Project 3: Inhibition of L1 to Alleviate Alzheimer's Disease Pathogenosis in Mouse Models

项目3：抑制L1以减轻小鼠模型中的阿尔茨海默氏病发病

• 批准号: 10581540
• 负责人: Vera Gorbunova
• 金额: $ 55.53万
• 依托单位: BROWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2026-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10581540
• 关键词: Age; Aging; Alzheimer' s Disease; Alzheimer' s disease brain; Alzheimer' s disease model; Alzheimer' s disease pathology; Alzheimer' s disease patient; Antibodies; Astrocytes; Automobile Driving; Binding Proteins; Biological Markers; Brain; Brain region; Breeding; Cardiovascular Diseases; Cell Aging; Cells; Chemicals; Chiroptera; Chromatin; Chronic; Clinical; Collaborations; Complementary DNA; Complex; Cytoplasm; DNA; DNA biosynthesis; Diabetes Mellitus; Elements; Engineering; Event; Family; Fibroblasts; Funding; Genetic; Genetic Recombination; Health; Human; Human Genome; Hybrids; Inflammaging; Inflammation; Inflammatory; Interferon Type I; Intervention; Knock-out; Knockout Mice; L1 Elements; Label; Link; Longevity; LoxP-flanked allele; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Microglia; Modeling; Mus; Mutate; Mutation; Nerve Degeneration; Neurofibrillary Tangles; Neurons; Nucleosides; Parasites; Pathogenesis; Pathology; Pathway interactions; Physical Performance; Physiology; Process; Proteins; RNA; RNA-Directed DNA Polymerase; Reporting; Repression; Resources; Retrotransposition; Retrotransposon; Reverse Transcriptase Inhibitors; Role; Senile Plaques; Sequence Analysis; Signal Transduction; Site; Source; Sterility; Stimulator of Interferon Genes; Testing; Time; Tissues; Transcriptional Activation; Transgenic Mice; Virus; Work; age related; aged; brain tissue; cellular pathology; clinical development; cognitive performance; cohort; experimental study; extracellular; genetic approach; genetic manipulation; healthspan; human disease; improved; inhibitor; longevity gene; mammalian genome; member; mouse model; novel; overexpression; pharmacologic; promoter; response; small hairpin RNA; systemic inflammatory response; transcriptomics

Retrotransposable elements (RTEs) make up a large fraction of mammalian genomes. They are repressed in young tissues but become reactivated during aging. The only family of RTEs capable of retrotransposition in the human genome is LINE-1 (L1). L1s can inflict damage by generating mutations and illegitimate recombination events. During the current funding cycle we, together with other PPG members, discovered another mechanism by which L1s cause pathology – by inducing inflammation. L1 transcriptional activation leads to accumulation of cDNA copies in the cytoplasm, where they activate cGAS-STING signaling, ultimately driving a type I interferon (IFN-I) response. Thus, L1s act like enemies within that awaken during aging and drive age-related pathologies. Remarkably, ‘sterile inflammation’ has emerged as a driver of multiple age-related pathologies, including Alzheimer’s disease (AD), cardiovascular diseases, cancer and diabetes. The brain has long been considered a ‘privileged’ site for L1 activation. Levels of SIRT6 protein, one of whose functions it to repress L1 elements, are lower in the brain of AD patients, further strengthening the link between L1 activation and AD pathology. In the next funding cycle, we propose to test the hypothesis that silencing of L1 by genetic or pharmacological approaches will alleviate age-related pathologies including AD. The collaborations among Projects and Cores in this PPG will allow us to comprehensively examine the role of L1 in AD pathogenesis using our mouse models as well as human neurons and astrocytes (Projects 1, 4). We will use three strategies to inactivate L1s: silencing with shRNA, overexpression of SIRT6, and pharmacological inhibition with nucleoside reverse transcriptase inhibitors. We generated two new mouse models: Annihilator mice in which L1 expression can be downregulated by chained shRNAs, and SIRT6 overexpressing mice (SIRT6-OE). Both constructs are floxed and integrated in the ROSA26 locus. Another approach will be to downregulate downstream inflammatory signaling by inhibiting STING. Our Specific Aims are: (1) Test the effects of genetic or pharmacological L1 inhibition on AD pathology in mouse models. In collaboration with Core C we will cross Annihilator and SIRT6-OE mice to MAPT and 5xFAD models of neurodegeneration. We will also treat MAPT and 5xFAD mice with the NRTI FTC and analyze the effect on lifespan and pathology with Project 1 and Cores B and C. (2) Determine the mechanisms responsible for the formation of cytoplasmic L1 cDNAs in brain tissue (in collaboration with Projects 1 and 4, which will work with human astrocytes and neurons, and Core B, who will provide technical resources). We will sequence the cytoplasmic L1 DNA from brain tissue, determine the mechanisms of its priming, intracellular localization, and identify binding proteins using mass spectrometry. (3) Determine the effects of STING inhibition on AD pathogenesis. We will breed STING knockout mice and mice with dampened STING activity to MAPT and 5xFAD models of neurodegeneration, with Core C. These proposed studies will address the underlying causes of multiple age-related pathologies and will open paths for novel clinical interventions in Alzheimer’s disease.

