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

基本信息

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
项目类别：
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&apos 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

项目摘要

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并开发有效的治疗方法是一项重大的医学挑战。人们花费了大量精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。已经发现了许多基因，每个基因都解释了一小部分家族病例，并且对我们了解散发病例的发病机制贡献甚微。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了解该疾病的关键。我们提出了一个新的假设：ALS 或 AD 患者大脑中普遍存在的 LINE1 (L1) 反转录转座子（可能来自基因组的许多位置）的表达增加，而不是单基因位点的突变，导致了疾病病理学。我们预测，L1 编码的 ORF1p RNA 结合蛋白的细胞水平随之增加，可能会增加其与对 ALS 或 AD 具有重要意义的蛋白质的结合，并促进它们在应激颗粒和其他细胞质聚集体中的隔离。此外，L1 ORF2 蛋白的相应增加可能是 ALS 患者血清中检测到的逆转录酶活性水平增加的原因。 LINE1 逆转录转座子是非病毒移动 DNA 元件，可使用 RNA 中间体通过“复制和粘贴”机制进行自我复制。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活性。 L1 逆转录转座还负责插入超过一百万个非自主 Alu 逆座子和数千个经过加工的假基因。细胞反过来又进化出了针对不受限制的逆转录转座的防御能力，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，这些防御有时会放松，导致某些体细胞类型的逆转录转座子活性增加，包括干细胞、一些癌症，尤其是人脑中的神经元细胞。使用免疫细胞化学、RNA 分析、免疫共沉淀和 L1 ORF2 逆转录酶活性功能测定，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），并将其与年龄匹配的对照进行比较，以确定如果 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 ORF1p 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。将分析 ORF1p 和疾病易感性蛋白的常见突变对共聚集的影响。