Base Excision DNA Repair in Premature Aging and Neurodegeneration

过早衰老和神经退行性疾病中的碱基切除 DNA 修复

• 批准号: 8335917
• 负责人: David Wilson
• 金额: $ 41.22万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8335917
• 关键词: APTX gene; Abdomen; Affect; Aging; Aging-Related Process; Alkylating Agents; Amino Acid Sequence; Animals; Ataxia; Atrophic; Azoxymethane; Base Excision Repairs; Behavior; Biochemical; Biological Aging; Biological Markers; Blood Chemical Analysis; Brain; C-terminal; C57BL/6 Mouse; Cell physiology; Cells; Cerebellum; Cessation of life; Chromosomes; Citrate (si)-Synthase; Cockayne Syndrome; Collaborations; Colon; Cutaneous; DNA; DNA Damage; DNA Repair; DNA copy number; Data; Defect; Development; Disease; Disease susceptibility; ERCC6 gene; Endoderm; Enzymes; Exhibits; Exposure to; Failure; Fatty acid glycerol esters; Free Radicals; Future; Genes; Growth; Hepatotoxicity; Human; In Vitro; Inborn Genetic Diseases; Incidence; Inner mitochondrial membrane; Lesion; Life Expectancy; Ligation; Link; Longevity; Malignant Neoplasms; Mammals; Mesoderm; Mitochondria; Mitochondrial DNA; Modeling; Molecular Epidemiology; Motor; Mus; Mutation; Myoblasts; N-terminal; Nerve Degeneration; Neurodegenerative Disorders; Neurologic; Neurons; Nuclear; Nucleic Acids; Organ; Organism; Outcome; Oxidative Stress; Pathway interactions; Patients; Peripheral; Phenotype; Photosensitivity; Play; Predisposition; Premalignant; Premature aging syndrome; Process; Production; Property; Protein Isoforms; Proteins; Purkinje Cells; RNA; Reaction; Recruitment Activity; Repair Complex; Role; Rupture; Scaffolding Protein; Series; Single Strand Break Repair; Skeletal Muscle; Specificity; Spinocerebellar Ataxias; Stretching; Symptoms; Tissues; Transcript; Weight; Work; XRCC1 gene; adenylate; age related; base; brain volume; cell type; disease phenotype; epidemiology study; in vitro activity; mitochondrial dysfunction; neuroblastoma cell; oculomotor; oxidative DNA damage; oxidative damage; repaired; sensory neuropathy; stem; telomere; theories; tyrosyl-DNA phosphodiesterase

Cockayne Syndrome (CS) is an autosomal recessive disorder, characterized by growth failure, neurological abnormalities, premature aging symptoms, and cutaneous photosensitivity, but no increased cancer incidence. CS is divided into two strict complementation groups: CSA (mutation in CKN1) and CSB (mutation in ERCC6). Of the patients suffering from CS, 80% have mutations in the CSB gene. We are pursuing the hypothesis that the primary role of CS proteins is to facilitate the repair of endogenous DNA damage, and we have evidence for a direct role of CSB in regulating BER efficiency. Our in vitro work has also helped define the biochemical properties of CSB, revealing that the protein interacts with a diverse range of nucleic acid substrates and likely has important ATP-dependent and ATP-independent functions. More recent results, obtained in collaboration with Dr. Vilhelm Bohr, suggest that CSB plays a direct role in not only nuclear BER, but in mitochondrial BER, likely by helping recruit, stabilize, and/or retain BER proteins in repair complexes associated with the inner mitochondrial membrane. Future work will continue to examine the in vitro activities of CSB on key DNA and RNA transaction intermediates, elucidate the contributions of the unique N- and C-terminal portions of the protein that likely impart functional specificity, and explore the possible role of CSB in processing endogenous DNA damage. XRCC1 is a critical scaffold protein that orchestrates efficient single-strand break repair (SSBR), an important subpathway of BER. Recent data has found an association of XRCC1 with proteins causally linked to human spinocerebellar ataxias - aprataxin and tyrosyl-DNA phosphodiesterase 1 - implicating SSBR in protection against neuronal cell loss and neurodegenerative disease. In addition, molecular epidemiology studies in humans indicate that impaired function in XRCC1 may be associated with increased cancer susceptibility. We have evaluated a series of chronological and biological aging parameters in XRCC1 heterozygous (HZ) mice, which are deficient for XRCC1 function. HZ and wild-type (WT) C57BL/6 mice exhibit a similar median lifespan of 26 months and a nearly identical maximal life expectancy of 37 months. However, a number of HZ animals (7 of 92) showed a propensity for abdominal organ rupture, which may stem from developmental abnormalities given the prominent role of XRCC1 in endoderm and mesoderm formation. For other end-points evaluated weight, fat composition, blood chemistries, condition of major organs, tissues and relevant cell types, behavior, brain volume and function, and chromosome and telomere integrity HZ mice exhibited by-and-large a normal phenotype. Treatment of animals with the alkylating agent azoxymethane resulted in both liver toxicity and an increased incidence of precancerous lesions in the colon of HZ mice. Our study therefore indicates that XRCC1 haploinsufficiency in mammals has little effect on chronological longevity and many key biological markers of aging in the absence of environmental challenges, but may adversely affect normal animal development or increase disease susceptibility to a relevant genotoxic exposure. Ataxia with oculomotor apraxia 1 (AOA1) is caused by mutation in the APTX gene, which encodes the DNA stand break repair protein aprataxin. Aprataxin removes 5-adenylate groups in DNA that arise from aborted ligation reactions. AOA1 is characterized by global cerebellar atrophy, highlighted by loss of Purkinje cells, ocular motor apraxia, and motor and sensory neuropathy. Strikingly, AOA1 patients lack the cancer susceptibility and other peripheral symptoms (e.g., immunological deficiencies) commonly associated with other inherited disorders stemming from a DNA repair defect. We have found that aprataxin localizes to mitochondria in human cells, and have identified an N-terminal amino acid sequence that targets certain isoforms of the protein to this intracellular compartment. Interestingly, transcripts encoding this unique N-terminal stretch are expressed in the human brain, with highest production in the cerebellum. Depletion of aprataxin in human SH-SY5Y neuroblastoma cells and primary skeletal muscle myoblasts results in mitochondrial dysfunction, as revealed by reduced citrate synthase activity and mitochondrial DNA (mtDNA) copy number. Moreover, mtDNA, not nuclear DNA, has higher levels of background DNA damage upon aprataxin knockdown, suggesting a direct role for the enzyme in mtDNA processing. These data indicate that aprataxin activity is indispensable for maintaining mitochondrial function and that there likely is a mitochondrial component to the disease phenotype of AOA1. Future studies are aimed at determining the reason behind the tissue selectivity of the disorder.

