Convergent signaling mechanism of estradiol to induce axon growth in developing hypothalamic neurons

CAMBIASSO, M.J.

Los Cocos, Córdoba, Argentina

Simposio; Non-classical mechanism of sexual steroid action on the nervous system. XXII Reunión Anual de la SAN; 2007

SAN

Convergent signaling mechanism of estradiol to induce axon growth in developing hypothalamic neurons María Julia Cambiasso Instituto de Investigación Médica Mercedes y Martín Ferreyra, INIMEC-CONICET. Córdoba, Argentina. Steroid hormones influence the cellular and synaptic organization of the central nervous system. Estrogen has been shown to exhibit growth- or neurite-promoting properties for its target neurons within the developing brain. These estrogenic actions were first demonstrated using cultured slices of developing hypothalamus and subsequently confirmed in other culture systems in the developing rodent brain. Previous work from our laboratory has shown that, in primary cultures of dissociated neurons, the neuritogenic effect of estradiol (E2) is differentially exerted depending on the genetic sex of the neurons, the region of the brain, the maturation of the neurons, and the glial environment in which they develop. At 16 days of embryonic age (E16), estradiol stimulated axogenesis only in hypothalamic neurons from males, and this effect was contingent on the presence of trophic factor(s) derived from astroglia obtained from a target region. The application of the nuclear estrogen receptor (ER) antagonists, like tamoxifen or ICI 182,780, did not prevent the axogenic effect of the hormone in vitro. Moreover, using a membrane-impermeant E2-albumin construct (E2BSA) the estrogenic effect was preserved suggesting that this effect is not exerted through the intracellular ER but depends on a membrane mechanism. A truncated ERa (» 46-kDa), but not ERb and GPR30, was localized in membrane fractions of hypothalamic tissue from E16 embryos. In our culture system E2 also induced an increase in the levels of tyrosine kinase receptor type B (TrkB), the specific receptor for the neurotrophin brain-derived neurotrophic factor (BDNF). Moreover, antisense oligonucleotide suppression of E2-dependent TrkB increase blocked the axogenic effect of E2, indicating a convergence of the pathways for E2 and neurotrophins. More recently, using a pharmacological approach, we studied the intracellular signaling cascades mediating the axogenic effect of estradiol in vitro. Treatment with an intracellular Ca2+ chelator, a Ca2+-dependent PKC inhibitor, or two specific inhibitors of mitogen-activated protein kinases (MAPK) completely inhibited the E2-induced axogenesis. E2 and the membrane impermeant construct E2BSA rapidly induced phosphorylation of extracellular signal-regulated kinases (ERK), which was blocked by the specific inhibitor of the ERK pathway UO126 but not by the ER antagonist ICI 182,780. Decrease of intracellular free Ca2+ or disruption of PKC activation by Ro 32-0432 attenuated ERK activation, indicating the confluence of signals in the MAPK pathway. Sub-cellular analysis of ERK demonstrated that the phospho-ERK signal is augmented in the nucleus after 15 min of E2 stimulation. We have also shown that E2 increased phoshorylation of CREB via ERK signaling. In summary, these studies demonstrates that E2, probably via a membrane-associated receptor, induces axonal growth by activating CREB phosphorylation through ERK signaling by a mechanism involving Ca2+ and PKC activation. Supported by CONICET and FONCyT.