We showed previously in neocortical explants, derived from developing wild-type and estrogen receptor (ER)-alpha gene-disrupted (ERKO) mice, that both 17alpha- and 17beta-estradiol elicit the rapid and sustained phosphorylation and activation of the mitogen-activated protein kinase (MAPK) isoforms, the extracellular signal-regulated kinases ERK1 and ERK2. We proposed that the ER mediating activation of the MAPK cascade, a signaling pathway important for cell division, neuronal differentiation, and neuronal survival in the developing brain, is neither ER-alpha nor ER-beta but a novel, plasma membrane-associated, putative ER with unique properties. The data presented here provide further evidence that points strongly to the existence of a high-affinity, saturable, 3H-estradiol binding site (K(d), approximately 1.6 nm) in the plasma membrane. Unlike neocortical ER-alpha, which is intranuclear and developmentally regulated, and neocortical ER-beta, which is intranuclear and expressed throughout life, this functional, plasma membrane-associated ER, which we have designated "ER-X," is enriched in caveolar-like microdomains (CLMs) of postnatal, but not adult, wild-type and ERKO neocortical and uterine plasma membranes. We show further that ER-X is functionally distinct from ER-alpha and ER-beta, and that, like ER-alpha, it is re-expressed in the adult brain, after ischemic stroke injury. We also confirmed in a cell-free system that ER-alpha is an inhibitory regulator of ERK activation, as we showed previously in neocortical cultures. Association with CLM complexes positions ER-X uniquely to interact rapidly with kinases of the MAPK cascade and other signaling pathways, providing a novel mechanism for mediation of the influences of estrogen on neuronal differentiation, survival, and plasticity.

We have been studying the role and mechanism of estrogen action in the survival and differentiation of neurons in the basal forebrain and its targets in the cerebral cortex, hippocampus, and olfactory bulb. Previous work has shown that estrogen-target neurons in these regions widely coexpress the mRNAs for the neurotrophin ligands and their receptors, suggesting a potential substrate for estrogen-neurotrophin interactions. Subsequent work indicated that estrogen regulates the expression of two neurotrophin receptor mRNAs in prototypic peripheral neural targets of nerve growth factor. We report herein that the gene encoding the neurotrophin brainderived neurotrophic factor (BDNF) contains a sequence similar to the canonical estrogen response element found in estrogentarget genes. Gel shift and DNA footprinting assays indicate that estrogen receptor-ligand complexes bind to this sequence in the BDNF gene. In vivo, BDNF mRNA was rapidly up-regulated in the cerebral cortex and the olfactory bulb of ovariectomized animals exposed to estrogen. These data suggest that estrogen may regulate BDNF transcription, supporting our hypothesis that estrogen may be in a position to influence neurotrophinmediated cell functioning, by increasing the availability of specific neurotrophins in forebrain neurons.Survival, differentiation, and maintenance of forebrain neurons are governed by several classes of local and target-derived neurotrophic factors. One critical class of growth and neurotrophic factors are members of the neurotrophin family of peptides. The neurotrophins, which include nerve growth factor, brain-derived neurotrophic factor (BDNF), and neurotrophin 3, are structurally and functionally related proteins, with distinct temporal and regional patterns of neural expression (1). While certain forebrain neurons respond to the neurotrophins in a ligand-specific manner, all three peptides appear to promote the survival and differentiation of basal forebrain cholinergic neurons in vitro (2, 3).Among the neurotrophins, BDNF, which was first described in 1989 (4), influences several neuronal subpopulations during development and adulthood. BDNF stimulates cell proliferation in the cochleovestibular ganglion (5) and enhances the survival and phenotype expression of basal forebrain cholinergic neurons (3), cerebral cortical neurons (6), and dopaminergic mesencephalic neurons (7). BDNF has also been shown to play a protective role in vivo, after injury, in diverse neuronal populations such as the basal forebrain cholinergic neurons (8, 9), nigrostriatal dopamine neurons (10), facial (11) and spinal (12)