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 ...