María Ángeles Arévalo

Selective estrogen receptor modulators decrease the production of interleukin‐6 and interferon‐γ‐inducible protein‐10 by astrocytes exposed to inflammatory challenge in vitro

Expression of proinflammatory molecules by glial cells is involved in the pathophysiological changes associated with chronic neurological diseases. Under pathological conditions, astrocytes release a number of proinflammatory molecules, such as interleukin‐6 (IL‐6) and interferon‐γ‐inducible protein‐10 (IP‐10). The ovarian hormone estradiol exerts protective effects in the central nervous system that, at least in part, may be mediated by a reduction of local inflammation. This study was designed to assess whether estradiol affects the production of IL‐6 and IP‐10 by primary cultures of newborn mice astrocytes exposed to lipopolysaccharide (LPS), a bacterial endotoxin known to cause neuroinflammation. In addition, the possible anti‐inflammatory effect of several selective estrogen receptor modulators (SERMs) was also assessed. LPS induced an increase in the expression of IL‐6 and IP‐10 mRNA levels in astrocytes and an increase in IL‐6 and IP‐10 protein levels in the culture medium. These effects of LPS were impaired by estradiol and by the four SERMs tested in our study: tamoxifen, raloxifene, ospemifene, and bazedoxifene. All SERMs tested showed a similar effect on IL‐6 and IP‐10 mRNA levels, but raloxifene and ospemifene were more effective than tamoxifen and bazedoxifene in reducing protein levels in LPS‐treated cultures. Finally, we report that news SERMs, ospemifene and bazedoxifene, exert anti‐inflammatory actions by a mechanism involving classical estrogen receptors and by the inhibition of LPS‐induced NFκB p65 transactivation. The results suggest that estrogenic compounds may be candidates to counteract brain inflammation under neurodegenerative conditions by targeting the production and release of proinflammatory molecules by astrocytes.

Selective Oestrogen Receptor Modulators Decrease the Inflammatory Response of Glial Cells

Neuroinflammation comprises a feature of many neurological disorders that is accompanied by the activation of glial cells and the release of pro‐inflammatory cytokines and chemokines. Such activation is a normal response oriented to protect neural tissue and it is mainly regulated by microglia and astroglia. However, excessive and chronic activation of glia may lead to neurotoxicity and may be harmful for neural tissue. The ovarian hormone oestradiol exerts protective actions in the central nervous system that, at least in part, are mediated by a reduction of reactive gliosis. Several selective oestrogen receptor modulators may also exert neuroprotective effects by controlling glial inflammatory responses. Thus, tamoxifen and raloxifene decrease the inflammatory response caused by lipopolysaccharide, a bacterial endotoxin, in mouse and rat microglia cells in vitro. Tamoxifen and raloxifene are also able to reduce microglia activation in the brain of male and female rats in vivo after the peripheral administration of lipopolysaccharide. In addition, tamoxifen decreases the microglia inflammatory response induced by irradiation. Furthermore, treatment with tamoxifen and raloxifene resulted in a significant reduction of the number of reactive astrocytes in the hippocampus of young, middle‐aged and older female rats after a stab wound injury. Tamoxifen, raloxifene and the new selective oestrogen receptor modulators ospemifene and bazedoxifene decrease the expression and release of interleukine‐6 and interferon‐γ inducible protein‐10 in cultured astrocytes exposed to lipopolysaccharide. Ospemifene and bazedoxifene exert anti‐inflammatory effects in astrocytes by a mechanism involving classical oestrogen receptors and the inhibition of nuclear factor‐kappa B p65 transactivation. These data suggest that oestrogenic compounds are candidates to counteract brain inflammation under neurodegenerative conditions by targeting the production and release of pro‐inflammatory molecules by glial cells.

Selective estrogen receptor modulators as brain therapeutic agents

Selective estrogen receptor modulators (SERMs), used for the treatment of breast cancer, osteoporosis, and menopausal symptoms, affect the nervous system. Some SERMs trigger neuroprotective mechanisms and reduce neural damage in different experimental models of neural trauma, brain inflammation, neurodegenerative diseases, cognitive impairment, and affective disorders. New SERMs with specific actions on neurons and glial cells may represent promising therapeutic tools for the brain.

