Mitochondrial Mechanisms of Estrogen Neuroprotection

Abstract

Mitochondria have become a primary focus in our search not only for the mechanism(s) of neuronal death but also for neuroprotective drugs and therapies that can delay or prevent Alzheimer’s disease and other chronic neurodegenerative conditions. This is because mitochrondria play a central role in regulating viability and death of neurons, and mitochondrial dysfunction has been shown to contribute to neuronal death seen in neurodegenerative diseases. In this article, we review the evidence for the role of mitochondria in cell death and neurodegeneration and provide evidence that estrogens have multiple effects on mitochondria that enhance or preserve mitochondrial function during pathologic circumstances such as excitotoxicity, oxidative stress, and others. As such, estrogens and novel non-hormonal analogs have come to figure prominently in our efforts to protect neurons against both acute brain injury and chronic neurodegeneration.

1. Mitochondrial and cell death mechanisms

Mitochondrial oxidative phosphorylation is essential for neurons to meet their high ATP demand, and neuronal viability is imperiled when this ATP production is even transiently diminished. In addition to a bioenergetic crisis, mitochondrial impairment also produces a concomitant increase in production of reactive oxygen species [1]. Mitochondrial failure is the key event in the pathogenic cascade leading to ischemia-induced cell death from both necrosis and apoptosis [1, 2]. Under conditions of oxidative stress and excessive cytoplasmic Ca²⁺ loading, mitochondria undergo a loss of the impermeability of the inner mitochondrial membrane that completely collapses the mitochondrial membrane potential (ΔΨm), a process called permeability transition. Such irreversible collapse of ΔΨm is accompanied by mitochondrial swelling and release of cytochrome c into the cytoplasm, where it activates certain caspases and induces apoptotic cell death [2, 3].

Normally, antioxidant defense systems reduce radical-induced damage by scavenging free radicals. However, accelerated mitochondrial radical production can overwhelm these defenses, inducing peroxidation of membrane lipids and impeding oxidative phosphorylation via inactivation of Fe–S clusters within the electron transport system. The resulting acute loss of ATP causes the transmembrane ion-dependent ATPases to fail, thereby inducing necrosis from osmotic collapse [4, 5]. In addition, less severe mitochondrial dysfunction initiates apoptosis in response to a variety of stressors. The excitotoxic process, for example, entails excessive stimulation of excitatory receptors, including NMDA and other voltage and metabotropic receptors, by exposure to elevated levels of dicarboxylate neurotransmitters, like glutamate, as well as several xentobiotics, like kainic acid. Excessive elevation of Ca²⁺ during glutamate excitotoxicity undermines mitochondrial integrity and accelerates free radical production, ultimately leading to cell death [6, 7]. As a result, mitochondria are seen as key modulators of neuronal viability during excitotoxicity [4, 5].

2. Mitochondria and Chronic Neurodegenerative Diseases

Chronic diseases with bioenergetic and oxidative etiologies that implicate mitochondrial dysfunction include Alzheimer’s disease (AD) and Parkinson’s disease (PD), as well as amyotrophic lateral sclerosis (ALS), Huntington’s disease and Freidreich’s ataxia, among many others [8]. Although the etiology of Alzheimer’s disease is multifactoral [9], a consistent finding is hypometabolism of glucose in those brain regions affected by the disease [10] that can be detected very early in the disease [11, 12]. Although hypometabolism could simply reflect neuronal loss in the effected regions, mitochondrial dysfunction can be more directly implicated in AD. Amyloid-β (Aβ ) causes an oxidative and bioenergetic crises [13–15], and in turn enhances the production of Aβ [16]. In addition, other aspects of normal mitochondrial physiology, such as intracellular distribution, could participate in AD progression secondary to the mitochondrial failure. For example, peri-nuclear mitochondria are more actively replicating than those elsewhere in the neuron, and are then distributed to the energetically demanding synapses. Impairment of normal axonal transport of mitochondria is likely due to the breakdown of microtubules from the hyperphosphorylation of the microtubule-associated protein, tau [17, 18].

