Magdalena Karolczak

Estrogenic Stimulation of Neurite Growth in Midbrain Dopaminergic Neurons Depends on cAMP/Protein Kinase A Signalling

Previous work from this laboratory indicates that the differentiation of mouse midbrain dopaminergic neurons is influenced by estrogen. These effects may be transmitted either through classical nuclear receptors or via “nongenomic” mechanisms, including the interaction with hypothetical membrane receptors coupled to distinct intracellular signalling pathways. The latter mechanism seems to be of particular interest for the observed interactions of estrogen with developing dopaminergic neurons, insofar as estrogen has been shown to increase intracellular calcium levels within seconds. This study focuses on signal transduction cascades that might be activated by estrogen during differentiation of dopaminergic cells. Treatment with 17β‐estradiol or a membrane‐impermeable estrogen‐BSA construct (E‐BSA) increased neurite growth and arborization of dopaminergic neurons. This effect was inhibited by antagonists of cAMP/protein kinase A (PKA) and calcium signalling pathways but not by the estrogen receptor antagonist ICI. In addition, estrogen exposure stimulated the phosphorylation of CREB in midbrain dopaminergic cells as studied by quantitative double‐labelling immunocytochemistry and gel shift assay. Again, this effect was antagonized only by the simultaneous treatment with inhibitors of the cAMP/PKA or calcium pathways and not by ICI pretreatment. These data together with our previous findings demonstrate that estrogen can interact with membrane binding sites on dopaminergic neurons, thereby stimulating the cAMP/PKA/phosphorylated cAMP‐responsive element binding protein (CREB) signalling cascade, most likely through the activation of calcium‐dependent kinases. In conclusion, rapid “nongenomic” estrogen signalling represents another mechanism, in addition to the activation of classical nuclear estrogen receptors, that is capable of influencing neuronal differentiation in the mammalian brain. J. Neurosci. Res. 59:107–116, 2000

Membrane receptors for oestrogen in the brain

Oestrogen is important for the development of neuroendocrine centres and other neural networks including limbic and motor systems. Later in adulthood, oestrogen regulates the functional performance of different neural systems and is presumably implicated in the modulation of cognitive efficiency. Although still a matter of controversial discussion, clinical and experimental studies point at a potential neuroprotective role of oestrogen. Concerning the concept of cellular oestrogen action, it is undisputed that it comprises the binding and activation of nuclear receptors. The last decades have, however, immensely broadened the spectrum of steroid signalling within a cell. Novel steroid‐activated intracellular signalling mechanisms were described which are usually termed ‘non‐classical’ or ‘non‐genomic’. The brain appears to be a rich source of this new mode of oestrogen action. Studies from the past years have pinpointed non‐classical oestrogen effects in many CNS regions. All available data support the view that non‐classical oestrogen action requires interactions with putative membrane binding sites/receptors. In this article, we aim at compiling the most recent findings on the nature and identity of membrane oestrogen receptors with respect to the brain. We also attempt to turn readers attention to the coupling of these ‘novel’ receptors to distinct intracellular signalling pathways.

Estrogen: A multifunctional messenger to nigrostriatal dopaminergic neurons

Gonadal steroids affect a wide variety of functions in the mammalian brain ranging from the regulation of neuroendocrine systems and the modulation of behavior to the stimulation of differentiation and plasticity of distinct neuronal populations and circuits. The last decades have also demonstrated that estrogen serves as a neuroprotective factor for distinct neurodegenerative disorders. Such neuroprotective effects of estrogen are most obvious for Parkinsons and Alzheimers disease. Despite this knowledge, little is known about the mechanisms and cellular targets by that estrogen might elicit its protective influence. In the past, we have intensively studied the effects of estrogen on midbrain dopaminergic neurons which represent the most affected cell population during Parkinsons disease. These studies were mainly performed on developing dopaminergic cells and revealed that estrogen is an important regulator of plasticity and function of this neuronal phenotype. Precisely, we found that dopaminergic neurons are direct targets for estrogen and that estrogen stimulates neurite extension/branching and the expression of tyrosine hydroxylase, the key enzyme in dopamine synthesis. Together with other in vivo studies, we might draw the conclusion that estrogen is required for the plasticity and activity of the developing and adult nigrostriatal system. The presence of the estrogen-synthesizing enzyme aromatase within the nigrostriatal system further supports this idea. Surprisingly, estrogen effects on nigrostriatal cell function are not only transmitted by classical nuclear estrogen receptors but also depend on nonclassical estrogen actions mediated through putative membrane receptors coupled to diverse intracellular signaling cascades. In the future, it has to be elucidated whether nonclassical mechanisms besides genomic actions also contribute to estrogen-mediated neuroprotection in the adult CNS.