17β-Oestradiol Anti-Inflammatory Effects in Primary Astrocytes Require Oestrogen Receptor β-Mediated Neuroglobin Up-Regulation

Neuroglobin (Ngb), so named after its initial discovery in brain neurones, has received great attention as a result of its neuroprotective effects both in vitro and in vivo. Recently, we demonstrated that, in neurones, Ngb is a 17β‐oestradiol (E2) inducible protein that is pivotal for hormone‐induced anti‐apoptotic effects against H2O2 toxicity. The involvement of Ngb in other brain cell populations, as well as in other neuroprotective effects of E2, is completely unknown at present. We demonstrate Ngb immunoreactivity in reactive astrocytes located in the proximity of a penetrating cortical injury in vivo and the involvement of Ngb in the E2‐mediated anti‐inflammatory effect in primary cortical astrocytes. Upon binding to oestrogen receptor (ER)β, E2 enhances Ngb levels in a dose‐dependent manner. Although with a lesser degree than E2, the pro‐inflammatory stimulation with lipopolysaccharide (LPS) also induces the increase of Ngb protein levels via nuclear factor‐(NF)κB signal(s). Moreover, a negative cross‐talk between ER subtypes and NFκB signal(s) has been demonstrated. In particular, ERα‐activated signals prevent the NFκB‐mediated Ngb increase, whereas LPS impairs the ERβ‐induced up‐regulation of Ngb. Therefore, the co‐expression of both ERα and ERβ is pivotal for mediating E2‐induced Ngb expression in the presence of NFκB‐activated signals. Interestingly, Ngb silencing prevents the effect of E2 on the expression of inflammatory markers (i.e. interleukin 6 and interferon γ‐inducible protein 10). Ngb can be regarded as a key mediator of the different protective effects of E2 in the brain, including protection against oxidative stress and the control of inflammation, both of which are at the root of several neurodegenerative diseases.

Role of estrogen receptor α in membrane-initiated signaling in neural cells: Interaction with IGF-1 receptor

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.

17β-Estradiol – A New Modulator of Neuroglobin Levels in Neurons: Role in Neuroprotection against H2O2-Induced Toxicity

Although discovered in 2000, neuroglobin (Ngb) functions are still uncertain. A contribution to the role played by Ngb in neurons could certainly derive from the identification of Ngb endogenous modulators. Here, we evaluate the possibility that Ngb could be regulated by 17β-estradiol (E2) signaling in both SK-N-BE human neuroblastoma cell line and mouse hippocampal neurons. 1 nM E2 rapidly induced a 300% increase in Ngb levels in both models. The E2 effect was specific, being not induced by testosterone or dihydrotestosterone. The E2-induced Ngb increase requires estrogen receptor (ER) β, but not ERα, as evaluated by the mimetic effect of ERβ-specific agonist DPN and by the blockage of E2 effect in ERβ-silenced SK-N-BE cells. Furthermore, both rapid (15 min) ERβ-dependent activation of p38/MAPK and transcriptional ERβ activity were required for the estrogenic regulation of Ngb. Finally, E2 exerted a protective effect against H2O2-induced neuroblastoma cell death which was completely prevented in Ngb-silenced cells. Overall, these data suggest that Ngb is part of the E2 signaling mechanism that is activated to exert protective effects against H2O2-induced neurotoxicity.

Role of astroglia in the neuroplastic and neuroprotective actions of estradiol

Astrocyte–neuron cross‐talk is an essential component of the mechanisms involved in the neuroendocrine and neuroprotective actions of estradiol. Astrocytes express estrogen receptors, show morphological and functional modifications in response to estradiol and participate in the hormonal regulation of synaptic plasticity and neuroendocrine events. In addition, estradiol interferes with the activation of astrocytes under pathological conditions, modulating the release of neurotrophic factors and inflammatory molecules by these cells. Furthermore, under neurodegenerative conditions, astrocytes synthesize estradiol, which acts as a local neuroprotectant. The actions of estradiol on astrocytes can be imitated by selective estrogen receptor modulators. Some of these molecules, which are free of the peripheral risks associated with estrogen therapy, exert estradiol‐like anti‐inflammatory actions on astrocytes and are potential therapeutic candidates for the control of reactive astrogliosis.

