Estrogen synthesis and signaling pathways during aging: from periphery to brain.

As age-related changes of endogenous estrogen production as well as estrogen receptor expression can be different depending on types of cell or tissue in human body, it is important to understand the tissue-specific roles of estrogen and estrogen receptors in age-related diseases. For example, reduction of endogenous estrogen levels increases risk of bone fracture, cardiovascular disease and Alzheimer's disease in postmenopausal women. Studies showed that estrogen therapy plays osteoprotective roles in both osteoporotic humans and rodents [ 104 ], while whether estrogen therapy can protect against heart disease or AD remain controversial [ 105 ]. Although it is unclear why the effeteness of estrogen therapy differs among those age-related diseases, literatures showed that healthy cells such as neurons response to estrogen treatment with positive and beneficial outcome while cells with unhealthy condition such as neurological healthy is compromised, estrogen explore might accelerate the disease process [ 106 ]. This bias of estrogen action is also confirmed in recent studies which showed that levels of local tissue-specific estrogen and estrogen receptors contribute the response to estrogen therapy in postmenopausal women [ 30 , 107 ]. Cardiovascular disease (CVD) is the leading cause of death in both sexes. The incidence of CVD is much lower in premenopausal women, who usually have lower blood pressure and a much lower risk of developing the disease, compared with age-matched men. However, this advantage for women gradually disappears after menopause with the cessation of ovarian function and reductions in estrogen levels; eventually the risk of CVD in postmenopausal women becomes higher than age-matched men [ 108 ]. In animal models of CVD, young females have lower rates of vascular injury, a slower progression to heart failure, and lower mortality than males, but these differences can be reduced or eliminated by estrogen deficiency or ovariectomy [ 109 ]. Some of the beneficial effects of estrogen that protect the cardiovascular system are mediated through ER-dependent mechanisms. For example, in mice, the effects of estrogens mediated by ERα can improve vascular function and reduce atherosclerosis [ 110 ]. Some studies also showed that estrogen can prevent cardiac fibrosis by blocking the fibroblast to myofibroblast transition via interactions with ERβ [ 111 ]. However, unlike ERβ plays an important neuroprotective role in the central nervous system, in the heart, ERα appears to play a prominent role in most of the other tissues. The critical role of ERa in heart is evidenced by findings from another study showed a increased the ratio of ERβ/ERα in both vascular endothelium and smooth muscle in aged female mice causes the reversal in the antioxidant effect of estrogen to a pro-oxidant profile and is responsible for the increased oxidative stress during aging [ 112 ]. In addition, estrogen can reduce ischemia and reperfusion injury and preserve cardiac function via GPR30 and transactivating epidermal growth factor receptors [ 113 ]. The effect of ERs on the cardiovascular function might be a tissue-specific since studies have found that a down-regulation of ERβ expression in aged human brain while both ERα and ERβ plays neuroprotective roles in against age-related cognitive decline [ 114 , 115 ]. However, there is conflicting evidence concerning the effects of estrogen treatment on CVD. Data from the Heart and Estrogen/Progestin Replacement Study (HERS-I, and the follow-up HERS-II) failed to confirm the protective effects on the heart [ 105 ], and the Women's Health Initiative (WHI) combined estrogen-progestin trial reported an increased risk for coronary heart disease in patients receiving estrogen supplements [ 116 ]. Questions remain regarding the effectiveness of the estrogen formulations used, dosing regimens, and routes of administration in those clinical trials, making it important to resolve the differences between the beneficial effects observed in the laboratory and the absence of such effects in clinical settings, at least as they pertain to estrogen-based therapeutic strategies for treating menopause-related cardiovascular risk factors. Postmenopausal women have a higher risk of developing osteoporosis, and the prevalence of osteoporosis in females increases significantly in postmenopausal populations after ovarian function is lost, and continues to increase with age throughout the postmenopausal period [ 117 ]. Similarly, female rodents develop an osteoporotic phenotype after ovariectomy, and lower circulating estrogen levels were found to be associated with increased rates of bone loss [ 118 ]. Bone density has been reported to increase with estrogen treatment in a dose- and duration-dependent manner, which then prevents osteoporotic fractures [ 119 ]. Estrogen protects women from osteoporosis by regulating bone metabolism. Osteroporosis is characterized by a progressive loss of bone tissue and is associated with low bone mass, compromised microarchitecture of the bone, and increased risk of fractures; estrogens have been shown be osteoprotective by inhibiting high bone turnover and preventing bone loss in both osteoporotic humans and rodents [ 104 ]. Although both ERα and ERβ expressed in bone, the molecular role of bone receptors in the osteoprotection induced by estrogen remains unclear. It has been suggested that estrogens protect bone loss by inhibition of bone resorption mediated by the osteoclastic ERα, through the shortened lifespan of osteoclasts in female mice [ 120 ]. In addition, estrogen is also promoting bone formation by increasing osteoblast. To study the cell- and tissue-specific ER in bone formation, a recent study using a mouse model with osteoblast-specific ERα inactivation and found significant bone loss in female by reducing bone formation in females, not in males [ 121 ]. While ERα can modulate gene transcriptional through classic (ERE biding) and nonclassic (non-ERE binding) pathways (see figure 4 , pathway 1), studies using gene knockout and knockin technologies in mice demonstrated that ERα-mediated osteroprotective actions is modulated through ERE biding signaling pathway in mice [ 122 ], suggesting a tissue-specific ER signaling in the bone turnover. As we mentioned above, ER can also be activated through ligand-independent signaling ( figure 4 , pathway 4). Indeed, recent studies found that mice with ovariectomy or overexpression of ERE did not change the mechanic loading-induced upregulation of osteogenic activity. Furthermore, mice with ERα gene knockout or specific inactivation of AF-1 in ERα showed a reduced response to mechanic loading-induced osteogenic activity [ 123 ], suggesting a ligand-independent pathway such as ERα-AP1 interaction, involved in the bone formation. Together, estrogen prevents bone loss and promotes bone formation through interactions with ERα with classic ER-dependent and ligend-independent signaling pathways. The bone-specific estrogen and ERα signaling pathways provides a potential strategy of new anti-osteoporotic drug development, such as a ideal selective estrogen receptor modulators for the prevention and treatment of postmenopausal osteoporosis and vertebral fractures with less side effects on uterus or breast where ERα also highly expressed. AD is both the most common neurodegenerative disease and the most common form of dementia. It is characterized by brain-specific senile plaques composed of aggregated amyloid β (Aβ) peptide and neurofibrillary tangles [ 124 ]. In both genders, epidemiological studies show an increased risk for AD with the age-related loss of sex steroid hormones. It is also commonly reported that AD is more prevalent in postmenopausal women relative to age-matched men [ 125 ]. The precipitous loss of ovarian estrogens and progestogens at menopause has been presumed to account for the increased AD susceptibility in women, but recent studies in both animals and humans suggest that depletion of brain-derived estrogen, rather than the circulating estrogen, is a more direct and significant risk factor for developing AD [ 30 ]. A number of observational studies have suggested that estrogen replacement therapy (ERT) might protect postmenopausal women against AD and age-associated cognitive decline [ 126 , 127 ]. However, the results from the Women's Health Initiative Memory Study (WHIMS) do not support the use of ERT to reduce the risk of AD in postmenopausal women older than 65 years [ 128 ]. Other randomized clinical trials in younger women offer a possible explanation for this discrepancy. For example, the Cache County Study reported that AD risk was only reduced in women using ERT long-term compared to short-term [ 129 ], suggesting that early initiation of ERT, most likely nearer the time of menopause, is essential for the beneficial effects on cognitive function in postmenopausal women. As mentioned above, one of the major arguments regarding these controversial findings is the timing of ERT. The complications associated with