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Role of estrogen receptor ?? in membrane-initiated signaling in neural cells: Interaction with IGF-1 receptor
Abstract
The mechanisms of action of estradiol in the nervous system involve nuclear-initiated steroid signaling and membrane-initiated steroid signaling. Estrogen receptors (ERs) are involved in both mechanisms. ERalpha interacts with the signaling of IGF-1 receptor in neural cells: ERalpha transcriptional activity is regulated by IGF-1 receptor signaling and estradiol regulates IGF-1 receptor signaling. The interaction between ERalpha and the IGF-1 receptor in the brain may occur at the plasma membrane of neurons and glial cells. Caveolin-1 may provide the scaffolding for the interaction of different membrane-associated molecules, including voltage-dependent anion channel, ERalpha and IGF-I receptor.
... The signalosomes are organized as multimolecular clusters whose components interact dynamically following spatiotemporal patterns (Hundsrucker and Klussmann, 2008;Wu and Fuxreiter, 2016;Kandy et al., 2021;Zaccolo et al., 2021). Our recent research in neuronal cells demonstrate that neuronal lipid rafts are the locus of a particular signalosome, the estrogen-receptor (ER)-signalosome, formed by a complex set of factors involved in cellular signaling and neuronal survival (Marin et al., 2009(Marin et al., , 2012Marin, 2011;Marin and Diaz, 2018). The main components of ER-signalosome are pro-survival receptors ERα (estrogen receptor α) and IGF-1Rβ (insulin-like growth factor 1 receptor β), scaffold proteins caveolin-1 and flotillin, prion protein (PrPc), pl-voltage dependent anion channel 1 (VDAC) (a plasmalemmal form of VDAC1) and ionotropic NMDAR and metabotropic mGluR5 glutamate receptors (Marin et al., 2008(Marin et al., , 2009Ramírez et al., 2009;Alonso and Gonzalez, 2012;Díaz and Marin, 2021). ...
... Our recent research in neuronal cells demonstrate that neuronal lipid rafts are the locus of a particular signalosome, the estrogen-receptor (ER)-signalosome, formed by a complex set of factors involved in cellular signaling and neuronal survival (Marin et al., 2009(Marin et al., , 2012Marin, 2011;Marin and Diaz, 2018). The main components of ER-signalosome are pro-survival receptors ERα (estrogen receptor α) and IGF-1Rβ (insulin-like growth factor 1 receptor β), scaffold proteins caveolin-1 and flotillin, prion protein (PrPc), pl-voltage dependent anion channel 1 (VDAC) (a plasmalemmal form of VDAC1) and ionotropic NMDAR and metabotropic mGluR5 glutamate receptors (Marin et al., 2008(Marin et al., , 2009Ramírez et al., 2009;Alonso and Gonzalez, 2012;Díaz and Marin, 2021). Further, the current evidence indicates that the neuronal ER-signalosome likely includes signal transducers such as monomeric G-protein, Ras, and tyrosine kinases such as Raf-1 involved in MEK/ERK signaling for ERα-mediated neuroprotection (Marin et al., 2003(Marin et al., , 2005Guerra et al., 2004). ...
... An emerging concept is that the potential disruption of lipid rafts-resident signalosomes, as a consequence of factors affecting the homeostasis of lipid rafts, contributes to the etiology of AD (Marin et al., 2013;Canerina-Amaro et al., 2017). A striking feature of neuronal ER-signalosome is that proapoptotic protein pl-VDAC shares a common cluster with survival factors ERα and IGF-1Rβ within lipid rafts (Marin et al., 2009;Ramírez et al., 2009;Alonso and Gonzalez, 2012). Our initial observations revealed that under resting conditions pl-VDAC remains inactive through the modulation of its phosphorylation state, a process that appears to be ERα-mediated (Herrera et al., 2011a,b;Canerina-Amaro et al., 2017;Marin and Diaz, 2018). ...
There exists considerable interest to unveil preclinical period and prodromal stages of Alzheimer's disease (AD). The mild cognitive impairment (MCI) is characterized by significant memory and/or other cognitive domains impairments, and is often considered the prodromal phase of AD. The cerebrospinal fluid (CSF) levels of β-amyloid (βA), total tau (t-tau), and phosphorylated tau (p-tau) have been used as biomarkers of AD albeit their significance as indicators during early stages of AD remains far from accurate. The new biomarkers are being intensively sought as to allow identification of pathological processes underlying early stages of AD. Fifty-three participants (75.4 ± 8.3 years) were classified in three groups as cognitively normal healthy controls (HC), MCI, and subjective memory complaints (SMC). The subjects were subjected to a battery of neurocognitive tests and underwent lumbar puncture for CSF extraction. The CSF levels of estrogen-receptor (ER)-signalosome proteins, βA, t-tau and p-tau, were submitted to univariate, bivariate, and multivariate statistical analyses. We have found that the components of the ER-signalosome, namely, caveolin-1, flotilin-1, and estrogen receptor alpha (ERα), insulin growth factor-1 receptor β (IGF1Rβ), prion protein (PrP), and plasmalemmal voltage dependent anion channel 1 (VDAC) could be detected in the CSF from all subjects of the HC, MCI, and SMC groups. The six proteins appeared elevated in MCI and slightly increased in SMC subjects compared to HC, suggesting that signalosome proteins undergo very early modifications in nerve cells. Using a multivariate approach, we have found that the combination of ERα, IGF-1Rβ, and VDAC are the main determinants of group segregation with resolution enough to predict the MCI stage. The analyses of bivariate relationships indicated that collinearity of ER-signalosome proteins vary Mesa-Herrera et al. ER-Signalosome Proteins in Prodromal AD depending on the stage, with some pairs displaying opposed relationships between HC and MCI groups, and the SMC stage showing either no relationships or behaviors similar to either HC or MCI stages. The multinomial logistic regression models of changes in ER-signalosome proteins provide reliable predictive criteria, particularly for the MCI. Notably, most of the statistical analyses revealed no significant relationships or interactions with classical AD biomarkers at either disease stage. Finally, the multivariate functions were highly correlated with outcomes from neurocognitive tests for episodic memory. These results demonstrate that alterations in ER-signalosome might provide useful diagnostic information on preclinical stages of AD, independently from classical biomarkers.
... How mERs are brought into the neuronal membrane is still unclear, although some data demonstrate the presence of these molecules in lipid raft domains, particularly structures with a peculiar lipid composition that allows the integration of different signalling proteins (17). In these domains, mERs are part of macromolecular complexes, or signalosomes, which may trigger neuroprotective responses in an orchestrated manner (18). ...
... Oestrogen binding to these membrane-associated ERs has been shown to induce (in a few minutes) the activation of distinct signalling pathways that participate in cognitive and neuronal survival processes (35). mER responses at the membrane level can be modulated by cross-talk with other membrane receptors, such as insulinlike growth hormone receptors (IGF-IR), metabotrophic glutamate receptors (mGluR), tyrosine kinase receptors, neurotransmitter receptors and porins (17,18,(36)(37)(38). These ER interactions modulate numerous signal transduction pathways, including the mitogenactivated protein kinase (MAPK) family, phosphatidylinositol-3 kinase/Akt (PI-3K/Akt), protein kinase A and C (PKA, PKC), the tyrosine kinase (Src) family and glycogen synthase kinase-3b (GSK3b) (39)(40)(41). ...
... IGF-I signalling through IGF-IR and ER has been largely involved in different actions to promote neuroprotection against AD (82). Indeed, numerous data have demonstrated that ERa and IGF-IR pathways cross-talk to promote beneficial effects against different neuronal impairments, and that this association is affected by ageing (18,83). ...
Oestrogens trigger several pathways at the plasma membrane that exert beneficial actions against neurodegenerative diseases, such as Alzheimer's disease (AD) and Parkinson's disease (PD). Part of these actions takes place in lipid rafts, which are membrane domains with a singular protein and lipid composition. These microdomains also represent a preferential site for signaling protein complexes, or signalosomes. A plausible hypothesis is that the dynamic interaction of signalosomes with different extracellular ligands may be at the basis of neuronal maintenance against different neuropathologies. Oestrogen receptors (ERs) are localized in neuronal lipid rafts, taking part of macromolecular complexes together with a voltage-dependent anion channel (VDAC), and other molecules. Estradiol binding to its receptor at this level enhances neuroprotection against amyloid-β degeneration through the activation of different signal transduction pathways, including VDAC gating modulation. Moreover, part of the stability and functionality of signaling platforms lays on the distribution of lipid hallmarks in these microstructures, which modulate membrane physicochemical properties, thus favoring molecular interactions. Interestingly, recent findings indicate a potential role of oestrogens in the preservation of neuronal membrane physiology related to lipid homeostasis. Thus, oestrogens and docosahexaenoic acid (DHA) may act synergistically to stabilise brain lipid structure by regulating neuronal lipid biosynthetic pathways, suggesting that part of the neuroprotective effects elicited by oestrogens occur through mechanisms aimed at preserving lipid homeostasis. Overall, oestrogen mechanisms of neuroprotection may occur not only by its interaction with neuronal protein targets through non-genomic and genomic mechanisms, but also through its participation in membrane architecture stabilization via "lipostatic" mechanisms. This article is protected by copyright. All rights reserved.
... Next, a series of studies from Raquel Marin's laboratory presented further evidence on this issue [105][106][107][108][109][110]. A first study on the toxic effect of amyloid Aβ peptides on septal (SN56) and hippocampal (HT22) neurons, on the one hand, proved another time that blocking VDAC in cell membranes by anti-porin antibodies means preventing an apoptotic development of cells. ...
... The topographic relationship of the molecules was further specified by demonstrating that both are integrated in caveolar lipid rafts. The Marin group has meanwhile presented additional data to demonstrate that the interaction of VDAC and mERα in caveolae from human cortex is altered in Alzheimer's disease [105][106][107][108][109][110], results in line with corresponding reports from other laboratories [111][112][113]. In addition, there are data on gadolinium [83,84] and aluminum [114] which may help in experiments on these issues. ...
... Keeping the channel closed by pre-incubation of cells with corresponding antibody preparations or anion channel blockers, e.g. DIDS, abolishes regulatory volume decrease (RVD) or apoptotic volume decrease (AVD) of hypotonically or toxically stimulated mammalian cells [28,83,84,[91][92][93][94][95][96]100,[105][106][107][108][109][110]. However, to assume that body wide distributed ...
... Raft-related proteins are frequently found in signaling clusters named signalosomes, that are rearranged depending on the extracellular ligand availability. For instance, we have previously characterized the estrogen receptor alpha (ERα) signalosome, formed by a complex set of factors involved in cellular signaling and neuronal survival [19][20][21][22]. Protein components of ER-signalosome includes flotillin and the ionotropic and metabotropic glutamatergic receptors, N-methyl-D-aspartate receptor (NMDAR) and mGluR5, respectively, suggesting the relevance of this signaling platforms in cognitive skills [18,19,[23][24][25]. ...
... For instance, we have previously characterized the estrogen receptor alpha (ERα) signalosome, formed by a complex set of factors involved in cellular signaling and neuronal survival [19][20][21][22]. Protein components of ER-signalosome includes flotillin and the ionotropic and metabotropic glutamatergic receptors, N-methyl-D-aspartate receptor (NMDAR) and mGluR5, respectively, suggesting the relevance of this signaling platforms in cognitive skills [18,19,[23][24][25]. ...
Long-chain polyunsaturated fatty acids (LCPUFA), essential molecules whose precursors must be dietary supplied, are highly represented in the brain contributing to numerous neuronal processes. Recent findings have demonstrated that LCPUFA are represented in lipid raft microstructures, where they favor molecular interactions of signaling complexes underlying neuronal functionality. During aging, the brain lipid composition changes affecting the lipid rafts’ integrity and protein signaling, which may induce memory detriment. We investigated the effect of a n-3 LCPUFA-enriched diet on the cognitive function of 6- and 15-months-old female mice. Likewise, we explored the impact of dietary n-3 LCPUFAs on hippocampal lipid rafts, and their potential correlation with aging-induced neuroinflammation. Our results demonstrate that n-3 LCPUFA supplementation improves spatial and recognition memory and restores the expression of glutamate and estrogen receptors in the hippocampal lipid rafts of aged mice to similar profiles than young ones. Additionally, the n-3 LCPUFA-enriched diet stabilized the lipid composition of the old mice’s hippocampal lipid rafts to the levels of young ones and reduced the aged-induced neuroinflammatory markers. Hence, we propose that n-3 LCPUFA supplementation leads to beneficial cognitive performance by “rejuvenating” the lipid raft microenvironment that stabilizes the integrity and interactions of memory protein players embedded in these microdomains.
... IGF-1 binds to the IGF-1 receptor and provokes intracellular signalling cascades [9]. There are accumulating evidence for an abundant expression of IGF-IR and ER in hippocampus [10,11]. The interaction of IGF-1 and E2 may promote neuroprotection under neurodegenerative conditions [12]. ...
... The interaction of IGF-1 and E2 may promote neuroprotection under neurodegenerative conditions [12]. It has been reported that IGF-1R and oestrogen receptors (ERa and ERb) are co-expressed in many neurons and glial cells in the CNS [11]. Pioneering work has shown that there is a cross-talk between IGF-1R and ER in cancer [13], and our previous paper reported that E2 up-regulated the expression of IGF-1 at human embryonic stem cells (hESCs) differentiation stage [14]. ...
Human embryonic stem cells (hESCs) can self-renew and differentiate into all cell lineages. E2 is known to exhibit positive effects on embryo development. Although the importance of E2 in many physiological processes has been reported, to date few researchers have investigated the effects of E2 on hESCs differentiation. We studied the effects of E2 on dopamine (DA) neuron induction of hESCs and its related signalling pathways using the three-stage protocol. In our study, 0.1 μM E2 were applied to hESCs-derived human embryoid bodies (hEBs) and effects of E2 on neural cells differentiation were investigated. Protein and mRNA level assay indicated that E2 up-regulated the expression of insulin-like growth factors (IGF)-1, ectoderm, neural precursor cells (NPC) and DA neuron markers, respectively. The population of hESC-derived NPCs and DA neurons was increased to 92% and 93% to that of DMSO group, respectively. Furthermore, yield of DA neuron-secreted tyrosine hydroxylase (TH) and dopamine was also increased. E2-caused promotion was relieved in single inhibitor (ICI or JB1) group partly, and E2 effects were repressed more stronger in inhibitors combination (ICI plus JB1) group than in single inhibitor group at hEBs, hNPCs and hDA neurons stages. Owing to oestrogen receptors regulate multiple brain functions, when single or two inhibitors were used to treat neural differentiation stage, we found that oestrogen receptor (ER)β but not ERα is strongly repressed at the hNPCs and hDA neurons stage. These findings, for the first time, demonstrate the molecular cascade and related cell biology events involved in E2-improved hNPC and hDA neuron differentiation through cross-talk between IGF-1 and ERβ in vitro.
... E2 and IGF1 work in tandem to reciprocally modulate and facilitate ER and IGFIR activation of the PI3KAkt and MAPKERK signaling cascades [96][97][98][99][100] . IGF1 shows differential sensitivities to the two estrogen receptor subtypes with ERα being more sensitive than ERβ [97,101] . Selective inhibition of IGF1R, for instance, downregulates ERα expression in the hypothalamus, hippocampus, and cerebral cortex, with the only significant changes of ERβ occurring in the cerebellum [38] . ...
... The present section does not, however, do justice to the complexity of the relation between estrogen and IGF1 receptors. A fuller explanation can be found in one of several reviews [37][38][39]101,109,117] . ...
Brain integrity and cognitive aptitude are often impaired in patients with diabetes mellitus, presumably a result of the metabolic complications inherent to the disease. However, an increasing body of evidence has demonstrated the central role of insulin-like growth factor 1 (IGF1) and its relation to sex hormones in many neuroprotective processes. Both male and female patients with diabetes display abnormal IGF1 and sex-hormone levels but the comparison of these fluctuations is seldom a topic of interest. It is interesting to note that both IGF1 and sex hormones have the ability to regulate phosphoinositide 3-kinase-Akt and mitogen-activated protein kinases-extracellular signal-related kinase signaling cascades in animal and cell culture models of neuroprotection. Additionally, there is considerable evidence demonstrating the neuroprotective coupling of IGF1 and estrogen. Androgens have also been implicated in many neuroprotective processes that operate on similar signaling cascades as the estrogen-IGF1 relation. Yet, androgens have not been directly linked to the brain IGF1 system and neuroprotection. Despite the sex-specific variations in brain integrity and hormone levels observed in diabetic patients, the IGF1-sex hormone relation in neuroprotection has yet to be fully substantiated in experimental models of diabetes. Taken together, there is a clear need for the comprehensive analysis of sex differences on brain integrity of diabetic patients and the relationship between IGF1 and sex hormones that may influence brain-health outcomes. As such, this review will briefly outline the basic relation of diabetes and IGF1 and its role in neuroprotection. We will also consider the findings on sex hormones and diabetes as a basis for separately analyzing males and females to identify possible hormone-induced brain abnormalities. Finally, we will introduce the neuroprotective interplay of IGF1 and estrogen and how androgen-derived neuroprotection operates through similar signaling cascades. Future research on both neuroprotection and diabetes should include androgens into the interplay of IGF1 and sex hormones.
... Indeed, these peculiar microenvironments may provide the optimal conditions to promote ER interactions in signalosomes, which are crucial for neuroprotective actions initiated at the plasma membrane against a variety of injuries [87]. In this sense, raft-located ERs have been identified in cerebral cognitive areas, including human hippocampus and cortical areas [88] as well as in mouse hippocampus and frontal cortex [89], reflecting the importance of ER location in these microenvironments related to cognitive functions. Some evidence indicates that raft-integrated caveolin-1 participates in membrane ER function in the brain related to its preservation and maintenance [90,91]. ...
