**5. Mitochondrial responses to estrogen**

II (Succinate Dehydrogenase), respectively. Electrons are shuttled through the complexes with oxygen acting as the final electron acceptor at Complex IV. Concurrently, hydrogen ions are pumped from the electronegative mitochondrial matrix into the more positively charged inner membrane space by Complexes I, III (cytochrome C reductase), and IV (cytochrome C oxidase). The protomotive force thus generated allows hydrogen ions to flow down their concentration gradient at Complex V (ATP Synthase), resulting in the formation of ATP. This chemiosmotic process is tightly regulated and highly efficient. Although ATP is the primary (and often most studied) product of cellular respiration, ROS and thermal energy/heat are also generated by mitochondria. ROS include chemical species produced by the incomplete

and hydroxyl radical (•OH). Mitochondrial ROS are thought to perform a variety of cell sig-

Mitochondria are highly dynamic organelles that are components of a constantly changing, dynamic mitochondrial network. Damaged or old mitochondria can be cleared from the cell by autophagy/mitophagy or bulk clearance and degraded by lysosomal enzymes [26]. Furthermore, mitochondria can also undergo fission and fusion. Fission is the process by which mitochondria bud off from the mitochondrial network. This is regulated by the proteins DRP-1 and FIZZ1. Fusion represents the incorporation of mitochondria in the mitochondrial network and is regulated by mitofusin1 (Mfn1), mitofusin2 (Mfn2), or optic atrophy 1 (OPA1) [27]. These regulatory proteins help to maintain the balance between fission and fusion that is required to preserve cell viability. In different disease states, however, this bal-

Manganese superoxide dismutase (MnSOD) and glutathione represent endogenous molecules that minimize mitochondrial-derived ROS. Mitochondrial injury occurs, however, when ROS formation exceeds the capacity for their removal by these antioxidant mechanisms. ROS biochemically modify other molecules to produce cytotoxic species that induce cellular injury [28]. For example, ROS induce the formation of 4-hydroxynonenal (HNE), a reactive lipid species that is associated with neuronal damage in brains of Parkinson's disease patients [29, 30]. ROS production also leads to cell death *via* the induction of apoptosis. Activation of the intrinsic apoptotic pathway occurs in response to a decrease in mitochondrial membrane potential, opening of the mPTP and release of cytochrome C [23]. Cytochrome C initiates the pro-apoptotic cascade by activating the initiator caspase 9, which in turn cleaves the final effector caspase 3. There are also a number of proteins that regulate apoptosis, including anti-apoptotic Bcl-2, and pro-apoptotic *Bax* and *Bad* proteins [31]. When cytosolic *Bax* binds to the outer mitochondrial membrane, it induces apoptosis by stimulating cytochrome C release. Further, the binding of *Bax* to Bcl-2 inhibits the antiapoptotic effects of Bcl-2, resulting in cell death. The dimerization and localization of this group of proteins modulate apoptosis under both basal and pathological conditions and

•−), hydrogen peroxide (H<sup>2</sup>

O2 ),

. These molecules include superoxide anion (O<sup>2</sup>

ance can be disrupted causing mitochondrial dysfunction and cell death.

naling functions under normal physiological conditions.

**4. Mitochondria, cell injury, and apoptosis**

can be modified by the cellular microenvironment.

reduction of O<sup>2</sup>

466 Mitochondrial Diseases

Both nuclear and mitochondrial genes are subject to regulation by estrogen [32]. Nuclear DNA encodes proteins that are incorporated in mitochondria and influence their function. For example, the binding of estrogen to nuclear ERα induces the expression of peroxisome proliferatoractivated receptor gamma coactivator 1-alpha (PGC1α) [33]. This protein plays an important role in mitochondrial biogenesis, a process by which new mitochondria are formed. The mitochondrion also contains its own maternally inherited DNA, which encodes 37 genes [25]. A recent study in MCF-7 cells showed that estrogen regulates mitochondrial RNA production under serum starvation conditions [34]. Subsequent to estrogen treatment, ERα translocated to the mitochondria and increased expression of mitochondrial tRNAs used to translate mitochondrial proteins. In GH4C1 pituitary cells, treatment with estrogen increases expression of mitochondrial-encoded RNA for subunit II of cytochrome C oxidase [35]. In female rats, levels of mitochondrial-encoded 16S RNA, a housekeeping gene used as a marker for mitochondrial number, are four times higher than males of the same age [36, 37]. Ovariectomy (OVX) in rats is characterized by an increase in liver and brain peroxide production and formation of 8-oxo-2′deoxyguanosine, a marker of mitochondrial DNA damage. These changes were associated with a reduction in the antioxidant protein GSH and MnSOD [36]. Estrogen treatment in OVX rats reversed these responses [36]. Collectively, these data suggest that increased estrogen levels regulate mitochondrial and nuclear anti-oxidant protein expression.

