**Estrogen and Brain Protection**

Xiaohua Ju, Daniel Metzger and Marianna Jung *University of North Texas Health Science Center USA* 

#### **1. Introduction**

In 1931, estrogen was originally discovered as a female sex hormone by Marrian and Butenandt (1931). Estrogen is responsible for maintaining female reproductive organs and functions. Beyond the effects on reproductive organs, the neuroprotective activities of estrogen have been identified by Simpkins et al. (1994) and thereafter by numerous other researchers (Viscoli et al., 2001). The simple classification of the mechanisms of estrogen is genomic and non-genomic processes. The genomic mechanisms of estrogen involve estrogen receptors located in DNA. Upon binding its receptors, estrogen stimulates the synthesis of a variety of neuro-modulatory proteins. A body of evidence indicates that estrogen receptors are not necessary for certain neuroprotective effects of estrogen. For example, estrogen scavenges harmful reactive free radical species (Dhandapani & Brann, 2002), inhibits apoptotic process (a certain type of cell death), and modulates signal transduction, all of which do not require nucleic estrogen receptors. Estrogen's neuroprotective properties may be the end result of well-orchestrated genomic and nongenomic processes.

There are three major forms of endogenous estrogens; 17β-estradiol, estrone, and estriol based on the hydroxyl or ketone ligand attached to the C17 position of the rightmost ring (D ring). Among these estrogens, 17β-estradiol (Figure 1) is the most potent, naturally occurring estrogen. Accordingly, 17β-estradiol has been the subject for neuroprotective properties in major neurodegenerative disorders such as stroke, Alzheimer's disease, Parkinson's disease, and ethanol withdrawal, and thus a topic of this book chapter.

Fig. 1. Chemical structures of 17β-estradiol, estriol, and estrone. Notice that 17β-estradiol has two hydroxyl (OH) groups, estriol has three hydroxyl groups, and estrone has one hydroxyl and one ketone group.

Estrogen and Brain Protection 141

another apoptotic protein, caspase-8. An increasing body of work has shown that Fas and Fas Ligand play an important role in the pathology of ischemic stroke (L. Liu et al., 2008; Rosenbaum et al., 2000). Both Fas and Fas ligand were upregulated by cerebral ischemia in brains of developing, as well as adult mice (Felderhoff-Mueser et al., 2000, 2003). Intriguingly, estrogen significantly reduced the level of Fas and the adaptor protein in mice undergoing post-ischemic stress (Jia et al., 2009). Furthermore, estrogen reduced the downstream apoptotic effectors such as caspase-8 and caspase-3. These findings suggest that estrogen protects against ischemia, in part, through its inhibitory effects on apoptosis

Estrogen also protects neurons from ischemia (Petito et al., 1987). Estrogen administered at physiological levels for two weeks before ischemia rescued the hippocampal neurons and ameliorated ischemia-induced cognitive deficits in female rats (Lebesgue, 2009). This study provides direct evidence that estrogen is neuroprotective against ischemia. There are at least two estrogen receptors in the brain, estrogen receptor-α and -β (Shughrue, 2004). Estrogen receptors are intracellular proteins which activate genomic as well as nongenomic effectors in neural cells (Maggi et al., 2004). Selective agonists for estrogen receptor-α or estrogen receptorβ was to were able to spare hippocampal neurons following ischemia. In addition, ICI 182780, a competitive antagonist for both estrogen receptors-α and -β, completely blocked estrogen's protection against post-ischemic stress (Miller et al., 2005). On the other hand, Lebesgue et al. (2009) found that a single injection of estrogen into the brain ventricle immediately after an ischemic event reduced both neuronal death and cognitive deficits. The genomic mechanism of estrogen is typically a slow process because it involves estrogen's receptors in the nuclei, affecting protein synthesis. Therefore, the rapid protection achieved by acute estrogen in

Above studies suggest that estrogen exerts neuroprotection against ischemia through its

