Simon McArthur

Estrogen Actions in the Brain and the Basis for Differential Action in Men and Women: A Case for Sex-Specific Medicines

The classic view of estrogen actions in the brain was confined to regulation of ovulation and reproductive behavior in the female of all mamamalian species studied, including humans. Burgeoning evidence now documents profound effects of estrogens on learning, memory, and mood as well as neurodevelopmental and neurodegenerative processes. Most data derive from studies in females, but there is mounting recognition that estrogens play important roles in the male brain, where they can be generated from circulating testosterone by local aromatase enzymes or synthesized de novo by neurons and glia. Estrogen-based therapy therefore holds considerable promise for brain disorders that affect both men and women. However, as investigations are beginning to consider the role of estrogens in the male brain more carefully, it emerges that they have different, even opposite, effects as well as similar effects in male and female brains. This review focuses on these differences, including sex dimorphisms in the ability of estradiol to influence synaptic plasticity, neurotransmission, neurodegeneration, and cognition, which, we argue, are due in a large part to sex differences in the organization of the underlying circuitry. There are notable sex differences in the incidence and manifestations of virtually all central nervous system disorders, including neurodegenerative disease (Parkinsons and Alzheimers), drug abuse, anxiety, and depression. Understanding the cellular and molecular basis of sex differences in brain physiology and responses to estrogen and estrogen mimics is, therefore, vitally important for understanding the nature and origins of sex-specific pathological conditions and for designing novel hormone-based therapeutic agents that will have optimal effectiveness in men or women.

DOSE- AND SEX-DEPENDENT EFFECTS OF THE NEUROTOXIN 6-HYDROXYDOPAMINE ON THE NIGROSTRIATAL DOPAMINERGIC PATHWAY OF ADULT RATS: DIFFERENTIAL ACTIONS OF ESTROGEN IN MALES AND FEMALES

Epidemiological and clinical studies provide growing evidence for marked sex differences in the incidence of certain neurological disorders that are largely attributed to the neuroprotective effects of estrogen. Thus there is a keen interest in the clinical potential of estrogen-related compounds to act as novel therapeutic agents in conditions of neuronal injury and neurodegeneration such as Parkinsons disease. Studies employing animal models of neurodegeneration in ovariectomised female rats treated with estrogen support this hypothesis, yet experimental evidence for sex differences in the CNS response to direct neurotoxic insult is limited and, as yet, few studies have addressed the role played by endogenously produced hormones in neuroprotection. Therefore, in this study we aimed to determine (1) whether the prevailing levels of sex steroid hormones in the intact rat provide a degree of protection against neuronal assault in females compared with males and (2) whether sex differences depend solely on male/female differences in circulating estrogen levels or whether androgens could also play a role. Using the selective, centrally administered neurotoxin 6-hydroxydopamine, which induces a lesion in the nigrostriatal dopaminergic pathway similar to that seen in Parkinsons disease, we have demonstrated a sexually dimorphic (male-dominant), dose-dependent susceptibility in rats. Furthermore, following gonadectomy, dopamine depletion resulting from a submaximal dose of 6-hydroxydopamine (1 microg) was reduced in male rats, whereas in females, ovariectomy enhanced dopamine depletion. Administration of the nonaromatizable androgen dihydrotestosterone to gonadectomized animals had no significant effect on 6-hydroxydopamine toxicity in either males or females, whereas treatment of gonadectomized males and females with physiological levels of estrogen restored the extent of striatal dopamine loss to that seen in intact rats, viz, estrogen therapy reduced lesion size in females but increased it in males. Taken together, our findings strongly suggest that there are sex differences in the mechanisms whereby nigrostriatal dopaminergic neurones respond to injury. They also reveal that the reported clinically beneficial effects of estrogen in females may not be universally adopted for males. While the reasons for this gender-determined difference in response to the activational action of estrogen are unknown, we hypothesize that they may well be related to the early organizational events mediated by sex steroid hormones, which ultimately result in the sexual differentiation of the brain.

