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.

Cockayne综合征（CS）是一种常染色体隐性疾病，其特征是生长衰竭，神经系统异常，过早衰老症状和皮肤光敏性，但癌症发病率没有增加。 CS分为两个严格的补体组：CSA（CKN1中的突变）和CSB（ERCC6中的突变）。 在患有CS的患者中，有80％的患者患有CSB基因突变。 我们追求的假设是，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效率方面的直接作用。 我们的体外工作还有助于定义了CSB的生化特性，表明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依赖性和非ATP依赖性功能。 与Vilhelm Bohr博士合作获得的最新结果表明，CSB不仅在核BER中起着直接的作用，而且在线粒体BER中起着直接的作用，这可能是通过帮助募集，稳定和/或保留与内部线粒体膜相关的维修复合物中的招募，稳定和/或保留BER蛋白。 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，阐明蛋白质的独特N和C末端部分的贡献，可能会赋予功能特异性，并探索CSB在处理内源性DNA损伤中的可能作用。 XRCC1是一种关键的支架蛋白，它可以编排有效的单链破裂修复（SSBR），这是BER的重要子路口。最近的数据发现，XRCC1与蛋白质的关系与人脊椎发子共济失调有关 - 阿普拉天蛋白和酪糖基-DNA磷酸二酯酶1-与SSBR有关保护神经元细胞丧失和神经退行性疾病。 此外，人类的分子流行病学研究表明，XRCC1功能受损可能与癌症易感性提高有关。 我们已经评估了XRCC1杂合（Hz）小鼠中的一系列年代和生物老化参数，这些参数缺乏XRCC1功能。 Hz和野生型（WT）C57BL/6小鼠的中位寿命相似，为26个月，最大预期寿命几乎相同37个月。 然而，许多Hz动物（92中的7个）显示出腹部器官破裂的倾向，这可能源于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。 对于其他终点，评估了体重，脂肪组成，血液化学，主要器官的状况，组织和相关细胞类型，行为，脑体积和功能以及染色体和端粒完整性HZ小鼠的大小表现出正常的表型。 用烷基化剂二甲烷对动物进行处理会导致肝毒性和HZ小鼠结肠前癌性病变的发生率增加。 因此，我们的研究表明，在没有环境挑战的情况下，XRCC1单倍症对年代寿命和衰老的许多关键生物学标记的影响很小，但可能会对正常的动物发育产生不利影响或增加对相关遗传毒性暴露的疾病的易感性。 用眼动子ARAXIA 1（AOA1）的共济失调是由APTX基因突变引起的，该基因编码了DNA支架破裂修复蛋白Aprataxin。 Aprataxin在DNA中除去了由中止的连接反应引起的5-腺苷酸基。 AOA1的特征是全球小脑萎缩，以purkinje细胞，眼运动失调以及运动和感觉神经病的损失突出。 令人惊讶的是，AOA1患者缺乏癌症的敏感性和其他与其他因DNA修复缺陷引起的遗传性疾病相关的外周症状（例如免疫缺陷）。 我们发现，Aprataxin将人类细胞中的线粒体定位于线粒体，并确定了N末端氨基酸序列，该氨基酸序列将蛋白质的某些同工型靶向该细胞内室。 有趣的是，编码这种独特的N末端拉伸的转录本在人的大脑中表达，在小脑中产生最高。 如柠檬酸酸盐合酶活性降低，线粒体功能障碍在人类SH-SY5Y神经母细胞瘤细胞和原发性骨骼肌成肌细胞中的Aprataxin耗竭会导致线粒体功能障碍，并导致线粒体DNA（MTDNA）拷贝数。 此外，mtDNA而非核DNA在Aprataxin敲低时具有较高的背景DNA损伤水平，这表明该酶在mtDNA加工中的直接作用。这些数据表明，Aprataxin活性对于维持线粒体功能是必不可少的，并且可能是AOA1疾病表型的线粒体成分。 未来的研究旨在确定该疾病的组织选择性背后的原因。