Estradiol-Mediated Axogenesis of Hypothalamic Neurons Requires ERK1/2 and Ryanodine Receptors-Dependent Intracellular Ca2+ Rise in Male Rats.

Lucas E Cabrera Zapata,María Julia Cambiasso,Mariana Bollo

doi:10.3389/fncel.2019.00122

Abstract

17β-estradiol (E2) induces axonal growth through extracellular signal-regulated kinase 1 and 2 (ERK1/2)-MAPK cascade in hypothalamic neurons of male rat embryos in vitro, but the mechanism that initiates these events is poorly understood. This study reports the intracellular Ca2+ increase that participates in the activation of ERK1/2 and axogenesis induced by E2. Hypothalamic neuron cultures were established from 16-day-old male rat embryos and fed with astroglia-conditioned media for 48 h. E2-induced ERK phosphorylation was completely abolished by a ryanodine receptor (RyR) inhibitor (ryanodine) and partially attenuated by an L-type voltage-gated Ca2+ channel (L-VGCC) blocker (nifedipine), an inositol-1,4,5-trisphosphate receptor (IP3R) inhibitor (2-APB), and a phospholipase C (PLC) inhibitor (U-73122). We also conducted Ca2+ imaging recording using primary cultured neurons. The results show that E2 rapidly induces an increase in cytosolic Ca2+, which often occurs in repetitive Ca2+ oscillations. This response was not observed in the absence of extracellular Ca2+ or with inhibitory ryanodine and was markedly reduced by nifedipine. E2-induced axonal growth was completely inhibited by ryanodine. In summary, the results suggest that Ca2+ mobilization from extracellular space as well as from the endoplasmic reticulum is necessary for E2-induced ERK1/2 activation and axogenesis. Understanding the mechanisms of brain estrogenic actions might contribute to develop novel estrogen-based therapies for neurodegenerative diseases.

Highlights

For many years, estrogens have been recognized as one of the main orchestrators of the sexual differentiation of the brain, acting during critical periods of development to organize neural circuits in a way that determines the modulatory/activational effects of gonadal hormones in adulthood
Our previous results suggested that E2-induced extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation is Ca2+-dependent
Results from ERK1/2 phosphorylation, Ca2+ imaging and neuronal growth consistently pointed to ryanodine receptor (RyR) as the Ca2+ channels necessary to mediate activation of the MAPK/ERK pathway and the final axogenic effect induced by E2

Summary

Introduction

Estrogens have been recognized as one of the main orchestrators of the sexual differentiation of the brain, acting during critical periods of development to organize neural circuits in a way that determines the modulatory/activational effects of gonadal hormones in adulthood. Estradiol-Induced Ca2+ Signaling and Axogenesis indicates that E2 is a reproductive hormone and a brain-derived neuroprotective factor, coordinating multiple signaling mechanisms that protect the brain from neurodegenerative diseases, affective disorders and cognitive decline (Arevalo et al, 2015). These beneficial actions in the brain have positioned estrogens as promising therapeutic compounds against different brain pathologies such as Parkinson and Alzheimer diseases, schizophrenia, multiple sclerosis, stroke, neuroinflammation, among others (Dye et al, 2012; Villa et al, 2016; McGregor et al, 2017; Giatti et al, 2018). Estrogens generate a wide diversity of rapid ‘‘non-classical’’ effects, which occur in a range from some seconds to a few minutes via membrane-initiated mechanisms (Arevalo et al, 2012), including the triggering of Ca2+ signals (Beyer and Raab, 1998; Picotto et al, 1999; Wong et al, 2012), and the activation of several signaling pathways, such as phospholipase C (PLC)/inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (Le Mellay et al, 1997; Chaban et al, 2004), nitric oxide synthase/nitric oxide (Kelly and Levin, 2001), adenylate cyclase/AMPc/protein kinase A (PKA; Beyer and Karolczak, 2000), phosphoinositide-3 kinase (PI3K; Garcia-Segura et al, 2010), PKC, and extracellular signal-regulated kinase 1 and 2 (ERK1/2) cascades (Wu et al, 2005)

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Results

Conclusion