Estradiol activates the CaMKKβ/AMPK pathway to enhance neurite outgrowth in cultured adult sensory neurons

Adult rat dorsal root ganglion (DRG) sensory neurons express estrogen receptors (ERs) α and β. Estrogen regulates multiple aspects of the nervous system including development, survival, and axonal outgrowth of DRG neurons. While previous studies have established estrogen's neuroprotective role in these neurons, the specific ER subtypes and downstream signaling pathways mediating these effects remain poorly defined. The objective of our study was to investigate the effects of 17 beta-estradiol (E2) on mitochondrial function and axonal regeneration of cultured DRG neurons and explore the pathways by which E2 acts. We observed that E2 treatment upregulated the levels of phosphorylated AMP-activated protein kinase (AMPK). E2 also increased the levels of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) and activating transcription factor 3 (ATF3), which are proteins involved in mitochondrial biogenesis and axonal regeneration. The Seahorse assay showed that E2 elevated basal respiration in cultured DRG neurons. Additionally, E2 treatment for 24 h significantly increased total neurite outgrowth of DRG neurons. Pharmacological inhibition of AMPK using Compound C inhibited E2-mediated increases in ATF3 expression and neurite outgrowth. The Ca²⁺/calmodulin-dependent protein kinase kinase β (CaMKKβ) inhibitor STO-609 blocked E2-mediated AMPK activation. Furthermore, we assessed whether these effects were mediated by ERα or ERβ by using the ERα selective agonist propyl pyrazole triol (PPT) and ERβ selective agonist diarylpropionitrile (DPN). PPT upregulated phosphorylated AMPK levels and increased total neurite outgrowth, whereas DPN was ineffective. The results demonstrate that E2 acts through ERα to promote neurite outgrowth via a pathway involving activation of CaMKKβ/AMPK in adult DRG neurons. Our findings identify ERα-mediated AMPK activation as a therapeutic target for enhancing neuronal regeneration and mitochondrial function in neurodegenerative disorders.