1 Estrogens in Neuropsychiatric Disorders: From Physiology to Pathophysiology

(1)

Introduction:

The Role of Estrogens in Human Neuropsychiatric Diseases

A large number of studies, which often focus on the effects of estrogen replacement therapy (ERT) in women, have reported beneficial actions of these hormones on various neurobiological and neuropa- thological parameters in health and disease (Fig. 1). It is likely that postmenopausal ERT helps reduce the risk of Alzheimer’s disease (Kawas et al. 1997), improves cognitive and affective functions (Schmidt et al. 1996), including postmenopausal depressive symp- toms (Halbreich 1997), and affects the course of other illnesses such as schizophrenia (Riecher-Rössler and Häfner 1993; Riecher-Rössler et al. 1994) and probably also stroke (Toung et al. 1998; Yang et al.

2000) and Parkinson’s disease (Blanchet et al. 1999; Saunders-Pull- man et al. 1999).

Unfortunately, these data have often not been clearly confirmed by appropriately designed double-blind studies due to inherent prob- lems such as the necessity for long-term evaluation and controlling for confounding factors. Moreover, a recent treatment study under rigorously controlled conditions failed to show positive effects of estrogen treatment in patients already affected by Alzheimer’s disease (Mulnard et al. 2000).

In contrast, a large number of preclinical neurobiological studies have unequivocally demonstrated neuroprotective effects of this group of hormones during numerous toxic states and under various different conditions (Behl and Holsboer 1999). Up to now, it is not at all clear which of the various effects of estrogens on cells of different systems (neuronal, vascular, immune, and others) contribute to what extent to the neuroprotective effects of these hormones. It is likely that several of these described actions represent similar cellu-

Estrogens in Neuropsychiatric Disorders:

From Physiology to Pathophysiology

Helmut Vedder and Christian Behl

(2)

lar mechanisms or that the effects, for example, on the vascular and the neuronal systems promote neuroprotection. Although the dual- ism between “slow classical receptor-mediated genomic effects”

and the rather rapidly occurring so-called “nongenomic” effects is still apparent, the existence of membrane receptors similar to the nuclear receptors has been suggested. It is possible that these may mediate the so-called “nongenomic” effects of these hormones (Weiss and Gurpide 1988; Fiorelli et al. 1996). On the other hand, estrogens are also able to induce alterations in the redox state or changes of the electrophysiological state of a cell and it is possible that these effects may result in changes in gene-expressions patterns.

Therefore, the network of the multiple activities of estrogens must be studied and new categories defined to characterize and to evaluate the data on the neuroprotective actions of these hormones. Hope- fully, this will be possible in the near future, to allow not only for a clearer understanding of the described effects but also to gain further perspective for the clinical use of estrogen compounds.

In this chapter, we focus on the basic mechanisms of the actions of estrogens, which are likely to play an important role in the pa- thology of neuropsychiatric diseases and to contribute to the im- provement of neuronal survival and function in general. Several of

Fig. 1. ERT affects the pathogenesis of various neuropsychiatric disorders

Alzheimer’s disease Estrogen Replacement Therapy

Mild cognitive impairment

Parkinson’s disease

Stroke

Cognition

Depression

Schizophrenia

(3)

these mechanisms of actions may be conclusively related to specific disease processes such as Alzheimer’s disease or to the toxic effects of free radicals in the course of stroke and other diseases. Moreover, estrogen-induced changes in neurotransmitter systems such as the NMDA excitatory system or the dopaminergic system suggest possible effects in diseases such as Parkinson’s disease and schizophrenia. In addition, even clinical studies support an interaction of estrogens with cholinergic functions and dysfunctions, since co- medication with estrogens has been shown to induce advantageous effects in the treatment of memory deficits, including those occurring in conjunction with Alzheimer’s disease (Schneider at al. 1997).

In a large number of preclinical and basic neurobiological studies, the neuroprotective potential of this group of hormones has been clearly shown (for recent reviews see: Behl and Holsboer 1999;

Green and Simpkins 2000). Several effects, including the interactions of estrogens with the cellular Ca⁺⁺metabolism or with important factors of cellular metabolism such as the signaling cascades involving cAMP and MAP-kinase, must be further examined in the future with regard to their neuroprotective potential.

Additional clinical studies still need to be conducted to clarify whether the protective effects of estrogens observed in vitro also apply to the in vivo situation, whether the basic neuroprotective effects are also detectable in the human system and which co-factors have to be controlled or modified to optimize treatment effects. In addition, we need to analyze whether novel estrogen derivatives lacking genomic activity can be designed and are more likely to be used in humans. Since the question of which particular functions contribute to the beneficial effects of estrogens is still open, the different mechanisms of actions under which neuroprotection occurs need to be analyzed and this knowledge applied in clinical treatment and trials.

Basic Mechanisms of Estrogen Action:

Genomic Effects of Estrogens via the “Classical” Estrogen Receptors

Estrogen receptors (ER) belong to the family of nuclear steroid receptors (Evans 1988) and, therefore, many of the effects of estrogens are mediated via the “classical genomic way of steroid hormone

(4)

action” (Fig. 2). Estrogens readily enter and cross the cellular membrane due to their lipophilicity and bind to the intracellular “high affinity estrogen hormone receptors” (ER). The formation of the “hormone-receptor complex” then leads to the binding of the complex to

“hormone-responsive elements” (HRE) at regulatory sequences of the genomic DNA after dissociation of HSP-90 shock protein and dimerization of receptors. This binding subsequently induces the regulation of the HRE-regulated genes, probably via transient induction of nuclear proto-oncogenes (Yamashita 1998).

In addition, a variety of co-activator and co-repressor proteins that further affect estrogen-responsive genes, the cellular occurrence, and the state of the unbound receptor molecule represent important factors for the effects of estrogens (Shibata et al. 1997). Other co-factors and co-repressors are also involved in the regulation of gene expression by steroid hormones, leading to complex actions of various elements converging on the changes in gene transcription (Shibata et al. 1997; Klinge 2000). Overall, these data show that the genomic effects of estrogens not only regulate the activity of single genes, but also induce more complex changes in the metabolism of the re-

Fig. 2. “Classical” genomic pathway of estrogen action as transcription factor

(5)

sponsive cell via the induction of gene-expression patterns (Russell 1996).

Up to now, at least two ERs, ER-α and ER-β, have been identified (for a recent review see: Gustafsson 2000). ER-α appears to be only weakly expressed in certain areas of the hippocampus, whereas ER-β is found more abundantly in this region (Shughrue et al. 1997). In addition to the hippocampus, estrogens may also affect other regions and various transmitter systems in the brain that express ERs, including the basal forebrain transmitter systems for acetylcholine and 5-HT and the dopamine and the norepinephrine system (Das and Chaudhuri 1995; Mudd et al. 1998; McEwen and Alves 1999). ER-induced gene expression via the classical pathway may participate in the neuroprotective actions of estrogens since the genes for the neurotrophin brain-derived neurotrophic factor (BDNF) (Singh et al.

1995; Sohrabji et al. 1995) are regulated by estrogens and have been demonstrated to exert neuroprotective properties. Some studies of the estrogen antagonist tamoxifen support a role of the classical ERs in neuroprotective paradigms (Singer et al. 1996; Green et al. 1997).

Despite this, these results are by far not applicable to all studies in this area and other mechanisms, including nongenomic effects, also contribute – most likely to a much larger extent – to the neuroprotective effects of these hormones (Behl and Holsboer 1999; Moos- mann and Behl 1999; Green and Simpkins 2000). In addition, most of the work up to now has been done on the ER-α. Therefore, further studies are required to clarify the role of other ERs in genomically- mediated neuroprotective actions of estrogens.

Interestingly, the transcription of the bcl-2 gene, which is significantly involved in apoptotic cell death, is affected by estrogens via a cAMP response element in the promotor region (Dong et al. 1999), showing that the genomic effects of these hormones are also mediated by mechanisms other than the classical binding of receptors to the hormone-responsive DNA elements. This means that estrogens are also able to indirectly modulate gene transcription by influenc- ing intracellular signaling pathways.