Mitochondria from AD subjects are hypofunctional [19], produce excessive reactive oxygen species [20, 21], and show a defect in respiratory complex IV (C-IV) [22, 23]. When inserted into transformed cells depleted of their endogenous mtDNA, mtDNA from AD patients produces a phenotype in the resulting cytoplasmic hybrids (cybrids) of increased oxidative stress, propensity towards apoptosis and C-IV impairment [24, 25], suggesting that some of the cellular pathology in AD reflects mitochondrial defects. While mitochondrial impairment can be a consequence as well as a primary cause of the disease [26], mitochondrial dysfunction is clearly involved in progressive neuronal death in AD and as such represents a viable therapeutic target.

3. Estrogens and Neuroproptection

The neuroprotective effects of estrogens have been widely described against a variety of toxicities including serum deprivation [27, 28], Aβ toxicity [29, 30] and oxidative stress [31–34], among others, in hippocampal, amygdala, cortical and mesencephalic neurons [35–37]. Similarly, in rodent animal models, estrogens have been shown to attenuate neuronal loss following induction of cerebral ischemia [38–41], following kainic acid administration [42], and in a contusion physical injury model [43].

Retrospective epidemiological studies in post-menopausal women indicate that hormone/estrogen replacement therapy is associated with a reduction in the risk of Alzheimer’s disease as well as a delay its the progression[44–47]. In addition, several clinical studies have reported improvement of cognitive functions in female AD patients receiving estrogen replacement therapy [48–54]. The positive and protective role of estrogens in female AD patients may be in part due to the effects on mitochondrial function and/or stability.

Neuroprotection can be achieved by non-genomic mechanisms independent of interactions with the classical estrogen receptors (ERs). In many of the studies cited above using diverse cell types and cytotoxic insults, naturally occurring 17β-estradiol (17β-E2) and its diastereomer, 17α-estradiol (17α-E2), which is almost inactive as a hormone, have been found to be equally neuroprotective. Similarly, studies with a variety of novel estrogen analogues have confirmed that the structure-activity relationship for neuroprotection with this class of compounds differs significantly from the structural requirements for ER-dependent gene transcription [55–59]. For example, the complete enantiomer of 17β-E2 (ent-17β-E2) has identical physiochemical properties as 17β-E2 except for interactions with other stereospecific molecules such as ERs. Ent-17β-E2 is reported to interact only weakly with uterine-derived ERs [60, 61] and lacks estrogenic effects on reproductive tissues in rodents [62, 63]. In fact, some reports indicate that ent-17β-E2 exerts slight anti-uterotrophic activity and can antagonize the uterotrophic effects of 17β-E2 [63, 64]. We have reported that ent-17β-E2 exerts neuroprotective effects both in vitro and in vivo in the absence of stimulation of other estrogen-responsive tissues [57]. Novel estrogen analogues such as ZYC3, 2-(1-adamantyl)-3-hydroxyestra-1,3,5(10)-trien-17-one, do not bind to estrogen receptors [59], yet possess both neuroprotective and vasoactive effects in an in vivo model of ischemic stroke, which offers the possibility of clinical application for stroke without the estrogen receptor mediated side effects of estrogens [65].

We defined the essential requirement for neuroprotection to be the A ring of estrogens [56] and have been able to enhance neuroprotective activity via chemical modification of the estrogen scaffold [59]. For example, increasing the stability of the A-ring phenolic radical yields an increased neuroprotective potency [59]. Other approaches to augment potency includes placement of constituents on the 2 and/or 4 position of the phenolic A ring of estradiol or estrone, unstaturation of the B or C rings of the planar steroid, synthesizing diastiomers or enantiomers of estrogens, and combinations of these approaches [59].

4. Estrogens and Mitochondrial Function

Therapeutic agents that are able to stabilize mitochondrial function during ischemia are expected to be effective neuroprotectants, preventing apoptosis by maintaining functionally intact mitochondria. A major challenge then in understanding the neuroprotective effects of estrogens and their potential for application in acute brain damage and more chronic neurodegenerative disease is determining their mitochondrial mechanism(s) of action, and the four possibilities discussed below are under active investigation.