When ischemic patients receive blood supply (reperfusion), the introducing blood itself can induce significant damage to the brain. The damage is largely attributable to very active harmful oxygen species such as the reactive superoxide anion (Peters et al., 1998; Sugawara et al., 2005). These oxygen species give rise to other damaging oxygen species, for example, hydroxyl ion and peroxynitrite (Mattson et al., 2000). Estrogen contains profound antioxidant properties that mediate its protective effects on neurons. Estrogen directly scavenges free radicals by oxidizing its hydroxyl group attached to the C3 position of A ring (left most ring) through an enzyme, NADPH. The A ring then becomes the phenoxyl radical ring, a certain type of a ring structure containing free radicals. The phenoxyl radical ring is converted to para-quinol ring by scavenging further free radicals like -OH. This para-quinol ring structure finally becomes the original A ring of 17β-estradiol through NADPH (Prokai et al., 2003; Prokai-Tatrai et al., 2008). The important point of this cyclic reaction is that 17βestradiol is rejuvenated after it absorbs harmful free radicals (Figure 2). Indeed, estrogen attenuated superoxide production in hippocampal neurons after stroke (Q.G. Zhang et al., 2009). In addition to this directly scavenging of free radicals, estrogen upregulates antioxidant enzymes and chelates redox-active metal ions. In terms of estrogen receptor, Zhang et al. (2009) suggested that the antioxidant effect of estrogen is independent of estrogen receptor-α. They found that estrogen deprivation abolished the antioxidant and

anti-apoptotic property and the mechanisms associated with estrogen receptors.

Lebesgue's study may indicate the non-genomic effects of estrogen.

associated with Fas (Jia et al., 2009).

**2.3 Oxidative stress** 

#### **2. Estrogen and ischemia**

#### **2.1 Introduction**

Stroke is the sudden loss of brain function that is attributed to ischemia which indicates a disturbance in the blood supply to the brain. The affected brain area is unable to function, resulting in an inability to move limbs, understand or formulate speech, or an inability to see the visual field. It is the leading cause of adult disability in the United States and Europe and the second leading cause of death worldwide (Feigin, 2005). Women have a higher risk, due to their longer lifespan and are also more likely to have fatal strokes than men (Bushnell, 2008). Especially women in the 45−54 age range (perimenopause) are reportedly at a higher risk for stroke (Towfighi et al., 2007). This study suggests that declining levels of ovarian hormones perpetuate the risk for this neurovascular disease. The depletion of ovarian hormones also alters stroke outcomes. In postmenopausal women, stroke-associated disability and fatality are worse compared to men (Niewada et al., 2005). If ovarian hormones influence stroke, it is not surprising to see sex differences in the severity of stroke. For instance, a smaller area of tissue death was found in young adult female mice (Park et al., 2006) compared to their age-matched males. Furthermore, the sex difference in stroke infarct (area of tissue death) was abolished when the female mice were ovariectomized, suggesting that ovarian steroids mediate the neuroprotection seen in younger females (Selvamani et al., 2010).

Among ovarian hormones, 17β-estradiol seems to possess greater protective properties than other ovarian hormones. 17β-estradiol mitigated brain inflammation (Suzuki et al., 2009) and blood-brain barrier dysfunction (R. Liu et al., 2005). 17β-estradiol increased the blood flow of the cerebrum (Pelligrino et al., 1998), the ability of neurons to transmit signals (synaptic plasticity), and cognitive function (Sherwin, 2007). By comparison to these protections in animal studies, human studies showed somewhat inconsistent results. In large clinical trials, such as the Women Estrogen Stroke Trial and the Women's Health Initiative, estrogen treatment failed to exert the beneficial effects on stroke incidence (Viscoli et al., 2001). Rather, the clinical study showed that estrogen treatment increased the stroke risk and worsened neurological outcomes in postmenopausal women (Viscoli et al., 2001). Similarly, the Women's Health Initiative study reported an increased risk for stroke following the treatment with estrogen or another female hormone progestin (synthetic progesterone) (Wassertheil-Smoller et al., 2003). Notably, many women in these clinical trials were postmenopausal for several years prior to the hormone treatment. The unexpected negative results might have been due to prolonged estrogen-withdrawal before estrogen was reintroduced (De et al., 2009). Other researchers suggested that differences in the duration of treatment, timing of administration, sex, age, and an ischemia model contributed to the inconsistent outcome of estrogen therapy (J. Li, 2011; Sherwin, 2009).

