Maria-Angeles Arevalo

The neuroprotective actions of oestradiol and oestrogen receptors

Hormones regulate homeostasis by communicating through the bloodstream to the bodys organs, including the brain. As homeostatic regulators of brain function, some hormones exert neuroprotective actions. This is the case for the ovarian hormone 17β-oestradiol, which signals through oestrogen receptors (ERs) that are widely distributed in the male and female brain. Recent discoveries have shown that oestradiol is not only a reproductive hormone but also a brain-derived neuroprotective factor in males and females and that ERs coordinate multiple signalling mechanisms that protect the brain from neurodegenerative diseases, affective disorders and cognitive decline.

Actions of estrogens on glial cells: Implications for neuroprotection.

Glial cells are directly or indirectly affected by estradiol and by different estrogenic compounds, such as selective estrogen receptor modulators. Acting on oligodendrocytes, astrocytes and microglia, estrogens regulate remyelination, edema formation, extracellular glutamate levels and the inflammatory response after brain injury. In addition, estradiol induces the expression and release of growth factors by glial cells that promote neuronal survival. Therefore, glial cells are important players in the neuroprotective and reparative mechanisms of estrogenic compounds.

Neuroprotective actions of estradiol revisited

Results from animal experiments showing that estradiol is neuroprotective were challenged 10 years ago by findings indicating an increased risk of dementia and stroke in women over 65 years of age taking conjugated equine estrogens. Our understanding of the complex signaling of estradiol in neural cells has recently clarified the causes of this discrepancy. New data indicate that estradiol may lose its neuroprotective activity or even increase neural damage, a situation that depends on the duration of ovarian hormone deprivation and on age-associated modifications in the levels of other molecules that modulate estradiol action. These studies highlight the complex neuroprotective mechanisms of estradiol and suggest a window of opportunity during which effective hormonal therapy could promote brain function and cognition.

Notch and NGF/p75NTR control dendrite morphology and the balance of excitatory/inhibitory synaptic input to hippocampal neurones through Neurogenin 3.

We have previously shown that dendrite morphology of cultured hippocampal neurones is controlled by Notch receptor activation or binding of nerve growth factor (NGF) to its low affinity receptor p75NTR, i.e. processes that up‐regulate the expression of the Homologue of enhancer of split 1 and 5. Thus, the increased expression of these genes decreases the number of dendrites, whereas abrogation of Homologue of enhancer of split 1/5 activity stimulates the outgrowth of new dendrites. Here, we show that Neurogenin 3 is a proneural gene that is negatively regulated by Homologue of enhancer of split 1/5. It also influences dendrite morphology. Hence, a deficit of Notch or NGF/p75NTRactivation can lead to the production of high levels of Neurogenin 3, which stimulates the outgrowth of new dendrites. Conversely, activation of either Notch or p75NTR depressed Neurogenin 3 expression, which not only decreased the number of dendrites but also favoured inhibitory (GABAergic) synaptogenesis, thereby diminishing the ratios of excitatory/inhibitory inputs. NGF also augmented the levels of mRNA encoding the vesicular inhibitory amino acid transporter, but it did not affect the fraction of GAD65/67‐positive neurones. Conversely, overexpression of Neurogenin 3 largely reduced the number of inhibitory synaptic contacts and, consequently, produced a strong increase in the ratios of excitatory/inhibitory synaptic terminals. Our results reveal a hitherto unknown contribution of NGF/p75NTR to dendritic and synaptic plasticity through Neurogenin 3 signalling.

Interactions of estradiol and insulin-like growth factor-I signalling in the nervous system: New advances

Estradiol and insulin-like growth factor-I (IGF-I) interact in the brain to regulate a variety of developmental and neuroplastic events. Some of these interactions are involved in the control of hormonal homeostasis and reproduction. However, the interactions may also potentially impact on affection and cognition by the regulation of adult neurogenesis in the hippocampus and by promoting neuroprotection under neurodegenerative conditions. Recent studies suggest that the interaction of estradiol and IGF-I is also relevant for the control of cholesterol homeostasis in neural cells. The molecular mechanisms involved in the interaction of estradiol and IGF-I include the cross-regulation of the expression of estrogen and IGF-I receptors, the regulation of estrogen receptor-mediated transcription by IGF-I and the regulation of IGF-I receptor signalling by estradiol. Current investigations are evidencing the role exerted by key signalling molecules, such as glycogen synthase kinase 3 and beta-catenin, in the cross-talk of estrogen receptors and IGF-I receptors in neural cells.

Interaction of estrogen receptors with insulin-like growth factor-I and Wnt signaling in the nervous system.

Estradiol signaling through estrogen receptors in the nervous system involves a variety of rapid membrane/cytoplasm-initiated events that are integrated with different mechanisms of transcriptional regulation. Here we review the role of glycogen synthase kinase 3 (GSK3) and beta-catenin in the coordination of membrane/cytoplasm-initiated and nuclear-initiated estrogen receptor signaling. Estradiol activates in vitro and in vivo the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway in neural cells. By activating this pathway through estrogen receptors, estradiol increases the levels of inactive GSK3beta (phosphorylated in serine 9). In turn, the inhibition of GSK3beta increases the stability of beta-catenin and its nuclear translocation. Then, beta-catenin exerts two different transcriptional effects: (i) regulates beta-catenin/T cell factor (TCF) mediated transcription in a similar but not identical way as Wnt ligands and (ii) regulates estrogen receptor mediated transcription after its association with estrogen receptor alpha. In addition, by the regulation of PI3K/Akt/GSK3/beta-catenin pathway, other factors such as insulin-like growth factor-I (IGF-I) regulate estrogen receptor mediated transcription. Therefore, GSK3 and beta-catenin allow the interaction of membrane/cytoplasm-initiated estrogen receptor signaling, IGF-I signaling, Wnt signaling and nuclear-initiated estrogen receptor signaling in the nervous system.