4.1. Historic Observations

Several studies have shown that estrogens and related compounds may exert direct or indirect effects on mitochondrial function. It has been reported that 17β-E2 inhibits mitochondrial F0F1-ATPase by binding to one of its subunits, the oligomycin sensitivity conferring protein [66, 67]. A testable model for cytoprotective activity is that such inhibition impedes the reverse reactions of the ATPase during ischemia, using ATP to generate a membrane potential, thereby preserving cytosolic ATP arising from glycolysis. Other investigators have found that estradiol augments sequestration of cytosolic Ca²⁺ by mitochondria in the presence of glutamate which, given the diffusion limited kinetics of the Ca²⁺ uniporter, is prima facie evidence of mitochondrial stabilization [68]. Moderating Ca²⁺-induced permeability transition during excitoxicity, and in so doing maintaining OXPHOS, would clearly be beneficial. In another study, 17β-E2 has been shown to stabilize mitochondrial function and thereby protect neural cells against the pro-apoptotic action of mutant presenilin-1 [69]. Anti-apoptotic action may also involve receptor mediated transactivation such as the upregulation of Bcl-2, known to occur upon estradiol exposure.

To further illuminate the protective effects of estrogens and other polycyclic phenols on mitochondrial integrity, we examined a number of markers of mitochondrial activity in SK-N-SH neuroblastoma cells exposed to 3-nitroproprionic acid (3-NPA). The latter inhibits oxidative phosphorylation by blocking electron entry into electron transport at the level of succinate dehydrogenase (respiratory Complex II), and so serves to model not only acute impaired energy production, seen during cerebral ischemia [38, 70, 71], but also more modest energy impairment associated with chronic neurodegenerative disease [72]. We reported that 17β-E2 prevents depletion of ATP, preserves ΔΨm, and inhibits production of reactive oxygen species [1] caused by 3-NPA [73]. Further, we made similar observations of the mitochondrial effects of estrogens when H₂O₂ was used to impair mitochondrial function [74].

Estrogens have been shown to protect neurons against various other mitochondrial toxins such as MPTP, an inhibitor of respiratory complex I [75–79]. Estrogens increase the expression of longevity-associated genes, including those encoding the antioxidant enzymes superoxide dismutase and glutathione peroxidase. As a result, mitochondria from females produce fewer reactive oxygen species [1] than those from males, an effect that may contribute to increased female longevity [80–82].

4.2. Nuclear Transcriptional Effects

Estrogens regulate both nuclear and mitochondrial protein expression, although this mechanism does not pertain to estrogen analogs that do not interact with ERs. For example, estrogens increase expression of glucose transporter subunits thereby promoting glucose transport into neurons [83, 84], and also increase expression of glycolytic enzymes including hexokinase, phosphofructokinase, phosphoglycerate kinase [85], and components of pyruvate dehydrogenase complex [86]. Moreover, estrogens also regulate enzymes involved in TCA cycle, such as aconitase 2 and malate dehydrogenase [86]. Estrogens enhance glycolytic activity that is coupled with an increase in glutamate turnover, evidenced by the increased expression of glutamate dehydrogenase and glutamate oxaloacetate transaminase-2 [86]. The latter impacts the generation of neurotoxic free ammonium and reduces excitotoxic free glutamate [87]. Consistent with their actions on glycolytic metabolism, estrogens also enhance the expression of F1 subunits of ATP synthase [86]. In toto these findings indicate that estrogens promote glucose utilization via regulation of nuclear encoded genes.

In addition, estrogens also directly regulate the expression of mitochondrial stress responsive factors encoded by nuclear DNA. The Bcl-2 family of proteins is important regulators of the mitochondrial pathway of apoptosis, determining whether the mitochondria initiate the cell death program by releasing proapoptotic factors such as cytochrome c. The family of Bcl-2 proteins consists of anti- and pro-apoptotic members and their interactions play a key role in regulating apoptosis in cells [88]. Estrogens increase anti-apoptotic proteins, Bcl-2 and Bcl-xL, which prevent permeability transition [68]. In primary neuronal cultures, estrogens upregulate expression of antiapoptotic Bcl-w and downregulate expression of proapoptotic bim in an ER-dependent manner [89].

Estrogens also enhance free radical defenses by increasing the expression and activity of glutaredoxin, gamma-glutamylcysteine synthetase, and MnSOD, all encoded by nuclear genes [88, 90–93]. In addition, an increase in peroxiredoxin-V was also found upon estrogen treatment [86]. The action of estrogens on glutaredoxin, gamma-glutamylcysteine synthetase, and MnSOD expression and activation are inhibited by the ER antagonist, ICI182780, suggesting an ER-dependent mechanism.

Recently, it has been demonstrated that estradiol stimulates transcription of nuclear respiratory factor-1 (NRF-1) via binding of E2-ectivated ER to the estrogen response element [94]. In turn, NRF-1 binds to its response elements and increases the transcription of mitochondrial transcription factor A (Tfam) and mitochondrial transcription factor B types 1 and 2 (TFBs), which are imported into mitochondria where mitochondrial DNA (mtDNA) replication and transcription are increased.