Increasing knowledge about the selective expression and co- expression of ERs and their pharmacology and the elucidation of the mechanisms of the neurotrophic activity of estrogens via their genomic effects suggests that these hormones and their receptors could prove to be important novel targets in the search for neuroprotective agents.

(6)

Basic Mechanisms:

The Importance of Nongenomic Effects for the Neuroprotective Actions of Estrogens

As already discussed, increasing data have revealed that other mechanisms of action beside the “classical” genomic effects of estrogen hormones are also effective:

• Under certain conditions, estrogen receptor antagonists are not, or only partly, effective in neutralizing the neuroprotective actions of these hormones (Green et al. 1997; Regan and Guo 1997;

Moosmann and Behl 1999; Culmsee et al. 1999).

• Moreover, protein (Goodman et al. 1996; Regan and Guo 1997; Sa- wada et al. 1998) or mRNA synthesis inhibitors (Goodman et al.

1996) do not interfere with the neuroprotective actions in several toxicity paradigms.

• Structure-activity studies demonstrate different structural require- ments for the effects on cellular ERs (Korenman 1969; Wiese et al.

1997) and the neuroprotective effects with regard to different cytotoxic paradigms, including free radical-induced cytotoxicity (Behl et al. 1997; Green et al. 1997; Moosman and Behl 1999) in different neuroprotection models. Moreover, cells such as the clonal mouse hippocampal cell line HT 22 that do not show a classical ER response are also protected by estrogens after cytotoxic challenge (Behl et al. 1995; Green et al. 1998; Vedder et al. 2000).

• Cell-free cytotoxic paradigmatic approaches such as the induction of lipid peroxidation in cell or brain homogenates also point to beneficial actions of estrogens via this pathway under in vivo conditions, excluding the necessity for a functioning cellular metabolism as required for the genomic actions (Sergeev et al. 1974;

Goodman et al. 1996; Vedder et al. 1999).

These results clearly demonstrate that both genomic actions of estrogens, including those via the steroid receptors, and a variety of other nongenomic effects mediate and contribute to the structural and functional neuroprotective effects of these hormones.

(7)

Modulatory Effects of Estrogens on Neurotransmitter Systems (Glutamate, Dopamine, Serotonin, Acetylcholine)

and Neuronal Excitability

In the central nervous system, a large number of cellular activities and functions are influenced by the excitatory status of the cell membrane and the effects of neurotransmitters that, to a large extent, modulate this state.

Glutamate, the major excitatory transmitter, may cause neuronal cell death under certain conditions, such as cerebral ischemia after activation of N-methyl-D-aspartate (NMDA) and kainate/α-amino- 3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) binding sites (Choi 1992; Choi 1996). Estrogens have been examined with regard to their interference with this type of cytotoxic paradigm and both interactions with NMDA-evoked excitotoxicity and membrane effects have been demonstrated in this context (Weaver et al. 1997;

Regan and Guo 1997). Additionally, genomic effects may also play a role in the inhibition of glutamate toxicity, since the antiestrogen tamoxifen was able to block the neuroprotective effects of the hormone – at least under certain experimental conditions (Singer et al. 1996).

Interestingly, there are also data on the enhancement of the activity of NMDA effects by estradiol, most likely in a region-specific manner (Wooley et al. 1997; Foy et al. 1999; Cyr et al. 2000), with a focus on the hippocampal CA 1 region, the frontal cortex, and the nucleus accumbens (Cyr et al. 2000). Moreover, estradiol has been demonstrated to potentiate the depolarizing influences of AMPA, kainate, and quisqualate, but not NMDA (Wong and Moss 1992), pointing to short-term actions of estrogens on non-NMDA receptors. Unfortunately, the data are not conclusive yet; thus, these underlying effects may be influenced by other yet undefined factors.

Inhibitory transmitters such as GABA or other excitability- decreasing substances may reduce the cytotoxicity of NMDA. Wei- land et al. (1992) showed that estrogens induce the mRNA levels for glutamic acid decarboxylase (GAD), the GABA-synthesizing enzyme in the CA1 pyramidal cell layer of the hippocampus. This effect may also contribute to a reduction in excitatory amino acid-evoked cytotoxicity by increasing the inhibitory neurotransmission via a genomic effect on the GABA-ergic system. Direct membrane effects have been demonstrated with regard to Ca⁺⁺ currents (Joels and Karst

(8)

1995; Mermelstein et al. 1996), probably related to the activation of G-proteins (Mermelstein et al. 1996). Recently, molecular biological data have demonstrated that both putative membrane and nuclear ERs for ER-β can be derived from a single transcript (Razandi et al.

1999), further supporting the concept of direct membrane effects of these steroids even via the same receptor, which mediates the effects of estrogen at the cell nucleus. Overall, the data on potential estrogen membrane receptors are presently not conclusive. Other results suggest that at least some of the membrane effects of estrogens might also be mediated by so-called caveolae, specific membrane structures (Toran-Allerand et al. 1999).

The dopaminergic system is a transmitter system which is involved in addiction and reward processes and acts as a neuromodula- tor (Raevskii 1997). Effects of estrogens on the dopaminergic system include a decrease in dopaminergic neurotransmission and a subsequent increase in dopaminergic binding sites (Di Paolo 1994; Bosse and DiPaolo 1996), supporting a physiological and possibly also a pathophysiological role of this mechanism in schizophrenia. More- over, estrogen treatment increases the concentrations of dopamine in the striatum (Becker 1999) and modulates the sensitivity and also the number of striatal D2 receptors (Lammers et al. 1999).

These effects have also been supported by functional studies on apomorphine- and haloperidol-induced dopamine-mediated behavior such as stereotypy and catalepsy (Häfner et al. 1991, Gattaz et al.

1992). These data clearly show effects of these hormones on the dopaminergic system and support a role of estrogens in the modulation of the pathophysiology of diseases such as schizophrenia, Parkin- son’s disease, and probably even addiction.

Effects of estrogens on the serotonergic system have also been described, showing a significant increase in the density of 5-HT 2A binding sites in different brain areas (Fink et al. 1998) and inhibition of serotonin reuptake in synaptosomal preparations of the rat cortex (Michel et al. 1987). Other actions may include direct effects of the hormones on serotoninergic neurons as suggested by autoradiogra- phy studies on the binding sites in the midbrain region (Stumpf and Jennes 1984). Moreover, a study on hormonal responses in postmenopausal women with or without estrogen replacement even suggested a modulation of serotonin agonist-evoked effects by these hormones (Halbreich et al. 1995).

Other effects of estrogens have been detected on the neuroten-

(9)

sin/neuromedin gene (Watters and Dorsa 1998). This gene is induced by estrogens in the preoptic area. Since the promoter region lacks an estrogen-responsive element, it is likely that the changes in gene-expression are mediated by other mechanisms, such as interaction with the cAMP system.

Specific Interactions of Estrogens with Disease Processes:

Effects of Estrogens on Cholinergic Functions

The cholinergic system consists of neurons which synthesize the transmitter acetylcholine and innervate most parts of the neocortex, including the hippocampal formation. Cholinergic neurons are mostly localized in the basal forebrain. One of the areas involved in the innervation of the hippocampal formation is the nucleus basalis magnocellularis Meynert. In the course of Alzheimer’s disease, cholinergic functions decrease with a concomitant impairment of mne- stic and cognitive functions.

Interestingly, estrogens influence cholinergic functions by several mechanisms (Gibbs and Aggarwal 1998; McEwen and Alves 1999).

The therapeutic relevance of these mechanisms is further supported by the additive effects of a co-medication with estrogens in the course of the treatment of Alzheimer patients with the acetylcholine- esterase inhibitor tacrine (Schneider et al. 1996).

The growth and the function of cholinergic neurons highly depends on the influence of nerve growth factor (NGF), a protein which is synthesized and secreted by neurons and glial cells. Interestingly, effects of estrogens have not only been detected with regard to cholinergic functions, but also with regard to the NGF system. Estrogen treatment promotes an upregulation of the acetylcholine-producing enzyme choline acetyltransferase (ChAT) and a subsequent downre- gulation of the receptors for NGF, p75, trkA, and the NGF protein it- self (Gibbs et al. 1994). Sohrabji et al. (1994) even demonstrated a reciprocal regulation of the two systems in PC 12 cells: long-term treatment with NGF induced a six-fold increase in estrogen binding, whereas subsequent co-treatment with estrogen and NGF led to an induction of the NGF receptor mRNA for trkA and down-regulation of the p75 mRNA. This was also confirmed by another study, even with a specificity of the effects for females (Gibbs and Pfaff 1992). Tak- en together, the data show an induction of ChAT by estrogens and

(10)

a down-regulation of the NGF system. In addition to the functional actions on the cholinergic system, synergistic neuroprotective effects have been shown after treatment with estrogens andNGF in a paradigm of serum-derived PC 12 cells (Gollapudi and Oblinger 1999).

