Interactions of Aromatase and Seladin-1: A Neurosteroidogenic and Gender Perspective

Abstract Aromatase and seladin-1 are enzymes that have major roles in estrogen synthesis and are important in both brain physiology and pathology. Aromatase is the key enzyme that catalyzes estrogen biosynthesis from androgen precursors and regulates the brain’s neurosteroidogenic activity. Seladin-1 is the enzyme that catalyzes the last step in the biosynthesis of cholesterol, the precursor of all hormones, from desmosterol. Studies indicated that seladin-1 is a downstream mediator of the neuroprotective activity of estrogen. Recently, we also showed that there is an interaction between aromatase and seladin-1 in the brain. Therefore, the expression of local brain aromatase and seladin-1 is important, as they produce neuroactive steroids in the brain for the protection of neuronal damage. Increasing steroid biosynthesis specifically in the central nervous system (CNS) without affecting peripheral hormone levels may be possible by manipulating brain-specific promoters of steroidogenic enzymes. This review emphasizes that local estrogen, rather than plasma estrogen, may be responsible for estrogens’ protective effects in the brain. Therefore, the roles of aromatase and seladin-1 and their interactions in neurodegenerative events such as Alzheimer’s disease (AD), ischemia/reperfusion injury (stroke), and epilepsy are also discussed in this review.


Introduction
Neuroactive steroids, or neurosteroids, are produced peripherally by exocrine glands, such as the ovaries and adrenal glands, and can cross the blood-brain barrier to influence neuronal signaling [1,2]. They play important roles in the neuroendocrine control of brain excitability based on their conversion to different metabolites, such as androstenediol and estradiol (E 2 ) [3][4][5][6].
Even though the brain is only 2% of body weight, it contains 25% of the total body cholesterol, the main precursor of all hormones [7]. Brain cholesterol is involved in myelin sheath formation, synaptogenesis, neurotransmission, and neurosteroidogenesis [8][9][10][11][12][13]. Although peripherally produced neurosteroids directly influence brain functions, it is well-established that the nervous system is also a steroidogenic tissue and expresses enzymes that are involved in the synthesis and metabolism of steroids. This unique ability of neurosteroidogenesis allows the brain to produce specific steroids required for neuroendocrine control and allows the brain to protect itself from neurodegeneration [14] (Fig. 1).
Two enzymes come into the foreground when discussing neuroprotective activity: Aromatase, which converts estrogen from androgen during the last step of estrogen biosynthesis from cholesterol, and seladin-1, which synthesizes cholesterol from desmosterol ( Table 1). The reason why these two enzymes are emphasized in neurodegeneration is hidden in the internal dynamics of the brain. It is well known that estrogens exert neurotrophic and neuroprotective effects by stimulating the expression of neurotrophins and cellsurvival factors, enhancing synaptic plasticity, and by acting as antioxidants. Estrogens are protective under numerous types of stressors, including oxidative stress, glutamate excitotoxicity, chemical lesions, traumatic or mechanical injuries, ischemia, iron toxicity, glucose or serum deprivation, and specialized disease related pathogens, such as amyloid beta (Aβ) and HIV proteins [15]. Aromatasemediated estrogen formation in the brain is known to have regulatory effects on synaptic plasticity, neural stem cell (NSC) proliferation, neurogenesis, newborn neuron migration, differentiation, survival, and neuroprotection [13,[15][16][17][18][19][20][21][22][23].
The expression of aromatase is regulated through the alternative use of multiple, promoter-specific first exons (reviewed in [24]).
These first exons, which remain untranslated, are spliced into the coding exons 2 through 10 of the aromatase gene, resulting in numerous aromatase transcripts, all of which code for the same protein (reviewed in [24]). Because the brain-tissue specific aromatase promoter (I.f ) is known, designing a drug to increase local estrogen levels in the brain by targeting brainspecific aromatase transcription is theoretically possible [25][26][27].

Aromatase and Brain Physiology and Pathology: Estrogendependent action
First, the role of aromatase in physiological events will be discussed before elaborating on its role in brain pathology (Fig. 2). Aromatase is the key enzyme catalyzing estrogen biosynthesis from androgen precursors, such as testosterone. It is mainly a member of the cytochrome P450 enzyme family and is encoded by the CYP19 gene [28].
When the aromatase gene was examined in detail, researchers discovered that it was encoded by different promoters in different tissues (reviewed in [24]). In vertebrate brains, aromatase is synthesized primarily through promoter I.f in the hypothalamus, hippocampus, and amygdala [27].
Studies conducted to understand the physiological significance of aromatase showed that aromatase knock-out (ArKO) male mice demonstrated aggressive behavior patterns [30], while depressive symptoms were observed in ArKO female rats [31] and women carrying the CYP19 polymorphism [32]. The chronic use of aromatase inhibitors in women with breast cancer causes damage to visual and spatial memory [33,34]

