Exploring a role for LINE1 retrotransposons in neurodegenerative disease

探索 LINE1 逆转录转座子在神经退行性疾病中的作用

• 批准号: 8678465
• 负责人: JOHN Lawrence GOODIER
• 金额: $ 8.1万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-01 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8678465
• 关键词: Accounting; Age; Alzheimer' s Disease; Amyloid beta-Protein; Amyotrophic Lateral Sclerosis; Antisense RNA; Area; Binding; Biological Assay; Biology; Brain; Cell Death; Cell Line; Cells; Chemicals; Co-Immunoprecipitations; Collaborations; Cytoplasmic Granules; DNA Methylation; Databases; Disease; Disease susceptibility; Elements; Epigenetic Process; Etiology; Future; Genes; Genetic Medicine; Genetic Transcription; Genome; Grant; Health; Hospitals; Human; Human Genome Project; Immunohistochemistry; Individual; Institutes; Investigation; Laboratories; Letters; Literature; Location; Malignant Neoplasms; Mediating; Medical; Minor; Monitor; Morphology; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neurology; Neurons; Nucleic Acids; ORF2 protein; Paste substance; Pathogenesis; Pathology; Patients; Predisposition; Process; Proteins; Pseudogenes; RNA; RNA Interference; RNA-Binding Proteins; RNA-Directed DNA Polymerase; Research; Retroposon; Retrotransposition; Retrotransposon; Reverse Transcriptase Polymerase Chain Reaction; Reverse Transcription; Ribonucleoproteins; Role; Sampling; Science; Serum; Somatic Cell; Source; Stem cells; Stress; Structure; Susceptibility Gene; Testing; Therapeutic; Tissue Sample; Tissues; Up-Regulation; Variant; Virus; aging population; base; brain tissue; cell type; chromatin remodeling; effective therapy; endonuclease; human DNA; immunocytochemistry; induced pluripotent stem cell; insight; interest; link protein; medical schools; mutant; novel; novel therapeutics; nucleoside inhibitor; particle; protein aggregate; protein aggregation; protein expression; protein misfolding; public health relevance; transposon/insertion element

DESCRIPTION (provided by applicant): Understanding the causes of neurodegenerative diseases with the aim of developing effective treatments is a significant medical challenge. Much effort has been expended identifying susceptibility genes for amyotrophic lateral sclerosis (ALS) and Alzheimer's (AD) disease. A number of genes have been found, each explaining a minor percentage of familial cases and contributing little to our understanding of the pathogenesis of sporadic cases. However, one common feature of many neurodegenerative diseases is protein aggregation, and it is widely thought that revealing the processes of cytoplasmic aggregate formation is key to better understanding the disease. We propose a novel hypothesis: rather than mutations at single gene locus, increased expression of ubiquitous LINE1 (L1) retrotransposons in the brains of ALS or AD patients, perhaps from many locations in the genome, contribute to the disease pathology. We predict that the consequent increase in cellular levels of the L1-encoded ORF1p RNA-binding protein could increase its binding to proteins of significance for ALS or AD and promote their sequestration in stress granules and other cytoplasmic aggregates. Furthermore, a corresponding increase in L1 ORF2 protein could account for increased levels of reverse transcriptase activity that have been detected in sera of ALS patients. LINE1 retrotransposons are non-viral mobile DNA elements that duplicate themselves by a "copy and paste" mechanism using an RNA intermediate. The Human Genome Project estimated that over 500,000 L1 copies occupy 17% of human DNA, although it is believed that only about 100 of these remain potentially active in any individual. L1 retrotransposition has also been responsible for the insertion of over a million non- autonomous Alu retroposons and thousands of processed pseudogenes. The cell in turn has evolved defenses against unrestricted retrotransposition, including DNA methylation, chromatin remodeling, nucleic acid editing, and RNA interference. However, recent investigations show that these defenses are occasionally relaxed, leading to increased retrotransposon activity in certain somatic cell types, including stem cells, some cancers, and notably neuronal cells in the human brain. Using immunocytochemistry, RNA analyses, coimmunoprecipitation, and a functional assay for L1 ORF2 reverse transcriptase activity, we will examine brain tissue and derived iPS cell lines, mainly from ALS patients but also including AD samples, and compare these with age-matched controls to ascertain if L1 RNA and protein expression is elevated in the disease state. We will assay for alteration in the size, number, and morphology of L1 ORF1p-mediated cytoplasmic RNA granule formation in patient samples and monitor for association and colocalization of ALS/AD-associated proteins with the L1. Common mutations in both ORF1p and disease susceptibility proteins will be assayed for their effects on coaggregation.

描述（由申请人提供）：了解神经退行性疾病的原因，以开发有效的治疗方法是一项重大的医学挑战。人们已经花费了大量的努力来鉴定肌萎缩侧索硬化症（ALS）和阿尔茨海默病（AD）的易感基因。许多基因已被发现，每一个解释了一小部分的家族性病例，并有助于我们了解散发病例的发病机制。然而，许多神经退行性疾病的一个共同特征是蛋白质聚集，人们普遍认为揭示细胞质聚集体形成的过程是更好地理解疾病的关键。我们提出了一个新的假设：ALS或AD患者脑中普遍存在的LINE 1（L1）逆转录转座子的表达增加，而不是单个基因位点的突变，可能来自基因组中的许多位置，有助于疾病病理学。我们预测，随之而来的L1编码的ORF 1 p RNA结合蛋白的细胞水平的增加可以增加其与ALS或AD的重要蛋白质的结合，并促进其在应激颗粒和其他细胞质聚集体中的螯合。此外，L1 ORF 2蛋白的相应增加可以解释ALS患者血清中检测到的逆转录酶活性水平的增加。 LINE 1反转录转座子是非病毒的移动的DNA元件，其使用RNA中间体通过“复制和粘贴”机制复制自身。人类基因组计划估计，超过500，000个L1拷贝占据了人类DNA的17%，尽管据信其中只有大约100个在任何个体中仍然具有潜在的活性。L1反转录转座还负责插入超过一百万个非自主的Alu反转录子和数千个加工的假基因。反过来，细胞也进化出了对不受限制的逆转录转座的防御，包括DNA甲基化、染色质重塑、核酸编辑和RNA干扰。然而，最近的研究表明，这些防御偶尔会放松，导致某些体细胞类型（包括干细胞、一些癌症，特别是人脑中的神经元细胞）中逆转录转座子活性增加。 使用免疫细胞化学，RNA分析，免疫共沉淀，和L1 ORF 2逆转录酶活性的功能测定，我们将检查脑组织和衍生的iPS细胞系，主要来自ALS患者，但也包括AD样本，并将这些与年龄匹配的对照组进行比较，以确定是否L1 RNA和蛋白质表达在疾病状态下升高。我们将检测患者样本中L1 ORF 1 p介导的细胞质RNA颗粒形成的大小、数量和形态的变化，并监测ALS/AD相关蛋白与L1的关联和共定位。ORF 1 p和疾病易感蛋白中的常见突变将被测定其对共聚集的影响。