PROJECT SUMMARY Currently, there are over 5.7 million Alzheimer’s disease (AD) patients in the United States, and this number is predicted to reach over 14 million by the year 2050. Despite decades of research, effective therapies for treating AD remain lacking, and recent clinical trials targeting β-amyloid plaques (Aβ) have been largely unsuccessful. Nearly two-thirds of AD patients are postmenopausal women who have lost the neuropr otective effects of estrogens. Studies on hormone replacement therapy raised hopes that 17β-estradiol (E2) might provide an effective treatment for preventing neuronal loss, but a large clinical trial found that conjugated estrogens caused an increased risk of breast cancer and thrombotic events. As an alternative, we have synthesized and characterized a non-steroidal estrogenic ligand, STX that selectively targets an unidentified receptor in the central nervous system (CNS), but importantly STX does not engage the “classical” nuclear estrogen receptors, which enables STX to avoid the unfavorable effects of estrogen. Orally administered STX crosses the blood brain barrier and activates neuroprotective signaling pathways in CNS neurons. STX improves mitochondrial function and enhances neuronal synaptic transmission. Moreover, STX protects against amyloid toxicity in cultured hippocampal neurons, while sustained oral STX protects against amyloid toxicity in a mouse model of AD. Therefore, STX has a high therapeutic potential, but identification of the receptor is needed for further development as a treatment for AD. Based on physiological / pharmacological data, we hypothesize that STX targets a Gq-coupled membrane estrogen receptor in CNS neurons to provide neuroprotective actions. We propose to isolate this receptor using photo-crosslinking and click chemistries and confirm its identity via in vivo CRISPR/Cas9 mutagenesis studies. We have designed and synthesized a novel bifunctional STX derivative (BF-STX) that contains a photo-crosslinkablediazirine group and an alkyne group, which permits specific tagging of STX-protein conjugates with a fluorophore and the isolation of STX-protein conjugates from cell lysates for proteomic analysis. Photo-crosslinked BF-STX labels POMC-expressing hypothalamic (mHypo43) cells in vitro. Therefore, we will: (1) photo-crosslink BF-STX to candidate receptors in mHypo43 cell lysates and isolate BF- STX-protein conjugates from the membrane fraction via click chemistry to azide-bearing beads and analyze by proteomics. The most promising hits will be validated by siRNA knock-down in mHypo43 cells followed by measuring the loss of STX signaling; and (2) validate promising receptor candidates in vivo using CRISPR/Cas9 mutagenesis in POMCCre mice. Single adeno-associated viral (AAV) vectors containing recombinase-dependent Staphylococcus aureus Cas9 and a single guide RNA against each candidate receptor will be targeted to POMC neurons. Molecular biological (single cell RT-PCR) and electrop...