逆转录转座元件（RTE）构成了哺乳动物基因组的很大一部分。他们被压抑在 年轻的组织，但在衰老过程中会重新激活。唯一能够逆转录转座的 RTE 家族 人类基因组是 LINE-1 (L1)。 L1 可以通过产生突变和非法重组造成损害 事件。在当前的融资周期中，我们与其他 PPG 成员一起发现了另一种机制 L1 通过诱导炎症来引起病理。 L1转录激活导致积累 细胞质中的 cDNA 拷贝，激活 cGAS-STING 信号传导，最终驱动 I 型干扰素 （干扰素-I）反应。因此，L1 的行为就像内部的敌人，在衰老过程中觉醒并引发与年龄相关的病理。 值得注意的是，“无菌炎症”已成为多种与年龄相关的病理的驱动因素，包括 阿尔茨海默病 (AD)、心血管疾病、癌症和糖尿病。长期以来，大脑一直被认为是 用于 L1 激活的“特权”站点。 SIRT6 蛋白的水平（其功能之一是抑制 L1 元件）为 L1 激活与 AD 病理之间的联系进一步加强。在 在下一个资助周期，我们建议测试通过遗传或药理学沉默 L1 的假设 方法将减轻包括 AD 在内的与年龄相关的病症。项目和核心之间的合作 该 PPG 将使我们能够使用小鼠模型全面检查 L1 在 AD 发病机制中的作用 以及人类神经元和星形胶质细胞（项目 1、4）。我们将使用三种策略来灭活 L1： shRNA、SIRT6 过表达以及核苷逆转录酶的药理学抑制 抑制剂。我们生成了两种新的小鼠模型： 歼灭者小鼠，其中 L1 表达可以下调 通过链式 shRNA 和 SIRT6 过表达小鼠 (SIRT6-OE)。两种结构都被固定并集成在 ROSA26 基因座。另一种方法是通过抑制下游炎症信号传导 蜇。我们的具体目标是： (1) 测试遗传或药理学 L1 抑制对 AD 病理学的影响 在小鼠模型中。与 Core C 合作，我们将把 Annihilator 和 SIRT6-OE 小鼠与 MAPT 和 5xFAD 杂交 神经变性模型。我们还将用 NRTI FTC 治疗 MAPT 和 5xFAD 小鼠并分析 项目 1 和核心 B 和 C 对寿命和病理学的影响。 (2) 确定负责的机制 用于在脑组织中形成细胞质 L1 cDNA（与项目 1 和 4 合作，这将起作用 与人类星形胶质细胞和神经元以及核心 B（将提供技术资源）。我们将排序 来自脑组织的细胞质 L1 DNA，确定其启动、细胞内定位和 使用质谱法鉴定结合蛋白。 (3)确定STING抑制对AD的影响 发病。我们将培育 STING 基因敲除小鼠以及 STING 活性减弱的 MAPT 和 5xFAD 小鼠 神经退行性变模型，核心 C。这些拟议的研究将解决神经退行性变的根本原因 多种与年龄相关的病理学，将为阿尔茨海默病的新型临床干预开辟道路。