PROJECT SUMMARY A major challenge for the development of effective, disease-modifying therapies for Alzheimer’s disease (AD) and related dementias (ADRD) has been our incomplete understanding of the molecular processes controlling pathogenesis. Important clues for the key molecular pathways controlling AD/ADRD pathogenesis are likely to be gained from the study of selective vulnerability of neurons to AD/ADRD. While different factors are likely to contribute to selective vulnerability, our central hypothesis is that cell-autonomous pathways in neurons contribute to selective vulnerability in AD/ADRD, and that these pathways are potential therapeutic targets to reduce neuronal vulnerability to disease. Therefore, there is an urgent need to uncover the neuronal pathways casually driving selective vulnerability, and to test their therapeutic potential. In order to uncover determinants of selective vulnerability in AD, we previously used single-nucleus RNA sequencing to provide the first molecular description of selectively vulnerable neurons in the human entorhinal cortex, a brain region affected early in AD by tau pathology and neuronal loss. Neuronal subtypes that were lost early in disease were also selectively affected by tau pathology. This work provided us with a list of differentially expressed genes between relatively vulnerable versus resilient neuronal populations. The next challenge is to determine which of these differentially expressed genes causally contributes to selective vulnerability. To establish a causal role of specific differentially expressed genes in selective vulnerability, we will leverage CRISPRi technology, which enables the control of expression levels of endogenous genes. CRISPRi was co-developed by MPI Dr. Kampmann, who also pioneered CRISPRi in human iPSC-derived neurons and optimized its use in mouse brains (see preliminary results). In a genome-wide CRISPRi modifier screen in human iPSC-derived neurons, we identified several pathways controlling levels of tau pathology. By comparing hits from the CRISPRi screen to genes that are differentially expressed between resilient and vulnerable neurons in the human AD brain, we uncovered candidate resilience factors, including an CUL5 E3 ubiquitin ligase complex (Aim 1) and candidate vulnerability factors, including key glycolytic enzymes (Aim 2). The goal of Aim 3 is to conduct a large-scale CRISPRi screen for factors controlling tau pathology directly in the brain of tauopathy mouse models. The focus of the proposed project is to uncover mechanisms underlying selective neuronal vulnerability in AD and ADRD. These mechanisms represent potential therapeutic targets, and future research will test the therapeutic potential of targeting the identified pathways. The experimental strategy we propose to establish here to uncover mechanisms underlying selective vulnerability to tauopathy will provide a blueprint that can be applied to many other neurodegenerative diseases.