科凯恩综合征 (CS) 是一种常染色体隐性遗传疾病，其特征是生长障碍、神经系统异常、过早衰老症状和皮肤光敏性，但不会增加癌症发病率。 CS分为两个严格的互补组：CSA（CKN1突变）和CSB（ERCC6突变）。 在患有CS的患者中，80%的CSB基因发生突变。 我们正在追求这样的假设：CS 蛋白的主要作用是促进内源性 DNA 损伤的修复，并且我们有证据表明 CSB 在调节 BER 效率方面具有直接作用。 我们的体外工作还帮助定义了 CSB 的生化特性，揭示了该蛋白质与多种核酸底物相互作用，并且可能具有重要的 ATP 依赖性和 ATP 独立功能。 与 Vilhelm Bohr 博士合作获得的最新结果表明，CSB 不仅在核 BER 中发挥着直接作用，而且在线粒体 BER 中发挥着直接作用，可能是通过帮助招募、稳定和/或保留与线粒体相关的修复复合物中的 BER 蛋白。线粒体内膜。 未来的工作将继续检查 CSB 对关键 DNA 和 RNA 交易中间体的体外活性，阐明可能赋予功能特异性的蛋白质独特 N 端和 C 端部分的贡献，并探索 CSB 在处理内源性 DNA 损伤。 XRCC1 是一种关键的支架蛋白，可协调有效的单链断裂修复 (SSBR)，这是 BER 的重要子通路。最近的数据发现，XRCC1 与与人类脊髓小脑共济失调有因果关系的蛋白质 - aprataxin 和酪氨酰 DNA 磷酸二酯酶 1 - 存在关联，表明 SSBR 可以防止神经元细胞丢失和神经退行性疾病。 此外，人类分子流行病学研究表明，XRCC1 功能受损可能与癌症易感性增加有关。 我们评估了 XRCC1 杂合 (HZ) 小鼠的一系列时间和生物衰老参数，这些小鼠缺乏 XRCC1 功能。 HZ 和野生型 (WT) C57BL/6 小鼠表现出相似的中位寿命（26 个月）和几乎相同的最大预期寿命（37 个月）。 然而，许多 HZ 动物（92 只中有 7 只）表现出腹部器官破裂的倾向，考虑到 XRCC1 在内胚层和中胚层形成中的重要作用，这可能源于发育异常。 对于评估体重、脂肪成分、血液化学、主要器官、组织和相关细胞类型的状况、行为、脑容量和功能以及染色体和端粒完整性的其他终点，HZ 小鼠总体上表现出正常的表型。 用烷化剂氧化偶氮甲烷治疗动物会导致 HZ 小鼠的肝脏毒性和结肠癌前病变发生率的增加。 因此，我们的研究表明，在没有环境挑战的情况下，哺乳动物中的 XRCC1 单倍体不足对实际寿命和衰老的许多关键生物标志物影响不大，但可能会对动物的正常发育产生不利影响或增加对相关基因毒性暴露的疾病易感性。 共济失调伴动眼神经失用 1 (AOA1) 是由 APTX 基因突变引起的，该基因编码 DNA 支架断裂修复蛋白 aprataxin。 Aprataxin 可去除 DNA 中因连接反应失败而产生的 5-腺苷酸基团。 AOA1 的特点是整体小脑萎缩，突出表现为浦肯野细胞丧失、眼部运动失用以及运动和感觉神经病变。 引人注目的是，AOA1 患者缺乏癌症易感性和其他外周症状（例如免疫缺陷），这些症状通常与 DNA 修复缺陷引起的其他遗传性疾病相关。 我们发现 aprataxin 定位于人类细胞的线粒体，并鉴定出 N 端氨基酸序列，该序列将蛋白质的某些亚型靶向该细胞内区室。 有趣的是，编码这种独特 N 末端延伸的转录本在人脑中表达，其中小脑的产量最高。 柠檬酸合酶活性和线粒体 DNA (mtDNA) 拷贝数降低表明，人 SH-SY5Y 神经母细胞瘤细胞和原代骨骼肌成肌细胞中 aprataxin 的消耗会导致线粒体功能障碍。 此外，mtDNA（而非核 DNA）在 aprataxin 敲低后具有更高水平的背景 DNA 损伤，表明该酶在 mtDNA 加工中具有直接作用。这些数据表明，aprataxin 活性对于维持线粒体功能是必不可少的，并且 AOA1 疾病表型可能存在线粒体成分。 未来的研究旨在确定该疾病组织选择性背后的原因。