Induction of cholinergic cell death by deposition and toxicity of ß-amyloid represents an important hallmark of Alzheimer’s disease (Hardy and Allsop 1991; Yankner 1996; Behl 1999). Interestingly, estrogens are able to effectively interact with ß-amyloid-induced cell death, most likely in a direct manner, by effectively interfering with free radical-mediated impairment of cellular functions (Behl et al.

1995, 1997; Behl and Holsboer 1999; Vedder et al. 2000).

Cross-Talk of Estrogens with Intracellular Factors and Signal Transduction Mechanisms

cAMP, CREB

A large number of peripheral influences on the cell converge at several intracellular transduction pathways. One of these pathways in- cludes the second messenger cyclic-AMP (cAMP). Changes in the intracellular concentrations of this molecule induced by the binding of extracellular mediators to their corresponding membrane receptors affects gene transcription via the cAMP response element-binding protein (CREB) (Hagiwara et al. 1996).

Estradiol has been shown to increase cAMP concentrations in different cellular systems (Minami et al. 1990; Gu and Moss 1996; Wat- ters and Dorsa 1998, Kelly et al. 1999), including hypothalamic neuronal cells (Gunaga and Menon 1973). Moreover, the functional character of these changes has been demonstrated by an increase in the phosphorylation of the CREB protein. With regard to the underlying receptor systems, no clear-cut results were obtained by the use of receptor antagonists (Gu et al. 1996; Watters and Dorsa 1998).

In a recent review, Green and Simpkins (2000) discussed the importance of the phosphorylation step of CREB as a convergence point not only for the cAMP pathway, but also for at least two other pathways, the MAP kinase pathway (Singh et al. 1999) and the CAM kinase pathway (Matsuno et al. 1997), which have also been shown to be activated after exposure to estradiol. They pointed out that an activation of the cAMP pathway is associated with a decreased susceptibility of neuronal cells to several types of apoptotic signals

(11)

(D’Mello et al. 1993; Kew et al. 1996; Campard et al. 1997; Kobayas- hi and Shinozawa 1997), indicating a role of this mechanism in the prevention of cell death. Moreover, increased phosphorylation of CREB is related to an increased resistance to ischemic injury (Wal- ton et al. 1996) and may activate the MAP kinase (Vossler et al.

1997). Interestingly, the transcription of the bcl-2 gene, which is strongly involved in apoptotic cell death, is also affected by estrogens (Garcia-Segura et al. 1998; Singer et al. 1998, Dong et al. 1999), most likely via a cAMP response element in the promotor region (Dong et al. 1999). Thereby, the cAMP pathway most likely contributes to genomic changes induced by estrogens. These effects may then induce or favor a state of decreased susceptibility of neuronal cells to damaging insults via cAMP increase, affording effective neuroprotection under certain conditions.

MAP Kinase and Other Kinases

Another important pathway for the intracellular transduction of signals is the mitogen-activated protein kinase (MAP ) pathway. After tyrosine phosphorylation of cellular proteins, the MAP kinases ERK- 1 (extracellular-signal-related kinase-1) and ERK-2 are activated and evoke further cellular effects, e. g., the cellular response to peptide growth factors such as nerve growth factor (NGF) or BDNF. These factors then act on nerve and other cells via the receptor molecules p75 or trkA. Subsequent cellular signaling takes place by interme- diate proteins such as ras and b-raf and subsequently affects the phosphorylation state of a variety of proteins via MAP kinase activity. Actions of estrogens on this pathway have been shown by at least two groups and under various different conditions and support a membrane specificity of the effects (Watters et al. 1997; Singh et al.

1999). These data indicate that such actions occur quite rapidly in the minute range and are not blocked by the estrogen receptor antagonist ICI 182.780 –, at least under in vitro conditions with primary neocortical neurons (Singh et al. 1999). Further available data on this issue allow for the speculation that the activated ER induces and increases the B-raf kinase, leading to the formation of an multimeric complex of ERs, hsp90, and B-raf kinase (Singh et al. 1999; Toran-Al- lerand et al. 1999).

We have recently been able to show that the activation of MAPK (ERK-1/ERK-2) by 17β-estradiol directly mediates the enhanced re-

(12)

lease of the nonamyloidogenic form of the amyloid precursor protein (APP) (Manthey et al. 2001). The metabolism of APP (amyloidogenic versus non-amyloidogenic processing) is believed to be one of the key events in the pathogenesis of Alzheimer’s disease.

Interestingly, very recent data (Honda et al. 2000) show that phosphatidylinositol 3-kinase (PI3-K) – as a result of ER receptor activation by incubation with 10 nM of 17β-estradiol – may lead to a phosphorylation of Akt/phosphokinase B, thereby significantly contributing to the neuroprotective effects of estrogens in glutamate-treated cultured cortical neurons via this pathway.

Bcl-2-Related Proteins

Cellular apoptosis describes a specific type of cell death, which starts from the cellular nucleus, and is characterized to a significant extent by the degradation of the cellular DNA. During gel electrophoretic analysis of the cellular material, the “laddering phenomenon” can be detected, which indicates the specific effects of DNA-degrading enzymes. The DNA degradation is executed by caspases, enzymes that exist in inactive pro-forms and are activated by apoptotic stimuli (Wolozin and Behl 2000).

The induction of apoptotic processes is controlled by specific inhibitors (bcl-XL, bcl-2) and promoters (bax, bad, bcl-Xs), which are members of the so-called Bcl-2 family of proteins. Not only does the occurrence of one or the other factors determine the fate of the cell, but their relative concentrations lead to a cellular state towards apoptosis, either under normal conditions or after challenge with toxic stimuli. Thereafter, more complex mechanisms take over and lead the cell into apoptosis (Merry and Korsmeyer 1997).

Inhibitors of apoptotic cell death such as bcl-2 may be affected by classical hormone action via the ERs, since several of their genes contain HREs (Dong et al. 1999; Perillo et al. 2000). Func- tionally, an increase in the mRNA of these proteins has been demonstrated after treatment with estrogens in neuronal NT 2 cells (Singer et al. 1998) and in the hypothalamus of female rats (Garcia- Segura et al. 1998).

With regard to both clinical and mechanistic aspects, the group of Dubal (1999) reported changes of the mRNA for bcl-2 in an animal model of stroke after administration of estrogens. Further data point to complex interactions between the cellular levels of the classical

(13)

receptors for estrogens (ER-α and ER-β), bcl-mRNA levels, and estrogen-induced changes in the ischemic penumbra (Dubal et al. 1999).

Nuclear Transcription Factor κB (NF-κB)

Nuclear Transcription Factor κB (NF-κB) is a redox-sensitive induc- ible transcription factor that positively regulates the expression of proimmune and proinflammatory genes, while glucocorticoids are potent suppressors of such responses (McKay and Cidlowski 1998).

With an in vitro approach, Shyamala and Guiot (1992) showed that estrogens activate NF-κB-specific proteins and postulated an interaction between these proteins and ERs. Further studies with gluco- corticoid receptors and NF-κB revealed that these effects are indeed mediated by physical interaction, resulting in a repression of NF-κB transactivation. Subsequently, this was also shown for estrogen, progesterone, and androgen receptors (McKay and Cidlowski 1998).

Functionally, Dodel et al. (1999) demonstrated that estradiol at- tenuated the amyloid-β and LPS-induced translocation of NF-κB in cultured rat astrocytes, pointing to a possible clinical relevance of this mechanism in Alzheimer’s disease. Because NF-κB is also thought to be directly involved in the survival of nerve cells under conditions of oxidative stress (Lezoualc’h and Behl 1998), an interaction between NF-κB and estrogens might even directly affect cell survival at a very basic cellular level.