Estrogen plays an important role in the regulation of apoptosis by stimulating Bcl-2 protein expression and translocation to the mitochondria. This is achieved *via* the Ca2+ regulated ERK pathway [38]. The regulation of both Bcl-2 and *Bax* expression by estrogen has been reported in THP-1 macrophages and human monocyte-derived macrophages. Pre-treatment with estrogen increased the Bcl-2: *Bax* ratio, thus increasing cell viability in the presence of proapoptotic stimuli. Estrogen treatment of cortical neurons has also been shown to inhibit glutamate toxicity and improve cell viability by upregulating Bcl-2 expression [39]. In SH-SY5Y neuroblastoma cells over-expressing ERβ, the receptor has been shown to interact with the pro-apoptotic protein *Bad* and prevent its binding to *Bax*, thereby inhibiting apoptosis [40]. These data suggest that both estrogen and ERs *per se* are anti-apoptotic and modulate disease pathogenesis. Estrogen also preserves cell viability by altering mitochondrial dynamics. In the myocardium of ischemia reperfusion injury rodents, OVX rodents display an increase in mitochondrial fusion after injury, which is reversed by estrogen treatment [41] Work in isolated cortical astrocytes from male and female postnatal day 1 mice shows that estrogen regulates fission and fusion genes in a gender-specific manner [42]. Further, estrogen stimulated mitochondrial biogenesis in skeletal muscle and adipocytes [43, 44]. These data suggest that estrogen can strengthen the mitochondrial network by increasing mitochondrial fusion, thus preserving mitochondrial function and cell viability.

Estrogen effects on mitochondrial function vary in different cell types. For example, estrogen binds specifically to the oligomycin-sensitivity conferring protein of ATP-synthase (Complex V) in brain mitochondria and inhibits ATP production [45]. In contrast, another study showed that the enzymatic activity of F0F1-ATPase, a Complex V subunit, is higher in mitochondria isolated from the heart than other tissues. Estrogen induced a further increase in cardiac ATPase activity implying a direct link between estrogen stimulation and ATP production [45, 46]. Estrogen can therefore exert different effects on mitochondria from different cell types. These differential responses may impact disease pathogenesis.

Mitochondrial structure in the heart is influenced by estrogen. The hearts of OVX rats that underwent I/R injury had lower levels of mitochondrial respiratory function and increased myocardial cell death compared to intact animals. Transmission electron microscopy showed that the mitochondria in cardiomyocytes from OVX rats were more disordered within the cell and structurally damaged compared to mitochondria from intact animals [41]. Interestingly, even male ERα KO mice that underwent cardiac I/R injury display lower coronary blood flow rates, increased calcium accumulation, and reduced nitrite production compared to non-ischemic hearts. Further, electron microscopic analysis revealed that the mitochondria from ERα KO mice were abnormally shaped [60]. These studies suggest that estrogen signaling plays a role in regulating mitochondrial structure in both females and males. In another model of I/R injury, female wild-type mouse hearts were shown to have better functional recovery and an attenuated inflammatory response compared to female ERα KO mice and wild-type male mice [61]. These data further suggest the importance of ER signaling as a cardioprotective mechanism in females. Effects of estrogen on mitochondrial function have been tested in a genetic model of hypertrophic cardiomyopathy (cTnT-Q92). Estrogen treatment improved ATP production, the mitochondrial respiratory ratio, and diastolic function in OVX cTnT-Q92 mice compared to untreated OVX mice [62]. OVX in cTnT-Q92 mice attenuated the expression of the mitochondrial biogenesis genes PGC1α, peroxisome proliferator-activated receptor alpha (PPARα), mitochondrial transcription factor A (tFAM), and the antioxidant protein nuclear respiratory factor 1 (NRF-1). Estrogen treatment improved cardiac mitochondrial organization and cristae structure and increased mitochondrial biogenesis. These data directly show that estrogen exerts cytoprotective effects at the level of the mitochondrion that translate into an improvement in cardiac function.

Estrogen and Mitochondrial Function in Disease http://dx.doi.org/10.5772/intechopen.73015 469

Macrophages contribute to the chronic inflammation associated with many diseases including CVD and neurodegeneration. Macrophages display plasticity in that they may adopt various phenotypes. The differentiation of these cells is highly dependent on the local microenvironment in which they are situated. M1 macrophages are pro-inflammatory and are induced by cytokines and lipopolysaccharide (LPS). M2 macrophages are anti-inflammatory, play a role in wound healing, and are induced by IL-4 and IL-13 [63, 64]. The metabolic characteristics of M1 and M2 macrophages are different. M1 macrophages rely on glycolysis for ATP formation while M2 macrophages are dependent on mitochondrial oxidative phosphorylation for energy [63, 64]. Damage to mitochondria induced by inflammatory stimuli can exacerbate cellular injury [65]. It was shown that both ERα knockout and mitochondrial dysfunction inhibit the IL-4 mediated conversion to macrophages from an M1 to an M2 phenotype [64, 66]. Treatment of macrophages with LPS/interferon-γ (IFN-γ) favors an increase in the M1 phenotype. In macrophages isolated from premenopausal women, estrogen treatment was shown to reduce the M1/M2 ratio in cells exposed to LPS/IFN-γ to a greater extent than macrophages isolated from postmenopausal women [67]. Recent studies from our group have shown that there is a significant decrease in ERα expression in macrophages from postmenopausal

**7. Estrogen, mitochondria, and inflammation**
