PROJECT SUMMARY Growing evidence links transposable/repetitive element (RE) transcripts with Alzheimer’s disease (AD), but the underlying mechanisms and disease relevance are unclear. In this project, we will test the hypothesis that the mechanism linking RE with AD is an age-dependent, global RE transcript increase that causes age/AD- related neuroinflammation. Our rationale is that: 1) aging is the key risk factor for AD; 2) RE transcripts derived from structural/retroviral sequences in the genome accumulate progressively with aging; 3) RE transcripts are prone to form double-stranded RNA (dsRNA) and/or complementary DNA (cDNA), both of which can cause neuroinflammation (a major driver of brain aging/AD that precedes pathology); 4) aging and AD are also linked with impairments in epigenetic control (e.g., hypo-methylation, which could facilitate RE transcription) and quality control systems like autophagy that degrade RE transcripts (which could potentiate RE-derived cDNA/dsRNA accumulation). Emerging data even suggest that RE-derived dsRNAs/cDNAs could spread via extracellular vesicles (EVs), a potential non-cell-autonomous explanation for the pathology observed in AD. These observations suggest a model that is highly consistent with the age-dependence of AD, in which global, age-related RE transcript increases lead to RE-derived dsRNAs/cDNAs that drive neuroinflammation and AD. In support of this idea, our preliminary data show that RE transcripts are hypomethylated in AD patients and correlate with neuroinflammation prior to pathology, and that inhibiting RE transcript buildup in human astrocytes/neurons may reduce inflammation. We also find evidence of RE in circulating EVs from AD patients. Based on these observations, we propose to (Aim 1) determine if age- rather than pathology-related RE transcript dysregulation links RE with AD by performing a large bioinformatics analysis of existing RNA-seq data, probing for RE in human/AD brains, and profiling global methylation (whole-genome bisulfite sequencing) in the same brains, as well as in samples from >100 subjects from a longitudinal study on brain aging, neuroinflammation and AD. In parallel (Aim 2), we will use patient-derived astrocytes and neurons to test the efficacy of clinically translatable treatments (e.g., methylation/autophagy activators) that reduce RE-derived dsRNAs and cDNAs for inhibiting age/AD-related neuroinflammation, and we will identify RE that may cause neuroinflammation by binding to cellular dsRNA and cDNA sensors (PKR and cGAS). Finally (Aim 3), we will determine if RE transcripts induce aged/AD-like neuroinflammation and accumulate in EVs in young astrocytes and neurons, and whether these EV-borne RE transcripts cause inflammation/toxicity in other cells. We also will use existing samples (as described above) to determine if RE in EVs are related to markers of systemic inflammation, neuroinflammation and AD in humans. These studies are specifically designed to ext...