Cellular Ca⁺⁺Levels

Intracellular Ca⁺⁺levels are under strict control by various cellular mechanisms (Racay et al. 1996). Multiple factors affect these concentrations, leading to morphological and functional changes in the cell (Sola et al. 1999). Thereby, Ca⁺⁺also functions as an important messenger system in the cell, even affecting cellular survival and cell death under various conditions (Leist and Nicotera 1998). Inter- estingly, Ca⁺⁺ concentrations seem to be able to directly influence cellular gene expression, including important genes such as the CREB gene (Bito et al. 1997; Hardingham and Bading 1998).

A lack of control of the intracellular Ca⁺⁺concentrations mostly results in an increase or a profound dysregulation of cellular Ca⁺⁺

concentrations and is induced under conditions of stroke or after treatment of cells with β-amyloid (Mattson et al. 1993). If the homeostatic mechanisms are not able to counteract this increase, the

(14)

loss of cellular functions due to the Ca⁺⁺dysregulation leads to cell death (Tymianski and Tator 1996; Leist and Nicotera 1998).

Estrogens may affect cellular Ca⁺⁺ levels by different mechanisms: direct membrane effects (Joels and Karst 1995; Mermelstein et al. 1996; Pozzo-Miller et al. 1999), interactions with different NMDA receptors (Wong and Moss 1992; Weaver at al. 1997; Pozzo- Miller et al. 1999), effects on intracellular mechanisms of Ca⁺⁺regulation via calmodulin (Hayashi et al. 1994), or direct effects on intracellular stores (Beyer and Raab 1998).

Functional aspects with regard to neuroprotection have been shown in the attenuation of Ca⁺⁺increases by estrogens after challenge of motor neurons with glutamate (Kruman et al. 1999) as well as in a model of cerebral ischemia in the gerbil (Chen et al.

1998). It is still not known whether the actions of estrogens on this cellular parameter are a relevant factor in neuroprotection or just an epiphenomenon in the course of other cellular events.

Structure-Related Intrinsic Neuroprotective Effects of Estrogens

Antioxidative Actions of 17β-Estradiol

Free radicals are a group of molecules which are generated at several places in the course of cellular metabolism, paticularly in energy- providing reactions in the mitochondria. These molecules are highly reactive and interact with a large number of cellular compounds and cause structural and functional changes in the cell (Halliwell and Gutteridge 1990; Sies 1997; Gutteridge and Halliwell 2000).

Usually, the intracellular amount of free radicals is controlled very strictly by a number of mechanisms, such as by different enzymes – superoxide dismutase, gluthatione peroxidase, and catalase – as well as by other components – ascorbic acid and vitamin E. Increases in free radical levels in the cell may either be evoked by impaired de- toxification mechanisms or by an increased production under pathological conditions such as degenerative disease processes or stroke (Coyle and Puttfarcken 1993).

Accumulating cell damage from the metabolism of free radicals has been connected to the aging process: this introduced the so- called “free radical hypothesis of aging” (Harman et al. 1976; Beck-

(15)

man and Ames 1998). Since free radicals are highly likely to play a substantial role in the pathophysiology of neurodegenerative diseases, including Alzheimer’s, exacerbation of cellular damage in the course of these diseases has to be postulated as an important factor, aggravating the “physiological changes of aging” to the more severe disease process of dementias and other neurodegenerative disorders (Harman et al. 1976).

This assumption is further supported by data showing that several products of oxidation reactions and mediators of oxidative stress can be found in association with the histopathological hallmarks of Alzheimer’s disease and vascular dementia, mainly in the “senile plaques”. Such oxidation end products include malondialdehyde, ad- vanced glycation endproducts (AGEs), carbonyls, nitrotyrosine, and various other oxidized molecules (Pappolla et al. 1992; Beal 1995;

Smith et al. 1996). The increased state of cellular oxidation as revealed by such an increase in the oxidation of proteins and lipids (Hajimohammadreza and Brammer 1996) may then lead to long- term changes such as alterations in enzyme activities and structural effects on membrane integrity and the oxidation of DNA with possible long-term mutagenic effects (Ames 1988).

Estrogens and related molecules have been demonstrated to exhi- bit intrinsic antioxidant activity, since they protect neuronal cells under in vitro conditions against free radical-induced cell death.

This is connected to specific properties of the molecule, e.g., the phenolic structure, since all components with such a structure act as efficient neuroprotectants under these conditions (Moosmann and Behl 1999) (Fig. 3). Modifications of this particular moiety by etherization (e.g., mestranol, methyl ether of ethinyl oestradiol) block the antioxidant activity of the molecule. Furthermore, various aromatic alcohols with intact phenolic groups but without ER-activating properties (e.g., dodecyl phenol) also possess intrinsic antioxidant neuroprotective activity and also prevent lipid peroxidation (Moosman and Behl 1999). Although the physiological relevance of the concentrations (nanomolar to millimolar) that are required for antioxidant neuroprotection in vitro are still a matter of debate, it is clear that the neuroprotective activity of estrogens can be structu- rally separated from their ER-activating classical properties as hormones.

Moreover, several factors may contribute to the efficacy of this mechanism: First, the concentration of estrogens is highly variable,

(16)

depending on the menstrual cycle, and might reach lower nanomo- lar concentrations under in vivo conditions. Secondly, in general, steroid compounds, including ovarian steroids, are concentrated several-fold in the brain in relation to the plasma levels. Moreover, the turnover rate of brain sequestration of blood-borne sex steroids is high compared to other steroid compounds such as corticosteroids (Pardridge et al. 1980). Thirdly, drugs used for effective treatment might be able to reach pharmacological levels rather than physiological concentrations due to an effective administration regimen.

And fourthly, other estrogen or phenolic compounds may be found, which require lower tissue concentrations for an effective interaction with free radical-induced cell damage such as the catecholes- trogens (Trepker et al. 2003).

Inhibition of Lipid Peroxidation

The central nervous system contains a substantial amount of membranes and fatty acids (Halliwell and Gutteridge 1990; Halliwell

Fig. 3. Estradiol is a phenolic free radical scavenger/antioxidant similar to vitamin E

(17)

1992). This implies an increased vulnerability of membrane lipid constituents in the CNS to oxidative injury, either directly by cellular free radicals (Coyle and Puttfarcken 1993; Olanow 1993; Bondy 1995; Frölich and Riederer 1995; Sies 1997) or via other indirect or exogenic mechanisms (Nohl 1993; Piotrowski et al. 1996). The increased vulnerability of neuronal membranes may even be due to the low amount of integral proteins compared to other tissues, as proposed recently (Moosmann and Behl 2000). In this recent study, it was shown that the peptide stretches enriched in phenolic amino acids (tyrosine) protect neurons against oxidative cell death.

As pointed out above, pro-oxidative mechanisms such as trauma, ischemia, and other illnesses increase the cellular load of free radicals (Olanow 1993; Bondy 1995). This increase in free radicals subsequently affects the membranes in the brain, resulting in increased lipid peroxidation (LPO), a loss of cellular compartimentalization (Nohl 1993) and finally in cell death. Therefore, LPO respresents an important cellular endpoint for a large number of toxic events in the CNS (Halliwell and Gutteridge 1990; Gutteridge 1995).

Different approaches showed interactions of estrogen hormones with the peroxidation of lipids in non-neuronal systems, including lipid fractions (Sugioka et al. 1987), microsomal liver preparations (Ruiz-Larrea et al. 1994), and blood constituents such as low density lipoproteins (Miller et al. 1996). Up to now, most of these studies have been conducted on systems outside the CNS.

Due to the importance of LPO processes in the CNS and the clinical relevance of their interaction with estrogens, we recently characterized the effects of estrogens on iron-induced LPO in different CNS-relevant systems: Hippocampal HT 22 cells and living rat neocortical brain cells were used as CNS-derived in vitro systems to study the direct cellular effectiveness of estrogens. Immortalized hippocampal cells of the HT 22 cell line have been used to evaluate the neuroprotective effects of estrogens after challenge with glutamate, iron, and hydrogen peroxide (Morimoto and Koshland 1990;

Behl et al. 1995; 1997). Primary cultures of rat brain cells represent model systems for the evaluation of mechanisms of neurodegeneration and steroid-mediated neuroprotection (Choi et al. 1990; Vedder et al. 1993). In the course of earlier studies, whole brain homogenates were used to characterize LPO in the rodent and in the human CNS, respectively. We examined the effects of estrogens on LPO in these systems and were able to show an inhibition of this important

(18)

pathophysiological mechanism in all different systems, including the human brain (Vedder et al. 1999)

These data are in line with other nonhuman data from the litera- ture showing the LPO-inhibiting effects of estrogens under different conditions, including the LPO evoked by β amyloid (Gridley et al.