4.3. Mitochondrial Transcriptional Effects

Akin to bacterial DNA, mtDNA is an intronless, circular molecule of about 16.6 kb that lacks histones and it encodes thirty-seven genes. Of the thousands of proteins in the mitochondrial proteome, mtDNA encodes thirteen proteins are the essential component of the enzyme complexes of the oxidative phosphorylation system. In mammals, the mitochondrial genome is maternally inherited and unlike the nuclear genome in non-dividing, terminally differentiated cells, mtDNA is continuously replicated during mitochondrial reproduction [95]. The strands of the DNA duplex can be distinguished as heavy (H) and light (L) strand based upon the G+T base composition which results in different buoyant densities of each stand. Most information is encoded on the H strand, with genes for two tRNAs, 14tRNAs, and 12 proteins. The L strand codes the other eight tRNAs and a single protein.

Many of the electron transport chain components encoded by mitochondrial genome are regulated by estrogens. Van Itallie and Dannies [96] found a 16-fold increase of cytochrome c oxidase subunit II mRNA upon 17β-E2 treatment in rat pituitary tumor cells. An estrogen-induced increase of cytochrome c oxidase subunit III transcript was also observed [97]. The estrogen-regulated mitochondrial encoded transcripts have been extended to all three subunits of the complex IV and subunits 6 and 8 of ATP synthase [98, 99]. More recently, Nilsen et al. [86] identified 4 of the 7 subunits of complex I encoded by mitochondrial genome were regulated by 17β-E2. Given the single promoter for each strand of mtDNA and the broad range of estrogen-regulated mitochondrial transcripts, the action of estrogens on mitochondrial transcription seems universal, not specific to any single gene.

Although it is not clear how estrogens regulate mitochondrial gene expression, studies have shown that enhancement of mitochondrial transcripts by estrogens can be blocked by ER antagonist, ICI182780, suggesting an ER-dependent mechanism [100, 101]. This notion is further supported by the newly-identified mitochondrial localization of ERs, especially ERβ [100, 102], and up-regulation of mitochondrial complex IV by the ERβ selective ligand, diarylpropionitrile (DPN), has been demonstrated [103]. The crystal structure of ERβ has been well described and it shares a highly conserved architecture with other nuclear receptors such as ERα. Although ERα and ERβ have nearly identical DNA-binding domain, increasing evidence indicates that they regulate the expression of a distinct set of genes [104, 105]. This distinction could be partly due to different compartmentalization of ERα and ERβ or recruitment of different coactivators and adaptor proteins. Most studies have been focused on the nuclear transcription regulation. However, extranuclear localization of both ER α and ERβ has been indicated [106–108]. In fact, increasing evidence has demonstrated that ERβ is mainly localized extranuclearly [100, 102, 106, 108–110]. Consistently, most of the genes modified in ERβ knock-out mice are mitochondrial structural proteins related to oxidative phosphorylation [105]. More direct evidence has been shown with ERβ knockdown in a murine hippocampal cell line through RNA interference. This study demonstrated a phenotype change in the mitochondrial functions, which included a substantial reduction in cellular vulnerability to oxidative insults due to a more stable ΔΨm [109].

4.4. Signaling

Estrogens can also regulate mitochondrial function indirectly through various signaling pathways. Accumulating evidence has shown that neuroprotective effects of 17β-estradiol involve transient activation of intracellular signaling pathways via G proteins [111, 112], extracellularly regulated kinases (phosphoinositol 3-kinase and protein kinase B/AKT, ERK and p38 MAP kinases) [113–115], phosphorylation of the cAMP response element-binding protein [116–119], and alterations in intracellular calcium levels [120–123]. Changes in the activity of these enzymes can regulate the phosphorylation of numerous intermediary signaling proteins such as Rsk, p38 and JNK, and nuclear transcriptional factors, cyclic AMP response element binding protein (CREB) and cfos/cjun, which may ultimately mediate cell survival changes (for review see [37]).