ERT may include hormone responsiveness with age, that is, during the menopause transition, neural sensitivity to sex steroid hormones may weaken, indicating a “critical period” around the time of menopause in which ERT needs to be prescribed to protect cognitive function [ 130 , 131 ]. Early ERT around menopause would allow the body to maintain hormone responsiveness by regulating hormone levels before the irreversible, age-related hormone fluctuations begin. It is also related to the condition of neuronal healthy since the epidemiological observation analysis showed a reduction in risk of AD in women who had estrogen therapy started at the time of menopause while estrogen increase risk of AD in those women began estrogen therapy at 10–15 years after menopause or who already developed AD [ 106 , 129 , 132 – 134 ]. Furthermore, our recent studies not only confirmed with the benefit effect of early ERT, but also showed that the level of brain estrogen level determines the effect of estrogen therapy in protect against AD pathology in animal model [ 107 ]. The level of expression, subcellular distribution and activities of ERα and ERβ can change during aging, which lead to the likely net outcome of a differential response to estrogen in the aging brain. The age-related expression of ERα and ERβ has been studied in rodent cerebral cortex and the result showed that ERα and ERβ undergo differential changes in expression during aging: ERα level did not change with aging while ERβ level decreases signicantly with advancing age in both sexes [ 114 ]. In female rats, the age-related decrease in ERβ expression in the cortex is correlated with a decline in cognitive function during aging [ 115 ]. The age-related changes of ERs are also evidenced by the subcellular distribution of ERα and ERβ, such as a significant attenuation of both ERα and ERβ expression in spine synapse complexes in hippocampal CA1 region in aged female rats compared to young rats [ 135 , 136 ], which is correlated to the decreased ability of E2 in promoting spine density in the hippocampus during aging [ 137 ]. However, the changes of ERs observed in the brain during ageing is a tissue-specific event since, instead of decrease of ERβ expression, a gradual increase in ERβ expression was observed in uterine arteries during aging even 10 years after of menopause onset, while there is only a slight increase in ERα expression during aging [ 138 ]. The age-related elevation of ERβ expression in uterine arteries of the postmenopausal women is significant positive associated with the proinflammatory effects of estrogen. The accumulation of Aβ, a proteolytic byproduct of amyloid precursor protein (APP) metabolism, is an important aspect of AD pathology [ 124 ]. APP is processed by two competing pathways, the amyloidogenic pathway through β-secretase (BACE1) and γ-secretase, which produce β-APPs and Aβ40/Aβ42 peptides, and the predominant non-amyloidogenic pathway via α-secretase, which produces neuroprotective α-APPs and several non-amyloidogenic peptides [ 139 ]. Estrogens can reduce Aβ production by favoring the non-amyloidogenic pathway through activation of MARK/ERK, inhibiting the amyloidonenic pathway by reducing the levels of BACE1, and promoting Aβ clearance by stimulating microglial phagocytosis and degradation as well as regulating levels of major enzymes involved in Aβ degradation [ 88 , 140 ]. Other mechanisms also contribute to the protective roles of estrogens in the context of AD. For example, estrogens can regulate the Bcl-2 protein family by increasing the expression of antiapoptotic Bcl-xL and Bcl-w, and suppressing the expression of proapoptotic Bim to protect against neuronal loss induced by Aβ-mediated toxicity [ 141 ]. Estrogens can also decrease the level of hyperphosphorylated tau (a major component of neurofibrillary tangles) by modulating the kinases and phosphatases involved in tau phosphorylation, such as GSK-3β, Wnt, or PKA pathways [ 142 ]. Notably, estrogen can enhance neural plasticity and affect related cognitive functions as well as regenerative potential of brain. As reviewed by Brinton [ 143 ], the benefit effects of estrogen on neural plasticity occur at three levels, such as cellular, morphological and synaptic function levels. Studies demonstrated that E2 can increase neurogenesis in various brain regions such as dentate gyrus of hippocampus [ 144 ]. These estrogen-induced newly generated neurons in the hippocampus contribute to brain region-specific type of learning and memory [ 145 , 146 ]. In addition, E2 can also