... Although still partially characterized, this complex is formed by caveolin-1 anchoring protein as a requirement for neuronal steroid membrane signaling [93], together with other proteins involved in neurotrophic and neuroprotective actions against neurodegeneration, the insulin growth factor 1 receptor (IGF-1R) and a voltage-dependent anion channel (VDAC). An interesting aspect is that this association appears to be affected by aging [88,96] and appears to be differentially induced by the availability of extracellular ligands, i.e., estradiol, insulin and insulin growth factor, thereby inducing the selective recruitment of different intracellular targets to modulate the final cell response [87,97]. A similar scenario may occur related to glutamatergic responses, as glutamate receptors are known to interact with caveolin and ER in signaling clusters as a main functional requirement [98,99]. ...
Natural selection clearly favors the accumulation and storage of lipids in humans, predisposing women to store excess fat in gluteal regions and predisposing males to store excess fat in visceral regions. In addition, gender differences are reported with respect to the concentrations of circulating lipids and lipoproteins, with lower concentrations of total cholesterol and low density lipoprotein (LDL)-cholesterol in premenopausal women than in men. This latter evidence renders gender differences in fat distribution and whole-body lipid metabolism of particular interest with respect to the incidence and prevalence of human diseases. Although the mechanisms underlying gender-related differences in body fat distribution and lipid homeostasis remain to be fully determined, the reported differences appear to principally reflect the actions of the sex steroid hormone estrogen on whole-body lipid metabolism. In the present review, we dissect the role played by 17-estradiol, the most active between estrogens, and by its receptors in regulating lipid homeostasis in adipose tissue, liver, and brain, evaluating the potential impact of this hormone in preventing lipid abnormalities.
... Indeed, these peculiar microenvironments may provide the optimal conditions to promote ER interactions in signalosomes, which are crucial for neuroprotective actions initiated at the plasma membrane against a variety of injuries [87]. In this sense, raft-located ERs have been identified in cerebral cognitive areas, including human hippocampus and cortical areas [88] as well as in mouse hippocampus and frontal cortex [89], reflecting the importance of ER location in these microenvironments related to cognitive functions. Some evidence indicates that raft-integrated caveolin-1 participates in membrane ER function in the brain related to its preservation and maintenance [90,91]. ...
... Although still partially characterized, this complex is formed by caveolin-1 anchoring protein as a requirement for neuronal steroid membrane signaling [93], together with other proteins involved in neurotrophic and neuroprotective actions against neurodegeneration, the insulin growth factor 1 receptor (IGF-1R) and a voltage-dependent anion channel (VDAC). An interesting aspect is that this association appears to be affected by aging [88,96] and appears to be differentially induced by the availability of extracellular ligands, i.e., estradiol, insulin and insulin growth factor, thereby inducing the selective recruitment of different intracellular targets to modulate the final cell response [87,97]. A similar scenario may occur related to glutamatergic responses, as glutamate receptors are known to interact with caveolin and ER in signaling clusters as a main functional requirement [98,99]. ...
Natural selection clearly favours the accumulation and storage of lipids in humans predisposing women to store excess fat in gluteal regions for reproduction and male in visceral region that allows specific intermittent activities typical for men. In addition, gender differences are reported in the concentration of circulating lipids and lipoproteins, with lower concentrations of total cholesterol and low density lipoprotein (LDL)-cholesterol in premenopausal women than in men. This latter evidence renders the gender differences in fat distribution and whole-body lipid metabolism of particular interest in the area of incidence and prevalence of human diseases. Although the mechanisms underlying gender-related differences in body fat distribution and lipid homeostasis remain to be fully determined, the differences reported appear to principally reflect the actions of the sex steroid hormone estrogens on whole-body lipid metabolism. In the present review, we dissect the role played by 17β-estradiol, the most active between estrogens, and by its receptors in regulating lipid homeostasis in adipose tissue, liver, and brain evaluating the potential impact of this hormone in preventing lipid abnormalities.
... This band has also been observed in previous studies (Rao, 1998;Marin et al., 2005;González et al., 2007), and may be the result of the duplication of two exons within the ERα gene, as previously reported in cancer cells (Pink et al., 1996). Although not shown here, a battery of antibodies directed to distinct epitopes of ERα consistently reproduced these results, indicating that this receptor is a high homologue to ERα, which is in agreement with previous studies in immortalized cells and neuronal tissues (reviewed in Marin et al., 2009). The purity of membrane isolates was confirmed by testing the distribution of cytosolic Hsp90, only observed in whole tissue extracts, and transmembrane α1 subunit of Na + /K + ATPase highly abundant at the membrane fractions. ...
... Although not explored in this work, other proteins may be part of this complex. Previous data in different neuronal types have reported the physical association of ERα with insulin growth factor-1 (IGF-1) receptor, and with the downstream proteins insulin receptor substrate-1 (IRS-1), and phosphatidilinositol-3-kinase (PI3K) that, together with the demonstrated ERα interaction with β-catenin, may represent different mechanisms of estrogen to promote cell survival (Garcia-Segura et al., 2007;Marin et al., 2009). ...
Membrane estrogen receptor Caveolae Caveolin-1 Human brain Alzheimer's disease Voltage-dependent anion channel (VDAC) is a mitochondrial porin also found in the neuronal membrane (pl-VDAC), where its function may be related to redox homeostasis and apoptosis. Murine models have evidenced pl-VDAC into caveolae in a complex with estrogen receptor alpha (mERα), which participates in neuroprotection against amyloid beta (Aβ), and whose integration into this hydrophobic domain remains unclear. Here, we have demonstrated in caveolae of human cortex and hippocampus the presence of pl-VDAC and mERα, in a complex with scaffolding caveolin-1 which likely provides mERα stability at the plasma membrane. In Alzheimer's disease (AD) brains, VDAC was accumulated in caveolae, and it was observed in dystrophic neurites of senile plaques, whereas ERα was expressed in astrocytes surrounding the plaques. Together with previous data in murine neurons demonstrating the participation of pl-VDAC in Aβ-induced neurotoxicity, these data suggest that the channel may be involved in membrane dysfunctioning observed in AD neuropathology.
... This band has also been observed in previous studies (Rao, 1998;Marin et al., 2005;González et al., 2007), and may be the result of the duplication of two exons within the ERα gene, as previously reported in cancer cells (Pink et al., 1996). Although not shown here, a battery of antibodies directed to distinct epitopes of ERα consistently reproduced these results, indicating that this receptor is a high homologue to ERα, which is in agreement with previous studies in immortalized cells and neuronal tissues (reviewed in Marin et al., 2009). The purity of membrane isolates was confirmed by testing the distribution of cytosolic Hsp90, only observed in whole tissue extracts, and transmembrane α1 subunit of Na + /K + ATPase highly abundant at the membrane fractions. ...
... Although not explored in this work, other proteins may be part of this complex. Previous data in different neuronal types have reported the physical association of ERα with insulin growth factor-1 (IGF-1) receptor, and with the downstream proteins insulin receptor substrate-1 (IRS-1), and phosphatidilinositol-3-kinase (PI3K) that, together with the demonstrated ERα interaction with β-catenin, may represent different mechanisms of estrogen to promote cell survival (Garcia-Segura et al., 2007;Marin et al., 2009). ...
Membrane estrogen receptor Caveolae Caveolin-1 Human brain Alzheimer's disease Voltage-dependent anion channel (VDAC) is a mitochondrial porin also found in the neuronal membrane (pl-VDAC), where its function may be related to redox homeostasis and apoptosis. Murine models have evidenced pl-VDAC into caveolae in a complex with estrogen receptor alpha (mERα), which participates in neuroprotection against amyloid beta (Aβ), and whose integration into this hydrophobic domain remains unclear. Here, we have demonstrated in caveolae of human cortex and hippocampus the presence of pl-VDAC and mERα, in a complex with scaffolding caveolin-1 which likely provides mERα stability at the plasma membrane. In Alzheimer's disease (AD) brains, VDAC was accumulated in caveolae, and it was observed in dystrophic neurites of senile plaques, whereas ERα was expressed in astrocytes surrounding the plaques. Together with previous data in murine neurons demonstrating the participation of pl-VDAC in Aβ-induced neurotoxicity, these data suggest that the channel may be involved in membrane dysfunctioning observed in AD neuropathology.
... Interestingly, age negatively correlates with serum levels of both IGF-1 and estrogen in humans, particularly females (Labrie et al., 1997;Leifke et al., 2000;Tanimoto et al., 2008). IGF-1 increases following 17b-estradiol exposure in rodent uterus (Murphy et al., 1987;Kapur et al., 1992), and IGF-1 can directly stimulate phosphorylation of the estrogen receptor (ER) to induce transcription in the absence of 17b-estradiol (Aronica and Katzenellenbogen, 1993), whereas IGF-1 receptor (IGF-1R) and ER cross talk has been reported in the uterus (Klotz et al., 2000(Klotz et al., , 2002 and brain (Marin et al., 2009). Surprisingly, potential cross talk between IGF-1/IGF-1R and 17b-estradiol/ER has not been explored in the skin. ...
... A number of studies report that IGF-1 can signal via the ERs (Aronica and Katzenellenbogen, 1993;Klotz et al., 2000Klotz et al., , 2002, in particular ERa, which is required for IGF-induced proliferation in the uterus (Klotz et al., 2002). Interactions between ERa and IGF-1 have also been demonstrated in numerous other organ systems, including the breast (Surmacz and Bartucci, 2004), brain (Marin et al., 2009), and liver (Della Torre et al., 2011). Far fewer associations have been made between ERb and IGF-1 (e.g., Rivadeneira et al., 2006). ...
Although it is understood that endogenous IGF-1 is involved in the wound repair process, the effects of exogenous IGF-1 administration on wound repair remain largely unclear. In addition, the signaling links between IGF-1 receptor (IGF-1R) and estrogen receptors (ERs), which have been elucidated in other systems, have yet to be explored in the context of skin repair. In this study, we show that locally administered IGF-1 promotes wound repair in an estrogen-deprived animal model, the ovariectomized (Ovx) mouse, principally by dampening the local inflammatory response and promoting re-epithelialization. Using specific IGF-1R and ER antagonists in vivo, we reveal that IGF-1-mediated effects on re-epithelialization are directly mediated by IGF-1R. By contrast, the anti-inflammatory effects of IGF-1 are predominantly via the ERs, in particular ERα. Crucially, in ERα-null mice, IGF-1 fails to promote healing, and local inflammation is increased. Our findings illustrate the complex interactions between IGF-1 and estrogen in skin. The fact that IGF-1 may compensate for estrogen deficiency in wound repair, and potentially other contexts, is an important consideration for the treatment of postmenopausal pathology.Journal of Investigative Dermatology advance online publication, 19 July 2012; doi:10.1038/jid.2012.228.
... Our findings, using one single injection of estrogenic compounds, support previous studies showing that acute treatments with estradiol, tamoxifen and raloxifene affect behavioral and synaptic parameters in the brain (González-Burgos et al., 2012;Kramár et al., 2009;Lagunas et al., 2011;Scott et al., In press;Smejkalova and Woolley, 2010;Walf and Frye, 2008;Woolley, 2007). These acute actions of estrogens may be mediated by membrane/cytoplasmic steroidsignaling mechanisms, which are involved in the rapid actions of estradiol in the brain (Marin et al., 2009;Roepke et al., 2011). Rapid ERα-mediated estrogenic actions in neurons include the formation of dendritic spines by the local reorganization of actin fibers at sites of spine formation (Sanchez et al., 2009). ...
... Rapid ERα-mediated estrogenic actions in neurons include the formation of dendritic spines by the local reorganization of actin fibers at sites of spine formation (Sanchez et al., 2009). Rapid ERα signaling also activates the PI3K pathway (Garcia-Segura et al., 2010;Marin et al., 2009), which is involved in spinogenesis (Cuesto et al., 2011;Kumar et al., 2005;Lee et al., 2011) and in the consolidation of some memory modalities (Fan et al., 2010;Horwood et al., 2006;Sui et al., 2008). Further molecular studies are needed to determine the signaling mechanisms that mediate the regulation of allocentric working memory and the morphology of dendritic spines in the prefrontal cortex by tamoxifen and raloxifene. ...
Estradiol and some selective estrogen receptor modulators (SERMs) are neuroprotective in a variety of experimental models of neurodegeneration, reduce the inflammatory response of glial cells, reduce anxiety and depression, promote cognition and modulate synaptic plasticity in the hippocampus of rodents. In this study we have assessed whether estradiol and two SERMs currently used in clinics, tamoxifen and raloxifene, affect medial prefrontal cortex function and morphology. Rats were ovariectomized and six days later some animals received a subcutaneous injection of the estrogenic compounds. In a first experiment animals were treated with estradiol benzoate or sesame oil vehicle. In a second experiment animals received raloxifene, tamoxifen or dimethyl sulfoxide as vehicle. Twenty four hours after the pharmacological treatment, animals were challenged to solve an allocentric working memory paradigm in a "Y" maze. Twenty trials consisting of a study phase and a test phase were conducted according to a delayed match-to-sample procedure in a single one-day session. Animals that were not submitted to behavioral test were used for Golgi analysis of the prefrontal cortex. Rats treated with estradiol benzoate, tamoxifen or raloxifene performed better in the Y maze and showed a significant increase in the numerical density of dendritic spines in secondary apical dendrites of layer III pyramidal neurons from the prelimbic/infralimbic prefrontal cortex, compared to their respective control groups. These findings suggest that estradiol, tamoxifen and raloxifene improve prefrontal cortex-related cognitive performance and modulate prefrontal cortex morphology in ovariectomized rats.
... Longitudinal growth and somatic maturation are mainly regulated by IGF1 binding to IGF1R and the subsequent IGF1R activation of main intracellular signaling pathways in target tissues [32,[45][46][47]. Intracellular signal of the IGF1R is reported to be regulated by cross-interaction with ER and differential activation of synergistic PI3K/AKT, GSK3, ERK and Wnt pathways, with these actions possibly being tissue/cell specific [37,[48][49][50][51]. To determine sex differences in putative IGF1 signaling pathway dysregulation in response to Pappa2 deficiency, we analyzed key intracellular pathways, including PI3K, AKT, AMPKα, ERK1/2, mTOR and GSK3β, among others, in the hypothalamus-pituitaryliver axis of Pappa2 ko/ko male and female mice, and the liver of mice treated with rhGH, rhIGF1 and rhPAPP-A2. ...
Background
Children with pregnancy-associated plasma protein-A2 ( PAPP-A2 ) mutations resulting in low levels of bioactive insulin-like growth factor-1 (IGF1) and progressive postnatal growth retardation have improved growth velocity and height following recombinant human (rh)IGF1 treatment. The present study aimed to evaluate whether Pappa2 deficiency and pharmacological manipulation of GH/IGF1 system are associated with sex-specific differences in growth-related signaling pathways.
Methods
Plasma, hypothalamus, pituitary gland and liver of Pappa2 ko/ko mice of both sexes, showing reduced skeletal growth, and liver of these mice treated with rhGH, rhIGF1 and rhPAPP-A2 from postnatal day (PND) 5 to PND35 were analyzed.
Results
Reduced body and femur length of Pappa2 ko/ko mice was associated with increases in: (1) components of IGF1 ternary complexes (IGF1, IGFBP5/ Igfbp5 , Igfbp3 , Igfals ) in plasma, hypothalamus and/or liver; and (2) key signaling regulators (phosphorylated PI3K, AKT, mTOR, GSK3β, ERK1/2 and AMPKα) in hypothalamus, pituitary gland and/or liver, with Pappa2 ko/ko females having a more prominent effect. Compared to rhGH and rhIGF1, rhPAPP-A2 specifically induced: (1) increased body and femur length, and reduced plasma total IGF1 and IGFBP5 concentrations in Pappa2 ko/ko females; and (2) increased Igf1 and Igf1r levels and decreased Ghr , Igfbp3 and Igfals levels in the liver of Pappa2 ko/ko females. These changes were accompanied by lower phospho-STAT5, phospho-AKT and phospho-ERK2 levels and higher phospho-AMPK levels in the liver of Pappa2 ko/ko females.
Conclusions
Sex-specific differences in IGF1 system and signaling pathways are associated with Pappa2 deficiency, pointing to rhPAPP-A2 as a promising drug to alleviate postnatal growth retardation underlying low IGF1 bioavailability in a female-specific manner.
... These observations have been confirmed in an ever-increasing number of studies, which collectively have led to consider GM1 gangliosides as key factors in neurotrophic and neuroprotective roles (Chiricozzi et al., 2020;Grassi et al., 2020). In order to delve further into the lipid raft modification in response to n-3 LCPUFA, we also analyzed the contents of flotillin-1, a scaffold protein is known to associate with lipid rafts and to participate in the domain stabilization of raft resident and transient signaling proteins (Marin et al., 2009;Marin & Diaz, 2018). This scaffolding structure stabilizes its location within membrane rafts, and it is therefore considered a canonical lipid raft marker. ...