1997) and iron sulfate (Goodman et al. 1996).

Effects of Estrogens on Immunological Activation in the CNS: Immunological Activation During

Neurodegeneration and Aging

Estrogens affect a large variety of immune functions (Grossman 1984; Cutolo et al. 1995), including macrophage functions (Miller and Hunt 1996). These effects become pathophysiologically relevant in autoimmune diseases such as multiple sclerosis (Kim et al. 1999;

Jansson and Holmdahl 1998). Another emerging focus is the immunological response in Alzheimer’s disease (McGeer and McGeer 1999) and its possible relevance for treatment (McGeer and McGeer 1996). Therefore, estrogens may influence microglial functions such as the expression and the secretion of cytokines in the brain (Mor et al. 1999). Interestingly, such effects may be mediated by the ER-β, which was detected in microglial cells in the brain (Mor et al. 1999).

Up to now, the role of this modulation of the inflammatory response by estrogens has not been elucidated. In addition, the local immune response has also been shown to induce tissue damage via oxidation reactions either directly through the inflammatory mediators or through secondary events (McGeer and McGeer 1999). Particularly, activated microglia detected in Alzheimer’s disease tissue may powerfully induce oxidative damage via the released inflammatory mediators (Mor et al. 1999).

Neuroprotection as a Vital Byproduct of the Homeostatic Actions of Estrogens – A Hypothesis

Studies of several parts of the effector mechanisms of estrogens indicate that these hormones might not only directly affect the cellular responses during physiological and pathological states, but exert stabilizing and homeostatic effects: With regard to the antioxidative

(19)

actions after a cellular load with hydrogen peroxide, a free-radical inducing agent, estrogens acted only at higher concentrations of this toxic substance, but showed no effects at lower concentrations (Ved- der et al. 2000). In neocortical neurons in culture, the dose-response profile for 17β-estradiol regulation of the macromorphological features exhibited a bimodal dose-response relationship whereas the dose-response profile for 17β-estradiol regulation of the micromor- phological features displayed the more characteristic dose-response of an inverted V-shaped function (Brinton et al. 1997). In hippocampal neurons, the modulation of electrophysiological changes on the NMDA response – the major part of an excitotoxic challenge – by estrogens comparatively depends on the extent of the increase in NMDA activation (Murphy and Segal 1996; Wooley and McEwen 1994).

Therefore, estrogens may only act under pathological conditions, supporting the homeostatic mechanisms in the cell and yielding a state of “decreased vulnerability” to damaging conditions and agents. This assumption fits with the observation that estrogens only delay illness processes such as Alzheimer’s disease and schizophrenia but are not able to directly neutralize the basic pathophysiological mechanisms. At the cellular level, they act on basic mechanisms such as modulation of excitotoxicity, free-radical deto- xification mechanisms, and changes in gene transcription. Co-factors such as CREB, GSH, and other intracellular compounds are required for the actions of estrogens. Moreover, the application of estrogens often increases the detoxifying and antiapoptotic actions of these factors. Future research will have to further examine and subsequently describe a possible unifying concept of these estrogen actions and to weigh the relative importance of the individual factors and mechanisms involved in the pathophysiological events of the different disease processes.

Outlook: From Preclinical to Clinical Neuroprotective Effects of Estrogens

A number of clinical data support a beneficial function of estrogens in health and disease. During the past few years, the effects of estrogens on the brain have gained increasing interest and has led to the ongoing characterization of the neuroprotective effects of this class

(20)

of hormones. Presently, a certain gap in our knowledge still exists re- garding the presumed clinical effects on neuropsychiatric diseases such as Alzheimer’s and Parkinson’s disease and schizophrenia, on one hand, and the large number of preclinical neurobiological fin- dings, on the other. Overall, authors consider estrogens more as preventive compounds rather than as drugs. This implies the necessity of advance administration or administration during the course of the illness and appears feasible since age-related disorders such as Alz- heimer’s disease develop over decades. Once the disease process has started, it may be difficult to halt the pathogenetic mechanisms by physiological modulators such as estrogen, even if high concentrations are used.

With respect to the basic neurobiological research, estrogens have been demonstrated to act as powerful neuroprotectants under a large variety of toxic challenge paradigms such as iron and amyloid-β induced neurotoxicity. Moreover, effects on transmitter systems such as the cholinergic and the glutamatergic system and on neuro- cellular morphology and functions also point to neuroprotective endpoints of the actions of these hormones. By examining the underlying cellular mechanisms in more detail, changes in second- messenger systems and a number of signaling cascades have been detected, including interactions with apoptosis-inducing and -inhibiting proteins and nuclear transcriptions factors such as NF-κB. The very basic effector mechanisms include, in addition to the classical genomic and the nongenomic effects, interactions with free radical detoxifying systems and the inhibition of the cellular LPO, an important pathophysiological process in the brain with its large amount of membranes.

The process of discovering further neuroprotective effects and their underlying mechanisms is still in progress. Presently, it does not seem that a general scheme of the effects is unraveling, a cir- cumstance which may contribute to the lack of transfer from the basic neurobiological knowledge to the clinical application of these neuroprotective effects. Clinical trials will have to show whether the concept of antioxidants as illness-preventive drugs or as a therapy principle for neurodegenerative disorders, including Alzheimer’s disease, holds promise for the future.

Overall, it is clear that the neuroprotective effects of estrogens represent more a general concept rather than just one or a few effects of one substance. The general applicability of the actions of estro-

(21)

gens on the damaged brain – and even on other tissues – is the ad- vantage of the underlying concept. On the basis of this concept, further research should give a more unifying picture of the pathways by which neuronal cell damage is conveyed under several pathophysiological conditions and identify the important effector points where interference with these processes could result in optimal and therefore the most effective neuroprotection.

Acknowledgement

The authors thank A. Tittmar for substantial help with the editing of the manuscript and A. Thum for valuable suggestions.

References

Ames BN (1988) Measuring oxidative damage in humans: relation to cancer and ageing. IARC Sci Publ 89: 407-416

Beal MF (1995) Aging, energy, and oxidative stress in neurodegenerative diseases.

Ann Neurol 38: 357-366

Becker JB (1999) Gender differences in dopaminergic function in striatum and nucleus accumbens. Pharmacol Biochem Behav 64: 803-812

Beckman KB, Ames BN (1998) The free radical theory of aging matures. Physiol Rev 78: 547-581

Behl C, Holsboer F (1999) The female sex hormone oestrogen as a neuroprotectant.

Trends Pharmacol Sci 20: 441-444

Behl C, Skutella T, Lezoualc’h F, Post A, Widmann M, Newton CJ, Holsboer F (1997) Neuroprotection against oxidative stress by estrogens: structure-activity relationship. Mol Pharmacol 51: 535-541

Behl C, Widmann M, Trapp T, Holsboer F (1995) 17beta-estradiol protects neurons from oxidative stress-induced cell death in vitro. Biochem Biophys Res Com- mun 216: 473-482

Beyer C, Raab H (1998) Nongenomic effects of oestrogen: embryonic mouse midbrain neurones respond with a rapid release of calcium from intracellular stores.

