PROJECT SUMMARY/ABSTRACT The accumulation of DNA Double-Strand Breaks (DSBs) in neurons is an early hallmark of Alzheimer’s disease (AD). Increased DSBs are also associated with aging, which is the largest risk factor for AD. AD is also a complex disease involving all major brain glial cell types. Thus, there is a critical need to understand the molecular mechanisms of DSB induced changes in both neurons and glia. The structural stability of the genome is paramount in maintaining a functional genome, and recently, the 3D organization of the genome has emerged as a major regulator of genome function. My overall hypothesis is that 3D genome reorganization and structural genome instability mediated by DSBs are principle drivers of AD pathogenesis and brain aging. My objective is to determine how and to what extent DSBs within the neurons impact genome organization and the glial response, with the goal of identifying molecular pathways that can be targeted as novel therapies for preventing or halting the progression of neurodegeneration. I will use mouse models that recapitulate the pre-symptomatic accumulation of DSBs in neurons and human iPSC models to determine the degree of the 3D genome disruption caused due to DSBs and the underlying molecular mechanisms. I will also identify the consequences of neuronal DSBs on the structural stability of the genome by measuring the frequency of DSB mediated chromosomal translocations in human AD neurons. Interestingly, normal neuronal activity causes DSBs at immediate early gene promoters. Also, neuronal hyperactivity has been reported in AD. I will test if IEGs are locations of frequent chromosomal translocations after neuronal hyperactivity induced in neuronal culture. Previous studies have shown that microglia transitions to a reactive state in response to neurodegeneration. To understand the role of the 3D genome organization in this microglia transition, I will use single cell Hi-C to measure the unique chromatin interactions that mediate the reactive microglia state. Subsequently, transcription factor predictions using integrative analysis of single cell Hi-C will be tested in iPSC derived microglia-neuron co-cultures for their potential to modulate the reactive microglia response. This approach will be extended to study oligodendrocyte response in the independent phase. I will work with my mentor Dr. Li-Huei Tsai, my mentorship committee, Dr. Bruce Yankner and Dr. Manolis Kellis, my technical support and advisory committee, Dr. Frederick Alt and Dr. Peter Fraser to carry out my proposed training plan. I will gain experience in iPSC technology and differentiation from the Tsai lab to modulate the disease associated microglia response to neuronal DSBs and work with Dr. Manolis Kellis to implement the computational pipelines for the transcription factor predictions. To bridge the gap in my training in the biology of AD and brain aging, I will audit relevant courses and receive additional mentoring from ...