G protein-coupled estrogen receptor is required for the neuritogenic mechanism of 17β-estradiol in developing hippocampal neurons.

Estradiol promotes neuritogenesis in developing hippocampal neurons by a mechanism involving the upregulation of neurogenin 3, a Notch-regulated transcription factor. In this study we have explored whether G-protein coupled estrogen receptor 1 (GPER) participates in this hormonal action. GPER agonists (17β-estradiol, G1, ICI 182,780) increased neurogenin 3 expression and neuritogenesis in mouse primary hippocampal neurons and this effect was blocked by the GPER antagonist G15 and by a siRNA for GPER. In addition, GPER agonists increased Akt phosphorylation in ser473, which is indicative of the activation of phosphoinositide-3-kinase (PI3K). G15 or GPER silencing prevented the estrogenic induction of Akt phosphorylation. Furthermore, the PI3K inhibitor wortmannin prevented the effect of G1 and estradiol on neurogenin 3 expression and the effect of estradiol on neuritogenesis. These findings suggest that GPER participates in the control of hippocampal neuritogenesis by a mechanism involving the activation of PI3K signaling.

Neurogenin 3 cellular and subcellular localization in the developing and adult hippocampus.

Neurogenin 3 (Ngn3), a proneural gene controlled by the Notch receptor, is implicated in the control of dendrite morphology and synaptic plasticity of cultured hippocampal neurons. Here we report the localization and subcellular distribution of Ngn3 in the hippocampus in vivo and in neuronal cultures. In situ hybridization showed Ngn3 mRNA expression in the pyramidal layer and dentate gyrus of adult mouse hippocampus. Immunohistochemistry studies revealed that Ngn3 localization is mostly cytoplasmic in the hippocampal eminence at embryonic day (E)17 and postnatal day (P)0. At P10 it is cytoplasmic in CA1–CA3 pyramidal neurons and nuclear in granule cells of the dentate gyrus. In the adult hippocampus Ngn3 is localized in the nucleus and cytoplasm of both pyramidal neurons and granule cells. During development of cultured hippocampal neurons, Ngn3 mRNA expression is higher at stages of neuronal polarization, as judged by reverse‐transcription polymerase chain reaction (RT‐PCR), and it is mostly cytoplasmic. The tracking of the subcellular localization of Ngn3 in neurons infected with a virus expressing myc‐Ngn3 suggests that the protein is quickly translocated to the cell nucleus after synthesis and then reexported to the cytoplasm. Treatment with leptomycinB, a potent and specific inhibitor of the exportin CRM1, induced its accumulation into the nucleus, suggesting that CRM1 mediates the nuclear export of Ngn3. These results suggest that Ngn3 may play a role in neuronal development by actions in the cytoplasm. J. Comp. Neurol. 518:1814–1824, 2010.

Formin1 Mediates the Induction of Dendritogenesis and Synaptogenesis by Neurogenin3 in Mouse Hippocampal Neurons

Neurogenin3, a proneural transcription factor controlled by Notch receptor, has been recently shown to regulate dendritogenesis and synaptogenesis in mouse hippocampal neurons. However, little is known about the molecular mechanisms involved in these actions of Ngn3. We have used a microarray analysis to identify Ngn3 regulated genes related with cytoskeleton dynamics. One of such genes is Fmn1, whose protein, Formin1, is associated with actin and microtubule cytoskeleton. Overexpression of the Fmn1 isoform-Ib in cultured mouse hippocampal neurons induced an increase in the number of primary dendrites and in the number of glutamatergic synaptic inputs at 4 days in vitro. The same changes were provoked by overexpression of Ngn3. In addition downregulation of Fmn1 by the use of Fmn1-siRNAs impaired such morphological and synaptic changes induced by Ngn3 overexpression in neurons. These results reveal a previously unknown involvement of Formin1 in dendritogenesis and synaptogenesis and indicate that this protein is a key component of the Ngn3 signaling pathway that controls neuronal differentiation.

An in vitro experimental model of neuroinflammation: the induction of interleukin-6 in murine astrocytes infected with Theiler’s murine encephalomyelitis virus, and its inhibition by oestrogenic receptor modulators

This paper describes an experimental model of neuroinflammation based on the production of interleukin‐6 (IL‐6) by neural glial cells infected with Theiler’s murine encephalomyelitis virus (TMEV). Production of IL‐6 mRNA in mock‐infected and TMEV‐infected SJL/J murine astrocytes was examined using the Affymetrix murine genome U74v2 DNA microarray. The IL‐6 mRNA from infected cells showed an eightfold increase in hybridization to a sequence encoding IL‐6 located on chromosome number 5. Quantitative real‐time reverse transcription PCR (qPCR) was used to study the regulation of IL‐6 expression. The presence of IL‐6 in the supernatants of TMEV‐infected astrocyte cultures was quantified by ELISA and found to be weaker than in cultures of infected macrophages. The IL‐6 was induced by whole TMEV virions, but not by Ad.βGal adenovirus, purified TMEV capsid proteins, or UV‐inactivated virus. Two recombinant inflammatory cytokines, IL‐1α and tumour necrosis factor‐α were also found to be potent inducers of IL‐6. The secreted IL‐6 was biologically active because it fully supported B9 hybridoma proliferation in a [3H]thymidine incorporation bioassay. The cerebrospinal fluid of infected mice contained IL‐6 during the acute encephalitis phase, peaking at days 2–4 post‐infection. Finally, this in vitro neuroinflammation model was fully inhibited, as demonstrated by ELISA and qPCR, by five selective oestrogen receptor modulators.