Estrogen receptor‐α is associated with the plasma membrane of astrocytes and coupled to the MAP/Src‐kinase pathway

Estrogens influence CNS development and a broad spectrum of neural functions. Several lines of evidence also suggest a neuroprotective role for estrogen. Different modes of estrogen action have been described at the cellular level involving classical nuclear estrogen receptor (ER)‐dependent and nonclassical membrane ER‐mediated rapid signaling. We have previously shown that nonclassical estrogen signaling is implicated in the control of dopamine cell function and protection. Since nonclassical interactions between estrogens and glia may contribute to these effects, our aim was to demonstrate the presence of membrane‐associated ERs and their putative coupling to intracellular signaling pathways in astrocytes. Confocal image analysis and fluorescence‐activated cell sorting (FACS) studies indicated the attachment of ER‐α but not ER‐β to the plasma membrane of astrocytes. ERs were located in the cell soma region and glial processes. FACS analysis revealed that only a subpopulation of midbrain astrocytes possesses membrane ER‐α. In FACS studies on ER‐α knockout astrocytes, only a few membrane ER‐positive cells were detected. The activation of membrane ERs appears to be coupled to the MAP‐kinase/Src signaling pathway as shown by Western blotting. In conclusion, our data provide good evidence that nonclassical estrogen action in astrocytes is mediated by membrane ER‐α. The physiological consequence of this phenomenon is not yet understood, but it might have a pivotal role in estrogen‐mediated protective effects on midbrain dopamine neurons.

Developmental Expression and Regulation of Aromatase– and 5α‐Reductase Type 1 mRNA in the Male and Female Mouse Hypothalamus

Androgen metabolites synthesized by neural aromatase and 5α–reductase are implicated in many aspects of mammalian brain development and, in particular, in the masculinization of distinct central nervous system structures and brain functions. The present study was designed to determine (1) the developmental profile of aromatase‐ and 5α–reductase type I mRNA expression in the mouse hypothalamus and (2) to relate ontogenetic sex differences in aromatase activity which have been described in the past to sex‐specific aromatase gene expression. In addition, we analysed the effect of androgens on the perinatal regulation of hypothalamic aromatase and 5α–reductase type I mRNA expression. By applying semiquantitative reverse transcription‐polymerase chain reaction analysis, we found hypothalamic aromatase mRNA expression to be developmentally regulated and to display sex differences at birth and on postnatal day 15 with higher mRNA levels in males. Newborn males and females, which were treated in utero with the androgen receptor antagonist cyproterone actetate, exhibited significantly reduced aromatase mRNA levels compared with untreated controls. In contrast to aromatase, expression levels of hypothalamic 5α–reductase mRNA did not reveal a clear‐cut developmental profile or sex differences, and no regulatory role for androgens in controlling 5α–reductase mRNA expression was found. In conclusion, these results demonstrate perinatal sex differences in hypothalamic aromatase‐ but not 5α–reductase gene expression and suggest that sex differences in perinatal aromatase activity are reflected by corresponding differences in mRNA levels. Androgens are found to control brain estrogen formation pretranslationally at the level of aromatase gene expression. Our findings imply that sex differences in androgen availability and responsiveness are important regulatory factors for aromatase expression in the developing male hypothalamus.

Estrogen stimulates the mitogen-activated protein kinase pathway in midbrain astroglia.

Estrogen stimulates the development of midbrain dopamine neurons predominantly by acting through membrane receptors coupled to Ca(2+)-signaling. In this report, we describe that estrogen activates extracellular signal-regulated kinases (ERK1/2) in midbrain astrocytes but not neurons. This effect was inhibited by BAPTA which interrupts Ca(2+)-signaling but not by antagonists specific for other signaling pathways. The activation of the MAP kinase pathway suggests a potential role for astrocytes in mediating estrogen effects in the midbrain.

