Selective non-nuclear estrogen receptor activation with PaPE-1 evokes neuroprotection involving mTOR/MEK in an in vitro model of hypoxic/ischemic injury

BACKGROUND: Hypoxic/ischemic brain injuries, including ischemic stroke and perinatal asphyxia, remain major causes of mortality and long-term neurological disability, establishing a demand for therapeutic strategies suitable against the multifactorial nature of underlying mechanisms. Estrogen receptors (ERs) signaling is known to exert neuroprotective effects, however, genomic ER activation is associated with serious adverse effects, including carcinogenesis and thromboembolisms. Pathway Preferential Estrogen-1 (PaPE-1), a compound that selectively activates the non-nuclear subset of ERs, may provide neuroprotection, thereby overcoming the deleterious effects. The aim of this study was to elucidate the molecular mechanisms underlying the neuroprotective effects of PaPE-1 in an in vitro model of hypoxic-ischemic neuronal injury, with particular emphasis on non-nuclear estrogen receptor signaling and its downstream pathways. METHODS: Primary mouse cortical neuronal cells were subjected to 6 hours of experimental hypoxic/ischemic injury, followed by 18 hours of post-treatment with PaPE-1. Subsequently, a variety of biochemical assessments were conducted, including measurements of neuronal viability, cell death, and formation of autophagy-related vesicles. Moreover, the influence of PaPE-1 was assessed with molecular methods, encompassing measurements of gene and protein expression level and a set of epigenetic-related parameters, for instance, assessment of global DNA/RNA methylation and locus-specific methylation of genes and miRNA expression. To dissect signaling pathways, selective pharmacological inhibitors targeting mTOR/MEK1/2 and autophagy regulators were applied. ER subtype involvement was examined using ER-selective antagonists and specific siRNA silencing. RESULTS: Non-nuclear ER activation with PaPE-1 attenuated maladaptive autophagy, RNA/DNA oxidative stress damage, and neuronal degeneration while contributing to the regulation of gene expression and epigenetic processes. Evocation of robust neuroprotection involved modulation of mTOR and MEK1/2 signaling, predominantly mediated by estrogen receptor 1 (ESR1). CONCLUSION: In conclusion, PaPE-1 exhibits a multitarget mode of action that provides broad-spectrum protection against hypoxic/ischemic neuronal injuries. Considering its complexity of action and confirmed strong, neuroprotective activity, this compound holds promise for broader evaluation across different brain cell types and in vivo models.