experimental study; gastrointestinal system; genome integrity; high risk; human data; in vivo; knock-down; male; men; monomer; motor behavior; mutant; nervous system disorder; neuron loss; olfactory bulb; olfactory nuclei; overexpression; phosphodiester; pollutant; pre-clinical; prevent; protein aggregation; repair enzyme; repaired; response; small hairpin RNA; synucleinopathy; theories; transmission process

The genome is the foremost determinant of inherent defenses, and loss of its integrity can culminate in pathological conditions. Cellular DNA repair systems have evolved to mitigate this risk, but are poorly understood in neurological disorders. We propose to examine if changes in DNA repair modify behavioral and histological outcomes in limbic-predominant Lewy body disorder, a neurological condition in which insoluble deposits of α-synuclein protein fill areas of the brain involved in olfaction, emotion, and memory. The repair of oxidized, damaged DNA is facilitated by apurinic/apyrimidinic endonuclease I (APE1). The pleiotropic APE1 gene has both a DNA repair function and a redox function—the former is impaired in brain tissue from Parkinson’s patients. It is known that oxidized α-synuclein enters the nucleus and elicits breaks in DNA, but the relationship between APE1 and α-synucleinopathy has not, until now, been scrutinized. We have collected pilot data to support the central hypothesis that APE1 tempers the pathological sequelae of exposure to preformed α-synuclein fibrils in cellular and animal models of limbic-predominant Lewy body disorders. The olfactory bulb/anterior olfactory nucleus (OB/AON) complex is one of the earliest brain regions to display inclusions in Lewy body disorders. Our pilot data reveal that male mice respond to fibril infusions in the OB/AON with loss of smell, dense limbic inclusions, and a decrease in APE1 expression within six months. In contrast, females respond with a global increase in APE1, do not develop smell deficits, and display fewer limbic inclusions. These sex differences parallel the 2 to 3-fold higher risk of Lewy body disorders in men compared to women. Our recent human postmortem work similarly reveals that gender modifies the impact of limbic Lewy body disease on APE1 expression in limbic brain regions. New pilot data also show that shRNA-mediated knockdown of APE1 increases inclusions in primary neurons. Here, we propose further mechanistic studies that will put the causality of the link between APE1 and α-synucleinopathy to the test. AIM 1. We will test the hypothesis that lentivirus-mediated APE1 knockdown in primary neuron/astrocyte and primary neuron cultures treated with α-synuclein fibrils will elicit loss of APE1 expression and endonuclease activity, greater DNA damage, and more protein aggregations and cell loss. We will employ two shRNA sequences to address this hypothesis and determine if overexpressing human WT APE1 prevents the knockdown phenotype, to ensure shRNA specificity. In AIM 2, we will test the hypothesis that APE1 mitigates the behavioral and histological impact of α-synuclein fibril infusions in the male and female rat OB/AON. We will contrast human wildtype APE1-overexpressing rats to transgenic rats that overexpress mutant APE1 with impaired DNA repair (D210A mutation) or loss of redox capacity (C65A mutation). Cognitive, anxiety, olfactory, and motor behavior will be assessed at monthly intervals. APE1 expression and activity, DNA damage markers, proteinopathic inclusions, and cell counts will be measured at 3, 6, and 12 months. If funded, these studies will be the first to examine the relationship between α-synucleinopathy and DNA repair capacity and to characterize sex differences in limbic Lewy pathology. The experiments are designed to either support or reject the notion that DNA reparatory mechanisms should be pursued as a rational target for stymieing α-synucleinopathic disease.

基因组是内在防御的最重要的决定因素，其完整性的丧失可导致病理性疾病。 条件细胞DNA修复系统已经进化到可以减轻这种风险，但在神经系统中了解甚少。 紊乱我们建议检查DNA修复的变化是否会改变边缘为主的路易体障碍的行为和组织学结果，路易体障碍是一种神经系统疾病，其中α-突触核蛋白蛋白的不溶性沉积物填充了大脑皮层的区域。 大脑负责嗅觉情感和记忆氧化损伤的DNA的修复是由 脱嘌呤/脱嘧啶核酸内切酶I（APE 1）。多效APE 1基因具有DNA修复功能和氧化还原功能 功能-前者在帕金森病患者的脑组织中受损。已知氧化的α-突触核蛋白进入 APE 1与α-synucleinopathy之间的关系至今尚未被详细研究。我们已经收集了初步数据来支持中心假设，即APE 1缓和了在边缘占主导地位的路易体疾病的细胞和动物模型中暴露于预先形成的α-突触核蛋白纤维的病理后遗症。嗅球/前嗅核（OB/AON）复合体是路易体疾病中最早显示包涵体的脑区之一。我们的试验数据显示，雄性小鼠对OB/AON中的原纤维输注有反应，在六个月内嗅觉丧失，边缘系统致密包涵体和APE 1表达减少。相比之下，女性的反应是APE 1的全球性增加，不会出现嗅觉缺陷，并且显示较少的边缘系统内含物。这些性别差异与男性患路易体疾病的风险比女性高2至3倍相平行。我们最近的人类尸检工作同样揭示了性别改变边缘路易体病对边缘脑区域APE 1表达的影响。新的试验数据还表明，shRNA介导的APE 1敲低增加了原代神经元中的包涵体。在这里，我们提出了进一步的机制研究，将把APE 1和α-突触核蛋白病之间的联系的因果关系进行测试。 AIM 1.我们将检验慢病毒介导的APE 1敲低在原代神经元/星形胶质细胞和原代胶质细胞中表达的假设。 用α-突触核蛋白原纤维处理的神经元培养物将引起APE 1表达和核酸内切酶活性的丧失、更大的DNA损伤以及更多的蛋白质聚集和细胞损失。我们将使用两个shRNA序列来解决这一假设，并确定是否过表达人WT APE 1阻止敲低表型，以确保shRNA特异性。 在AIM 2中，我们将检验APE 1减轻α-突触核蛋白原纤维的行为和组织学影响的假设。 在雄性和雌性大鼠OB/AON中输注。我们将人类野生型APE 1过表达大鼠与转基因大鼠进行对比， 过表达突变型APE 1并伴有DNA修复受损（D210 A突变）或氧化还原能力丧失（C65 A）的大鼠 突变）。认知、焦虑、嗅觉和运动行为将每月进行一次评估。将在3、6和12个月时测量APE 1表达和活性、DNA损伤标记物、蛋白质病性包涵体和细胞计数。 如果获得资助，这些研究将是第一个研究α-突触核蛋白病和DNA修复能力之间的关系，并描述边缘路易病理学的性别差异。这些实验旨在支持或拒绝DNA修复机制应作为阻止α-突触核蛋白病的合理靶点的观点。