Identifying Novel Estrogenic Mitochondrial Targets in Hypothalamic Proopiomelanocortin Neurons by Chemoproteomics

Loss of estrogens at menopause is linked to impaired brain metabolism and increased risk of Alzheimer’s disease (AD). However, estrogen replacement therapies are limited due to the deleterious effects of estrogen on peripheral organs and an increased risk of vascular dementia. We have developed a non-steroidal estrogenic compound, STX, which does not bind to the classical estrogen receptors α and β, but mimics estrogenic signaling in the central nervous system (CNS) without eliciting the peripheral reproductive actions. STX is protective against neurodegeneration in stroke and AD models, but its molecular targets are unknown. Here, we identified and validated neural targets of STX using chemoproteomic, molecular biological, electrophysiological and metabolic assays in hypothalamic proopiomelanocortin (POMC) neurons. Chemoproteomic profiling identified voltage-dependent anion channels (VDAC1-3) as major intracellular binding partners in mHypo43 (POMC) cells. Based on quantitative single-cell PCR, Vdac2 was identified as the dominant isoform in female hypothalamic POMC neurons. Seahorse metabolic flux analyses showed that STX potently increased glycolysis, oxidative respiration and mitochondrial ATP production in mHypo43 cells. Nanomolar concentrations of STX enhanced VDAC2 voltage-dependent gating in reconstituted lipid membranes and shifted the low-conductance states toward anion selectivity, consistent with increased ATP flux. Together, these findings identify VDAC proteins as previously unrecognized mitochondrial targets of STX and demonstrate that STX can directly modulate VDAC2 channel properties and cellular bioenergetics. These findings establish a STX–VDAC mitochondrial mechanism in an estrogen-responsive neuronal system and provide a molecular framework for future studies examining how this pathway contributes to the neuroprotective actions of STX in neurodegenerative diseases.