PROJECT SUMMARY Excessive cell death contributes to tauopathies including Alzheimer's disease (AD), which are pathologically characterized by the aberrant accumulation of the microtubule associated protein tau and its oligomerization into neurofibrillary tangles (NFT). To identify therapeutic targets for neurodegenerative tauopathies such as Alzheimer's disease, it is vital to elucidate molecular mechanisms controlling cellular response to proteotoxic stress due to tau accumulation. We have examined how BCL-2 family member BOK, which chiefly appears bound to the inositol-3-phosphate (IP3R) calcium transporter in the endoplasmic reticulum (ER), induces apoptosis in response to select ER stress stimuli. We found BOK mediates calcium transfer from ER to mitochondria and is key to forming mitochondrial-ER contact sites (MERCs), also called mitochondrial ER- associated membranes (MAMs). MAMs are central signaling hubs, mediating cell processes like calcium transfer and metabolic homeostasis; many are regulated by BOK. Our objective is to directly address how BOK regulates tau-induced cell death in neurons through specific MAM subsets, including the ion-MAM, which mediates calcium transfer, and the phago-MAM, which is where autophagosomes are produced. Aim 1 will determine the role of BOK in ion-MAM regulation of tauopathy by examining calcium levels and cell death in BOK deficient cortical neurons challenged by mutant tau-induced ER stress. Aim 2 will determine the role of BOK in phago-MAM regulation of tauopathy by analyzing the impact of BOK and MAM formation on autophagy in neurons challenged by mutant tau. Aim 3 will determine the physiological consequences of loss of BOK on tauopathy-induced neurodegeneration. This project is conceptually innovative in our recognition that BOK regulates MAMs, which are altered in patients diagnosed with dementia. The project is technically innovative in our use of 2Phatal, two- photon chemical apoptotic targeted ablation, to study cell death in tauopathies. This technique uses a femtosecond-pulsed laser to induce dose-dependent apoptosis of individual cells in mouse brains in vivo without collateral damage. Our hypothesis is that BOK's ability to impact ER-stress induced apoptosis, calcium regulation, and MAM formation suggests a critical but undefined role in the production of autophagic membranes and the regulation of neuronal cell death in tauopathy-induced neurodegeneration. This project will elucidate the metabolic pathways regulated by BOK to reveal potential mechanisms to prevent tau-mediated death of neurons, providing targets for therapeutic development. This proposal is submitted in response to PAR-23-179, Small Research Grant Program for the Next Generation of Researchers in AD/ADRD Research (R03 Clinical Trial Optional). The proposed project leverages the Principal Investigator's prior discoveries and expertise on the multi-domain, pro-apoptotic BCL-2 family member BOK to examine a novel role in autop...