rapidly increase dendritc spine number or dendritic spine contacts in hippocampus, themedial amygdala and hypothalamus, and thus enhance performance on a hippocampal-dependent memory task in monkeys [ 147 , 148 ]. Furthermore, E2 is a potent and efficacious potentiator of synaptic transmission in hippocampal system [ 149 ]. Together, E2 plays important roles in promoting neurogenesis and neuronal plasticity in order to keep healthy cognitive function and protect against cognitive decline in females during aging. PD is the second most common neurodegenerative disease, after AD. In contrast to higher incidence of AD, epidemiological observations found lower risk of PD in women. One might expect is the difference in the age at onset of the diseases. The average age at onset of PD is 60 years old with 10% of PD are younger than 40 years old. In addition, recent neuropathology evidence suggest a 5–20 years long preclinical period before the motor manifestations in PD patients [ 150 ]. In AD, the most common onset of the disease is at age of 65 or older which is further away from the onset of menopause. The estrogen protection of PD and AD is also evidenced by findings of women with early age at menopause have higher risk of dementia [ 151 ] and a delayed onset of PD in women with higher number of pregnancies, longer fertile life and longer cumulative length of pregnancies [ 152 ]. Furthermore, estrogen replacement therapy showed improvement of motor symptoms in female PD patients [ 153 ]. Together, it might explain the epidemiological observations of lower incidence of PD and higher risk of AD in women and the protective role of estrogens. While ERa, ERb and GPR30 all been identified in the striatum and substantia nigra, the most affect brain regions in PD [ 154 ], ERα and ERβ could have distinct roles in neuroprotection against insult-induced dopamine cell death. Studies found that ERα is the dominant receptor involved in neuroprotection in PD, while ERβ plays little role in neuroprotection [ 155 , 156 ]. Compare to wild type control mice, mice with genetic depletion of ERα, not for ERβ depletion, are more vulnerable to insult-induced cell toxicity and 17β-estradiol failed to rescue the dopamine neuronal death caused by the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in PD model, suggesting an ER-specific role in PD pathology. In addition to the ER-dependent PD pathology, two major ER-dependent signaling pathways are also involved in the neuroprotective roles of estrogen against PD [ 157 ]. One is the extracellular signal-regulated kinase (ERK1/2) pathway and the other is the phosphatidylinositol 3 kinase (PI3K)/Akt pathway. In PD animal model studies, estrogen treatment protect against cell death and inhibitor of the PI3K/Akt pathway (LY294002) blocked the survival effects of estrogen in dopamine neurons, while inhibition of the MAPK/ERK signaling was ineffective [ 158 ]. The mechanism by which ERs activate ERK1/2 and Akt signaling involves multiple interactions with signaling proteins, such as calveolin proteins, G proteins, Src, and the p85a regulatory subunit of PI3K [ 159 ]. These two pathways converge downstream to some common targets. For example, both pathways can inhibit the pro-apoptotic protein Bad through phosphorylation [ 160 ]. Furthermore, the activation of ERK1/2 and PI3K/Akt pathway can also activate various transcription factors such as cAMP-response element-binding protein (CREB), which finally induces the expression of target genes such as Bcl-2 [ 161 , 162 ]. Together, maintaining reproductive level of endogenous estrogen in females might prevent women to develop PD. Recent animal studies further demonstrated that ERα plays neuroprotection in PD via estrogen-dependent and --independent signaling pathways. These newly reported evidences further revealed molecular mechanisms of tissue- and disease-specific effects of estrogen on PD.

Although the roles of estrogens in gonadal organs are well understood, recent studies have begun to demonstrate that localized estrogen production plays tissue-specific roles, with or without dependency on circulating estrogen. The cell- and tissue-specific actions of estrogen and ERs are directly involved various age-related diseases. Nevertheless, it remains unclear how the local synthesis of estrogens and their respective receptor functions are controlled in normal aging. We believe that estrogens are no longer just sex hormones, but important therapeutic targets for preventing diseases as disparate as osteoporosis, heart disease, and neurodegeneration.

