Not all DNA damage is toxic, as cells can induce programmed DNA damage to regulate core cell biological processes, such as DNA demethylation. Altered DNA demethylation has been observed in Alzheimer’s Disease and Alzheimer’s Disease Related Dementias (AD+ADRD), including frontotemporal dementia (FTD), and age-related cognitive dysfunction. Yet, the role of active DNA demethylation in AD+ADRD remains unclear. We recently discovered that programmed DNA damage occurs within neuronal enhancers and likely plays key roles in active DNA demethylation and epigenetic remodeling (Wu, W,* Hill, SE,* Nathan, WJ* et al., Nature 2021). Preliminary data suggest that transcription controls sites of programmed DNA damage and that AD+ADRD-linked proteins are involved, including TARDBP/TDP-43. Loss of TDP-43, a pathologic hallmark of FTD, Limbic-Predominant Age-related TDP-43 Encephalopathy (LATE), and dysregulated in many AD cases, decreased programmed DNA damage or repair, while depletion of XRCC1, a DNA repair protein linked to a familial neurodegeneration, increased repair. This suggests that programmed DNA damage is carefully regulated in aging to prevent AD+ADRD and neurodegeneration. In this proposal, I will investigate how programmed DNA damage is regulated and the consequences of dysregulation in AD+ADRD and age-related neurodegeneration. The K99 phase research will be performed mainly at the NIH with primary mentor Dr. Ward, whose lab uses mice and iPSC-derived neurons to study neurodegenerative diseases including FTD. To supplement my previous training in iPSC-derived neurons, CRISPR, and live cell-imaging, I will also learn advanced genomics (from co-mentor Dr. Nussenzweig and collaborator Dr. Chen), single-particle PALM imaging (from co-mentor Dr. Lippincott-Schwartz) and in vivo mouse models (from Dr. Ward and collaborator Dr. Petrucelli). This additional training and immersion into the DNA repair field will enable my long-term goal to study how neurons regulate programmed DNA damage, chromatin architecture, and DNA repair in AD+ADRD disease states. For Aim 1 (K99 phase), I will use genomics and live-cell imaging to examine the role of transcription in shaping programmed DNA damage sites. In Aim 2 (R00 phase), I will apply these techniques to examine the consequences of diminished programmed DNA damage or repair upon loss of TDP-43. Through studying the mechanisms underlying repair of programmed DNA damage and the role of TDP-43, we will gain additional insight into AD+ADRD pathogenesis. In Aim 3 (K99/R00), studies will investigate the consequences of increased DNA repair by sequencing enhancers in neurons from aged or XRCC1-deficient mouse brains for induced mutations and then conducting an in vivo CRISPRi mouse screen targeting neuronal enhancers to test their essentiality. This proposal facilitates the development of a unique perspective from which to launch an independent career and expand our understanding of programmed DNA damage and its role i...