Role of astrocytes in the neuroprotective actions of 17β-estradiol and selective estrogen receptor modulators.

Neuroprotective actions of 17β-estradiol (estradiol) are in part mediated by direct actions on neurons. Astrocytes, which play an essential role in the maintenance of the homeostasis of neural tissue, express estrogen receptors and are also involved in the neuroprotective actions of estradiol in the brain. Estradiol controls gliosis and regulates neuroinflammation, edema and glutamate transport acting on astrocytes. In addition, the hormone regulates the release of neurotrophic factors and other neuroprotective molecules by astrocytes. In addition, reactive astrocytes are a local source of neuroprotective estradiol for the injured brain. Since estradiol therapy is not free from peripheral risks, alternatives for the hormone have been explored. Some selective estrogen receptor modulators (SERMs), which are already in use in clinical practice for the treatment of breast cancer, osteoporosis or menopausal symptoms, exert similar actions to estradiol on astrocytes. Therefore, SERMs represent therapeutic alternatives to estradiol for the activation of astroglia-mediated neuroprotective mechanisms.

Gonadal hormones and the control of reactive gliosis

Astrocytes and microglia respond to central nervous system (CNS) injury with changes in morphology, proliferation, migration and expression of inflammatory regulators. This phenomenon is known as reactive gliosis. Activation of astrocytes and microglia after acute neural insults, such as stroke or traumatic CNS injury, is considered to be an adaptive response that contributes to minimize neuronal damage. However, reactive gliosis may amplify CNS damage under chronic neurodegenerative conditions. Progesterone, estradiol and testosterone have been shown to control reactive gliosis in different models of CNS injury, modifying the number of reactive astrocytes and reactive microglia and the expression of anti-inflammatory and proinflammatory mediators. The actions of gonadal hormones on reactive gliosis involve different mechanisms, including the modulation of the activity of steroid receptors, such as estrogen receptors α and β, the regulation of nuclear factor-κB mediated transcription of inflammatory molecules and the recruitment of the transcriptional corepressor c-terminal binding protein to proinflammatory promoters. In addition, the Parkinsons disease related gene parkin and the endocannabinoid system also participate in the regulation of reactive gliosis by estradiol. The control exerted by gonadal hormones on reactive gliosis may affect the response of neural tissue to trauma and neurodegeneration and may contribute to sex differences in the manifestation of neurodegenerative diseases. However, the precise functional consequences of the regulation of reactive gliosis by gonadal hormones under acute and chronic neurodegenerative conditions are still not fully clarified.

In vitro myelination by oligodendrocyte precursor cells transfected with the neurotrophin-3 gene

Oligodendrocyte precursor cells require exogenous neurotrophin‐3 (NT‐3) for differentiation into oligodendrocytes. We transfected precursor cells with the gene for NT‐3 and looked for changes in their development into myelin‐forming cells. The expression of NT‐3 in transfected cells was demonstrated by reverse transcription followed by PCR as well as by Northern blots. Direct synthesis of the neurotrophin product and its release to the culture supernatants were also shown by specific ELISA. Transfection converts precursor cells into actively dividing cells that can incorporate 3H‐thymidine into DNA. In the absence of growth factors, a parallel increase in the survival of the transfected cultures was also demonstrated by the MTT test. The final demonstration of biological changes in transfected versus untreated cells was a 10‐fold increase in myelin basic protein production observed in Western blots and the direct observation by phase‐contrast and electron microscopy of myelin membranes in cocultures with hippocampal neurons. We discuss the future use of this transfected cells in regeneration and functional recovery in experimental models of multiple sclerosis.