#### **2.2 Apoptosis**

Apoptosis is a type of cell death that normally occurs to replace aged or injured cells with newer cells. However, excessive or defective apoptosis is often present at regions affected by stroke (Dirnagl et al., 1999). Fas is a receptor protein that triggers apoptotic cell death upon the binding of its ligand (Fas ligand). The structure of Fas contains a particular region, called 'death domain'. There is a cytoplasmic protein that favors to associate with the death domain of Fas. Therefore, it is called Fas-associated death domain adaptor protein. When this adaptor protein binds to the death domain of Fas, it subsequently activates

Stroke is the sudden loss of brain function that is attributed to ischemia which indicates a disturbance in the blood supply to the brain. The affected brain area is unable to function, resulting in an inability to move limbs, understand or formulate speech, or an inability to see the visual field. It is the leading cause of adult disability in the United States and Europe and the second leading cause of death worldwide (Feigin, 2005). Women have a higher risk, due to their longer lifespan and are also more likely to have fatal strokes than men (Bushnell, 2008). Especially women in the 45−54 age range (perimenopause) are reportedly at a higher risk for stroke (Towfighi et al., 2007). This study suggests that declining levels of ovarian hormones perpetuate the risk for this neurovascular disease. The depletion of ovarian hormones also alters stroke outcomes. In postmenopausal women, stroke-associated disability and fatality are worse compared to men (Niewada et al., 2005). If ovarian hormones influence stroke, it is not surprising to see sex differences in the severity of stroke. For instance, a smaller area of tissue death was found in young adult female mice (Park et al., 2006) compared to their age-matched males. Furthermore, the sex difference in stroke infarct (area of tissue death) was abolished when the female mice were ovariectomized, suggesting that ovarian steroids mediate the neuroprotection seen in younger females

Among ovarian hormones, 17β-estradiol seems to possess greater protective properties than other ovarian hormones. 17β-estradiol mitigated brain inflammation (Suzuki et al., 2009) and blood-brain barrier dysfunction (R. Liu et al., 2005). 17β-estradiol increased the blood flow of the cerebrum (Pelligrino et al., 1998), the ability of neurons to transmit signals (synaptic plasticity), and cognitive function (Sherwin, 2007). By comparison to these protections in animal studies, human studies showed somewhat inconsistent results. In large clinical trials, such as the Women Estrogen Stroke Trial and the Women's Health Initiative, estrogen treatment failed to exert the beneficial effects on stroke incidence (Viscoli et al., 2001). Rather, the clinical study showed that estrogen treatment increased the stroke risk and worsened neurological outcomes in postmenopausal women (Viscoli et al., 2001). Similarly, the Women's Health Initiative study reported an increased risk for stroke following the treatment with estrogen or another female hormone progestin (synthetic progesterone) (Wassertheil-Smoller et al., 2003). Notably, many women in these clinical trials were postmenopausal for several years prior to the hormone treatment. The unexpected negative results might have been due to prolonged estrogen-withdrawal before estrogen was reintroduced (De et al., 2009). Other researchers suggested that differences in the duration of treatment, timing of administration, sex, age, and an ischemia model contributed to the inconsistent outcome of estrogen therapy (J. Li, 2011; Sherwin, 2009).

Apoptosis is a type of cell death that normally occurs to replace aged or injured cells with newer cells. However, excessive or defective apoptosis is often present at regions affected by stroke (Dirnagl et al., 1999). Fas is a receptor protein that triggers apoptotic cell death upon the binding of its ligand (Fas ligand). The structure of Fas contains a particular region, called 'death domain'. There is a cytoplasmic protein that favors to associate with the death domain of Fas. Therefore, it is called Fas-associated death domain adaptor protein. When

this adaptor protein binds to the death domain of Fas, it subsequently activates

**2. Estrogen and ischemia** 

**2.1 Introduction** 

(Selvamani et al., 2010).

