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 ß-amyloid plaques (Aß) 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 17ß-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 electrophysiological experiments will be conducted to
validate the reduction in mRNA expression and loss of physiological responses to STX. The results from these
studies will help to develop STX as a novel therapeutic for treating postmenopausal women and AD.