However, persistent activation of multiple kinases sends a death signal. It has been observed that both glutamate and okadaic acid (OA) [124, 125] persistently activate JNK, p38 MAPK and ERK1/2 pathways. Activation of the different MAPK pathways occurs simultaneously, and there is crosstalk between ERK1/2 and p38 that is mediated by various protein phosphatases [126, 127]. Moreover, it has been demonstrated that PKC phosphorylates both ERK1/2 and p38 MAPK [128–130] providing a link between PKC and MAPK signaling. In the presence of functioning protein phosphatases, estrogens, via maintenance of protein phosphatase activity [131, 132], or specific inhibitors of ERK1/2 or PKC [133] prevent glutamate death signaling. However, in the face of broad protein phosphatase inhibition by OA, profound activation of multiple death-inducing kinases cannot be overcome with either estrogens or specific kinase inhibitors. 17β-Estradiol has been shown to block the persistent activation of both ERK and PKC in a variety of insults including ischemia and ethanol withdraw induced cytotoxicity [113, 115, 134–137].

The influence of these signaling effects of estrogens on mitochondrial function is only now being clarified. Phosphorylation events on the outer membrane of the mitochondria are well defined. Bcl-2 can be modulated by dimerization with proapoptotic family members (i.e. Bax, BAD, Bid) and by phosphorylation. The dynamic phosphorylation and dephosphorylation of Bcl-2 causes conformational changes within the protein and has been suggested to serve as a survival sensor during stressful or cytotoxic conditions [138]. Bcl-2 phosphorylation by a variety of kinases, such as PKC, ERK, Akt, PI-3 K, and others, is a cell survival signal; while dephosphorylation by PP2A and/or PP1 is associated with cell death [139]. BAD, a BH3-proapoptotic Bcl-2 family member, acts at a key nodal point in the mitochondrial apoptotic pathway in that unphosphorylated BAD binds and inactivates antiapoptotic Bcl-2 homologues. This elicits release of cytochrome c from mitochondria and consequent caspase activation cascades. PP1, PP2A, and calcineurin have been shown to dephosphorylate and activate BAD [140] and BAX [141–143]. In addition, upon dephosphorylation by PP1 and/or PP2A, BAX is thought to insert into the outer mitochondrial membrane which disrupts membrane stability and release cytochrome c.

It is also becoming clear that there is mitochondrial trafficking of an increasing number of nuclear receptors and transcription factors including but not limited to ER α, ERβ, glucocorticoid receptor, PPAR γ2, AP1, CREB, NF-κB, p53, TFAM, TFB1M, and TFB2M (for review see [144]) that have direct transcriptional effects on mitochondrial encoded proteins. For example, CREB has been shown to be present in the matrix or inner membrane of the mitochondria [145]. In addition, upon phosphorylation of external CREB by PKA, pCREB is transported into the mitochondria by TOM proteins and induces the synthesis of mitochondrial encoded subunits of oxidative phosphorylation [146]. Since estrogens have shown to mediate the phosphorylation of various signaling molecules including CREB, it is possible that estrogens may be modulating mitochondrial function and/or transcription via the singling molecules discussed in this section. Our studies indicate ERβ, along with Tom20 and/or Hsp90 are localized to the mitochondria [147]. Receptor preference may depend on ligand induced conformational change in the receptor, and studies show that estrogen increases mitochondria localization of ERβ [100]. The dynamic state of ERβ with and without ligand may facilitate its trafficking based on hydrophobic interactions, which could be mitigated by oxidative stress.

4.5. Direct Antioxidant Effects

Estrogens have long been recognized as antioxidants in a variety of in vivo and in vitro models. This is important as many neurodegenerative disorders and brain injuries involve oxidative stress, in part because of the abundance of polyunsaturated fatty acids in neuronal membranes which increases susceptibility to peroxidative damage. Estrogens have only weak radical scavenging activity [148–150], but are able to inhibit oxidative stress markers such as lipid peroxidation [59, 151–154], protein oxidation [155], and DNA damage [31, 156–158]. In cell-free systems, estrogens inhibit iron-induced lipid peroxidation [159], LDL oxidation, cholesterol oxidation, and conjugated diene formation [160–169]. Given the modest activity of estrogens as mass action radical scavengers, these potent antioxidant activities are likely due to a novel redox cycling mechanism (see below). We [28] and others [55, 58] have shown that estrogens, with higher capacity to donate a hydrogen radical from the phenolic hydroxyl group on the steroid A ring, are more potent neuroprotectants, underscoring the oxidative etiology of neuronal death.