Ligand-specific conformational change of the G-protein–coupled receptor ALX/FPR2 determines proresolving functional responses

Significance Inflammation is a crucial host defense response but can cause chronic disease if unregulated. Several endogenous anti-inflammatory and proresolving circuits balance and modulate inflammation, including a mechanism centered on the formyl peptide receptor (FPR) family. One receptor, ALX/FPR2, recognizes both proinflammatory and proresolving signals. We have investigated this unusual molecular mechanism finding that anti-inflammatory, but not proinflammatory signals, activate homodimers of this receptor. This triggers intracellular changes culminating in the release of anti-inflammatory mediators such as IL-10. Heterodimers of ALX with other FPR receptors can transduce proapoptotic signals. These results explain how both the development and resolution of inflammation may be integrated by the same receptor system and show how drugs can be developed that have only anti-inflammatory effects. Formyl-peptide receptor type 2 (FPR2), also called ALX (the lipoxin A4 receptor), conveys the proresolving properties of lipoxin A4 and annexin A1 (AnxA1) and the proinflammatory signals elicited by serum amyloid protein A and cathelicidins, among others. We tested here the hypothesis that ALX might exist as homo- or heterodimer with FPR1 or FPR3 (the two other family members) and operate in a ligand-biased fashion. Coimmunoprecipitation and bioluminescence resonance energy transfer assays with transfected HEK293 cells revealed constitutive dimerization of the receptors; significantly, AnxA1, but not serum amyloid protein A, could activate ALX homodimers. A p38/MAPK-activated protein kinase/heat shock protein 27 signaling signature was unveiled after AnxA1 application, leading to generation of IL-10, as measured in vitro (in primary monocytes) and in vivo (after i.p. injection in the mouse). The latter response was absent in mice lacking the ALX ortholog. Using a similar approach, ALX/FPR1 heterodimerization evoked using the panagonist peptide Ac2-26, identified a JNK-mediated proapoptotic path that was confirmed in primary neutrophils. These findings provide a molecular mechanism that accounts for the dual nature of ALX and indicate that agonist binding and dimerization state contribute to the conformational landscape of FPRs.

Annexin A1: A Central Player in the Anti-Inflammatory and Neuroprotective Role of Microglia

The brain microenvironment is continuously monitored by microglia with the detection of apoptotic cells or pathogens being rapidly followed by their phagocytosis to prevent inflammatory responses. The protein annexin A1 (ANXA1) is key to the phagocytosis of apoptotic leukocytes during peripheral inflammatory resolution, but the pathophysiological significance of its expression in the CNS that is restricted almost exclusively to microglia is unclear. In this study, we test the hypothesis that ANXA1 is important in the microglial clearance of apoptotic neurons in both noninflammatory and inflammatory conditions. We have identified ANXA1 to be sparingly expressed in microglia of normally aged human brains and to be more strongly expressed in Alzheimer’s disease. Using an in vitro model comprising microglial and neuronal cell lines, as well as primary microglia from wild-type and ANXA1 null mice, we have identified two distinct roles for microglial ANXA1: 1) controlling the noninflammatory phagocytosis of apoptotic neurons and 2) promoting resolution of inflammatory microglial activation. In particular, we showed that microglial-derived ANXA1 targets apoptotic neurons, serving as both an “eat me” signal and a bridge between phosphatidylserine on the dying cell and formyl peptide receptor 2 on the phagocytosing microglia. Moreover, inflammatory activation of microglia impairs their ability to discriminate between apoptotic and nonapoptotic cells, an ability restored by exogenous ANXA1. We thus show that ANXA1 is fundamental for brain homeostasis, and we suggest that ANXA1 and its peptidomimetics can be novel therapeutic targets in neuroinflammation.

