Project Summary: Alzheimer’s disease (AD) is a devastating neurodegenerative condition that is of increasing importance to an aging global population. Despite decades of research, there are currently no effective treatments for AD, and the molecular causes for sporadic forms of AD remain elusive. AD pathologies are intimately tied to the aging brain; and understanding sources of age-related dysfunction in brain cell types are key for developing therapeutic interventions. Increasingly, retrotransposable elements (RTEs), including the active human RTE LINE1 (L1), are conspicuously linked to aging dysfunctions that closely resemble changes seen in AD, including increased sterile inflammation, epigenetic instability and ultimately neurotoxicity. This presents the enticing opportunity of pursuing L1 as a druggable target to halt runaway neuroinflammation and neurodegeneration in AD. To this end, this proposal utilizes a trifecta of state-of-the-art human in vitro modeling systems, including aged induced neurons (iNs), induced pluripotent stem cell (iPSC) derived astrocytes, and glial enriched organoids to investigate the aging- and cell type-dependent consequences of L1 activation in disease relevant cell types. Using a cohort of AD patients and healthy age-matched controls derived neurons, the Gage team will test the contribution of L1 activity to two sporadic AD pathologies: neuronal senescence, and global relaxation of chromatin. Next, the team will directly test the necessity of L1 activation for the novel finding of spontaneous neuroinflammation in aged AD neuronal cultures. Finally, to avoid a narrow neuron-centric model of AD, we will extend our analyses to include patient-derived astrocytes and glial-enriched brain organoids to understand the consequences of L1 induced inflammation in other cell types of the brain and in 3D tissue specific contexts. This proposal provides ample opportunity for close collaborations with other participating project members. Patient cell lines and derived neural cells can be readily provided to collaborators for targeted analyses on a consistent set of clinically verified human samples. Sequencing and genetic analyses are paired with collaborating strategies in both mouse and fly models of AD, providing a platform for verification of results and future mechanistic research. The Gage team’s strategy involves L1 targeting vectors designed by collaborating cores to ensure reliable interventions and readouts across model systems. This project, in tandem with the efforts of collaborators, will provide the most thorough examination of consequences of L1 activation on the AD brain to date, potentially pioneering new therapeutic strategies for this disease of immense concern.