Recently, we have identified additional structural modifications that improve neuroprotective potency while eliminating or reducing estrogen receptor binding [59]. For example, neuroprotection is enhanced by as much as 200-fold through addition of ba ulky hydrophobic moiety at the C2 or C4 position of the phenolic A ring. In the same screening, we were also able to establish a correlation between neuroprotective potency and inhibition of iron-induced lipid peroxidation [59]. The lipophilicity of estrogens leads to their accumulation in the hydrophobic plasma membranes and affects membrane fluid [170–173]. With a logP of 4.008, E2 is localized to the lipid environment of membranes, placing them at the site of key peroxidation events thereby forestalling oxidative damage. By preserving membrane integrity, which is key for OXPHOS, these molecules preserve mitochondrial function, and so moderate the injurious bioenergetic and oxidative sequales of mitochondrial failure. The lipid membrane is also the site of various signal transduction processes including PI3K/Akt signaling and phosphotidylserine flipping in apoptosis.

Oxidative stress is also a key player in normal physiological conditions. The redox state of the cell is a primary determinant for cell survival, and influences parameters such as the ratio of reduced and oxidized glutathione, oxidative state of proteins, and differentiation status. Previous work on neuroprotection has shown a synergistic interaction between E2 and glutathione (GSH) [28, 174], as well as between the E2 phenoxy radical (E2O•) with other antioxidants such as α-tocopherol in vitro [175]. Estradiol has also been reported to elicit significant increases in GSH levels in HT-22, primary hippocampal, and primary neocortial cells [176]. Quinol derivitives of estradiol capable of redox cycling have also been shown to be neuroprotective [177].

Estrogen’s role in maintaining the redox (homeostasis) state of cell has implications on many cellular processes. However, estrogens may also interact with proteins with sulfhydryl-containing (redox sensitive) cysteine groups. Oxidative modifications to key proteins, such as NMDA receptors [178, 179], ATPase[[180, 181], ryanodine receptors [181], , Keap1 [182], GAPDH [183] and tau [184] is a response to cell stress and in some cases alters signaling pathways. Many signaling pathways are redox sensitive and include AP-1, CREB, Erk, HIF-1, NFκB, JNK1/SAPK, PKB, PKC, ARE (Keap) [185], to name a few (for review, see [186]). Interestingly, estrogens are known to affect many of these same pathways, including MAPK [137], NFκB [187], CREB [188], PKC [189], ARE [190], and PKB/Akt [191].

Thiol redox state has wide implications throughout the cell, including the mitochondria [192, 193]. Gender difference were noted above, and isolated mitochondria from female rats produced less peroxides and mitochondrial DNA damage compared to males and ovariectomized animals no doubt because the former contained higher levels of reduced GSH, plus greater activities of Mn-superoxide dismutase (MnSOD), and GSH peroxidase [194]. Diethylstilbestrol [195] and E2 [196] have been shown to increase manganese SOD activity, and E2 rreplacement in the ovariectomized rats restored mitochondrial function to levels comparable to intact female animals [186]. Aside from these classic antioxidant enzymes, E2 has also been shown to induce protein thiol/disulfide oxidoreductases, which include protein disulfide isomerase (PDI), thioredoxin, and glutaredoxin [78, 197], and thereby indirectly affect thiol-dependent regeneration systems. In rat bone marrow, ovariectomy causes a decrease in total and reduced glutathione, and declines in activities of GSH reductase and thioredoxin reductase, which were normalized by E2 administration [198].

5. Conclusions

Estrogens have both direct and indirect beneficial effects on mitochondria that serve to preserve function under pathogenic circumstances. Development of non-hormonal analogs allows separation of cytoprotection via transactivation from pharmacological activities. As such, estrogens and the novel analogs are playing a central role in our effort to protect neurons against acute brain injury and chronic neurodegeneration. While the mechanism(s) of the beneficial effects of estrogens are likely multifaceted, the cytoprotective data reviewed here encourage the notion that targeting mitochondrial instability should be considered as a strategy to delay or prevent Alzheimer’s disease and other chronic neurodegenerative conditions.

Table 1.

Estrogen Interaction with Mitochondria

Direct	Indirect
Stabilization of the ΔΨm	Increased nuclear mediated transcription
Inhibition of F0F1 – ATPase	Increased mitochondrial mediated transcription
Augment mitochondrial Sequestration of Ca+2	Activation of intracellular signaling proteins