The Size and Distribution of Midbrain Dopaminergic Populations are Permanently Altered by Perinatal Glucocorticoid Exposure in a Sex- Region- and Time-Specific Manner

Central dopaminergic (DA) systems appear to be particularly vulnerable to disruption by exposure to stressors in early life, but the underlying mechanisms are poorly understood. As endogenous glucocorticoids (GCs) are implicated in other aspects of neurobiological programming, this study aimed to characterize the effects of perinatal GC exposure on the cytoarchitecture of DA populations in the substantia nigra pars compacta (SNc) and the ventral tegmental area (VTA). Dexamethasone was administered non-invasively to rat pups via the mothers’ drinking water during embryonic days 16–19 or postnatal days 1–7, with a total oral intake circa 0.075 or 0.15 mg/kg/day, respectively; controls received normal drinking water. Analysis of tyrosine hydroxylase-immunoreactive cell counts and regional volumes in adult offspring identified notable sex differences in the shape and volume of the SNc and VTA, as well as the topographical organization and size of the DA populations. Perinatal GC treatments increased the DA population size and altered the shape of the SNc and VTA as well as the organization of the DA neurons by expanding and/or shifting them in a caudal direction. This response was sexually dimorphic and included a feminization or demasculinization of the three-dimensional cytoarchitecture in males, and subtle differences that were dependent on the window of exposure. These findings demonstrate that inappropriate perinatal exposure to GCs have enduring effects on the organization of midbrain DA systems that are critically important for normal brain function throughout life.

Identification of an essential endogenous regulator of blood-brain barrier integrity, and its pathological and therapeutic implications.

The blood–brain barrier (BBB), a critical guardian of communication between the periphery and the brain, is frequently compromised in neurological diseases such as multiple sclerosis (MS), resulting in the inappropriate passage of molecules and leukocytes into the brain. Here we show that the glucocorticoid anti-inflammatory messenger annexin A1 (ANXA1) is expressed in brain microvascular endothelial cells, where it regulates BBB integrity. In particular, ANXA1−/− mice exhibit significantly increased BBB permeability as a result of disrupted interendothelial cell tight junctions, essentially related to changes in the actin cytoskeleton, which stabilizes tight and adherens junctions. This situation is reminiscent of early MS pathology, a relationship confirmed by our detection of a selective loss of ANXA1 in the plasma and cerebrovascular endothelium of patients with MS. Importantly, this loss is swiftly restored by i.v. administration of human recombinant ANXA1. Analysis in vitro confirms that treatment of cerebrovascular endothelial cells with recombinant ANXA1 restores cell polarity, cytoskeleton integrity, and paracellular permeability through inhibition of the small G protein RhoA. We thus propose ANXA1 as a critical physiological regulator of BBB integrity and suggest it may have utility in the treatment of MS, correcting BBB function and hence ameliorating disease.

Sex dimorphisms in the neuroprotective effects of estrogen in an animal model of Parkinson's disease

The incidence of certain neurological disorders, including Parkinsons disease, appears to be more prevalent in men. Studies involving estrogen treatment of ovariectomised rodents attribute this largely to the neuroprotective effects of estrogen. However, a neuroprotective role for physiological levels of circulating hormones in males and females is less clear. Using the 6-hydroxydopamine (6-OHDA) model of Parkinsons disease to lesion the nigrostriatal dopaminergic (NSDA) pathway, we have shown that in females, endogenously produced estrogen is neuroprotective, whereas in males, gonadal factors increase striatal 6-OHDA toxicity. Intriguingly, estrogen, but not dihydrotestosterone, a nonaromatizable androgen, reversed the effects of orchidectomy on lesion size, raising the novel the hypothesis that enhanced male susceptibility may be attributable to the effects of endogenous testosterone only after its aromatization to estrogen. Thus, estrogen appears to exert opposite effects in the NSDA in males and females, being neuroprotective in females, but not in males, where it may even exacerbate neurodegenerative responses, with important implications for the clinical potential of estrogen-related compounds as neuroprotective agents. Preliminary experiments support the hypothesis that sex differences in the adult NSDA may result from the organisational actions of gonadal steroids during the critical neonatal period for the masculinization of the brain. Further studies are needed to determine whether this early organisation of a sexually differentiated neural circuitry may contribute to the emergence of neurodegenerative conditions such as Parkinsons disease.