"Lipid raft aging" in nerve cells represents an early event in the development of aging-related neurodegenerative diseases, such as Alzheimer's disease. Lipid rafts are key elements in synaptic plasticity, and their modification with aging alters interactions and distribution of signaling molecules, such as glutamate receptors and ion channels involved in memory formation, eventually leading to cognitive decline. In the present study, we have analyzed, in vivo, the effects of dietary supplementation of n-3 LCPUFA on the lipid structure, membrane microviscosity, domain organization, and partitioning of ionotropic and metabotropic glutamate receptors in hippocampal lipid raffs in female mice. The results revealed several lipid signatures of "lipid rafts aging" in old mice fed control diets, consisting in depletion of n-3 LCPUFA, membrane unsaturation, along with increased levels of saturates, plasmalogens, and sterol esters, as well as altered lipid relevant indexes. These changes were paralleled by increased microviscosity and changes in the raft/non-raft (R/NR) distribution of AMPA-R and mGluR5. Administration of the n-3 LCPUFA diet caused the partial reversion of fatty acid alterations found in aged mice and returned membrane microviscosity to values found in young animals. Paralleling these findings, lipid rafts accumulated mGluR5, NMDA-R, and ASIC2, and increased their R/NR proportions, which collectively indicate changes in synaptic plasticity. Unexpectedly, this diet also modified the lipidome and dimension of lipid rafts, as well as the domain redistribution of glutamate receptors and acid-sensing ion channels involved in hippocampal synaptic plasticity, likely modulating functionality of lipid rafts in memory formation and reluctance to age-associated cognitive decline.
... In the cortex and hippocampus, mERα, along with caveolin 1, cooperates with important cellular mediators such as insulin growth factor-1 receptor beta (IGF-IRβ) and a voltagedependent anion channel (VDAC) [90][91][92]. A decrease in mER-signalosome complexes and insufficient mER-VDAC phosphorylation in the frontal cortex during menopause have been connected with cognitive deficits and the promotion of Aβ toxicity [22]. ...
Nuclear- and membrane-initiated estrogen signaling cooperate to orchestrate the pleiotropic effects of estrogens. Classical estrogen receptors (ERs) act transcriptionally and govern the vast majority of hormonal effects, whereas membrane ERs (mERs) enable acute modulation of estrogenic signaling and have recently been shown to exert strong neuroprotective capacity without the negative side effects associated with nuclear ER activity. In recent years, GPER1 was the most extensively characterized mER. Despite triggering neuroprotective effects, cognitive improvements, and vascular protective effects and maintaining metabolic homeostasis, GPER1 has become the subject of controversy, particularly due to its participation in tumorigenesis. This is why interest has recently turned toward non-GPER-dependent mERs, namely, mERα and mERβ. According to available data, non-GPER-dependent mERs elicit protective effects against brain damage, synaptic plasticity impairment, memory and cognitive dysfunctions, metabolic imbalance, and vascular insufficiency. We postulate that these properties are emerging platforms for designing new therapeutics that may be used in the treatment of stroke and neurodegenerative diseases. Since mERs have the ability to interfere with noncoding RNAs and to regulate the translational status of brain tissue by affecting histones, non-GPER-dependent mERs appear to be attractive targets for modern pharmacotherapy for nervous system diseases.
... Receptor tyrosine kinases observed to reside in lipid rafts include tropomyosinrelated kinase A (TrkA) receptor and the low-affinity p75 neurotrophin receptor (p75NTR), which are receptors for Nerve Growth Factor (NGF) [63], IGF-1R (insulin growth factor-1 receptor) [64], EGFR (epidermal growth factor receptor) [65] or PDGFR (platelet-derived growth factor receptor) [66,67], amongst others. Alternatively, RTKs that are not lipid rafts resident proteins, may translocate to rafts after activation, as it was initially demonstrated for glial-derived neurotrophic factor (GDNF)-mediated activation of the Ret RTK [2]. ...
A wealth of evidence accumulated over the last two decades has unambiguously linked lipid rafts to neurodegenerative diseases, in particular to Alzheimer’s disease (AD). These microdomains are highly dynamic membrane platforms with differentiated physicochemical and molecular properties compared to the surrounding membrane microenvironment, and are the locus for a number of central processes in neuronal physiology. Most recent evidence pinpoint to lipid rafts as main players in AD neuropathology. It is now widely accepted that lipid rafts actively participate in the processing of amyloid precursor protein to generate amyloid beta peptides, a main component of amyloid plaques. Current evidence have highlighted the existence of severe alterations in the molecular structure and functionality of lipid rafts in the frontal cortex of human brains affected by Alzheimer’s disease. An exceptionally interesting observation is that lipid raft destabilization can be demonstrated even at the earliest stages of AD neuropathology. In the present review, we will first elaborate on the structure and function of these multifaceted subcellular structures and second to focus on the impact of their alterations in neuronal pathophysiology along the onset and progression of AD continuum.
... The loss of circulating E2, loss of E2 neuroprotection, and degradation of ERα is connected to E2 modulation of voltage-dependent anion channel (VDAC) gating, for example during Ab toxicity, and deterioration of ERα signalosome integrity. VDAC channels have been found accompanying ERα lipid raft signalosomes across both murine and human neurons from several brain regions, most notably for the purposes of this review, in the hippocampus [130][131][132]. Specifically, the loss of E2 may be correlated with the deregulation of VDAC gating, which subsequently precedes AD pathogenesis [133], while E2 activation of the ERα signalosome inhibits the dephosphorylation of VDACs, leading to neuroprotection against Ab-mediated degradation in neurons [104,134]. ...
17β-Estradiol (estradiol or E2) is a steroid hormone that has been broadly applied as a neuroprotective therapeutic for a variety of neurodegenerative and cerebrovascular disorders such as ischemic stroke, Alzheimer's disease, and Parkinson's disease. Several laboratory and clinical studies have reported that estrogen replacement therapy (ERT) had no effect against these diseases in elderly postmenopausal women, and at worst, increased their risk of onset and mortality. This review focuses on the growing body of data from in vitro and animal models characterizing the potential underlying mechanisms and signaling pathways that govern successful neuroprotection by ERT, including the roles of E2 receptors in mediating neuroprotection, E2 genomic regulation of apoptosis-related pathways, membrane-bound receptor-mediated non-genomic signaling pathways, and the antioxidant mechanisms of E2. Also discussed is current evidence for a critical period of effective treatment with estrogen following natural or surgical menopause and the outcomes of E2 administration within the advantageous time period. The known mechanisms governing the duration of the critical period include depletion of E2 receptors, the switch to a ketogenic metabolic profile by neuronal mitochondria, and a decrease in acetylcholine that accompanies E2 deficiency. Also summarized are the major clinical trials and observational studies concerning postmenopausal hormone therapy (HT), to compare their outcomes with respect to neurological disease and discuss their relevance to the critical period hypothesis. Finally, potential controversies and future directions for this field are discussed throughout the review.
... Many of these moieties are combined to tune the ability of an overall amphiphilic lipid to prefer lipid rafts or non-raft domains, or maybe be confined to regions where both domains co-exist (the edges of lipid rafts). From this perspective, it is interesting that some proteins are only acylated (e.g., G proteins [54], Src family members [51,76,85]), some are only isoprenylated (G proteins [59,84], lamins [26]), while others are simultaneously acylated and isoprenylated (Ras-family GTPases) [7,66]. This may ensure the protein is localized to lipid rafts, non-raft domains, or the interface between raft and non-raft domains, respectively. ...
Lipid rafts are nanometer-sized subdomains of the plasma membrane containing higher concentrations of cholesterol, phosphatidylinositols, and sphingolipids. Their lipid constituents have less conformational freedom and compact together more efficiently. Furthermore, lipids and proteins embedded within lipid rafts collide with molecular neighbors more frequently. Similar to how oil droplets form “floating islands” on water, lipid rafts form “islands” in plasma membranes. It is now clear that the integrity of these lipid rafts is essential in order to have proper signaling by many immune, neuronal, and endocrine receptor complexes (e.g., serpentine receptors and many multi-chain receptor complexes including those for antigens, major histocompatibility complex-restricted peptides, the TNF-family, GH family, the IL-2R family and the IL-6 family) and acylated proteins (e.g., src kinases, ras, and protein arginine methyltransferase 8). The fact that these receptors co-purify with detergent-resistant membranes supports theories that they reside within lipid rafts. Because lipid rafts are too small to see directly, and change their size and position easily, they are incredibly hard to visualize, especially in intact cells. We attempted to use fluorescence resonance energy transfer (FRET, an effect observed only when two components are less than 10 nm apart) to investigate the influence of the lipid raft nanoenvironment on the assembly of Class II cytokine receptor complexes, especially interferon receptors. In this chapter, we summarize our observations that (1) receptor preassembly is required for biological function, (2) the interaction between receptor chains requires both the presence of Jak1 and their co-nanolocalization within lipid rafts, (3) a sequence-supported structural analysis of Janus kinases that suggests a significant influence of phospholipids on Janus kinase function, and (4) critical observations made by others. Altogether, we present a model that lipid rafts shape the conformation of Jak1, which in turn controls how it interacts with multiple cytokine receptors, permitting their interaction and consequently their biological function. The increased viscosity of lipid rafts compared to non-raft domains may help stabilize interactions within the receptor complex.
... ERα mRNA levels are increased shortly after the induction of ischemic injury both in the presence and absence of estradiol (Dubal et al., 1999). In addition, estradiol induces the expression of IGF-1 (Kapur et al., 1992;Michels et al., 1993) and IGF-1R (Cardona-Gómez et al., 2000;El-Bakri et al., 2004) in the brain and activates the signaling cascade of IGF-1R via ERα (Kahlert et al., 2000;Marin et al., 2009). On the other hand, IGF-1R activation is essential for several actions of estradiol in the brain, such as neuroprotection in the hippocampus . ...
Perinatal asphyxia (PA) remains as one of the most important causes of short-term mortality, psychiatric and neurological disorders in children, without an effective treatment. In previous studies we have observed that the expression of different neurodegenerative markers increases in CA1 hippocampal area of 4-months-old male rats born by cesarean section and exposed for 19 min to PA. We have also shown that a late treatment with 17β estradiol (daily dose of 250 μg/kg for 3 days) was able to revert the brain alterations observed in those animals. Based on these previous results, the main aim of the present study was to explore the mechanism by which the estrogenic treatment is involved in the reversion of the chronic neurodegenerative conditions induced by PA. We demonstrated that estradiol treatment of adult PA exposed animals induced an increase in estrogen receptor (ER) α and insulin-like growth factor receptor (IGF-1R) protein levels, an activation of the phosphatidylinositol 3-kinase/Akt/glycogen synthase kinase 3 beta/β-catenin signaling pathway and an increase in Bcl-2/Bax ratio in the hippocampus in comparison to PA exposed animals treated with vehicle. Taking together, our data suggest that the interaction between ERα and IGF-IR, with the subsequent downstream activation, underlies the beneficial effects of estradiol observed in late treatment of PA.
... It has been postulated that part of E2 neuroprotective actions triggered in ERα signalosomes against Aβ toxicity occur by the activation of different signal transduction pathways, including a voltage-dependent anion channel (VDAC) gating modulation. This channel appears associated with ERα in neuronal lipid rafts from a wide variety of brain regions, including septum, hippocampus, and cortex from, both, murine and human origins (Marin et al., 2007(Marin et al., , 2009Ramirez et al., 2009), where it participates in different pathogenesis including AD (Thinnes, 2015). In raft fractions of cortical neurons, VDAC interacting with Aβ promotes the channel dephosphorylation in tyrosine residues, a phenomenon that promotes VDAC gating, and enhances neuronal death (Fernandez-Echevarria et al., 2014). ...
Estrogens (E2) exert a plethora of neuroprotective actions against aged-associated brain diseases, including Alzheimer's disease (AD). Part of these actions takes place through binding to estrogen receptors (ER) embedded in signalosomes, where numerous signaling proteins are clustered. Signalosomes are preferentially located in lipid rafts which are dynamic membrane microstructures characterized by a peculiar lipid composition enriched in gangliosides, saturated fatty acids, cholesterol, and sphingolipids. Rapid E2 interactions with ER-related signalosomes appear to trigger intracellular signaling ultimately leading to the activation of molecular mechanisms against AD. We have previously observed that the reduction of E2 blood levels occurring during menopause induced disruption of ER-signalosomes at frontal cortical brain areas. These molecular changes may reduce neuronal protection activities, as similar ER signalosome derangements were observed in AD brains. The molecular impairments may be associated with changes in the lipid composition of lipid rafts observed in neurons during menopause and AD. These evidences indicate that the changes in lipid raft structure during aging may be at the basis of alterations in the activity of ER and other neuroprotective proteins integrated in these membrane microstructures. Moreover, E2 is a homeostatic modulator of lipid rafts. Recent work has pointed to this relevant aspect of E2 activity to preserve brain integrity, through mechanisms affecting lipid uptake and local biosynthesis in the brain. Some evidences have demonstrated that estrogens and the docosahexaenoic acid (DHA) exert synergistic effects to stabilize brain lipid matrix. DHA is essential to enhance molecular fluidity at the plasma membrane, promoting functional macromolecular interactions in signaling platforms. In support of this, DHA detriment in neuronal lipid rafts has been associated with the most common age-associated neuropathologies, namely AD and Parkinson disease. Altogether, these findings indicate that E2 may participate in brain preservation through a dual membrane-related mechanism. On the one hand, E2 interacting with ER related signalosomes may protect against neurotoxic insults. On the other hand, E2 may exert lipostatic actions to preserve lipid balance in neuronal membrane microdomains. The different aspects of the emerging multifunctional role of estrogens in membrane-related signalosomes will be discussed in this review.
... 113 CAV1 is required for estrogen receptor (ER) activation of the metabotrophic glutamate 114 receptor, mGluR1 in hippocampal neurons, potentially acting in long-term depression 115 ( Takayasu et al., 2010). CAV1 provides a scaffold for both the ER voltage-dependent anion 116 receptor and one of its interactors, the IGF-1 receptor ( Maggi et al., 2002;Marin et al., 2009), a 117 known positive regulatory pathway for corticospinal projection neuron axonal outgrowth 118 ( Ozdinler and Macklis, 2006). 119 ...
The neocortex is composed of many distinct subtypes of neurons that must form precise subtype-specific connections to enable the cortex to perform complex functions. Callosal projection neurons (CPN) are the broad population of commissural neurons that connect the cerebral hemispheres via the corpus callosum (CC). Currently, how the remarkable diversity of CPN subtypes and connectivity is specified, and how they differentiate to form highly precise and specific circuits, are largely unknown. We identify in mouse that the lipid-bound scaffolding domain protein Caveolin 1 (CAV1) is specifically expressed by a unique subpopulation of Layer V CPN that maintain dual ipsilateral frontal projections to premotor cortex. CAV1 is expressed by over 80% of these dual projecting callosal/frontal projection neurons (CPN/FPN), with expression peaking early postnatally as axonal and dendritic targets are being reached and refined. CAV1 is localized to the soma and dendrites of CPN/FPN, a unique population of neurons that shares information both between hemispheres and with premotor cortex, suggesting function during postmitotic development and refinement of these neurons, rather than in their specification. Consistent with this, we find that Cav1 function is not necessary for the early specification of CPN/FPN, or for projecting to their dual axonal targets. CPN subtype-specific expression of Cav1 identifies and characterizes a first molecular component that distinguishes this functionally unique projection neuron population, a population that expands in primates, and is prototypical of additional dual and higher-order projection neuron subtypes.
... It has been postulated that part of E2 neuroprotective actions triggered in ERα signalosomes against Aβ toxicity occur by the activation of different signal transduction pathways, including a voltage-dependent anion channel (VDAC) gating modulation. This channel appears associated with ERα in neuronal lipid rafts from a wide variety of brain regions, including septum, hippocampus, and cortex from, both, murine and human origins (Marin et al., 2007(Marin et al., , 2009Ramirez et al., 2009), where it participates in different pathogenesis including AD (Thinnes, 2015). In raft fractions of cortical neurons, VDAC interacting with Aβ promotes the channel dephosphorylation in tyrosine residues, a phenomenon that promotes VDAC gating, and enhances neuronal death (Fernandez-Echevarria et al., 2014). ...
... Previous studies demonstrated that estrogens also play an essential role in pubertal growth via the GH/IGF-I axis. 26 Indeed, estrogens stimulate GH secretion, which induces hepatic synthesis of IGF-I. 27 In our study, after puerarin administration, the level of IGF-1 increased. ...
Objective:
To investigate serum bone biomarkers in rats with intrauterine growth restriction (IUGR) in order to determine the effects of puerarin on bone metabolism.
Methods:
A rat model of IUGR was induced using a low protein diet during pregnancy. The offspring were given puerarin or an identical volume of saline via subcutaneous abdominal injection. All rats were studied at 1, 3, and 8 weeks of age. Serum biomarkers of bone formation, including insulin-like growth factor-1 (IGF-1), bone-specific alkaline phosphatase (BALP), osteocalcin (OC), osteoprotegerin (OPG), receptor-activator of nuclear factor-κB Iigand (RANKL), as well as blood levels of calcium and phosphorus were measured.
Results:
Serum BALP, OPG, IGF-1, and OC levels, as well as the OPG/RANKL ratio, were lower in the IUGR group compared with the control group at 1 week of age (P = 0.024, 0.011, 0.014, 0.004, and 0.002, respectively). At 3 weeks of age, the serum BALP and OC levels were higher in the protein-restricted group compared with the control group (P = 0.003 and 0.001, respectively). A comparison between the IUGR plus puerarin intervention group and the IUGR group revealed differences in the levels of BALP and IGF-1 at 3 weeks of age (P = 0.008 and 0.003, respectively). In addition, serum OPG and OC levels and the OPG/RANKL ratio were higher at 8 weeks of age (P = 0.044, 0.007, and 0.016, respectively). No differences in serum calcium and phosphorus levels were observed among the three groups.
Conclusion:
Our study demonstrates that the bone microenvironment of the fetus can be altered by a low protein maternal diet and that puerarin can reverse these effects. These results indicate that the nutritional environment plays an important role in early skeletal development and that the bone turnover of IUGR rats can be altered by puerarin treatment.