**2.2 Apoptosis** 

another apoptotic protein, caspase-8. An increasing body of work has shown that Fas and Fas Ligand play an important role in the pathology of ischemic stroke (L. Liu et al., 2008; Rosenbaum et al., 2000). Both Fas and Fas ligand were upregulated by cerebral ischemia in brains of developing, as well as adult mice (Felderhoff-Mueser et al., 2000, 2003). Intriguingly, estrogen significantly reduced the level of Fas and the adaptor protein in mice undergoing post-ischemic stress (Jia et al., 2009). Furthermore, estrogen reduced the downstream apoptotic effectors such as caspase-8 and caspase-3. These findings suggest that estrogen protects against ischemia, in part, through its inhibitory effects on apoptosis associated with Fas (Jia et al., 2009).

Estrogen also protects neurons from ischemia (Petito et al., 1987). Estrogen administered at physiological levels for two weeks before ischemia rescued the hippocampal neurons and ameliorated ischemia-induced cognitive deficits in female rats (Lebesgue, 2009). This study provides direct evidence that estrogen is neuroprotective against ischemia. There are at least two estrogen receptors in the brain, estrogen receptor-α and -β (Shughrue, 2004). Estrogen receptors are intracellular proteins which activate genomic as well as nongenomic effectors in neural cells (Maggi et al., 2004). Selective agonists for estrogen receptor-α or estrogen receptorβ was to were able to spare hippocampal neurons following ischemia. In addition, ICI 182780, a competitive antagonist for both estrogen receptors-α and -β, completely blocked estrogen's protection against post-ischemic stress (Miller et al., 2005). On the other hand, Lebesgue et al. (2009) found that a single injection of estrogen into the brain ventricle immediately after an ischemic event reduced both neuronal death and cognitive deficits. The genomic mechanism of estrogen is typically a slow process because it involves estrogen's receptors in the nuclei, affecting protein synthesis. Therefore, the rapid protection achieved by acute estrogen in Lebesgue's study may indicate the non-genomic effects of estrogen.

Above studies suggest that estrogen exerts neuroprotection against ischemia through its anti-apoptotic property and the mechanisms associated with estrogen receptors.

#### **2.3 Oxidative stress**

When ischemic patients receive blood supply (reperfusion), the introducing blood itself can induce significant damage to the brain. The damage is largely attributable to very active harmful oxygen species such as the reactive superoxide anion (Peters et al., 1998; Sugawara et al., 2005). These oxygen species give rise to other damaging oxygen species, for example, hydroxyl ion and peroxynitrite (Mattson et al., 2000). Estrogen contains profound antioxidant properties that mediate its protective effects on neurons. Estrogen directly scavenges free radicals by oxidizing its hydroxyl group attached to the C3 position of A ring (left most ring) through an enzyme, NADPH. The A ring then becomes the phenoxyl radical ring, a certain type of a ring structure containing free radicals. The phenoxyl radical ring is converted to para-quinol ring by scavenging further free radicals like -OH. This para-quinol ring structure finally becomes the original A ring of 17β-estradiol through NADPH (Prokai et al., 2003; Prokai-Tatrai et al., 2008). The important point of this cyclic reaction is that 17βestradiol is rejuvenated after it absorbs harmful free radicals (Figure 2). Indeed, estrogen attenuated superoxide production in hippocampal neurons after stroke (Q.G. Zhang et al., 2009). In addition to this directly scavenging of free radicals, estrogen upregulates antioxidant enzymes and chelates redox-active metal ions. In terms of estrogen receptor, Zhang et al. (2009) suggested that the antioxidant effect of estrogen is independent of estrogen receptor-α. They found that estrogen deprivation abolished the antioxidant and

Estrogen and Brain Protection 143

2007). In lipopolysaccharide-induced brain inflammation, estrogen suppressed both resident microglial activation and the recruitment of peripheral T and B cells (Vegeto et al., 2001). These studies provide empirical evidence that the anti-inflammatory effect of estrogen plays