Altered Mesencephalic Dopaminergic Populations in Adulthood as a Consequence of Brief Perinatal Glucocorticoid Exposure

Early exposure to stressors is strongly associated with enduring effects on central nervous system function, but the mechanisms and neural substrates involved in this biological ‘programming’ are unclear. This study tested the hypothesis that inappropriate exposure to glucocorticoid stress hormones (GCs) during critical periods of development permanently alters the mesencephalic dopaminergic populations in the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc). Using a rat model, the synthetic GC dexamethasone was added to the maternal drinking water during gestational days 16–19 or over the first week of postnatal life. In adulthood, the effects upon tyrosine hydroxylase immunopositive (TH+) cell numbers in the midbrain, and monoamine levels in the forebrain, of the adult offspring were assessed and compared with control offspring whose dams received normal drinking water. In the VTA, both prenatal and postnatal dexamethasone treatment increased TH+ cell numbers by approximately 50% in males and females. Although prenatal dexamethasone treatment also increased TH+ cell numbers in the SNc by 40–50% in males and females, postnatal treatment affected females only by increasing TH+ cell numbers by approximately 30%. In comparison, similar changes were not detected in the monoamine levels of the dorsolateral striatum, nucleus accumbens or infralimbic cortex of either males or females, which is a feature likely to reflect adaptive changes in these pathways. These studies demonstrate that the survival or phenotypic expression of VTA and SNc dopaminergic neurones is profoundly influenced by brief perinatal exposure to GCs at times when endogenous levels are normally low. These findings are the first to demonstrate permanent changes in the cytoarchitecture within midbrain dopamine nuclei after perinatal exposure to stress hormones and implicate altered functionality. Thus, they have significance for the increasing use of GCs in perinatal medicine and indicate potential mechanisms whereby perinatal distress may predispose to the development of a range of psychiatric conditions in later life.

Independent influences of sex steroids of systemic and central origin in a rat model of Parkinson's disease: A contribution to sex-specific neuroprotection by estrogens

This review considers evidence which reveals considerable complexity and sex differences in the response of the nigrostriatal dopaminergic (NSDA) system to hormonal influences. This pathway degenerates in Parkinsons disease (PD) and sex hormones contribute to sex differences in PD, where men fare worse than women. Here we discuss evidence from animal studies which allows us to hypothesize that, contrary to expectations, the acclaimed neuroprotective property of physiological concentrations of estradiol arises not by promoting NSDA neuron survival, but by targeting powerful adaptive responses in the surviving neurons, which restore striatal DA functionality until over 60% of neurons are lost. Estrogen generated locally in the NSDA region appears to promote these adaptive mechanisms in females and males to preserve striatal DA levels in the partially injured NSDA pathway. However, responses to systemic steroids differ between the sexes. In females there is general agreement that gonadal steroids and exogenous estradiol promote striatal adaptation in the partially injured NSDA pathway to protect against striatal DA loss. In contrast, the balance of evidence suggests that in males gonadal factors and exogenous estradiol have negligible or even harmful effects. Sex differences in the organization of NSDA-related circuitry may well account for these differences. Compensatory mechanisms and sexually dimorphic hard-wiring are therefore likely to represent important biological substrates for sex dimorphisms. As these processes may be targeted differentially by systemic steroids in males and females, further understanding of the underlying processes would provide valuable insights into the potential for hormone-based therapies in PD, which would need to be sex-specific. Alternatively, evidence that estrogen generated locally is protective in the injured male NSDA pathway indicates the great therapeutic potential of harnessing central steroid synthesis to ameliorate neurodegenerative disorders. A clearer understanding of the relative contributions and inter-relationships of central and systemic steroids within the NSDA system is an important goal for future studies.

Oestrogen and immunomodulation: new mechanisms that impact on peripheral and central immunity.

The regulation of the immune response to infection or tissue damage is a complex interplay of multiple factors, but it has long been recognised that steroid hormones can exert powerful modulatory effects at all levels of the innate and adaptive immune systems. Although most attention has been paid to glucocorticoids given their widespread clinical use, it is becoming increasingly clear that sex steroid hormones, and in particular the principle female sex steroid oestrogen, exerts potent effects upon the immune response. In this review, we will discuss the latest findings on the impact of oestrogen upon various cellular components of the immune system, and how this hormone can offer new opportunities to pharmacologically harness the immune response.