... IGF-1 receptor and ERs are co-localized in hippocampal neurons (Cardona-Gómez et al., 2000). ERα interacts with IGF-1 receptor through the p85 catalytic subunit of PI3K and the insulin receptor substrate (IRS)-1 in an estradiol-dependent process (Mendez et al., 2003;Marin et al., 2009). The interaction of ERα with the IGF-1 receptor signaling allows the synergic activation of PI3K/Akt pathway in the hippocampus and cerebral cortex, among other brain regions Cardona-Gómez et al. (2002) and the consequent inhibition of glycogen synthase kinase (GSK)-3β, resulting in the stabilization of β-catenin, allowing its translocation to the cell nucleus and its regulation of transcriptional activity (Cardona-Gomez et al., 2004;Mendez and Garcia-Segura, 2006;Wandosell et al., 2012) (Fig. 3). ...
Estradiol participates in the regulation of the function and plasticity of synaptic circuits in key cognitive brain regions, such as the prefrontal cortex and the hippocampus. The mechanisms elicited by estradiol are mediated by the regulation of transcriptional activity by nuclear estrogen receptors and by intracellular signaling cascades activated by estrogen receptors associated with the plasma membrane. In addition, the mechanisms include the interaction of estradiol with the signaling of other factors involved in the regulation of cognition, such a brain derived neurotrophic factor, insulin-like growth factor-1 and Wnt. Modifications in these signaling pathways by aging or by a long-lasting ovarian hormone deprivation after menopause may impair the enhancing effects of estradiol on synaptic plasticity and cognition.
Copyright © 2015. Published by Elsevier Inc.
... 4). Oestradiol induces the interaction of ERα with the p85 catalytic subunit of PI3K, probably in specific plasma membrane domains, such as the lipid rafts 116 . This allows the formation of a multimolecular complex composed of ERα, IGF1R and components of the IGF1R signalling pathway, such as insulin receptor substrate 1 (IRS1), PI3K, AKT and GSK3β 98,117 . ...
Hormones regulate homeostasis by communicating through the bloodstream to the body's 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.
... The neuroprotective actions of E2 in the brain also involve activation of intracellular signaling pathways via GPR30 (Gingerich et al., 2010;Liu, 2012). Although GPR30 might be primarily responsible for activation of intracellular signaling pathways, membraneassociated ER-α and ER-β can also mediate these effects (Bao, 2011;Kelly, 2009;Kuo et al., 2010;Marin, 2009). It has been shown that E2-induced activation of ERK pathway is required for E2 neuroprotection against glutamate-induced excitotoxicity in primary cortical neurons (Singer, 1999) and in hippocampal CA1 neurons during global ischemia (Jover-Mengual, 2007). ...
Extensive studies from the past decade have completely revolutionized our understanding about the role of astrocytes in the brain from merely supportive cells to an active role in various physiological functions including synaptic transmission via cross-talk with neurons and neuroprotection via releasing neurotrophic factors. Particularly, numerous studies have reported that astrocytes mediate the neuroprotective effects of 17β-estradiol (E2) and selective estrogen receptor modulators (SERMs) in various clinical and experimental models of neuronal injury. Astrocytes contain two main glutamate transporters, glutamate aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1), that play a key role in preventing excitotoxic neuronal death, a process associated with most neurodegenerative diseases. E2 has shown to increase expression of both GLAST and GLT-1 mRNA and protein and glutamate uptake in astrocytes. Growth factors such as transforming growth factor α (TGF-α) appear to mediate E2-induced enhancement of these transporters. These findings suggest that E2 exerts neuroprotection against excitotoxic neuronal injuries, at least in part, by enhancing astrocytic glutamate transporter levels and function. Therefore, the present review will discuss proposed mechanisms involved in astrocyte-mediated E2 neuroprotection, with a focus on glutamate transporters.
... E2 elicits either organizational effects during embryonic and neonatal development or the regulation of the gene expression in the brain during the later stages of life cycle (Garcia-Segura et al., 2001;Woolley, 2007;DonCarlos et al., 2009). Furthermore, different experimental models of neurodegeneration have shown that E2 acts as a neuroprotective factor promoting integrity of neuronal structure and/or function (Maggi et al., 2004;Suzuki et al., 2006;Marin et al., 2009;reviewed in Barha and Galea, 2010;Gillies and McArthur, 2010;Foster, 2011). E2 is proposed to mediate neuroprotection via a multimodal mechanism that involves: genomic and rapid (non-genomic) signaling, antioxidant actions, and regulation of mitochondrial bioenergetics (reviewed in Scott et al., 2012). ...
... The immune complexes were adsorbed and precipitated using protein A-Sepharose beads (Sigma Aldrich), washed three times with lysis buffer, and denatured by boiling for 5 min in sample buffer. Parallel immunoprecipitations were performed using pituitary cell lysate as the positive control to detect ER␣ with the known interactor IGF-I receptor, (40) using the total extract from the Hela ER-negative cell line as negative control. The membranes were blocked with 5% nonfat dry milk diluted in 0.1% Tween-20 Tris-buffered saline and incubated overnight with the primary antibodies to FGFR (1/200 -1 g/ml). ...
In the present work we investigated the effect of 17β-estradiol (E2) and basic fibroblast growth factor (FGF2) on the lactotroph cell proliferative response and the related membrane-initiated signalling pathway. Anterior pituitary mixed cell cultures of random cycling 3-month-old female rats were treated with 10nM of E2, E2 membrane-impermeable conjugated (E2-BSA), PPT (ERα agonist) and DPN (ERβ agonist) alone or combined with FGF2 (10ng/ml) for 30min or 4h. Although our results showed that the uptake of BrdU into the nucleus of lactotrophs was not modified by E2 or FGF2 alone, a significant increase in the lactotrophs uptake of BrdU was observed after E2/FGF2 co-incubation, with this effect being mimicked by PPT/FGF2. These proliferative effects were blocked by ICI182780 or PD98059. The involvement of membrane ER in the proliferative response of prolactin cells induced by the steroid and FGF2 co-incubation was confirmed using E2-BSA, and the association between ERα and FGF receptor was observed after E2/FGF2 treatment by immunoprecipitation. A significant increase in the ERK1/2 expression was noted after E2, E2-BSA, PPT and FGF2 alone, which was more noticeable after E2-BSA/FGF-2, E2/FGF2 or PPT/FGF2 treatments. This study provides evidence that E2 and FGF2 exert a cooperative effect on the lactotroph proliferation principally by signalling initiated at the plasma membrane triggering a genomic effect mediated by MEK/ERK1/2, a common signalling pathway, which finally regulates the lactotroph population thus contributing to pituitary plasticity.
... Increased data have reported the involvement of signalosomes in different neuroprotective activities against a variety of injuries ( Marin et al., 2009). Thus, present available evidences have demonstrated that two important receptors involved in neuronal preservation, growth and differentiation, estrogen receptor (ER) and insulin-growth factor-1 receptor (IGF-1R), interact in lipid rafts to promote neuroprotective events . ...
Lipid rafts are the preferential site of numerous membrane signaling proteins which are involved in neuronal functioning and survival. These proteins are organized in multiprotein complexes, or signalosomes, in close contact with lipid classes particularly represented in lipid rafts (i.e. cholesterol, sphingolipids and saturated fatty acids), which may contribute to physiological responses leading to neuroprotection. Increasing evidence indicates that alteration of lipid composition in raft structures as a consequence of neuropathologies, such as Alzheimer's disease (AD) and Parkinson's disease (PD), causes a dramatic increase in lipid raft order. These phenomena may correlate with perturbation of signalosome activities, likely contributing to neurodegenerative progression. Interestingly, significant disruption of stable raft microenvironments has been already observed in the first stages of either AD or PD, suggesting that these alterations may represent early events in the neuropathological development. In this regard, the search for biochemical markers, such as specific metabolic products altered in the brain at the first steps of the disease, presently represents an important challenge for early diagnosis strategies. Alterations of these biomarkers may be reflected in either plasma or cerebrospinal fluid, thus representing a potential strategy to predict an accurate diagnosis. We propose that pathologically-linked lipid raft markers may be interesting candidates to be explored at this level, although it has not been studied so far to what extent alteration of different signalosome components may be reflected in peripheral fluids. In this mini-review, we will discuss on relevant aspects of lipid rafts that contribute to the modulation of neuropathological events related to AD and PD. An interesting hypothesis is that anomalies on raft biomarkers measured at peripheral fluids might mirror the lipid raft pathology observed in early stages of AD and PD.
... In astrocytes, activation of ERa enhances mGlur1-dependent increases in intracellular calcium (Kuo et al., 2009). Furthermore, ERa in caveolin-rich regions of neuronal and glial cells interacts directly with the IGF-1 receptor and a voltagedependent anion channel (VDAC) present in cell membranes and in mitochondria (Marin et al., 2009). Thus far, these ER interactions with caveolin, glutamate receptors, and VDAC have been implicated in E2 neuroprotection against b-amyloid (Marin et al., 2012;Spampinato et al., 2012), but their role in cerebral ischemia has not been determined. ...
... These results suggest that the present neuroprotective effects of 17bE2 on spinal cord motoneurons might also involve the activation of the two subtypes of ER expressed by these cells. Although ERa is mainly localized in the nucleus of spinal cord motoneurons under basal conditions, this ER subtype might also contribute to Akt activation by the fact that in neurons and astrocytes exposure to 17bE2 is able to transiently increase membrane levels of ERa (Bondar et al. 2009; Dominguez and Micevych 2010); moreover, a small ERa population may still remain associated with the plasma membrane and be implicated in some membrane/cytoplasm-initiated signaling events (Marin et al. 2009). ERa has also been implicated in the ability of 17bE2 to induce embryonic stem cells to differentiate into motoneurons (Gonzalez et al. 2011); thus the possibility remains that, in addition to its neuroprotective effects, the estrogen is causing progenitors/neuroblasts in the explant to express motoneuron markers. ...
Amyotrophic lateral sclerosis (ALS) is an adult-onset degenerative disorder characterized by motoneuron death. Clinical and experimental studies in animal models of ALS have found gender differences in the incidence and onset of disease, suggesting that female hormones may play a beneficial role. Cumulative evidence indicates that 17β-estradiol (17βE2) has a neuroprotective role in the central nervous system. We have previously developed a new culture system by using rat spinal cord embryonic explants in which motoneurons have the singularity of migrating outside the spinal cord, growing as a monolayer in the presence of glial cells. In this study, we have validated this new culture system as a useful model for studying neuroprotection by estrogens on spinal cord motoneurons. We show for the first time that spinal cord motoneurons express classical estrogen receptors and that 17βE2 activates, specifically in these cells, the Akt anti-apoptotic signaling pathway and two of their downstream effectors: GSK-3β and Bcl-2. To further validate our system, we demonstrated neuroprotective effects of 17βE2 on spinal cord motoneurons when exposed to the proinflammatory cytokines TNF-α and IFN-γ. These effects of 17βE2 were fully reverted in the presence of the estrogen receptor antagonist ICI 182,780. Our new culture model and the results presented here may provide the basis for further studies on the effects of estrogens, and selective estrogen receptor modulators, on spinal cord motoneurons in the context of ALS or other motoneuron diseases.
... Immunohistochemical techniques identified ERα and ERβ associated with the membrane and in the membrane fraction [11,24,75]. While it has been suggested that membrane ERs (mERs) are attached to the inner leaflet of the cell membrane [143], surface biotinylation, a method of labeling membrane proteins, demonstrated that membrane-associated ERα has an exposed extracellular portion [13,39,49]. ...
Over the years, our ideas about estrogen signaling have greatly expanded. In addition to estradiol having direct nuclear actions that mediate transcription and translation, more recent experiments have demonstrated membrane-initiated signaling. Both direct nuclear and estradiol membrane signaling can be mediated by the classical estrogen receptors, ERα and ERβ, which are two of the numerous putative membrane estrogen receptors. Thus far, however, only ERα has been shown to play a prominent role in regulating female reproduction and sexual behavior. Because ERα is a ligand-gated transcription factor and not a typical membrane receptor, trafficking to the cell membrane requires post-translational modifications. Two necessary modifications are palmitoylation and association with caveolins, a family of scaffolding proteins. In addition to their role in trafficking, caveolin proteins also serve to determine ERα interactions with metabotropic glutamate receptors (mGluRs). It is through these complexes that ERα, which cannot by itself activate G proteins, is able to initiate intracellular signaling. Various combinations of ERα-mGluR interactions have been demonstrated throughout the nervous system from hippocampus to striatum to hypothalamus to dorsal root ganglion (DRG) in both neurons and astrocytes. These combinations of ER and mGluR allow estradiol to have both facilitative and inhibitory actions in neurons. In hypothalamic astrocytes, the estradiol-mediated release of intracellular calcium stores regulating neurosteroid synthesis requires ERα-mGluR1a interaction. In terms of estradiol regulation of female sexual receptivity, activation of ERα-mGluR1a signaling complex leads to the release of neurotransmitters and alteration of neuronal morphology. This review will examine estradiol membrane signaling (EMS) activating a limbic-hypothalamic lordosis regulating circuit, which involves ERα trafficking, internalization, and modifications of neuronal morphology in a circuit that underlies female sexual receptivity.
... As stated above, GPR30 is a transmembrane protein making it ideal for transducing an intracellular signaling cascade. However, because both ER ␣ and ER  can localize to the plasma membrane, they are not excluded as mediators of estrogen-induced rapid signaling [139][140][141][142][143][144][145][146][147] . Indeed ICI 182,780-sensitive ERs (ER ␣ and ER  ) are responsible for neuroprotective signaling initiated by a membrane impermeable form of estrogen [148] . ...
This review is intended to assess the state of current knowledge on the role of estrogen receptors (ERs) in the neuroprotective effects of estrogens in models for acute neuronal injury and death. We evaluate the overall evidence that estrogens are neuroprotective in acute injury and critically assess the role of ERα, ERβ, GPR 30, and nonreceptor-mediated mechanisms in these robust neuroprotective effects of this ovarian steroid hormone. We conclude that all three receptors, as well as nonreceptor-mediated mechanisms can be involved in neuroprotection, depending on the model used, the level of estrogen administrated, and the mode of administration of the steroid. Also, the signaling pathways used by both ER-dependent and ER-independent mechanisms to exert neuroprotection are considered. Finally, further studies that are needed to parse out the relative contribution of receptor versus nonreceptor-mediated signaling are discussed.
... E2 elicits either organizational effects during embryonic and neonatal development or the activational control of gene expression during the later stages of life cycle by regulating synaptic transmission, neuronal survival, neuronal, and glial differentiation (Garcia-Segura et al., 2001;Woolley, 2007;Don-Carlos et al., 2009). In addition, different experimental models of neurodegeneration have shown that E2 acts as a neuroprotective factor promoting neuronal survival and tissue integrity (Maggi et al., 2004;Suzuki et al., 2006;Marin et al., 2009). ...
Besides its crucial role in many physiological events, 17β-estradiol (E2) exerts protective effects in the central nervous system. The E2 effects are not restricted to the brain areas related with the control of reproductive function, but rather are widespread throughout the developing and the adult brain. E2 actions are mediated through estrogen receptors (i.e., ERα and ERβ) belonging to the nuclear receptor super-family. As members of the ligand-regulated transcription factor family, classically, the actions of ERs in the brain were thought to mediate only the E2 long-term transcriptional effects. However, a growing body of evidence highlighted rapid, membrane initiated E2 effects in the brain that are independent of ER transcriptional activities and are involved in E2-induced neuroprotection. The aim of this review is to focus on the rapid effects of E2 in the brain highlighting the specific role of the signaling pathway(s) of the ERβ subtype in the neuroprotective actions of E2.
... Given that juvenile play is a highly motivated behavior, dopamine antagonists consistently reduce juvenile play, as one would expect (Niesink and Van Ree, 1989;Siviy and Panksepp, 2011). Interestingly, dopamine and IGF1 both belong to a growing group of ligands capable of activating estrogen receptors in a steroid hormoneindependent manner (Mani et al., 2009;Marin et al., 2009). Given the sexually dimorphic nature of play, increased dopamine and IGF1 during juvenile play could potentially mediate sex-specific changes through activation of estrogen receptors, which have a masculinizing effect (see below). ...
This review summarizes what is presently known about the function, sexual differentiation, and neural circuitry of juvenile rough-and-tumble play. Juvenile rough-and-tumble play is a unique motivated behavior that is widespread throughout the mammalian order and usually occurs more often in males. Immediate early gene studies indicate that cortical and subcortical circuits, many of which are sensitive to sex steroid hormones, mediate juvenile play. Sex differences in rough-and-tumble play are controlled in part by neonatal exposure to androgens or their estrogenic metabolites. Studies indicate that testicular androgens during play are also necessary to stimulate male-like levels of play initiation. The resemblance of rough-and-tumble play to aggression and sexual behavior has led some to question whether male-typical adult behavior is contingent upon the experience of play. Attempts to control the amount of play through social isolation show that social experience during adolescence is critical for male-typical adult behaviors to be expressed. This well-established finding, together with evidence that play induces neural plasticity, supports the hypothesis that juvenile play contributes to male-typical brain development that ultimately enables the expression of adult social and reproductive behavior.
... In order to mediate their biological effects, estrogens interact with classical intracellular estrogen receptors (ER) a (Green et al., 1986) and b (Gustafsson, 1999), which function as ligand-dependent transcription factors. Although it is known that this hormone activates membrane-associated ER (Pietras and Szego, 1977;Bulayeva et al., 2004;Marin et al., 2009) there are several intriguing questions not elucidated about this topic, being a very active area of original researches and reviews (Warner and Gustafsson, 2006;Levin, 2009bLevin, , 2011. ...
In the present work we aimed at identifying ERα in the plasma membrane of normal anterior pituitary cells and investigated if 17β-estradiol was able to induce their subcellular redistribution. Our results show that about 8% of anterior pituitary cells expressed ERα in the plasma membrane, with the geometrical mean fluorescence intensity being increased after steroid hormone treatment. 17β-Estradiol and the selective ERα agonist PPT induced an increase of ERα expression in the plasma membrane and activated the PKCα/ERK 1/2 pathway in a time-course not compatible with genomic actions, thus supporting the notion of membrane-initiated effects. These findings suggest that 17β-estradiol stimulates the translocation of endogenous ERα to the plasma membrane, consequently modulating this ER pool and leading to cellular biological effects in normal anterior pituitary gland.
