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: 10333663
• 负责人: Vera Gorbunova
• 金额: $ 57.26万
• 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/10333663
• 关键词: Address; 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; Cardiovascular Diseases; Cell Aging; Cells; Chemicals; Chiroptera; Chromatin; Chronic; Clinical; Collaborations; Complementary DNA; Complex; Cytoplasm; DNA; Diabetes Mellitus; Elements; Engineering; Event; Family; Fibroblasts; Funding; Genetic; Genetic Recombination; Health; Human; Human Genome; Hybrids; Inflammaging; Inflammation; Inflammatory; Interferon Type I; Interferons; 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; Pharmacology; Physical Performance; Physiology; Process; Proteins; RNA; RNA-Directed DNA Polymerase; Reporting; 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; 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; 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.

逆转录转座因子（RTEs）在哺乳动物基因组中占很大比例。他们被压抑在 年轻的组织，但在衰老过程中被重新激活。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PPG的其他成员一起发现了另一种机制 L1通过诱导炎症导致病理学改变。L1转录激活导致L1的积累， 细胞质中的cDNA拷贝，在那里它们激活cGAS-STING信号传导，最终驱动I型干扰素 （IFN-I）应答。因此，L1的行为就像在衰老过程中醒来的敌人，并驱动与年龄相关的病理。 值得注意的是，“无菌性炎症”已经成为多种与年龄相关的病理学的驱动因素，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 L1激活的“特权”部位。SIRT 6蛋白的水平，其功能之一是抑制L1元件， 在AD患者的大脑中，L1激活水平较低，进一步加强了L1激活与AD病理学之间的联系。在 下一个资助周期，我们建议测试的假设，沉默的L1基因或药理学 这些方法将减轻包括AD在内的年龄相关的病理。项目和核心之间的协作 这个PPG将使我们能够使用我们的小鼠模型全面地检查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消除1：沉默 用shRNA、SIRT 6的过表达和核苷逆转录酶的药理学抑制 抑制剂的我们建立了两种新的小鼠模型：Annihilator小鼠，其中L1表达可以下调 通过链式shRNAs和SIRT 6过表达小鼠（SIRT 6-OE）。这两种结构都是floxed的，并集成在 ROSA 26基因座。另一种方法是通过抑制下游炎症信号传导来下调下游炎症信号传导。 刺痛。我们的具体目的是：（1）检测遗传或药理学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Annihilator和SIRT 6-OE小鼠与MAPT和5xFAD杂交， 神经退化的模型。我们还将用NRTI FTC处理MAPT和5xFAD小鼠，并分析它们的生长情况。 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， 与人类星形胶质细胞和神经元，以及核心B，谁将提供技术资源）。我们将对 细胞质L1 DNA从脑组织，确定其启动机制，细胞内定位， 使用质谱法鉴定结合蛋白。(3)确定STING抑制对AD的影响 发病机制我们将培育STING敲除小鼠和对MAPT和5xFAD具有抑制STING活性的小鼠。 神经退行性疾病模型，核心C。这些拟议的研究将解决以下问题的根本原因： 多种与年龄相关的病理，并将开辟新的临床干预阿尔茨海默病的路径。