Age increase of estrogen receptor-α (ERα) in cortical astrocytes impairs neurotrophic support in male and female rats.

Rodent models show decreased neuronal responses to estradiol (E2) during aging (E2-desensitization) in association with reduced neuronal estrogen receptor (ER)-α, but little is known about age changes of E2-dependent astrocytic neurotrophic support. Because elevated expression of astrocyte glial fibrillary acidic protein (GFAP) is associated with impaired neurotrophic activity and because the GFAP promoter responds to ERα, we investigated the role of astrocytic ERα and ERß in impaired astrocyte neurotrophic activity during aging. In vivo and in vitro, ERα was increased greater than 50% with age in astrocytes from the cerebral cortex of male rats (24 vs 3 months), whereas ERß did not change. In astrocytes from 3-month-old males, experimentally increasing the ERα to ERß ratio induced the aging phenotype of elevated GFAP and impaired E2-dependent neurite outgrowth. In 24-month-old male astrocytes, lowering ERα reversed the age elevation of GFAP and partially restored E2-dependent neurite outgrowth. Mixed glia (astrocytes to microglia, 3:1) of both sexes also showed these age changes. In a model of perimenopause, mixed glia from 9- to 15-month rats showed E2 desensitization: 9-month regular cyclers retained young-like ERα to ERß ratios and neurotrophic activity, whereas 9-month noncyclers had elevated ERα and GFAP but low E2-dependent neurotrophic activity. In vivo, ERα levels in cortical astrocytes were also elevated. The persisting effects of ovarian acyclicity in vitro are hypothesized to arise from steroidal perturbations during ovarian senescence. These findings suggest that increased astrocyte ERα expression during aging contributes to the E2 desensitization of the neuronal responses in both sexes.

17ß-estradiol and progesterone regulate expression of ß-amyloid clearance factors in primary neuron cultures and female rat brain.

The accumulation of ß-amyloid protein (Aß) is a key risk factor in the development of Alzheimer's disease. The ovarian sex steroid hormones 17ß-estradiol (E(2)) and progesterone (P(4)) have been shown to regulate Aß accumulation, although the underlying mechanism(s) remain to be fully elucidated. In this study, we investigate the effects of E(2) and P(4) treatment on the expression levels of Aß clearance factors including insulin-degrading enzyme, neprilysin, endothelin-converting enzyme 1 and 2, angiotensin-converting enzyme, and transthyretin, both in primary neuron cultures and female rat brains. Our results show that E(2) and P(4) affect the expression levels of several Aß clearance factors in dose- and time-dependent manners. Most notably, expression of insulin-degrading enzyme is significantly increased by both hormones in cultured neurons and in vivo and is inversely associated with the soluble Aß levels in vivo. These findings further define sex steroid hormone actions involved in regulation of Aß, a relationship potentially important to therapeutic approaches aimed at reducing risk of Alzheimer's disease.

Differential responses of progesterone receptor membrane component-1 (Pgrmc1) and the classical progesterone receptor (Pgr) to 17ß-estradiol and progesterone in hippocampal subregions that support synaptic remodeling and neurogenesis.

Progesterone (P4) and estradiol (E2) modulate neurogenesis and synaptic remodeling in the hippocampus during the rat estrous cycle and in response to deafferenting lesions, but little is known about the steroidal regulation of hippocampal progesterone receptors associated with these processes. We examined the neuronal expression of progesterone receptor membrane component-1 (Pgrmc1) and the classical progesterone receptor (Pgr), by in situ hybridization and immunohistochemistry. Pgr, a transcription factor, has been associated with synaptic remodeling and other major actions of P4, whereas Pgrmc1 is implicated in P4-dependent proliferation of adult neuroprogenitor cells and with rapid P4 effects on membranes. Ovariectomized adult rats were given E2, P4, or E2+P4 on two schedules: a 4-d model of the rodent estrous cycle and a 30-d model of postmenopausal hormone therapy. Pgr was hormonally responsive only in CA1 pyramidal neurons, and the induction of Pgr by E2 was partly antagonized by P4 only on the 30-d schedule. In CA3 pyramidal and dentate gyrus (DG) neurons, Pgr was largely unresponsive to all hormone treatments. In contrast to Pgr, Pgrmc1 was generally induced by E2 and/or P4 throughout the hippocampus in CA1, CA3, and DG neurons. In neuroprogenitor cells of the DG (immunopositive for bromodeoxyuridine and doublecortin), both Pgrmc1 and Pgr were detected. The differential regulation of hippocampal Pgrmc1 and Pgr by E2 and P4 may guide drug development in hormonal therapy for support of neurogenesis and synaptic regeneration.

Progesterone influence on neurite outgrowth involves microglia.

Progesterone (P4) antagonizes estradiol (E2) in synaptic remodeling in the hippocampus during the rat estrous cycle. To further understand how P4 modulates synaptic plasticity, we used entorhinal cortex lesions, which induce E2-dependent neurite sprouting in the hippocampus. In young ovariectomized rats, the E2-dependent entorhinal cortex lesion-induced sprouting was attenuated by concurrent treatment with P4 and E2. Microglial activation also showed the E2-P4 antagonism. These findings extend reports on the estrous cycle synaptic remodeling without lesions by showing the P4-E2 antagonism during simultaneous treatment with both E2 and P4. Glial mechanisms were analyzed with the wounding-in-a-dish model of cocultured glia and embryonic d-18 cortical neurons from rat. In cocultures of mixed glia (astrocytes plus 30% microglia), P4 antagonized the E2-dependent neurite outgrowth (number and length) and neuron viability in the presence of E2, as observed in vivo. However, removal of microglia (astrocyte-neuron coculture) abolished the antagonism of E2 by P4 on neuron sprouting. The P4 receptor antagonists ORG-31710 and RU-486 blocked the antagonism of P4 on E2-dependent sprouting. These findings suggest a new role for microglia in P4 antagonism of E2 in neuronal plasticity and show its dependence on progesterone receptors. These findings are also relevant to the inclusion of progestins in hormone therapy, which is controversial in relation to cognitive declines during aging and in Alzheimer's disease.

N-myc oncogene expression in neuroblastoma is driven by Sp1 and Sp3.

Regulation of N-myc oncogene expression is an important determinant of the biological behavior of neuroblastoma. The N-myc promoter contains several potential binding sites for transcription factors of the Sp1 family. Mutation of a CT-box motif contained within a 26 bp region required for N-myc downregulation by retinoic acid decreased basal transcriptional activity and altered DNA-protein interactions of the promoter, while mutations flanking this motif did neither. On super-shift, this region was shown to recruit Sp1 and Sp3 transcription factor proteins, while a functionally significant CT-box mutation resulted in their replacement by NF-1 transcription factor. Lysates from Drosophila S2 cells expressing exogenous Sp1, Sp3, and NF-1 proteins were able to partially mimic gel shift complexes seen with neuroblastoma nuclear extract and either wild type or mutant probes. Transient transfections of S2 cells showed that both individually and together, Sp1 and Sp3 were able to trans-activate a wild type CT-box-driven luciferase reporter construct in a dose-dependent manner. Transfection of the wild type but not mutant CT-box oligonucleotide was able to decrease endogenous N-myc expression in neuroblastoma cells. Together these results suggest that the CT-box element serves a critically functional role, and in the basal state, allows for N-myc trans-activation by Sp1 and Sp3. Moreover when mutated, the CT-box may still function as a binding motif for alternate transcription factors such as NF-1 that can allow persistent N-myc expression.