... The presence of the classical ␣ and  ERs and the signaling pathways involved in estrogen regulation of hormone secretion, cell proliferation, and cell death have been reported in pituitary gland (47). In addition to cytoplasmic/nuclear localization of ER, in several cell types there are also small pools of ER␣ and - at the plasma membrane (mER) (28,34) that can be activated and rapidly trigger multiple signal transduction cascades through direct interactions of ER with various proteins, including growth factor-dependent kinases and adaptor proteins (30,43). This multiprotein complex leads to the activation of many downstream signaling molecules, such as protein kinase C (PKC) (38), protein kinase A (3) , nitric oxide (NO) (24), mitogen-activated protein kinase (5), phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt) (12), and increased calcium levels in different tissues (6) . ...
Considering that estradiol is a major modulator of prolactin (PRL) secretion, the aim of the present study was to analyze the role of membrane estradiol receptor-α (mERα) in the regulatory effect of this hormone on the PRL secretion induced by thyrotropin-releasing hormone (TRH) by focusing on the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway activation. Anterior pituitary cell cultures from female rats were treated with 17β-estradiol (E(2), 10 nM) and its membrane-impermeable conjugated estradiol (E(2)-BSA, 10 nM) alone or coincubated with TRH (10 nM) for 30 min, with PRL levels being determined by RIA. Although E(2), E(2)-BSA, TRH, and E(2)/TRH differentially increased the PRL secretion, the highest levels were achieved with E(2)-BSA/TRH. ICI-182,780 did not modify the TRH-induced PRL release but significantly inhibited the PRL secretion promoted by E(2) or E(2)-BSA alone or in coincubation with TRH. The PI3K inhibitors LY-294002 and wortmannin partially inhibited the PRL release induced by E(2)-BSA, TRH, and E(2)/TRH and totally inhibited the PRL levels stimulated by E(2)-BSA/TRH, suggesting that the mER mediated the cooperative effect of E(2) on TRH-induced PRL release through the PI3K pathway. Also, the involvement of this kinase was supported by the translocation of its regulatory subunit p85α from the cytoplasm to the plasma membrane in the lactotroph cells treated with E(2)-BSA and TRH alone or in coincubation. A significant increase of phosphorylated Akt was induced by E(2)-BSA/TRH. Finally, the changes of ERα expression in the plasmalemma of pituitary cells were examined by confocal microscopy and flow cytometry, which revealed that the mobilization of intracellular ERα to the plasma membrane of lactotroph cells was only induced by E(2). These finding showed that E(2) may act as a modulator of the secretory response of lactotrophs induced by TRH through mER, with the contribution by PI3K/Akt pathway activation providing a new insight into the mechanisms underlying the nongenomic action of E(2) in the pituitary.
Estrogen exerts neuroprotective activity under different experimental conditions through classical nuclear receptors, but mainly receptors expressed at the cell surface. Transducing mechanisms activated by these membrane estrogen receptors in the brain have been intensely investigated and, among others, interaction with G-protein coupled, metabotropic glutamate (mGlu) receptors has been considered. Besides mediating physiological estrogen functions, such as regulation of hormone production or sexual behavior in the hypothalamus, mGlu receptors, specifically mGlu1 receptor subtype, take part to the protective effect of estrogen in a model of neuronal toxicity induced by β-amyloid peptide. Coupling of estrogen receptor to mGlu1 receptor is supported by co-immunoprecipitation, similar neuroprotective effect induced by either receptor activation, lack of additivity when the two receptors are activated at the same time and prevention of the protective effect when antagonists of the other receptor are used, i.e. reduction of the protective effect of estrogen by the mGlu1 receptor antagonist and vice versa. In addition, the phosphatidylinositol-3 kinase/Akt pathway may represent the common signaling pathway to produce neuroprotection. These data introduce a novel view of the mechanisms underlying the neuroprotective activity of estrogen and open new perspectives also for future pharmacological interventions.
Olfactory deficits are observed early in the course of chronic neurological disorders including Alzheimer׳s disease (AD). Estrogen treatment in post-menopausal women reduced the incidence of olfactory dysfunction, raising the possibility that estrogen treatment can cure olfactory deficits in preclinical stages of AD. In this study, we examined the estradiol׳s effects on neurite outgrowth in explant cultures of mouse olfactory epithelium (OE). We found that neurons in OE cultures treated with 100pM 17-β estradiol (estradiol) had significantly longer neurite outgrowth than cultures treated with ethanol alone (vehicle). The OE neurons expressed estrogen receptors alpha (ERα) and ER beta (ERβ). Estrogen treatment upregulated both ERα and ERβ expression in OE culture. Treatment of OE cultures with propyl pyrazole triol (PPT), a selective agonist for ERα increased neurite outgrowth to comparable extent as estradiol treatment. In contrast, 2,3-bis-4-hydroxyphenyl (DPN), a specific agonist for ERβ, had no effect on neurite outgrowth. Furthermore, estradiol treatment increased neurite outgrowth in OE cultures derived from ERβ-deficient/knockout mice and wild-type littermates, but not in ERα-deficient/knockout mice. These data suggest that ERα mediates the neurite outgrowth promoting effects of estradiol in OE cultures. We propose that olfactory dysfunction in chronic neurological disorders, where estrogen deficiency is a risk factor, is an indicator of compromised axonal regeneration of olfactory sensory neurons.
Estradiol-induced plasticity involves changes in dendritic spine density and in the relative proportions of the different dendritic spine types that influence neurons and neural circuits. Such events affect brain structures that control the timing of neuroendocrine and behavioral processes, influencing both reproductive and cognitive functions during the estrous cycle. Accordingly, to investigate the dendritic spine-related plastic changes that may affect the neural processes involved in mating, estradiol-mediated dendritic spine plasticity was studied in type II cells situated in the ventrolateral portion of the ventromedial hypothalamic nucleus (VMN) of female, adult rats. The rats were assigned to four different groups (n = 6) in function of their stage in the estrous cycle: proestrus, estrus, metaestrus, and diestrus. Dendritic spine density and the proportions of the different spine types on type II neurons were analyzed in the ventrolateral region of the VMN of these animals. Dendritic spine density on primary dendrites of VMN type II neurons was significantly lower in metaestrus than in diestrus, proestrus and estrus (with no differences between these latter stages). However, a significant variation in the proportional density of the different spine types was found, with a higher proportion of thin spines in diestrus, proestrus and estrus than in metaestrus. Likewise, a higher proportion of mushroom spines was seen in diestrus and proestrus than in metaestrus, and a higher proportion of stubby spines in estrus than in diestrus and metaestrus. Very few branched spines were found during proestrus and they were not detected during estrus or metaestrus.
It has become widely accepted that along with their ability to directly regulate gene expression, estrogens also influence cell signalling and cell function via rapid membrane-initiated events. Many of these signalling processes are dependent on estrogen receptors (ER) localized to the plasma membrane. However, the mechanisms by which ER are able to trigger cell signalling when targeted to the membrane surface have to be determined yet. Lipid rafts seem to be essential for the plasma membrane localization of ER and play a critical role in their membrane-initiated effects. In this review, we briefly recapitulate the localization and function of ER in different cell types and mostly discuss the possible role of lipid rafts in this context. Further studies in this field may disclose new promising therapeutic avenues by the disruption of lipid rafts in those diseases in which membrane ER activation has been demonstrated to play a pathogenetic role.
Voltage-dependent anion channel (VDAC) is a mitochondrial protein abundantly found in neuronal lipid rafts. In these membrane domains, VDAC is associated with a complex of signaling proteins that trigger neuroprotective responses. Loss of lipid raft integrity may result in disruption of multicomplex association and alteration of signaling responses that may ultimately promote VDAC activation. Some data have demonstrated that VDAC at the neuronal membrane may be involved in the mechanisms of amyloid beta (Aβ)-induced neurotoxicity, through yet unknown mechanisms. Aβ is generated from amyloid precursor protein (APP), and is released to the extracellular space where it may undergo self-aggregation. Aβ aggregate deposition in the form of senile plaques may lead to Alzheimer's disease (AD) neuropathology, although other pathological hallmarks (such as hyper-phosphorylated Tau deposition) also participate in this neurodegenerative process. The present study demonstrates that VDAC1 associates with APP and Aβ in lipid rafts of neurons. Interaction of VDAC1 with APP was observed in lipid rafts from the frontal and entorhinal cortex of human brains affected by AD at early stages (I-IV/0-B of Braak and Braak). Furthermore, Aβ exposure enhanced the dephosphorylation of VDAC1 that correlated with cell death. Both effects were reverted in the presence of tyrosine phosphatase inhibitors. VDAC1 dephosphorylation was corroborated in lipid rafts of AD brains. These results demonstrate that Aβ is involved in alterations of the phosphorylation state of VDAC in neuronal lipid rafts. Modulation of this channel may contribute to the development and progression of AD pathology.
Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by memory loss and disordered cognition. Women have a higher AD incidence than men, indicating that the declining estrogen levels during menopause may influence AD pathogenesis. However, the mechanism underlying estrogen's neuroprotective effect is not fully clarified and is complicated by the presence of several distinct estrogen receptor (ER) types and the identification of a growing number of ER splice variants. Thus, a deeper analysis of ERs could elucidate the role of estrogen in age-related cognitive changes. Intracellular calcium signaling cascades play a pivotal role in ERα neuroprotection against AD. The ERα-mediated inhibition of Death domain-associated protein (Daxx) translocation and the combination of membrane ERα and caveolin in caveolae may protect against AD. Moreover, the voltage-dependent anion channel (VDAC)/ERα association may be important for maintaining channel inactivation and may be relevant in neuronal preservation against Aβ injury. Additionally, ERα may prevent glutamate excitotoxic injury by Aβ through estrogen signaling mechanisms. ERα and IGF-IR co-activation may mediate neuroprotection, and many other growth factors and intracellular signaling responses triggered by ERα may also play important roles in this process. Furthermore, details regarding the genes and mRNA variants of ERα that are expressed in different parts of the human organs have been clarified recently. Therefore, here we review the literature to clarify the neuroprotective role of ERα. This review focuses on the potential mechanisms mediated by ERα in the intracellular signaling events in nervous system cells, thereby clarifying ERα-mediated protections against AD.
Parkinson's disease is a neurodegenerative disorder caused by the selective loss of dopaminergic (DA) neurons. In this study, we investigated the protective roles of glial cell line-derived neurotrophic factor (GDNF) and 17β-estradiol (E2) in the neuron cell line MN9D following treatment with 6-hydroxydopamine. This result showed that phosphorylation of protein kinase B (Akt) was significantly increased in treated MN9D cells following co-application of GDNF and E2 compared with only GDNF or E2. Moreover, GDNF enhanced the E2-induced translocation of estrogen receptor α (ERα) from the cytosol to the membrane. Immunoprecipitation experiments showed that the translocated ERα interacted with neural cadherin (N-cadherin) in the membrane. Site-directed mutagenesis of Tyr860 (Y860) in N-cadherin inhibited its interaction with ERα. Combined with the fact that GDNF can stimulate N-cadherin Y860 phosphorylation, we hypothesize that N-cadherin is a novel anchor for ERα, and phosphorylation at Y860 further increases ER's capacity to activate the neuroprotective phosphatidyl inositol-3 kinase/Akt pathway. This study provides evidence that co-application of GDNF and E2 exert important protective effects on DA neurons by increasing the interaction between ERα and N-cadherin.
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 β-catenin, in the cross-talk of estrogen receptors and IGF-I receptors in neural cells.
Numerous studies indicate that estrogens are crucial in normal brain functioning and preservation against different injuries. At the neuronal membrane, estrogens, binding to estrogen receptors (ERs) or other surface targets, exert rapid actions involving a plethora of signaling pathways that may converge in neuronal survival. Emerging work reveals that at least part of these actions may require the compartmentalization of ERs in signaling platforms, composed of macromolecular signaling proteins and particular lipid composition integrated in lipid rafts. These particular microstructures may provide the optimal microenvironment to trigger multiple ER interactions that may be crucial for neuroprotection against different brain impairments, such as Alzheimer's disease (AD). In this order of ideas, recent evidence has demonstrated that a membrane ER (mER) physically interacts with a voltage-dependent anion channel (VDAC) in lipid rafts from septal, hippocampal and cortical neurons, and these interactions may have important consequences in the alternative mechanisms developed by estrogens to achieve neuroprotection against amyloid beta (Aβ)-induced toxicity. This review includes a survey of some of the rapid mechanisms developed by estrogen to prevent neuronal death, and the ER interactions that are involved in the structural maintenance and signal transduction mechanisms important for neuronal survival against AD neuro-pathology. A special emphasis is put on the biological relevance of neuronal membrane VDAC in Aβ-related neurotoxicity, and the potential modulation of this channel as a part of a signaling complex with mER, which may be modified in AD brains.
Pretreatment with 10 nm 17β-estradiol (17βE2) or 100 μm of the metabotropic glutamate 1 receptor (mGlu1R) agonist, dihydroxyphenylglycine (DHPG), protected neurons against N-methyl-d-aspartate (NMDA) toxicity. This effect was sensitive to blockade of both estrogen receptors and mGlu1R by their respective antagonists. In contrast, 17βE2 and/or DHPG, added after a low-concentration NMDA pulse (45 μm), produced an opposite effect, i.e. an exacerbation of NMDA toxicity. Again this effect was prevented by both receptor antagonists. In support of an interaction of estrogen receptors and mGlu1R in mediating a neurotoxic response, exacerbation of NMDA toxicity by 17βE2 disappeared when cultures were treated with DHPG prior to NMDA challenge, and conversely, potentiation of NMDA-induced cell death by DHPG was prevented by pretreatment with 17βE2. Addition of calpain III inhibitor (10 μm), 2 h before NMDA, prevented the increased damage induced by the two agonists, an affect that can be secondary to cleavage of mGlu1R by calpain. Accordingly, NMDA stimulation reduced expression of the full-length (140 kDa) mGluR1, an effect partially reversed by calpain inhibitor. Finally, in the presence of NMDA, the ability of 17βE2 to stimulate phosphorylation of AKT and ERK was impaired. Pretreatment with calpain inhibitor prevented the reduction of phosphorylated ERK but had no significant effect on phosphorylated AKT. Accordingly, the inhibition of ERK signaling by U0126 (1 μm) counteracted the effect of calpain inhibition on 17βE2-induced exacerbation of NMDA toxicity. The present data confirm the dual role of estrogens in neurotoxicity/neuroprotection and highlight the role of the timing of exposure to estrogens.
Lipid Rafts: Keys to Signaling Platforms in NeuronsEstrogen Receptors Are Part of Signaling Platforms in Neuronal RaftsRole of Lipid Raft ERα–VDAC Interactions in Neuronal Preservation Against Aβ ToxicityDisruption of ERα–VDAC Complex in AD BrainsFuture StudiesReferences
We have previously shown that neurons in the basal forebrain colocalize the neurotrophin receptor p75NGFR and estrogen receptors. The present study was designed to examine (1) if neural neurotrophin targets respond to estrogen as a general phenotypic feature and (2) if NGF receptor mRNAs are regulated by estrogen, using a prototypical target of NGF, the dorsal root ganglion (DRG) (sensory) neuron. We demonstrate, for the first time, the presence of estrogen receptor mRNA and protein (binding sites) in adult female rat DRG. Moreover, estrogen receptor mRNA expression, while present in DRG neurons from both the ovariectomized (OVX; estrogen deficient) and intact female rat, was downregulated, as in the adult CNS, during proestrus (high estrogen levels) and in OVX animals replaced with proestrus levels of estrogen, as compared to OVX controls. In contrast, although the mRNAs for the NGF receptors p75NGFR and trkA were also expressed in DRG neurons from OVX and intact animals, expression of both NGF receptor mRNAs was upregulated in sensory neurons during proestrus, as compared to the OVX condition. Estrogen replacement, on the other hand, resulted in a transient downregulation of p75NGFR mRNA and a time-dependent upregulation of trkA mRNA. Estrogen regulation of NGF receptor mRNA in adult peripheral neural targets of the neurotrophins supports the hypothesis that estrogen may regulate neuronal sensitivity to neurotrophins such as NGF and may be an important mediator of neurotrophin actions in normal neural function and following neural trauma.
Until recently, steroid hormones were believed to act only on cells containing intracellular receptors. However, recent evidence suggests that steroids have specific and rapid effects at the cellular membrane. Using whole-cell patch-clamp techniques, 17 beta-estradiol was found to reduce Ba2+ entry reversibly via Ca2+ channels in acutely dissociated and cultured neostriatal neurons. The effects were sex-specific, i.e., the reduction of Ba2+ currents was greater in neurons taken from female rats. 17 beta-Estradiol primarily targeted L-type currents, and their inhibition was detected reliably within seconds of administration. The maximum reduction by 17 beta-estradiol occurred at picomolar concentrations. 17 beta-Estradiol conjugated to bovine serum albumin also reduced Ba2+ currents, suggesting that the effect occurs at the membrane surface. Dialysis with GTP gamma S prevented reversal of the modulation, suggesting that 17 beta-estradiol acts via G-protein activation. 17 alpha-Estradiol also reduced Ba2+ currents but was significantly less effective than 17 beta-estradiol. Estriol and 4-hydroxyestradiol were found to reduce Ba2+ currents with similar efficacy to 17 beta-estradiol, whereas estrone and 2-methoxyestriol were less effective. Tamoxifen also reduced Ba2+ currents but did not occlude the effect of 17 beta-estradiol. These results suggest that at physiological concentrations, 17 beta-estradiol can have immediate actions on neostriatal neurons via nongenomic signaling pathways.