Eur J Neurosci 10: 255-262

Bito H, Deisseroth K, Tsien RW (1997) Ca²± dependent regulation in neuronal gene expression. Curr Opin Neurobiol 7: 419-429

Blanchet PJ, Fang J, Hyland K, Arnold LA, Mouradian MM, Chase TN (1999) Short- term effects of high-dose 17beta-estradiol in postmenopausal PD patients: an crossover study. Neurology 53: 91-95

Bondy SC (1995) The relation of oxidative stress and hyperexcitation to neurolog- ical disease. Proc Soc Exp Biol Med 208: 337-345

Bosse R, DiPaolo T (1996) The modulation of brain dopamine and GABAA receptors by estradiol: a clue for CNS changes occurring at menopause. Cell Mol Neuro- biol 16: 199-212

(22)

Brinton RD, Proffitt P, Tran J, Luu R (1997a) Equilin, a principal component of the estrogen replacement therapy pre-marin, increases the growth of cortical neurons via an NMDA receptor-dependent mechanism. Exp Neurol 147: 211-220 Brinton RD, Tran J, Proffitt P, Montoya M (1997b) 17beta-estradiol enhances the outgrowth and survival of neocortical neurons is culture. Neurochem Res 22:

1339-1351

Campard PK, Crochemore C, Rerie F, Monnier D, Kock B, Loefiier JP (1997) PACAP type I receptor activation promotes cerebellar neuron survival through the CAMP/PKA s’tgnaling pathway. DNA Cell Biol 16: 323-333

Chen J, Adachi N, Liu K, Arai T (1998) The effects of 17beta-estradiol on ischemia- induced neuronal damage in the gerbil hippocampus. Neuroscience 87: 817-822 Choi DW (1992) Excitotoxic cell death. J Neurobiol 23: 1261-76

Choi DW (1996) Ischemia-induced neuronal apoptosis. Curr Opin Neurobiol 6:

667-672

Choi DW, Monyer H, Giffard RG, Goldberg MP, Christiane CW (1990) Acute brain injury, NMDA receptors, and hydrogen ions: observations in cortical cell cultures. Adv Exp Med Biol 268: 501-504

Coyle JT, Puttfarcken P (1993) Oxidative stress, glutamate, and neurodegenerative disorders. Science 262: 689-695

Culmsee C, Vedder H, Ravati A, Junker V, Otto D, Ahlemeyer B, Krieg J-C, Kriegl- stein J (1999) Neuroprotection by estrogens in a mouse model of focal cerebral ischemia and in cultured neurons: evidence for a receptor-independent antioxidative mechanism. J Cereb Blood Flow Metab 19: 1263-1269

Cutolo M, Sulli A, Seriolo B, Accardo S, Masi AT (1995) Estrogens, the immune response and autoimmunity. Clin Exp Rheumatol 13: 217-226

Cyr M, Calon F, Morissette M, Grandbois M, DiPaolo T, Callier S (2000a) Drugs with estrogen-like potency and brain activity: potential therapeutic application for the CNS. Cur Pharm Des 6: 1287-1312

Cyr M, Ghribi O, DiPaolo T (2000b) Regional and selective effects of oestradiol and progesterone on NMDA and AMPA receptors in the rat brain. J Neuroendocri- nol 12: 445-452

Das A, Chaudhuri SK (1995) Effects of sex steroids on the concentrations of some brain neurotransmitters in male and female rats: some new observations. Indi- an J Physiol Pharmacol 39: 223-230

DiPaolo T (1994) Modulation of brain dopamine transmission by sex steroids. Rev Neurosci 5: 27-41

D’Mello SR, Galli C, Ciotti T, Calissano P (1993) Induction of apoptosis in cerebellar granule neurons by low potassium: inhibition of death by insulin-like growth factor I and cAMP. Proc Natl Acad Sci USA 90: 10,989-10,993

Dodel RC, Du Y, Bales KR, Gao F, Paul SM (1999) Sodium salicylate and 17beta- estradiol attenuate nuclear transcription factor NF-κB translocation in cultured rat astro-glial cultures following exposure to amyloid A beta(1-40) and lipopoly- saccharides. J Neurochem 73: 1453-1460

Dong L, Wang W, Wang F, Stoner M, Reed JC, Harigai M, Samudio L, Kladde MP, Vyhlidal C, Safe S (1999) Mechanisms of transcriptional activation of bcl-2 gene expression by 17beta-estradiol in breast cancer cells. J Biol Chem 274:

32099-32107

(23)

Dubal DB, Shughrue PJ, Wilson ME, Merchenthaler I, Wise PM (1999) Estradiol modulates bcl-2 in cerebral ischemia: a potential rote for estrogen receptors. J Neurosci 19: 6385-6393

Evans RM (1988) The steroid and thyroid hormone receptor superfamily. Science 240: 889-895

Fink G, Sumner BE, McQueen JK, Wilson H, Rosie R (1998) Sex steroid control of mood, mental state and memory. Clin Exp Pharmacol Physiol 25: 764-775 Fiorelli G, Gori F, Frediani U, Franceschelli F, Tanini A, Tosti-Guerra C, Benvenuti

S, Gennari L, Becherini L, Brandi ML (1996) Membrane binding sites and nongenomic effects of estrogen in cultured human pre-osteoclastic cells. J Steroid Biochem Mol Biol 59: 233-240

Foy MR, Xu J, Xie X, Brinton RD, Thompson RF, Berger TW (1999) 17beta-estradiol enhances NMDA receptor-mediated EPSPs and long-term potentiation. J Neu- rophysiol 81: 925-929

Frölich L, Riederer P (1995) Free radical mechanisms in dementia of Alzheimer type and the potential for antioxidative treatment. Arzneimittelforschung. 45:

443-446

Garcia-Segura LM, Cardona-Gomez P, Naftolin F, Chowen JA (1998) Estradiol upre- gulates Bcl-2 expression in adult brain neurons. Neuroreport 9: 593-597 Gattaz WF, Behrens S, De Vry J, Häfner H (1992) Estradiol inhibits dopamine me-

diated behavior in rats – an animal model of sex-specific differences in schizophrenia. Fortschr Neurol Psychiatr 60: 8-16

Gibbs RB (1994a) Estrogen and nerve growth factor-related systems in brain: effects an basal forebrain cholinergic neurons and implications for learning and memory processes and aging. Ann NY Acad Sci 743: 165-196

Gibbs RB, Aggarwal P (1998) Estrogen and basal forebrain cholinergic neurons: implications for brain aging and Alzheimer’s disease-related cognitive decline.

Horm Behav 34: 98-111

Gibbs RB, Pfaff DW (1992) Effects of estrogen and fimbria/fornix transection on p75NGFR and ChAT expression in the medial septum and diagonal band of Bro- ca. Exp Neurol 116: 23-39

Gibbs RB, Wu D, Hersh LB, Pfaff DW (1994b) Effects of estrogen replacement on the relative levels of choline acetyltransferase, trkA, and nerve growth factor messenger RNAs in the basal forebrain and hippocampal formation of adult rats. Exp Neurol 129: 70-80

Gollapudi L, Oblinger MM (1999) Estrogen and NGF synergistically protect termi- nally diffentiated, ERalpha-transfected PC12 cells from apoptosis. J Neurosci Res 56: 471-481

Goodman Y, Bruce AJ, Cheng B, Mattson MP (1996) Estrogens attenuate and corticosterone exacerbates excitotoxicity, oxidative injury, and amyloid beta- peptide toxicity in hippocampal neurons. J Neurochem 66: 1836-44

Green PS, Simpkins JW (2000) Neuroprotective effects of estrogens: potential mechanisms of action. Int J Devl Neuroscience 18: 347-358

Green PS, Bishop J, Simpkins JW (1997a) 17alpha-estradiol exerts neuroprotective effects on SK-N-SH cells. J Neurosci 17: 511-515

(24)

Green PS, Gordon K, Simpkins JW (1997b) Phenolic A ring requirement for the neuroprotective effects of steroids. J Steroid Biochem Mol Biol 63: 229-235

Green PS, Gridley KE, Simpkins JW (1998) Nuclear estrogen receptor-independent neuroprotection by estratrienes: a novel interaction with glutathione. Neuro- science 84: 7-10

Gridley KE, Green PS, Simpkins JW (1997) Low concentrations of estradiol reduce beta-amyloid (25-35)-induced toxicity, lipid peroxidation and glucose utilization in human SK-N-SH neuroblastoma cells. Brain Res 778: 158-165

Grossman CJ (1984) Regulation of the immune system by sex steroids. Endocr Rev 5: 435-455

Gu G, Rojo AA, Zee MC, Jianhua Y, Simerly RB (1996a) Hormonal regulation of CREB phosphorylation in the anteroventral periventricular nucleus. J Neurosci 16: 3035-3044

Gu Q, Moss RL (1996b) 17β-estradiol potentiates kainate-induced currents via activation of the cAMP cascade. J Neurosci 16: 3620-3629

Gunaga KP, Menon KM (1973) Effect of catecholamines and ovarian hormones on cyclic AMP accumulation in rat hypothalamus. Biochem Biophys Res Commun 54: 440-448

Gustafsson JA (2000) An update on estrogen receptors. Semin Perinatol 24: 66-69 Gutteridge JM (1995) Lipid peroxidation and antioxidants as biomarkers of tissue

damage. Clin Chem 41: 1819-1828

Gutteridge JM, Halliwell B (2000) Free radicals and antioxidants in the year 2000.