Developmental Sex Differences in Estrogen Receptor-β mRNA Expression in the Mouse Hypothalamus/Preoptic Region

Estrogens play a significant role during mammalian brain development and are required for the masculinization of neuronal circuits involved in sex-specific behaviors and neuroendocrine functions. Cellular estrogen signalling is transmitted through nuclear estrogen receptors (ER) which are divided into two subforms: the ER-α as well as the recently cloned ER-β have been demonstrated in the hypothalamus. In the present study, we have analyzed the sex-specific expression of ER-β mRNA in the pre- and postnatal mouse hypothalamus/preoptic region (Hyp/POA) by semiquantitative RT-PCR. The ER-β mRNA was detectable as early as embryonic day (E) 15 in the diencephalon of both sexes. In males, levels of mRNA expression in the Hyp/POA increased until birth and remained high throughtout postnatal (P) development, whereas in females, such an increase was not observed. Significantly higher mRNA levels were detected in the male Hyp/POA from E17 until P15. Perinatal sex differences in ER-β mRNA expression coincide with higher estrogen-forming rates in the male Hyp/POA. At present, no direct evidence is available which demonstrates that estrogen signalling through ER-β is involved in brain development. However, data from our and other studies suggest a potential role for this signal transduction pathway for brain differentiation.

Cell type-specificity of nonclassical estrogen signaling in the developing midbrain

Estrogens have widespread biological functions in the CNS involving the coordination of developmental processes, the regulation of cell physiology, and the control of neuroendocrine systems. In the midbrain, estrogens promote the survival, maturation, and function of neurons and, in particular, of dopamine cells. Aside from classical signaling through nuclear estrogen receptors, we have provided evidence that cellular transmission of estrogen effects in the midbrain comprises a complex intracellular signaling scenario. The major conclusion drawn from our studies is that estrogens interact with yet unidentified membrane receptor complexes which stimulate the phospholipase C and induce the formation of inosite-tri-phosphate (IP(3)). This causes a rapid and transitory rise in intracellular free calcium. The modulation of calcium homeostasis is the primary nonclassical physiological response to estrogens in all cell types. Surprisingly, a different secondary downstream signaling cascade seems to be activated in each estrogen-responsive cell population, i.e. phosphatidylinositol-3 kinase (PI3-kinase) in GABAergic and cAMP/ protein kinase A (PKA) in dopaminergic neurons, mitogen-activated protein kinase (MAP-kinase) in astrocytes. The precise biological role of estrogens for the different cell types is still fragmentary. We assume that estrogens positively influence intracellular signaling mechanisms which are important for cell differentiation and survival. It remains to be elucidated what determines the cell type-specificity of these estrogen responses.

Ontogenetic expression and splicing of estrogen receptor-α and β mRNA in the rat midbrain

Abstract Several studies have shown that estrogen is important for the differentiation of midbrain dopaminergic neurons. This is supported by the previous demonstration of estrogen synthesis in the perinatal ventral midbrain. The present study attempts to characterize the expression pattern of nuclear estrogen receptors (ER-α/β) mRNAs in the ventral rat midbrain during development. By applying primers specific for the hormone-binding domain, ER-α mRNA was detected from embryonic day (E) 14 until postnatal day (P) 20, whereas considerable levels of ER-β mRNA were found from P3 to P20. In contrast, primers spanning the DNA-binding domain demonstrated the presence of transcripts for ER-α as well as ER-β after birth. These findings indicate that both ERs are expressed in the developing midbrain. The presence of ER-α transcripts devoid of the DNA-binding region is discussed in the context of ‘non-genomic’ estrogen signaling possibly by membrane receptors.

Clock gene mRNA and protein rhythms in the pineal gland of mice.

In vertebrates, the rhythmic transcription of clock genes, regulated by their own gene products, provides the basis for self‐sustaining circadian clockworks. These endogenous clocks are lost in most mammalian tissues, but not in the central pacemaker of the hypothalamic suprachiasmatic nucleus (SCN). An interesting model system to understand this phylogenetic shift in function of clock gene products is the rodent pineal gland, as its intrinsic clockwork was replaced during evolution by an input‐dependent oscillator. By means of immunohistochemistry, immunoblotting and real time PCR, we investigated the day/night expression profiles of all major clock genes and their products in the pineal gland of one melatonin‐proficient and one melatonin‐deficient mouse strain. All clockwork elements known to be indispensable for a sustained rhythm generation in the SCN were also found in the pineal organ of both mouse strains. Only mPer1 mRNA and PER1 protein accumulation coincides with timecourses of many other pineal genes and their products, which are cyclicAMP inducible. Here, presented data together with the known mechanisms for regulation of the mPer1 gene in the rodent pineal gland forward the idea that in this tissue PER1 may have a trigger function for initiating the cycles of the clockworks transcriptional/translational feedback loops.