Caveolae are plasma membrane invaginations where key signaling elements are concentrated. In this report, both biochemical and histochemical analyses demonstrate that the amyloid precursor protein (APP), a source of Abeta amyloid peptide, is enriched within caveolae. Caveolin-1, a principal component of caveolae, is physically associated with APP, and the cytoplasmic domain of APP directly participates in this binding. The characteristic C-terminal fragment that results from APP processing by alpha-secretase, an as yet unidentified enzyme that cleaves APP within the Abeta amyloid sequence, was also localized within these caveolae-enriched fractions. Further analysis by cell surface biotinylation revealed that this cleavage event occurs at the cell surface. Importantly, alpha-secretase processing was significantly promoted by recombinant overexpression of caveolin in intact cells, resulting in increased secretion of the soluble extracellular domain of APP. Conversely, caveolin depletion using antisense oligonucletotides prevented this cleavage event. Our current results indicate that caveolae and caveolins may play a pivotal role in the alpha-secretase-mediated proteolysis of APP in vivo.
The rodent and primate basal forebrain is a target of a family of endogenous peptide signaling molecules, the neurotrophins--nerve growth factor, brain-derived neurotrophic factor, and neurotrophin 3--and of the gonadal steroid hormone estrogen, both of which have been implicated in cholinergic function. To investigate whether or not these ligands may act on the same neurons in the developing and adult rodent basal forebrain, we combined autoradiography with 125I-labeled estrogen and either nonisotopic in situ hybridization histochemistry or immunohistochemistry. We now report colocalization of intranuclear estrogen binding sites with the mRNA and immunoreactive protein for the low-affinity nerve growth factor receptor, which binds all three neurotrophins, and for the cholinergic marker enzyme choline acetyltransferase (acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6). Colocalization of estrogen and low-affinity nerve growth factor receptors implies that their ligands may act on the same neuron, perhaps synergistically, to regulate the expression of specific genes or gene networks that may influence neuronal survival, differentiation, regeneration, and plasticity. That cholinergic neurons in brain regions subserving cognitive functions may be regulated not only by the neurotrophins but also by estrogen may have considerable relevance for the development and maintenance of neural substrates of cognition. If estrogen-neurotrophin interactions are important for survival of target neurons, then clinical conditions associated with estrogen deficiency could contribute to the atrophy or death of these neurons. These findings have implications for the subsequent decline in those differentiated neural functions associated with aging and Alzheimer disease.
Caveolae undergo a cyclic transition from a flat segment of membrane to a vesicle that then returns to the cell surface. Here we present evidence that this cycle depends on a population of protein kinase C-alpha (PKC-alpha) molecules that reside in the caveolae membrane where they phosphorylate a 90-kD protein. This cycle can be interrupted by treatment of the cells with phorbol-12,13-dibutyrate or agents that raise the concentration of diacylglycerol in the cell. Each of these conditions displaces PKC-alpha from caveolae, inhibits the phosphorylation of the 90-kD protein, and prevents internalization. Caveolae also contain a protein phosphatase that dephosphorylates the 90-kD once PKC-alpha is gone. A similar dissociation of PKC-alpha from caveolae and inhibition of invagination was observed when cells were treated with histamine. This effect was blocked by pyrilamine but not cimetidine, indicating the involvement of histamine H1 receptors. These findings suggest that the caveolae internalization cycle is hormonally regulated.
Until recently, steroid hormones were believed to act only on cells containing intracellular receptors. However, recent evidence suggests that steroids have specific and rapid effects at the cellular membrane. Using whole-cell patch-clamp techniques, 17 beta-estradiol was found to reduce Ba2+ entry reversibly via Ca2+ channels in acutely dissociated and cultured neostriatal neurons. The effects were sex-specific, i.e., the reduction of Ba2+ currents was greater in neurons taken from female rats. 17 beta-Estradiol primarily targeted L-type currents, and their inhibition was detected reliably within seconds of administration. The maximum reduction by 17 beta-estradiol occurred at picomolar concentrations. 17 beta-Estradiol conjugated to bovine serum albumin also reduced Ba2+ currents, suggesting that the effect occurs at the membrane surface. Dialysis with GTP gamma S prevented reversal of the modulation, suggesting that 17 beta-estradiol acts via G-protein activation. 17 alpha-Estradiol also reduced Ba2+ currents but was significantly less effective than 17 beta-estradiol. Estriol and 4-hydroxyestradiol were found to reduce Ba2+ currents with similar efficacy to 17 beta-estradiol, whereas estrone and 2-methoxyestriol were less effective. Tamoxifen also reduced Ba2+ currents but did not occlude the effect of 17 beta-estradiol. These results suggest that at physiological concentrations, 17 beta-estradiol can have immediate actions on neostriatal neurons via nongenomic signaling pathways.
Neurotrophic effects of estradiol and insulin-like growth factor-I were assessed in primary cultures from fetal rat hypothalamus. Cultured neurons were immunostained with an antibody for the microtubule-associated protein-2. While both estradiol and insulin-like growth factor-I increased the number of microtubule-associated protein-2-immunoreactive neurons and the extension of immunoreactive processes, the effect of these two factors was not additive. The estradiol-induced increases in neuronal numbers and extension of neuronal processes were blocked by either the estrogen receptor antagonist ICI 182,780 or by an anti-sense oligonucleotide to the estrogen receptor. Furthermore, incubation of the cultures with an anti-sense oligonucleotide directed against the insulin-like growth factor-I messenger RNA also blocked the effect of estradiol. In turn, the effects of insulin-like growth factor-I were blocked by the estrogen receptor antagonist ICI 182,780 and by the anti-sense oligonucleotide to the estrogen receptor. These findings suggest that estradiol-induced activation of the estrogen receptor in developing hypothalamic cells requires the presence of insulin-like growth factor-I, and that both estradiol and insulin-like growth factor-I use the estrogen receptor as a mediator of their trophic effects on hypothalamic neurons.
Caveolin, a 21-24-kDa integral membrane protein, is a principal component of caveolae membranes. We have suggested that caveolin functions as a scaffolding protein to organize and concentrate certain caveolin-interacting proteins within caveolae membranes. In this regard, caveolin co-purifies with a variety of lipid-modified signaling molecules, including G-proteins, Src-like kinases, Ha-Ras, and eNOS. Using several independent approaches, it has been shown that a 20-amino acid membrane proximal region of the cytosolic amino-terminal domain of caveolin is sufficient to mediate these interactions. For example, this domain interacts with G-protein alpha subunits and Src-like kinases and can functionally suppress their activity. This caveolinderived protein domain has been termed the caveolin-scaffolding domain. However, it remains unknown how the caveolin-scaffolding domain recognizes these molecules. Here, we have used the caveolin-scaffolding domain as a receptor to select random peptide ligands from phage display libraries. These caveolin-selected peptide ligands are rich in aromatic amino acids and have a characteristic spacing in many cases. A known caveolin-interacting protein, Gi2alpha, was used as a ligand to further investigate the nature of this interaction. Gi2alpha and other G-protein alpha subunits contain a single region that generally resembles the sequences derived from phage display. We show that this short peptide sequence derived from Gi2alpha interacts directly with the caveolin-scaffolding domain and competitively inhibits the interaction of the caveolin-scaffolding domain with the appropriate region of Gi2alpha. This interaction is strictly dependent on the presence of aromatic residues within the peptide ligand, as replacement of these residues with alanine or glycine prevents their interaction with the caveolin-scaffolding domain. In addition, we have used this interaction to define which residues within the caveolin-scaffolding domain are critical for recognizing these peptide and protein ligands. Also, we find that the scaffolding domains of caveolins 1 and 3 both recognize the same peptide ligands, whereas the corresponding domain within caveolin-2 fails to recognize these ligands under the same conditions. These results serve to further demonstrate the specificity of this interaction. The implications of our current findings are discussed regarding other caveolin- and caveolae-associated proteins.
Dendritic spines are of major importance in information processing and memory formation in central neurons. Estradiol has been shown to induce an increase of dendritic spine density on hippocampal neurons in vivo and in vitro. The neurotrophin brain-derived neurotrophic factor (BDNF) recently has been implicated in neuronal maturation, plasticity, and regulation of GABAergic interneurons. We now demonstrate that estradiol down-regulates BDNF in cultured hippocampal neurons to 40% of control values within 24 hr of exposure. This, in turn, decreases inhibition and increases excitatory tone in pyramidal neurons, leading to a 2-fold increase in dendritic spine density. Exogenous BDNF blocks the effects of estradiol on spine formation, and BDNF depletion with a selective antisense oligonucleotide mimics the effects of estradiol. Addition of BDNF antibodies also increases spine density, and diazepam, which facilitates GABAergic neurotransmission, blocks estradiol-induced spine formation. These observations demonstrate a functional link between estradiol, BDNF as a potent regulator of GABAergic interneurons, and activity-dependent formation of dendritic spines in hippocampal neurons.
Estrogen elicits a selective enhancement of the growth and differentiation of axons and dendrites (neurites) in the developing brain. Widespread colocalization of estrogen and neurotrophin receptors (trk) within estrogen and neurotrophin targets, including neurons of the cerebral cortex, sensory ganglia, and PC12 cells, has been shown to result in differential and reciprocal transcriptional regulation of these receptors by their ligands. In addition, estrogen and neurotrophin receptor coexpression leads to convergence or cross-coupling of their signaling pathways, particularly at the level of the mitogen-activated protein (MAP) kinase cascade. 17beta-Estradiol elicits rapid (within 5-15 min) and sustained (at least 2 h) tyrosine phosphorylation and activation of the MAP kinases, extracellular-signal regulated kinase (ERK)1, and ERK2, which is successfully inhibited by the MAP kinase/ERK kinase 1 inhibitor PD98059, but not by the estrogen receptor (ER) antagonist ICI 182,780 and also does not appear to result from estradiol-induced activation of trk. Furthermore, the ability of estradiol to phosphorylate ERK persists even in ER-alpha knockout mice, implicating other estrogen receptors such as ER-beta in these actions of estradiol. The existence of an estrogen receptor-containing, multimeric complex consisting of hsp90, src, and B-Raf also suggests a direct link between the estrogen receptor and the MAP kinase signaling cascade. Collectively, these novel findings, coupled with our growing understanding of additional signaling substrates utilized by estrogen, provide alternative mechanisms for estrogen action in the developing brain which could explain not only some of the very rapid effects of estrogen, but also the ability of estrogen and neurotrophins to regulate the same broad array of cytoskeletal and growth-associated genes involved in neurite growth and differentiation. This review expands the usually restrictive view of estrogen action in the brain beyond the confines of sexual differentiation and reproductive neuroendocrine function. It considers the much broader question of estrogen as a neural growth factor with important influences on the development, survival, plasticity, regeneration, and aging of the mammalian brain and supports the view that the estrogen receptor is not only a ligand-induced transcriptional enhancer but also a mediator of rapid, nongenomic events.
Nerve growth factor (NGF) binding to its receptors TrkA and p75NTR enhances the survival, differentiation, and maintenance of neurons. Recent studies have suggested that NGF receptor activation
may occur in caveolae or caveolae-like membranes (CLM). This is an intriguing possibility because caveolae have been shown
to contain many of the signaling intermediates in the TrkA signaling cascade. To examine the membrane localization of TrkA
and p75NTR, we isolated caveolae from 3T3-TrkA-p75 cells and CLM from PC12 cells. Immunoblot analysis showed that TrkA and p75NTR were enriched about 13- and 25-fold, respectively, in caveolae and CLM. Binding and cross-linking studies demonstrated that
the NGF binding to both TrkA and p75NTRwas considerably enriched in CLM and that about 90% of high affinity binding to TrkA was present in CLM. When PC12 cells were
treated with NGF, virtually all activated (i.e. tyrosine phosphorylated) TrkA was found in the CLM. Remarkably, in NGF-treated cells, it was only in CLM that activated TrkA
was coimmunoprecipitated with phosphorylated Shc and PLCγ. These results document a signaling role for TrkA in CLM and suggest
that both TrkA and p75NTRsignaling are initiated from these membranes.
Estrogen may mediate some of its effects on hippocampal function through the alpha isoform of the estrogen receptor (ERα). By light microscopy, ERα‐immunoreactivity (‐I) is found in the nuclei of scattered inhibitory γ‐aminobutyric acid (GABA)ergic interneurons. However, several lines of evidence indicate that estrogen also may exert some of its effects through rapid nongenomic mechanisms, possibly by binding to plasma membranes. Thus, to determine whether ERα is found in extranuclear sites in the hippocampal formation (HF), four different antibodies to ERα were localized by immunoelectron microscopy in proestrous rats. Ultrastructural analysis revealed that in addition to interneuronal nuclei, ERα‐I was affiliated with the cytoplasmic plasmalemma of select interneurons and with endosomes of a subset of principal (pyramidal and granule) cells. Moreover, ERα labeling was found in profiles dispersed throughout the HF, but slightly more numerous in CA1 stratum radiatum. Approximately 50% of the ERα‐labeled profiles were unmyelinated axons and axon terminals that contained numerous small, synaptic vesicles. ERα‐labeled terminals formed both asymmetric and symmetric synapses on dendritic shafts and spines, suggesting that ERαs arise from sources in addition to inhibitory interneurons. About 25% of the ERα‐I was found in dendritic spines, many originating from principal cells. Within spines, ERα‐I often was associated with spine apparati and/or polyribosomes, suggesting that estrogen might act locally through the ERα to influence calcium availability, protein translation, or synaptic growth. The remaining 25% of ERα‐labeled profiles were astrocytes, often located near the spines of principal cells. Collectively, these results suggest that ERα may serve as both a genomic and nongenomic transducer of estrogen action in the HF. J. Comp. Neurol. 429:355–371, 2001. © 2000 Wiley‐Liss, Inc.
The neuroblastoma cell line SK-ER3, which is stably transfected with the estrogen receptor (ER), was used to study the effect of insulin and insulin-like growth factors (IGF-I and IGF-II) on growth and morphological differentiation induced by estrogens. The data demonstrate that insulin and related growth factors control the growth and morphological differentiation of the cell line expressing the ER, but not of the parental cell line. Effects elicited by the growth factors in SK-ER3 cells can be blocked by ER antagonists. Transient transfection studies further confirm an effect of the IGFs in modulation of ER-activated promoters. The results presented support the hypothesis of the existence of cross-talk between membrane and intracellular receptors and provide evidence for physiological consequences of the activation of such a pathway of communication. The present study is of particular interest with regard to the theory of prenatal involvement of the ER in maturation of nerve cells. It could, in fact, be hypothesized that IGF-I and IGF-II, present in high concentrations in the developing brain, might activate the ER expressed in several embryonic brain nuclei.
Insulin is a well known mitotic agent for neuroblastoma cells. Human SK-N-BE neuroblastoma cells stably transfected with the estrogen receptor, however, undergo growth arrest and differentiation when treated with insulin. These effects were shown to be due to an insulin-dependent activation of the unliganded estrogen receptor. Here, we demonstrate that this activation involves the AF-2 COOH-terminal domain of the estrogen receptor and that the communication between estrogen and insulin receptor systems occurs via selected and specific transduction signals. In fact, by the use of dominant negative and dominant positive mutants we demonstrate that p21ras is essential for insulin and estrogen receptor coupling. With pharmacological tools, we prove that PI 3'kinase does not contribute to this cross-talk and that protein kinase C triggers transduction signals that act in synergism with p21ras. These results prove the intricacy of all these intracellular paths of communication. The finding that, in neuroblastoma cells, selected signal transduction systems are involved in the insulin-dependent activation of estrogen receptor is of particular interest considering that estrogen receptor might restrict the role played by insulin during the differentiation of neural cells and interfere with its proliferative potential while allowing its regulation of other functions related to cell survival.
Recently, we demonstrated that estradiol (E2) modulates cross-talk between protein tyrosine kinases and norepinephrine (NE) receptor signaling in the hypothalamus (HYP) and preoptic area (POA), brain areas that govern female reproductive function. We are now investigating the identity of protein tyrosine kinase(s) that modify NE receptor signaling in the HYP and POA. Incubation of POA and HYP slices with insulin-like growth factor I (IGF-I), which signals via a receptor (IGF-IR) with endogenous tyrosine kinase activity, enhances NE-stimulated cAMP accumulation only in tissue derived from ovariectomized, E2-primed animals. JB-1, an antagonist for IGF-IR, prevents the IGF-I enhancement of NE-stimulated cAMP accumulation in both POA and HYP slices. IGF-I enhances NE-stimulated cAMP accumulation via modulation of α1-adrenoceptor potentiation of adenylyl cyclase. Binding studies in membranes demonstrate that ovariectomized, E2-primed animals show a significant increase in the density of[ 125I]IGF-I-binding sit...
Caveolae undergo a cyclic transition from a flat segment of membrane to a vesicle that then returns to the cell surface. Here we present evidence that this cycle depends on a population of protein kinase C-alpha (PKC-alpha) molecules that reside in the caveolae membrane where they phosphorylate a 90-kD protein. This cycle can be interrupted by treatment of the cells with phorbol-12,13-dibutyrate or agents that raise the concentration of diacylglycerol in the cell. Each of these conditions displaces PKC-alpha from caveolae, inhibits the phosphorylation of the 90-kD protein, and prevents internalization. Caveolae also contain a protein phosphatase that dephosphorylates the 90-kD once PKC-alpha is gone. A similar dissociation of PKC-alpha from caveolae and inhibition of invagination was observed when cells were treated with histamine. This effect was blocked by pyrilamine but not cimetidine, indicating the involvement of histamine H1 receptors. These findings suggest that the caveolae internalization cycle is hormonally regulated.