Collectively, cumulative evidence indicates that the convergence of endocrine changes, especially estrogen, impacts the pathophysiology of stroke and ischemic injury. It appears that estrogen protects against ischemia through multiple factors associated with apoptosis, inflammation, redox, and estrogen receptors. Understanding these mechanisms may

Alzheimer's disease is characterized as a gradual failure of memory, cognition, and bodily functions, ultimately leading to death. Although the exact etiology and mechanisms are unknown, the abnormal accumulation of a particular protein, called Amyloid β, has long been proposed as the most likely culprit in the pathogenesis of this disease (Hardy & Selkoe, 2002; Tanzi & Bertram, 2005). In a healthy brain, Amyloid β remains at a steady-state level as a result of the metabolic balance between production of Amyloid β from amyloid precursor protein and removal by cellular uptake and proteolytic degradation (Saido, 1998; Selkoe, 2000). Such a dynamic equilibrium, however, could be altered by genetic or environmental factors that may lead to Alzheimer's disease. It has been hypothesized that Amyloid β is folded into a oligomeric form or a fibrillar (cable-like strings) form (Yamin et al., 2008), both of which are more neurotoxic than Amyloid β itself. Of several different Amyloid β peptides produced, products of Amyloid β-40 and Amyloid β-42 residues are the most common constituents of amyloid plaques, and are widely accepted as the primary trigger for Alzheimer's disease (St George-Hyslop, 2000). In brains with early onset Alzheimer's disease, Amyloid β excessively accumulates. This may be due to the mutations of presenelin genes, which provoke the overproduction of Amyloid β from amyloid precursor protein (Hardy, 2004). In late-onset Alzheimer's disease, which constitutes more than 90% of the disease, the excess accumulation of Amyloid β has been associated with

Women are more likely to develop Alzheimer's disease after adjusting for age (Andersen et al., 1999). After menopause, the decline of estrogen levels in the brain may render neurons more susceptible to age-related neurodegenerative processes (Coffey et al., 1998). Estrogen therapy, when initiated at the onset of menopause, has reduced the risk or delayed the onset of Alzheimer's disease in women (LeBlanc et al., 2001; Zandi et al., 2002). A recent randomized control trial indicated that estrogen treatment had a beneficial effect on verbal memory in men with mild cognitive impairment (Sherwin et al., 2011 in press). However, clinical studies of estrogen therapy in non-demented and menopausal women have yielded inconclusive results (Craig & Murphy, 2010; Sano et al., 2008). In addition, estrogen administration induced beneficial effects on neuronal function and survival through improving mitochondrial function in healthy neurons (Brinton, 2008). When neurons became unhealthy, estrogen exposure had a detrimental effect (Brinton, 2008). This discrepancy may be due to differences in neurological health, age, hormonal status, the severity of symptoms, the type of menopause (surgical vs. natural), and the type of estrogen compound used (Brinton, 2009). Also, the age when estrogen therapy is initiated, may in part determine the

ultimately contribute to better research and therapeutic strategies for stroke therapy.

abnormal Amyloid β degrading proteases (Nalivaeva et al., 2008).

a protective role in immune responses to stroke.

**3. Estrogen and Alzheimer's disease** 

**3.1 Introduction** 

neuroprotective effects on the hippocampus without affecting estrogen receptor-α mediated effect on the uterus. At the very least, these findings indicate that estrogen protects against ischemia through antioxidant properties.

Fig. 2. Schematic illustration of the free radical scavenging antioxidant activity of 17βestradiol. 17β-estradiol captures •OH, producing the phenoxyl radical and then bioreversible quinol. The quinol is rapidly converted to the parent estrogen via a NAD(P)Hdependent reductive aromatization to perpetuate the antioxidant action. During this process, •OH is detoxified to H2O (Prokai et al., 2003; Prokai-Tatrai et al., 2008).