Estrogen signaling is primarily mediated through ERs. ERs include classic receptors as ERα and ERβ, members of the nuclear receptor superfamily acting as transcription factors and modulate the transcription of target genes, and membrane receptors such as GPR30 (an orphan G-protein coupled receptor) and ER-X [ 38 , 39 ]. Although both types of ER transduce estrogen signals into a large variety of physiological responses in various organs, the nuclear ERs initiate the biological events in a slow motion such as in hours or even days, while the cell membrane ERs triggers an intracellular signaling cascade response in a much fast manner such as in seconds. In combination of tissue and cell-specific distribution of ERs, it is critical to understand the function and ER signaling in physiological and pathological condition during ageing. ERα and ERβ colocalize in many cell types, including the endothelium, epithelium, muscle, bone, cartilage, hematopoietic cells, neurons, and glia ( Table 1 ), although each receptor exhibits a distinct pattern of tissue-specific distribution throughout the body. Although it is unclear how these patterns are dictated, it is known that ERα and ERβ are encoded by separate genes ( ESR1 and ESR2 , respectively) located on different chromosomes [ 40 – 44 ], and there are biological functional cross-talking between presence of the two receptors in certain cell types and tissues [ 45 ]. Is also known that some age-related changes of ERα and ERβ expression are tissue-specific and we will discuss in details for each age-related diseases later in the review. ERα is primarily expressed in the gonadal organs (uterus, ovary, prostate, testes, and breast), but is also present at lower levels in other tissues such as bone, liver, kidney, adipose tissue, and brain. ERβ is primarily expressed in non-gonadal tissues – colon, bone marrow, vascular endothelium, lung, bladder, and brain [ 46 ]. In the brain, both ERα and ERβ are widely distributed and are expressed in both neuronal and non-neuronal cell types. Although overlapping expression of ERα and ERβ has been observed in many brain regions, as summarized in table 1 , the spatiotemporal distribution patterns of ERα and ERβ are distinct, as are the levels of expression. ERα is the most abundant subtype in the hypothalamus and amygdala, key regions involved with the neuroendocrine system, and helps to regulate the autonomic nervous system and emotional reactions. ERβ levels are highest in the hippocampus, claustrum, and cerebral cortex, and are lower in the hypothalamus [ 47 , 48 ]. Gene knockout studies in mice and pharmacological studies with ER subtype-selective agonists in vivo have shown a significant correlation between the tissue distribution of ERα or ERβ and their functions [ 46 ]. ERα plays an essential role in the neuroendocrine and reproductive systems. By contrast, Studies using ERα and ERβ knockout mice have shown that ERα, but not ERβ, is required for both negative and positive feedback regulation of gonadotropin-releasing hormone neuron firing by estrogen [ 49 ]. ERβ is primarily involved in mood and cognitive activities; treatment with E2 or an ERβ agonist induced anti-anxiety-like behavior in wild-type mice, but not ERβ knockout animals, and cognitive activity is significantly reduced in ERβ knockout mice compared with wild-type mice [ 50 ]. However, there is a great functional overlap between ERα and ERβ in various tissues. For example, both ERα and ERβ are indispensable for normal ovarian function and estrogen-mediated cardioprotective actions [ 51 , 52 ]. GPR30, an orphan G protein coupled receptor, has been identified by several independent groups as membrane estrogen receptor which can trigger rapid estrogen non-genomic signaling independent of ERα and ERβ [ 53 ]. However, because CPR30 not only binds to estrogens but also other substances such as chemokine [ 54 ], whether or not GPR30 is a genuine novel estrogen receptor remains controversial. Furthermore, studies showed that higher concentration of E2 was needed to activate GPR30 under certain experimental condition [ 55 ] while some protection of E2 in mitochondrial and pancreatic islets are unrelated to GPR30 [ 56 , 57 ]. GPR30 is expressed in many brain regions, including the hippocampus, cortex, hypothalamus, specific nuclei within the midbrain, and the Purkinje layer of the cerebellum, as well as the adrenal medulla, renal pelvis, and ovaries [ 38 , 58 ]. ER-X expression is highly regulated during development, and has been shown to be enriched in the fetal baboon brain and in the neocortex, uterus, and lungs of - postnatal rodents [ 59 ]. The expression of ER-X peaks 7–10 days after birth and declines over the first postnatal month. In adults, ER-X expression is almost undetectable, but re-emerges after ischemic injury or in animal models of AD [ 59 ].