Abstract Although oestrogen [17β-estradiol (E2)]-related neuroprotection has been demonstrated in different models, the involvement of non-classical oestrogen receptors (ERs) remains unexplored. Using the SN56 cholinergic cell line, we present evidence indicating that an ER associated with the plasma membrane participates in oestrogen-dependent inhibition of cell death induced by amyloid-β peptide (Aβ) toxicity. Similarly to E2 alone, a 15-min exposure to estradiol-horseradish peroxidase (E-HRP) significantly reduced Aβ-induced cell death. This effect was decreased by the ER antagonist ICI 182,780 as well as by MC-20 antibody directed to a region neighbouring the ligand-binding domain of ERα. Using confocal microscopy on unpermeabilized SN56 cells exposed to MC-20 antibody, we identified a protein at the plasma membrane level. Western blot analysis of purified SN56 cell membrane fractions using MC-20 antibody revealed the presence of one band with the same electrophoretic mobility as intracellular ERα. Using conjugated forms of the steroid, E-HRP and E2 conjugated to bovine serum albumin-FITC, we demonstrated by confocal microscopy that SN56 cells contain surface binding sites for E2. Binding of both conjugates was blocked by pre-incubation with E2 and decreased by either ICI 182,780 or MC-20 antibody in a concentration-dependent manner. Thus, a membrane-related ER that shares some structural homologies with ERα may participate in oestrogen-mediated neuroprotection.
Cyclic AMP response element-binding protein (CREB) is a transcription factor that has been implicated in the activation of a number of genes. We reported that CREB levels decline following a severe hypoglycemic episode in the hippocampus and cortex in the male rat brain. The present experiment was undertaken to investigate whether 17β-estradiol prevents the decline in CREB-immunoreactive cells following seizure in female rats. Rats were divided into four groups: ovariectomized (OVX), ovariectomized and insulin-treated (OVX-I), estrogen-replaced (E2), and estrogen-replaced and insulin-treated (E2-I). Generalized seizures were induced by injections with insulin (12.5 IU/kg, intraperitoneally) and animals were recovered by administration of glucose within 5 min of the occurrence of seizure. Control animals were injected with saline instead of insulin. All animals were perfused 90 min after recovery and the brains were processed for CREB immunoreactivity. CREB-positive neurons were counted using a computer-assisted program. Insulin treatment of OVX rats caused a significant decline in CREB-positive neurons in the CA1, CA3, and dentate gyrus compared to OVX rats. Estrogen treatment of OVX rats significantly increased CREB-positive neurons in the CA1 and dentate gyrus and attenuated the insulin-induced decline of CREB-positive neurons in all three regions compared to OVX rats. In conclusion, estrogens appear to induce CREB expression and attenuate its decline in the hippocampus following a severe hypoglycemic episode. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 961–967, 1997
Recently, we demonstrated that estradiol (E2) modulates cross-talk between protein tyrosine kinases and norepinephrine (NE) receptor signaling in the hypothalamus (HYP) and preoptic area (POA), brain areas that govern female reproductive function. We are now inves- tigating the identity of protein tyrosine kinase(s) that modify NE receptor signaling in the HYP and POA. Incubation of POA and HYP slices with insulin-like growth factor I (IGF-I), which signals via a receptor (IGF-IR) with endogenous tyrosine kinase activity, enhances NE-stimulated cAMP accumulation only in tissue derived from ovari- ectomized, E2-primed animals. JB-1, an antagonist for IGF-IR, pre- vents the IGF-I enhancement of NE-stimulated cAMP accumulation in both POA and HYP slices. IGF-I enhances NE-stimulated cAMP accumulation via modulation of a1-adrenoceptor potentiation of ad- enylyl cyclase. Binding studies in membranes demonstrate that ovari- ectomized, E2-primed animals show a significant increase in the den- sity of (125I)IGF-I-binding sites in both POA and HYP compared with ovariectomized control animals. Neither the IC50 for ( 125 I)IGF-I dis- placement by IGF-I nor the levels of IGF-I binding proteins in serum or brain tissue are affected by E2. RIA results showed that E2 does not modify serum or brain IGF-I levels. These results indicate that E2 regulation of NE receptor function in the POA and HYP involves increased expression of IGF-IR, and that after E2 treatment, IGF-IR activation augments a1-adrenoceptor signaling. (Endocrinology 142: 599 - 607, 2001)
At the beginning phase, the reconstitution of an anion-selective channel, from mitochondria, came as a surprise. The existence of large channels in the outer membrane, favoring anions, fits nicely with the major function of mitochondria, energy transduction. Substrates and products are mostly negatively charged molecules, such as pyruvate, adenosine diphosphate (ADP), adenosine triphosphate (ATP), phosphate, etc. The reconstitution into planar phospholipid membranes of large voltage gated channels, called VDAC (voltage-dependent anion-selective channel), indicated that these channels are not static structures but dynamic and under regulation. VDAC channels form extremely conductive pathways in phospholipid membranes. Such channels are the primary pathway for the flow of metabolites across the mitochondrial outer membrane. Their well-conserved properties include a variety of regulatory mechanisms that could restrict the flow of metabolites between the cytoplasm and the mitochondrion. Such a bottleneck could limit such things, as energy production and mitochondrial growth and reproduction. Thus, VDAC may play an important role in these and other processes. The importance of VDAC and its properties to mitochondrial function is strongly indicated by the remarkable conservation of its structure and functional properties. Discovery of new regulatory mechanisms and their remarkable conservation provides further evidence for an important and elaborate regulatory system.
Caveolin 1, a component of caveolae, regulates signalling pathways compartmentalization interacting with tyrosine kinase receptors and their substrates. The role of caveolin 1 in the Insulin Receptor (IR) signalling has been well investigated. On the contrary, the functional link between caveolin 1 and IGF-I Receptor (IGF-IR) remains largely unknown. Here we show that (1) IGF-IR colocalizes with caveolin 1 in the lipid rafts enriched fractions on plasmamembrane in R-IGF-IRWT cells, (2) IGF-I induces caveolin 1 phosphorylation at the level of tyrosine 14, (3) this effect is rapid and results in the translocation of caveolin 1 and in the formation of membrane patches on cell surface. These actions are IGF-I specific since we did not detect caveolin 1 redistribution in insulin stimulated R− cells overexpressing IRs.
• The regulation of Maxi Cl− channels by 17-oestradiol and non-steroidal triphenylethylene antioestrogens represents a rapid, non-classical effect of these compounds. In the present study we have investigated the signalling pathways used for the regulation of Maxi Cl− channel activity by oestrogens and antioestrogens in C1300 neuroblastoma cells. • Whole-cell Maxi Cl− currents were readily and reversibly activated by tamoxifen, toremifene and the membrane-impermeant ethyl-bromide tamoxifen, only when applied to the extracellular medium. • Pre-treatment of C1300 cells with oestrogen or cAMP prevented the antioestrogen-induced activation of Maxi Cl− channels. The inhibitory effect of 17-oestradiol and cAMP was abolished by the kinase inhibitor staurosporine. • Current activation was unaffected by the removal of intracellular Ca2+ and Mg2+, but was completely abolished in the presence of okadaic acid. These results are consistent with the participation of an okadaic acid-sensitive serine/threonine protein phosphatase in the activation of Maxi Cl− channels. However, neither oestrogen or antioestrogen treatment modified the total activity of the two major serine/threonine phosphatases, PP1 and PP2A, in C1300 cells. • Although the role of these Maxi Cl− channels remains unknown, our findings suggest strongly that their modulation by oestrogens and antioestrogens is linked to intracellular signalling pathways.
Caveolae have been implicated in the transcytosis of macromolecules across endothelial cells and in the receptor-mediated uptake of 5-methyltetrahydrofolate. Structural studies indicate that caveolae are decorated on their cytoplasmic surface by a unique array of filaments or strands that form striated coatings. To understand how these nonclathrin-coated pits function, we performed structural analysis of the striated coat and searched for the molecular component(s) of the coat material. The coat cannot be removed by washing with high salt; however, exposure of membranes to cholesterol-binding drugs caused invaginated caveolae to flatten and the striated coat to disassemble. Antibodies directed against a 22 kd substrate for v-src tyrosine kinase in virus-transformed chick embryo fibroblasts decorated the filaments, suggesting that this molecule is a component of the coat. We have named the molecule caveolin. Caveolae represent a third type of coated membrane specialization that is involved in molecular transport.
Recent work has shown that estrogen receptor mRNA and protein co-localize with neurotrophin receptor systems in the developing basal forebrain. In the present study we examined the potential for reciprocal regulation of estrogen and neurotrophin receptor systems by their ligands in a prototypical neurotrophin target, the PC12 cell. Using in situ hybridization histochemistry, RT-PCR and a modified nuclear exchange assay, we found both estrogen receptor mRNA and estrogen binding in PC12 cells. Moreover, while estrogen binding was relatively low in naive PC12 cells, long-term exposure to NGF enhanced estrogen binding in these cells by sixfold. Furthermore, concurrent exposure to estrogen and NGF differentially regulated the expression of the two NGF receptor mRNAs. The expression of trkA mRNA was up-regulated, while p75NGFR mRNA was down-regulated transiently. The present data indicate that NGF may increase neuronal sensitivity to estrogen, and that estrogen, by differentially regulating p75NGFR and trkA mRNA, may alter the ratio of the two NGF receptors, and, consequently, neurotrophin responsivity. In view of the widespread co-localization of estrogen and neurotrophin receptor systems in the developing CNS, the reciprocal regulation of these receptor systems by NGF and estrogen may have important implications for processes governing neural maturation and the maintainance of neural function.
We are interested in examining mechanisms underlying estrogen actions during neuronal differentiation in the central nervous system (CNS). Our research has focused on one possible mechanism, the developmental interactions between estrogen and the neurotrophins (nerve growth factor [NGF], brain derived neurotrophic factor [BDNF] and neurotrophin-3 [NT-3]). Using combined isotopic and non-isotopic in situ hybridization, we found that neurons in developmental estrogen targets (e.g., the cerebral cortex), co-localized mRNAs for the neurotrophins (NGF or BDNF) with their cognate receptors (p75NGFR [the pan-neurotrophin receptor] and trkA or trkB [the tyrosine kinase receptors]), suggesting a localization of neurotrophin-autocrine loops to these estrogen-sensitive neurons. In contrast, the basal forebrain, which is estrogen-sensitive in the adult and during development, only expressed neurotrophin receptor mRNAs, suggesting that this region was not an autocrine neurotrophin target. We examined the potential for developmental estrogen-neurotrophin interactions, using a model neurotrophin-sensitive system, i.e., differentiating PC12 cells. NGF significantly increased estrogen receptor density in PC12 cells. Reciprocally, estrogen up-regulated trkA mRNA and transiently down-regulated p75NGFR mRNA, suggesting that estrogen may increase the efficiency of NGF binding in PC12 cells. Similar estrogen-dependent regulation of NGF receptor mRNAs were also observed in the adult dorsal root ganglia, suggesting that estrogen may regulate NGF sensitivity in adult neurotrophin targets as well. Such estrogen-neurotrophin interactions may have an important role during differentiation and in the adult, following injury.
The neuroblastoma cell line SK-ER3, which is stably transfected with the estrogen receptor (ER), was used to study the effect of insulin and insulin-like growth factors (IGF-I and IGF-II) on growth and morphological differentiation induced by estrogens. The data demonstrate that insulin and related growth factors control the growth and morphological differentiation of the cell line expressing the ER, but not of the parental cell line. Effects elicited by the growth factors in SK-ER3 cells can be blocked by ER antagonists. Transient transfection studies further confirm an effect of the IGFs in modulation of ER-activated promoters. The results presented support the hypothesis of the existence of cross-talk between membrane and intracellular receptors and provide evidence for physiological consequences of the activation of such a pathway of communication. The present study is of particular interest with regard to the theory of prenatal involvement of the ER in maturation of nerve cells. It could, in fact, be hypothesized that IGF-I and IGF-II, present in high concentrations in the developing brain, might activate the ER expressed in several embryonic brain nuclei.
We have previously shown that neurons in the basal forebrain colocalize the neurotrophin receptor p75NGFR and estrogen receptors. The present study was designed to examine (1) if neural neurotrophin targets respond to estrogen as a general phenotypic feature and (2) if NGF receptor mRNAs are regulated by estrogen, using a prototypical target of NGF, the dorsal root ganglion (DRG) (sensory) neuron. We demonstrate, for the first time, the presence of estrogen receptor mRNA and protein (binding sites) in adult female rat DRG. Moreover, estrogen receptor mRNA expression, while present in DRG neurons from both the ovariectomized (OVX; estrogen deficient) and intact female rat, was downregulated, as in the adult CNS, during proestrus (high estrogen levels) and in OVX animals replaced with proestrus levels of estrogen, as compared to OVX controls. In contrast, although the mRNAs for the NGF receptors p75NGFR and trkA were also expressed in DRG neurons from OVX and intact animals, expression of both NGF receptor mRNAs was upregulated in sensory neurons during proestrus, as compared to the OVX condition. Estrogen replacement, on the other hand, resulted in a transient downregulation of p75NGFR mRNA and a time-dependent upregulation of trkA mRNA. Estrogen regulation of NGF receptor mRNA in adult peripheral neural targets of the neurotrophins supports the hypothesis that estrogen may regulate neuronal sensitivity to neurotrophins such as NGF and may be an important mediator of neurotrophin actions in normal neural function and following neural trauma.
Alterations in the amino acid sequence of the estrogen receptor (ER) have been shown to have dramatic effects on its function. Recently, mutant ERs have been isolated from both clinical samples and established breast cancer cell lines, primarily through the use of the polymerase chain reaction (PCR). All previously reported mutations have given rise to either alterations or truncations of the ER protein. We determined the structure of a novel 80 kDa ER which is expressed in an estrogen independent subclone of the MCF-7 human breast cancer cell line (MCF-7:2A). This 80 kDa ER was initially detected by Western blot analysis using a variety of ER specific antibodies. PCR mapping and partial PCR mediated subcloning of the ER cDNA were used to demonstrate that this protein was an ER containing an in-frame duplication of exons 6 and 7. This type of duplication has not been previously described for any members of the steroid receptor superfamily. Karyotype analysis coupled with fluorescence in situ hybridization (FISH) demonstrated that MCF-7:2A cells contained 4-5 copies of the ER gene in contrast to 2 copies in MCF-7:WS8 cells. The ER gene was localized by FISH analyses in both the MCF-7:WS8 and MCF-7:2A cells on chromosome 6, which is the source of the ER in normal human cells. The relative expression level of 2:1 is consistent with DNA gene dosage analysis. Genomic PCR was then used to demonstrate that the 80 kDa ER mRNA was not derived from the trans-splicing of two ER mRNAs but was the result of a genomic rearrangement in which exons 6 and 7 were duplicated in an in-frame fashion. This variant ER may prove to be useful in elucidating the mechanism of estrogen action in breast cancer cells.
Estrogen enhances the growth and differentiation of neurites within the developing forebrain. A critical issue is whether these developmental actions of estrogen are mediated directly or indirectly by means of autocrine responses or local paracrine mechanisms, through interactions with growth factors, such as the neurotrophins, and their receptors. Support for the latter hypothesis comes from our recent observations of co-expression of estrogen receptor mRNA with the mRNAs for the neurotrophins and their receptors; differential and reciprocal up-regulation of estrogen and NGF receptor mRNA and protein expression by estrogen in adult female rat sensory neurons, PC12 cells; and cerebral cortical cultures; and putative estrogen response elements in the NGF, BDNF, trkA and p75 genes. Estrogen and the neurotrophins may influence each other's actions by regulating receptor and ligand availability or by reciprocal regulation at the level of signal transduction or gene transcription. The neurotrophins may serve as regulatory "switches" for the apparent developmentally-regulated, differential pattern of estrogen receptor regulation by its ligand, whereby their ability to increase estrogen receptor levels significantly may be sufficient to override the intrinsic suppressive action of estrogen on its receptor. Estrogen and the neurotrophins, acting in concert and reciprocally, may stimulate the synthesis of proteins required for neuronal differentiation, survival and maintenance of function.
Estrogen treatment of ovariectomized rats rapidly increases immunoreactivity for the phosphorylated form of the cAMP response element binding protein (CREB)in neurons of the preoptic area and the bed nucleus of the stria terminalis. These effects were detected within 15 minutes after estrogen exposure. Since the antisera used for these studies detect CREB phosphorylation at ser133, which is important for transcriptional activation these data provide a possible explanation for estrogen's effects on neuronal genes lacking estrogen response elements (EREs) but which contain cAMP response elements (CREs). These data also provide evidence for non-genomic effects of steroid hormones involving protein kinase associated signal transduction pathways traditionally associated with effects at the cell membrane.
Insulin is a well known mitotic agent for neuroblastoma cells. Human SK-N-BE neuroblastoma cells stably transfected with the estrogen receptor, however, undergo growth arrest and differentiation when treated with insulin. These effects were shown to be due to an insulin-dependent activation of the unliganded estrogen receptor. Here, we demonstrate that this activation involves the AF-2 COOH-terminal domain of the estrogen receptor and that the communication between estrogen and insulin receptor systems occurs via selected and specific transduction signals. In fact, by the use of dominant negative and dominant positive mutants we demonstrate that p21ras is essential for insulin and estrogen receptor coupling. With pharmacological tools, we prove that PI 3'kinase does not contribute to this cross-talk and that protein kinase C triggers transduction signals that act in synergism with p21ras. These results prove the intricacy of all these intracellular paths of communication. The finding that, in neuroblastoma cells, selected signal transduction systems are involved in the insulin-dependent activation of estrogen receptor is of particular interest considering that estrogen receptor might restrict the role played by insulin during the differentiation of neural cells and interfere with its proliferative potential while allowing its regulation of other functions related to cell survival.