#### **2.4 Inflammation\Immune response**

Inflammation is a critical event that occurs upon ischemic insults. Post-stroke events include the stimulation and subsequent degeneration of lymphoid organs such as the spleen and thymus (Offner et al., 2009). The activation of these lymphoid organs likely leads to immunocyte translocation into brain, exacerbating the evolving brain ischemia (Ajmo et al., 2008). Proinflammatory genes are rapidly induced in brain after ischemic injury, including genes synthesizing TNF-α (X. Wang et al., 1994), IL-6 (X. Wang et al., 1995), IL-1β (X. Wang et al., 1994), and interferon inducible protein-10 (IP-10) (X. Wang et al., 1998). The subsequent degeneration of lymphoid organs leads to immunodepression. Humans who survive the initial brain insult, may succumb to fatal infection due to the immunodepression (Dirnagl et al., 2007; Meisel et al., 2005).

Estrogen deficiency during menopause is associated with a proinflammatory phenotype, namely 'T cell expansion' in bone marrow that secretes inflammatory proteins such as IL-1, TNF-α, and IL-6 (Pfeilschifter et al., 2002). In a study done by Zhang et al. (2010), estrogen partially restored immune reactivity in ovariectomized females by increasing spleen cell population and cytokine responses (B. Zhang et al., 2010). In agreement, estrogen induced anti-inflammatory cytokines in the spleen after traumatic brain injury (Bruce-Keller et al.,

neuroprotective effects on the hippocampus without affecting estrogen receptor-α mediated effect on the uterus. At the very least, these findings indicate that estrogen protects against

Fig. 2. Schematic illustration of the free radical scavenging antioxidant activity of 17βestradiol. 17β-estradiol captures •OH, producing the phenoxyl radical and then

bioreversible quinol. The quinol is rapidly converted to the parent estrogen via a NAD(P)Hdependent reductive aromatization to perpetuate the antioxidant action. During this process, •OH is detoxified to H2O (Prokai et al., 2003; Prokai-Tatrai et al., 2008).

Inflammation is a critical event that occurs upon ischemic insults. Post-stroke events include the stimulation and subsequent degeneration of lymphoid organs such as the spleen and thymus (Offner et al., 2009). The activation of these lymphoid organs likely leads to immunocyte translocation into brain, exacerbating the evolving brain ischemia (Ajmo et al., 2008). Proinflammatory genes are rapidly induced in brain after ischemic injury, including genes synthesizing TNF-α (X. Wang et al., 1994), IL-6 (X. Wang et al., 1995), IL-1β (X. Wang et al., 1994), and interferon inducible protein-10 (IP-10) (X. Wang et al., 1998). The subsequent degeneration of lymphoid organs leads to immunodepression. Humans who survive the initial brain insult, may succumb to fatal infection due to the immunodepression

Estrogen deficiency during menopause is associated with a proinflammatory phenotype, namely 'T cell expansion' in bone marrow that secretes inflammatory proteins such as IL-1, TNF-α, and IL-6 (Pfeilschifter et al., 2002). In a study done by Zhang et al. (2010), estrogen partially restored immune reactivity in ovariectomized females by increasing spleen cell population and cytokine responses (B. Zhang et al., 2010). In agreement, estrogen induced anti-inflammatory cytokines in the spleen after traumatic brain injury (Bruce-Keller et al.,

ischemia through antioxidant properties.

**2.4 Inflammation\Immune response** 

(Dirnagl et al., 2007; Meisel et al., 2005).

2007). In lipopolysaccharide-induced brain inflammation, estrogen suppressed both resident microglial activation and the recruitment of peripheral T and B cells (Vegeto et al., 2001). These studies provide empirical evidence that the anti-inflammatory effect of estrogen plays a protective role in immune responses to stroke.

Collectively, cumulative evidence indicates that the convergence of endocrine changes, especially estrogen, impacts the pathophysiology of stroke and ischemic injury. It appears that estrogen protects against ischemia through multiple factors associated with apoptosis, inflammation, redox, and estrogen receptors. Understanding these mechanisms may ultimately contribute to better research and therapeutic strategies for stroke therapy.