A historical look to the future. Ann NY Acad Sci 899: 136-147

Häfner H, Behrens S, De Vry J, Gattaz WF (1991a) An animal model for the effects of estradiol on dopamine-mediated behavior: implications for sex differences in schizophrenia. Psychiatry Res 38: 125-134

Häfner H, Behrens S, De Vry J, Gattaz WF (1991b) Oestradiol anheances the vulnerability threshold for schizophrenia in women by an early effect on dopaminergic neurotransmission. Evidence from an epidemiological study and from animal experiments. Eur Arch Psychiatry Clin Neurosci 241: 65-68

Hagiwara M, Shimomura A, Yoshida K, Imaki J (1996) Gene expression and CREB phosphorylation induced by cAMP and Ca2+ in neuronal cells. Adv Pharmacol 36: 277-285

Hajimohammadreza I, Brammer M (1990) Brain membrane fluidity and lipid peroxidation in Alzheimer’s disease. Neurosci Lett 112: 333-337

Halbreich U, Rojansky N, Palter S, Tworek H, Hissin P, Wang K (1995) Estrogen augments serotonergic activity in postmenopausal women. Biol Psychiatry 37:

434-441

Halliwell B (1992) Reactive oxygen species and the central nervous system. J Neu- rochem 59: 1609-1623

Halliwell B, Gutteridge JM (1990) Role of free radicals and catytic metal ions in human disease: an overview. Methods Enzymol 186: 1-85

Hardingham GE, Bading H (1998) Nuclear calcium: a key regulator of gene expression. Biometals 11: 345-358

Hardy J, Allsop D (1991) Amyloid deposition as the central event in the etiology of Alzheimer’s disease. Trends Pharmacol Sci 12: 383-388

(25)

Harman D, Eddy DE, Noffsinger J (1976) Free radical theory of aging: inhibition of amyloidosis in mice by antioxidants; possible mechanism. J Am Geriatr Soc 24:

203-210

Hayashi T, Ishikawa T, Yamada K, Kuzuya M, Naito M, Hidaka H, Iguchi A (1994) Biphasic effect of estrogen on neuronal constitutive nitric oxide synthase via Ca(2+)-calmodulin dependent mechanism. Biochem Biophys Res Commun 203:

1013-1019

Honda K, Sawada H, Kihara T, Urushitani M, Nakamizo T, Akaike A, Shimohama S (2000) Phosphatidylinositol 3-kinase mediates neuroprotection by estrogen in cultured cortical neurons. J Neurosci Res 60: 321-327

Jansson L, Holmdahl R (1998) Estrogen-mediated immunosuppression in autoimmune diseases. Inflamm Res 47. 290-301

Joels M, Karst H (1995) Effects of estradiol and progesterone on voltage-gated calcium and potassium conductances in rat CA1 hippocampal neurons. J Neurosci 15: 4289-4297

Kawas C, Resnick S, Morrison A, Brookmeyer R, Corrada M, Zonderman A, Bacal C, Lingle DD, Metter E (1997) A prospective study of estrogen replacement therapy and the risk of developing Alzheimer’s disease: the Baltimore Longitu- dinal Study of Aging. Neurology 48: 1517-1521

Kelly MJ, Lagrange AH, Wagner EJ, Ronnekleiv OK (1999) Rapid effects of estrogen to modulate G protein-coupled receptors via activation of protein kinase A and protein kinase C pathways. Steroids 64: 64-75

Kew JN, Smith DW, Sofroniew MV (1996) Nerve growth factor withdrawal induces the apoptotic death of developing septal cholinergic in vitro: protection by cyclic AMP analogue and high potassium. Neuroscience 70: 329-339

Kim S, Liva SM, Dalal MA, Verity MA, Voskuhl RR (1999) Estriol ameliorates autoimmune demyelinating disease: implications for multiple sclerosis. Neuro- logy 52: 1230-1238

Klinge CM (2000) Estrogen receptor interaction with co-acivators and co-repressors.

Steroids 65: 227-251

Kobayashi Y, Shinozawa T (1997) Effect of dibutyryl cAMP and several reagents an apoptosis in PC12 cells induced by sialoglycopeptide from bovine brain. Brain Res 778: 309-317

Korenman SG (1969) Comparative binding affinity of estrogens and its relation to estrogenic potency. Steroids 13: 163-177

Kruman II, Pedersen WA, Springer JE, Mattson MP (1999) ALS-linked Cu/Zn-SOD mutation increases vulnerability of motor neurons to excitotoxicity by a mechanism involving increased oxidative stress and perturbed calcium homeostasis. Exp Neurol 160: 28-39

Lammers CH, D’Souza U, Qin ZH, Lee SH, Yajima S, Mouradian MM (1999) Regu- lation of striatal dopamine receptors by estrogen. Synapse 34: 222-227

Leist M, Nicotera P (1998) Calcium and neuronal death. Rev Physiol Biochem Phar- macol 132: 79-125

Lezoualc’h F, Behl C (1998) Transcription factor NF-kB: friend or foe of neurons?

Mol Psychiatry 3: 15-20

Matsuno A, Takekoshi S, Sanno N, Utsunomiya H, Ohsugi Y, Saito N, Kanemitsu H, Tamura A, Nagashima T, Osamura RY, Watanabe K (1997) Modulation of

(26)

protein kinases and microtubule-associated proteins and changes in ultrastruc- ture in female rat pituitary cells: effects of estrogen and bromocriptine. J Histo- chem Cytochem 45: 805-813

Mattsson MP, Rydel RE, Lieberburg I, Smith-Swintoskky VL (1993) Altered calcium signaling and neuronal injury: stroke and Alzheimer’s disease as examples. Ann NY Acad Sci 28: 679-721

McEwen BS, Alves SE (1999) Estrogen actions in the central nervous system. Endocr Rev 20: 279-307

McGeer EG, McGeer PL (1999) Brain inflammation in Alzheimer disease and the therapeutic inplications. Curr Pharm Des 5: 821-836

McGeer PL, McGeer EG (1996) Anti-inflammatory drugs in the fight against Alz- heimer’s disease. Ann NY Acad Sci 777: 213-220

McKay LI, Cidlowski JA (1998) Cross-talk between nuclear factor-κB and the steroid hormone receptors: mechanisms of mutual antagonism. Mol Endocrinol 12: 45- 56

Mermelstein PG, Becker JB, Surmeier DJ (1996) Estradiol reduces calcium currents in rat neostriatal neurons via a membrane receptor. J Neurosci 16: 595-604 Merry DE, Korsmeyer SJ (1997) Bcl-2 gene family in the nervous system. Annu Rev

Neurosci 20: 245-267

Michel MC, Rother A, Hiemke C, Graf R (1987) Inhibition of synaptosomal high- affinity uptake of dopamine and serotonin by estrogen agonists and antagonists.