While evidence has accumulated in favor of cAMP-associated genomic involvement in long-term synaptic plasticity, the mechanisms downstream of the activated nucleus that underlie these changes in neuronal function remain mostly unknown. Dendritic spines, the locus of excitatory interaction among central neurons, are prime candidates for long-term synaptic modifications. We now present evidence that links phosphorylation of the cAMP response element binding protein (CREB) to formation of new spines; exposure to estradiol doubles the density of dendritic spines in cultured hippocampal neurons, and concomitantly causes a large increase in phosphorylated CREB and in CREB binding protein. Blockade of cAMP-regulated protein kinase A eliminates estradiol-evoked spine formation, as well as the CREB and CREB binding protein responses. A specific antisense oligonucleotide eliminates the phosphorylated CREB response to estradiol as well as the formation of new dendritic spines. These results indicate that CREB phosphorylation is a necessary step in the process leading to generation of new dendritic spines.
Insulin-like growth factor I (IGF-I) is an important modulator of cell growth and plasticity in the CNS. Expression of the IGF-I receptor mRNA in brain peaks at times of active cell development perinatally and remains detectable, albeit at lower levels, in the adult. While both autoradiographic and in situ hybridization studies show a wide and specific distribution of IGF-I receptor throughout the adult rat brain, nothing is yet known about its subcellular localization, a critical issue that will help clarify the biological role of this trophic factor in the adult brain. The present study describes the subcellular localization of IGF-I receptor immunoreactivity in the cerebellar cortex and the hypothalamic arcuate nucleus by using electron microscopic immunocytochemistry. In the cerebellum, IGF-I receptor immunoreactivity is present postsynaptically in the dendrites and soma of the Purkinje cell and presynaptically in axon terminals impinging upon the Purkinje cell soma, as well as in mossy fibre rosettes in the cerebellar glomeruli. Neurons in the mediobasal hypothalamus also contain IGF-I receptors located pre- and postsynaptically. Endothelial cells, astroglial end-feet surrounding micro vessels thoughout all the brain parenchyma, tanycytes of the third ventricle and oligodendrocytes in the cerebellar white matter are also rich in IGF-I receptors. These results strongly support previous observations that IGF-I is a neuromodulator in the adult brain, probably acting as both a pre- and a postsynaptic messenger. They also suggest that glial cells may be involved in the actions of IGF-I in the adult brain.
Gonadal hormones exert a critical influence over the architecture of specific brain areas affecting the formation of neuronal contacts. Cellular mechanisms mediating gonadal hormone actions on synapses have been studied extensively in the rat arcuate nucleus, a hypothalamic center involved in the feed-back regulation of gonadotropins. Gonadal steroids exert organizational and activational effects on arcuate nucleus synaptic connectivity. Perinatal testosterone induces a sexual dimorphic pattern of synaptic contacts. Furthermore, during the preovulatory and ovulatory phases of the estrous cycle there is a transient disconnection of inhibitory synaptic inputs to the somas of arcuate neurons. This synaptic remodeling is induced by estradiol, blocked by progesterone, and begins with the onset of puberty in females. Astroglia appear to play a significant role in the organizational and the activational hormone effects on neuronal connectivity by regulating the amount of neuronal membrane available for the formation of synaptic contacts and by releasing soluble factors, such as insulin-like growth factor I (IGF-I), which promote the differentiation of neural processes. Recent evidence indicates that gonadal steroids and IGF-I may interact in their trophic effects on the neuroendocrine hypothalamus. Estradiol and IGF-I promote the survival and morphological differentiation of rat hypothalamic neurons in primary cultures. The effect of estradiol depends on IGF-I, while the effects of both estradiol and IGF-I depend on estrogen receptors. Furthermore, estrogen activation of astroglia in hypothalamic tissue fragments depends on IGF-I receptors. These findings indicate that IGF-I may mediate some of the developmental and activational effects of gonadal steroids on the brain and suggest that IGF-I may activate the estrogen receptor to induce its neurotrophic effects on hypothalamic cells. In addition, IGF-I levels in the neuroendocrine hypothalamus are regulated by gonadal steroids. IGF-I levels in tanycytes, a specific astroglia cell type present in the arcuate nucleus and median eminence, increase at puberty, are affected by neonatal androgen levels, show sex differences, and fluctuate in accordance to the natural variations in plasma levels of ovarian steroids that are associated with the estrous cycle. These changes appear to be mediated by hormonal regulation of IGF-I uptake from blood or cerebrospinal fluid by tanycytes. These results suggest that tanycytes may be involved in the regulation of neuroendocrine events in adult rats by regulating the availability of IGF-I to hypothalamic neurons. In summary, IGF-I and different forms of neuron-astroglia communication are involved in the effects of estradiol on synaptic plasticity in the hypothalamic arcuate nucleus.
The neuroblastoma SK-ER3 cell line obtained by stable transfection of the human SK-N-BE cell line is proposed as a model for the study of estrogen receptor activity in cells of neural origin. In the SK-ER3 cell line the estrogen receptor, once activated, initiates a differentiation program leading to growth arrest, morphological changes, and acquisition of the dopaminergic phenotype. In the absence of estrogens, this program can be triggered by IGF-I, which can activate the unliganded estrogen receptor via the ras-pathway. It is proposed that this model system might recapitulate the events occurring in vivo during the differentiation of the nervous system and that IGF-I may play an important role in the activation of estrogen receptor at the very early stage of brain development affecting the differentiation of a number of hypothalamic and extrahypothalamic brain regions.
Cyclic AMP response element-binding protein (CREB) is a transcription factor that has been implicated in the activation of a number of genes. We reported that CREB levels decline following a severe hypoglycemic episode in the hippocampus and cortex in the male rat brain. The present experiment was undertaken to investigate whether 17beta-estradiol prevents the decline in CREB-immunoreactive cells following seizure in female rats. Rats were divided into four groups: ovariectomized (OVX), ovariectomized and insulin-treated (OVX-I), estrogen-replaced (E2), and estrogen-replaced and insulin-treated (E2-I). Generalized seizures were induced by injections with insulin (12.5 IU/kg, intraperitoneally) and animals were recovered by administration of glucose within 5 min of the occurrence of seizure. Control animals were injected with saline instead of insulin. All animals were perfused 90 min after recovery and the brains were processed for CREB immunoreactivity. CREB-positive neurons were counted using a computer-assisted program. Insulin treatment of OVX rats caused a significant decline in CREB-positive neurons in the CA1, CA3, and dentate gyrus compared to OVX rats. Estrogen treatment of OVX rats significantly increased CREB-positive neurons in the CA1 and dentate gyrus and attenuated the insulin-induced decline of CREB-positive neurons in all three regions compared to OVX rats. In conclusion, estrogens appear to induce CREB expression and attenuate its decline in the hippocampus following a severe hypoglycemic episode.
The alpha-estrogen receptor (ER alpha) transcriptional activity can be regulated either by binding to the cognate ligand or by intracellular signaling pathways responsive to a variety of factors acting through cell membrane receptors. Studies carried out in HeLa and COS-1 cells demonstrated that the cross-coupling between estrogen and growth factor receptors is mediated by p21ras and requires phosphorylation of a specific serine residue (Ser 118 in the human ER alpha and Ser 122 in mouse ER alpha) located in the ER alpha N-terminal activation function 1 (AF-1). Likewise, in the SK-N-BE neuroblastoma cell line p21ras is involved in the cross-coupling between insulin and ER alpha receptors. However, in this cell line Ser 122 is not necessary for insulin-dependent activation of unliganded ER alpha. In addition, after insulin activation, the electrophoretic mobility associated to serine hyperphosphorylation of ER alpha in SK-N-BE and in COS-1 cells is different. Our study rules out the possibility of tyrosine phosphorylation in unliganded ER alpha activation by means of transactivation studies of ER alpha tyrosine mutants and analysis of Tyr phosphorylation immunoreactivity. The two cofactors for steroid receptors RIP 140 and SRC-1 do not seem to be specifically involved in the insulin-induced ER alpha transactivation. The present study demonstrates the possibility of an alternative, cell-specific pathway of cross-coupling between intracellular and membrane receptors, which might be of importance for the understanding of the physiological significance of this mode of activation in the nervous system.
Evidence is emerging that oestrogen, besides acting via classical nuclear receptors, can rapidly influence the physiology of nerve cells through other mechanisms. Oestrogens have been shown to modulate the differentiation and function of embryonic midbrain dopaminergic neurones by stimulating neurite outgrowth, expression of tyrosine hydroxylase mRNA, dopamine uptake and release in spite of the fact that dopaminergic cells in the prenatal midbrain do not express the classical oestrogen receptor. This study therefore intended to unravel possible signal transduction pathways activated by oestrogen which might be associated with the above oestrogen effects. As a physiological second-messenger mechanism, we studied the influence of oestrogen on fluctuations of intracellular Ca2+ levels [Ca2+]i by microspectrofluorimetry of the Ca2+-sensitive indicator Fura-2, in primary cultures from embryonic mouse midbrains. 17Beta-estradiol (10 nM-1 pM) but not 17alpha-estradiol increased [Ca2+]i within 1-3 s in a dose-dependent way. Removal of extracellular Ca2+ abrogated K+-stimulated Ca2+ rise but did not affect 17beta-estradiol stimulation. Pretreatment of cells with thapsigargin (1 microM, 10 min), an inhibitor of Ca2+-pumping ATPases in the endoplasmic reticulum, abolished the 17beta-estradiol effect but not the K+-stimulated [Ca2+]i rise. Oestrogen effects on [Ca2+]i were completely mimicked by using a membrane-impermeant oestrogen-BSA construct. In order to identify oestrogen-sensitive cells, some cultures were subsequently immunostained for microtubule-associated protein II, tyrosine hydroxylase, or GABA. All oestrogen-sensitive cells were immunocytochemically characterized as neurones, and about half of these responsive neurones was found to be dopaminergic or GABAergic. These results demonstrate that 17beta-estradiol is capable of rapidly modulating physiological parameters of developing midbrain neurones by directly interacting with specific membrane binding sites coupled to a signal transduction mechanism that causes a calcium release from intracellular Ca2+ stores. It is suggested that oestrogen effects on differentiation and function of midbrain dopaminergic neurones are mediated by intracellular Ca2+ signalling.
The cell biology of caveolae is a rapidly growing area of biomedical research. Caveolae are known primarily for their ability to transport molecules across endothelial cells, but modern cellular techniques have dramatically extended our view of caveolae. They form a unique endocytic and exocytic compartment at the surface of most cells and are capable of importing molecules and delivering them to specific locations within the cell, exporting molecules to extracellular space, and compartmentalizing a variety of signaling activities. They are not simply an endocytic device with a peculiar membrane shape but constitute an entire membrane system with multiple functions essential for the cell. Specific diseases attack this system: Pathogens have been identified that use it as a means of gaining entrance to the cell. Trying to understand the full range of functions of caveolae challenges our basic instincts about the cell.
To investigate the physiological consequences of the increase in spine density induced by estradiol in pyramidal neurons of the hippocampus, we performed simultaneous whole cell recordings and Ca2+ imaging in CA1 neuron spines and dendrites in hippocampal slices. Four- to eight-days in vitro slice cultures were exposed to 17beta-estradiol (EST) for an additional 4- to 8-day period, and spine density was assessed by confocal microscopy of DiI-labeled CA1 pyramidal neurons. Spine density was doubled in both apical and basal dendrites of the CA1 region in EST-treated slices; consistently, a reduction in cell input resistance was observed in EST-treated CA1 neurons. Double immunofluorescence staining of presynaptic (synaptophysin) and postsynaptic (alpha-subunit of CaMKII) proteins showed an increase in synaptic density after EST treatment. The slopes of the input/output curves of both alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) postsynaptic currents were steeper in EST-treated CA1 neurons, consistent with the observed increase in synapse density. To characterize NMDA-dependent synaptic currents and dendritic Ca2+ transients during Schaffer collaterals stimulation, neurons were maintained at +40 mV in the presence of nimodipine, picrotoxin, and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). No differences in resting spine or dendritic Ca2+ levels were observed between control and EST-treated CA1 neurons. Intracellular Ca2+ transients during afferent stimulation exhibited a faster slope and reached higher levels in spines than in adjacent dendrites. Peak Ca2+ levels were larger in both spines and dendrites of EST-treated CA1 neurons. Ca2+ gradients between spine heads and dendrites during afferent stimulation were also larger in EST-treated neurons. Both spine and dendritic Ca2+ transients during afferent stimulation were reversibly blocked by D, L-2-amino-5-phosphonovaleric acid (D,L-APV). The increase in spine density and the enhanced NMDA-dependent Ca2+ signals in spines and dendrites induced by EST may underlie a threshold reduction for induction of NMDA-dependent synaptic plasticity in the hippocampus.
Estrogen modulates a variety of functions, most of which can be explained by the classical genomic mechanism of action. However, a number of estrogen's actions appear to be incompatible with this mechanism and fall into the category of nongenomic. In the hippocampus, application of 17beta-estradiol rapidly enhances the amplitude of kainate-induced currents of CA1 neurons. The potentiation resulted from a cyclic adenosine monophosphate-dependent phosphorylation process rather than a direct allosteric modulation of AMPA/kainate receptors. To initiate this potentiation, estrogen is required on both sides of the plasma membrane. Extracellularly, estrogen appears to activate a G-protein-coupled receptor, whereas the intracellular action of estrogen appears to be a modulation of the balance between phosphorylation and dephosphorylation. The binding sites responsible for the potentiation are genetically or pharmacologically distinct from both estrogen receptors alpha and beta. These findings provide support for the concept of a novel mechanism of action for estrogen.
Insulin-like growth factor-I (IGF-I) has trophic and plastic effects on neurons and glial cells and modulates neuroendocrine events by acting at the level of the hypothalamus. IGF-I and estrogen signaling interact to regulate in vitro hypothalamic neuronal survival and differentiation. In vivo, IGF-I levels fluctuate in the rat hypothalamic arcuate nucleus during the estrous cycle in parallel with a phasic remodeling of synaptic contacts and glial cell processes. Both the fluctuation of IGF-I levels and the synaptic and glial changes are induced by estrogen. The possible role of IGF-I in the regulation of arcuate nucleus synaptic plasticity has been assessed in the present study by intracerebroventricular administration to cycling female rats of a specific IGF-I receptor antagonist. In agreement with previous findings, the number of synaptic inputs to arcuate neuronal somas in control rats showed a significant decrease between the morning of proestrus and the morning of estrus. This decline in synaptic inputs and the accompanying increase in glial ensheathing of neuronal somas were blocked by the IGF-I receptor antagonist. In contrast, the IGF-I receptor antagonist did not affect the basal number of synapses or the morphology of synaptic terminals or length of the synaptic contacts. These findings indicate that IGF-I receptor activation may be involved in the phasic remodeling of arcuate nucleus synapses during the estrous cycle. Res.
During transduction of an apoptotic (death) signal into the cell, there is an alteration in the permeability of the membranes of the cell's mitochondria, which causes the translocation of the apoptogenic protein cytochrome c into the cytoplasm, which in turn activates death-driving proteolytic proteins known as caspases. The Bcl-2 family of proteins, whose members may be anti-apoptotic or pro-apoptotic, regulates cell death by controlling this mitochondrial membrane permeability during apoptosis, but how that is achieved is unclear. Here we create liposomes that carry the mitochondrial porin channel (also called the voltage-dependent anion channel, or VDAC) to show that the recombinant pro-apoptotic proteins Bax and Bak accelerate the opening of VDAC, whereas the anti-apoptotic protein Bcl-x(L) closes VDAC by binding to it directly. Bax and Bak allow cytochrome c to pass through VDAC out of liposomes, but passage is prevented by Bcl-x(L). In agreement with this, VDAC1-deficient mitochondria from a mutant yeast did not exhibit a Bax/Bak-induced loss in membrane potential and cytochrome c release, both of which were inhibited by Bcl-x(L). Our results indicate that the Bcl-2 family of proteins bind to the VDAC in order to regulate the mitochondrial membrane potential and the release of cytochrome c during apoptosis.
We have previously shown that 17-beta-estradiol protects neurons in the dentate gyrus from kainic acid-induced death in vivo. To analyse whether this effect is mediated through estrogen receptors and through cross-talk between steroid and insulin-like growth factor (IGF) systems, we have concomitantly administered antagonists of estrogen receptor (ICI 182,780) or the IGF-I receptor (JB1) with estradiol. In addition, we have also administered IGF-I with or without the estrogen receptor antagonist. JB1 (20 microg/ml), ICI 182,780 (10(-7) M), and IGF-I (100 microg/ml) were delivered into the left lateral ventricle of young ovariectomized rats via an Alzet osmotic minipump (0.5 microl/hr) for 2 weeks. All rats received kainic acid (7 mg/Kg b.w.) or vehicle i.p. injections at day 7 after minipump implant. Also on day 7, rats treated i.c. v.with only ICI 182,780 or JB1 received a single i.p. injection of 17-beta-estradiol (150 microg/rat) or vehicle. On day 14 after minipump implant, the rats were killed, brains processed, and the number of surviving hilar neurons estimated by the optical disector technique. Both IGF-I and estradiol treatments resulted in over 90% survival of hilar neurons. The neuroprotective action of estradiol was blocked by ICI 182,780 and by JB1. Furthermore, IGF-I enhancement of neuronal survival was significantly reduced by ICI 182,780. These results indicate that in this model of hippocampal lesion, the neuroprotective effect of estradiol depends both on estrogen receptors and IGF-I receptors, while the protection exerted by IGF-I depends also on estrogen receptors. In conclusion, an interaction of estrogen receptor and IGF-I receptor signalling may mediate neuroprotection in the adult rat hippocampus.