Biochem Pharmacol 36: 3175-3180

Miller CP, Jirkovsky I, Hayhurst DA, Adelman SJ (1996) In vitro antioxidant effects of estrogens with a hindered 3-OH function on the copper-induced oxidation of low density lipoprotein. Steroids 61: 305-308

Miller L, Hunt JS (1996) Sex steroid hormones and macrophage function. Life Sci 59:

1-14

Minami T, Oomura Y, Nabekura J, Fukuda A (1990) 17beta-estradiol depolarization of hypothalamic neurons is mediated by cyclic AMP. Brain Res 519: 301-307 Moosmann B, Behl C (1999) The antioxidant neuroprotective effects of estrogens

and phenolic compounds are from their estrogenic properties. Proc Natl Acad Sci USA 96: 8867-8872

Moosmann B, Behl C (2000) Cytoprotective antioxidant function of tyrosine and tryptophan residues in transmembrane proteins. Eur J Biochem 267: 5687-5692 Mor G, Nilsen J, Horvath T, Bechmann I, Brown S, Garcia-Segura LM, Naftolin F (1999) Estrogen and microglia: a regulatory system that affects the brain. J Neu- robiol 40: 484-496

Morimoto BH, Koshland DE (1990) Induction and expression of long- and short-term neurosecretory potentiation in a neural cell line. Neuron 5: 875-880

Mudd LM, Torres J, Lopez TF, Montague J (1998) Effects of growth factors and estrogen on the development of septal cholinergic neurons from the rat. Brain Res Bull 45: 137-142

Mulnard RA, Cotman CW, Kawas C, van Dyck CH, Sano M, Doody R, Koss E, Pfeif- fer E, Jin S, Gamst A, Grundman M, Thomas R, Thal LJ (2000) Estrogen replacement therapy for treatment of mild to moderate Alzheimer disease: a ran-

(27)

domized controlled trial. Alzheimer’s Disease Cooperative Study. JAMA 283:

1007-1015

Murphy DD, Segal M (1996) Regulation of dendritic spine density in cultured rat hippocampal neurons by steroid hormones. J Neurosci 16: 4059-4068

Nohl H (1993) Involvement of free radicals in ageing: a consequence or cause of senescence. Brit Med Bull 49: 653-667

Olanow CW (1993) A radical hypothesis for neurodegeneration. Trends Neurosci 16:

439-444

Pappolla MA, Omar RA, Kim KS, Robakis NK (1992) Immunhistochemical evidence of oxidative stress in Alzheimer’s disease. Am J Pathol 140: 621-628

Pardridge WM, Moeller TL, Mietus LJ, Oldendorf WH (1980) Blood-brain barrier transport and brain sequestration of steroid hormones. Am J Physiol 239:

E96-E102

Perillo B, Sasso A, Abbondanza C, Palumbo G (2000) 17beta-estradiol inhibits apoptosis in MCF-7 cells, inducing bcl-2 expression via two estrogen-responsive elements present in the coding sequence. Mol Cell Biol 20: 2890-2901

Piotrowski J, Pietras T, Kurmanowska Z, Nowak D, Marczak J, Marks-Konczalik J, Mazerant P (1996) Effect of paraquat intoxication ond ambroxol treatment on hydrogen peroxide production and lipid peroxidation in selected organs of the rat. J Appl Toxicol 16: 501-507

Pozzo-Miller LD, Inoue T, Murphy DD (1999) Estradiol increases spine density and NMDA-dependent Ca²± transisents in spines of CA1 pyramidal neurons from hippocampal slices. J Neurophysiol 81: 1404-1411

Racay P, Kaplan P, Lehotsky J (1996) Control of Ca2+ homeostasis in neuronal cells.

Gen Physiol Biophys 15: 193-210

Raevskii KS (1997) Brain dopamine receptors: structure, functional role, and modulation by psychotropic substances. Vopr Med Khim 43: 553-565

Razandi M, Pedram A, Greene GL, Levin ER (1999) Cell membrane and nuclear estrogen receptors (ERs) originate from a single transcript: studies of ERalpha and ERbeta expressed in Chinese hamster ovary cells. Mol Endocrinol 13: 307-319 Regan RF, Guo Y (1997) Estrogens attenuate neuronal injury due to hemoglobin, chemical hypoxia, and excitatory amino acids in murine cortical cultures. Brain Res 764: 1333-1340

Riecher-Rössler A, Häfner H (1993) Schizophrenia and oestrogens – is there an as- ssociation? Eur Arch Psych Clin Neurosci 242: 323-328

Riecher-Rössler A, Häfner H, Stummbaum M, Maurer K, Schmidt R (1994) Can estradiol modulate schizophrenic symptomatology? Schizophr Bull 20: 203-214 Ruiz-Larrea B, Leal A, Lacort M, de Groot H (1994) Antioxidant effects of estradiol and 2-hydroxyestradiol on iron-induced lipid peroxidation of rat liver microso- mes. Steroids 59: 383-388

Russell SR (1996) Nuclear hormone receptors and the Drosophila ecdysone response. Biochem Soc Symp 62: 111-121

Saunders-Pullman R, Gordon-Elliott J, Parides M, Fahn S, Saunders HR, Bressman S (1999) The effect of estrogen replacement an early Parkinson’s disease. Neu- rology 52: 1417-1421

(28)

Sawada H, Ibi M, Kihara T, Urushitani M, Akaike A, Shimohama S (1998) Estradiol protects mesencephalic dopaminergic neurons from oxidative stress-induced neuronal death. J Neurosci Res 54: 707-719

Schmidt R, Fazekas F, Reinhart B, Kapeller P, Fazekas G, Orfenbacher S, Eber B, Schumacher M, Freidl W (1996) Estrogen replacement therapy in older women:

a neuropsychological and brain MRI study. J Am Geriatr Soc 44: 1307-1313 Schneider LS, Farlow MR, Henderson VW, Pogoda JM (1996) Effects of estrogen re-

placement therapy on response to tacrine in patients with Alzheimer’s disease.

Neurology 46: 1580-1584

Sergeev PV, Vladimirov IA, Seifulla RD, Denisov IP, Rudnev IN (1974) The role of the chemical structure of steroid hormones in inhibiting peroxidative lipid oxidation in mitochondrial membranes. Vopr Med Khim 20: 359-362

Shibata H, Spencer TE, Onate SA, Jenster G, Tsai SY, Tsai MJ, O’Malley BW (1997) Role of co-activators and co-repressors in the mechanism of steroid/thyroid receptor action. Recent Prog Horm Res 52: 141-164

Shughrue PJ, Lane MV, Merchenthaler I (1997) Comparative distribution of estrogen receptor-alpha and -beta mRNA in the rat central nervous system. J Comp Neurol 388: 507-525

Shyamal G, Guiot MC (1992) Activation of κB-specific proteins by estradiol. Proc Natl Acad Sci USA 89: 10628-10632

Sies H (1997) Oxidative stress: oxidants and antioxidants. Exp Physiol 82: 291-295 Singer CA, Rogers KL, Dorsa DM (1998) Modulation of Bcl-2 expression: a potential component of estrogen protection in NT2 neurons. Neuroreport 9: 2565-2568 Singer CA, Rogers KL, Strickland TM, Dorsa DM (1996) Estrogen protects primary

cortical neurons from glutamate toxicity. Neurosci Lett 212: 13-16

Singh M, Meyer EM, Simpkins JW (1995) The effect of ovariectomy and estradiol replacement on brain-derived neurotrophic factor mRNA expression in cortical an hippocampal brain regions of female Sprague Dawley rats. Endocrinology 136:

2320-2324

Singh M, Setalo G Jr, Guan X, Warren M, Toran-Allerand CD (1999) Estrogen-induced activation of mitogen-activated protein kinase in cerebral cortical ex- plants: convergence of estrogen and neurotrophin signaling pathways. J Neuro- sci 19: 1179-1188

Smith MA, Perry G, Richey PL, Sayre LM, Anderson VE, Beal MF, Kowall N (1996) Oxidative damage in Alzheimer’s. Nature 382: 120-121

Sohrabji F, Greene LA, Miranda RC, Toran-Allerand CD (1994) Reciprocal regulation of estrogen and NGF receptors by their ligands in PC12 cells. J Neurobiol 25: 974-988

Sohrabji F, Miranda R, Torran-Allerand CD (1995) Identification of a putative estrogen response element in the gene encoding brain-derived neurotrophic factor.

Proc Natl Acad Sci USA 92: 11110-11114

Sola C, Barron S, Tusell JM, Serratosa J (1999) The Ca2+/calmodulin signaling system in the neural response to excitability. Involvement of neuronal and glial cells. Prog Neurobiol 58: 207-232

Stumpf WE, Jennes L (1984) The A-B-C (Allocortex-Brainstem-Core) circuitry of endocrine-autonomic integration and regulation: a proposed hypothesis on the