FUNCTION
Androgen → Estrogen Normally synthesized in the nerve cells and regulates neuronal differentiation, neural and synaptic activity and plasticity, neurogenesis, memory and cognitive functions by producing local estrogen. Its expression increased as an acute response to neurodegenerative damage. Its expression decreased in Alzheimer's Disease.
Desmosterol → Cholesterol Normally synthesized in the nerve cells, provides membrane barrier structure and protects neurons from apoptotic cell death by inhibiting caspase-3 activity, Aβ toxicity and oxidative stress.
Its expression increased as an acute response to neurodegenerative damage. Its expression decreased in Alzheimer's Disease. Figure 1. Neuroendocrine and neurosteroidogenic functions of the brain. The brain contains 25% of the total body cholesterol, the main precursor of all hormones [7] and has unique ability of neurosteroidogenesis, which allows the brain to produce specific steroids required for neuroendocrine control and to protect itself from neurodegeneration.
to produce aromatase, glial cells begin to produce de novo aromatase synthesis around the damage site [18,23,41]. Neurotoxic and mechanical lesions, head trauma, ischemic insults, such as middle cerebral artery occlusion (MCAO), and global brain ischemia result in de novo enzyme expression in reactive astrocytes.
Increased aromatase expression in astrocytes after brain injury is followed by a significant increase in enzymatic activity and increased levels of E 2 and estrogen receptor-alpha (ERα) in the brain. Therefore, it can be said that aromatase-mediated estrogen conversion from steroid precursors is an endogenous defense mechanism of the brain against neurodegeneration [41,[43][44][45][46].
In fish, they persist during adulthood and maintain neural progenitor properties [52][53][54][55][56][57]. When a hippocampal lesion is formed, astrocytes express aromatase and produces estrogen, which then acts as a messenger and allows astrocytes to communicate with the RGCs. RGCs are progenitor cells that express aromatase and are located in the subventricular zone around the brain ventricles. Studies observed that RGCs migrate to damaged sites in the brain and accumulate around lesions. Treatment of these cells with an aromatase inhibitor, letrozole, or silencing of the cholesterol carrier steroidogenic acute regulatory protein (StAR) with siRNA has been shown to decrease the number of RGCs and induce apoptotic cell death. This data confirms that aromatase expression and local estrogen production regulate hippocampal neurogenesis [58][59][60].
It is well-known that Aβ accumulates in AD brains. In hippocampal neurons, estrogen has been shown to protect neuronal cells from Aβ-mediated cell death by decreasing Aβ production and increasing Aβ clearance (as reviewed in [61]). Additionally, E 2 reduces Aβ-induced calcium elevation (Ca 2+ ) in hippocampal neurons [62] and prevents the hyperphosphorylation of tau, another pathological hallmark of AD (as reviewed in [63]). Importantly, brains of late-stage AD patients have significantly reduced aromatase expression, particularly in vulnerable brain regions that are also deprived of estrogen protection [64,65]. However, clinical trials using hormone replacement therapy (HRT) and selective estrogen receptor modulators (SERM) to mimic the neuronal protective activity of estrogen in AD patients were unsuccessful and were abandoned due to the side effects.
Factors, such as age, basal cognitive activity, genetic background of the patients, disease stage, and duration of treatment were different among patients and likely contributed to the failure of HRT and SERM treatment [23,[63][64][65]. When the reasons of this failure were studied at the cellular level, local estrogens and aromatase were also found to be responsible [66]. Studies in hippocampal brain slices have revealed that local concentration of estrogen is six times higher than serum concentration [67].
Neuronal estrogen receptor (ER) expression Figure 2. Aromatase in brain physiology and pathology. Brain aromatase is normally synthesized in nerve cells and regulates neuronal differentiation, neural and synaptic activity and plasticity, neurogenesis, memory, and cognitive functions by producing estrogen locally [13,14,16,17,[20][21][22][23]. Aromatase knock-out (ArKO) male mice demonstrated aggressive behavior patterns [30], while depressive symptoms were observed in ArKO female rats [31] and women carrying the CYP19 polymorphism [32]. The chronic use of aromatase inhibitors in women with breast cancer causes damage to visual and spatial memory [33,34]. Glial aromatase expression increases in the early stages of neurodegenerative damage and triggers protective local estrogen synthesis [18,23,41]. Neurotoxic and mechanical lesions, head trauma, ischemic insults, such as middle cerebral artery occlusion (MCAO), and global brain ischemia result in de novo enzyme expression in reactive astrocytes. Aromatase expression and local estrogen production also regulate hippocampal neurogenesis [58][59][60].
is known to be mediated by local estrogen synthesis, and neuronal estrogen can only be provided by local aromatase activity. When local aromatase expression and, therefore, local estrogen synthesis is inhibited, neuronal ER expression and the protective effect of estrogen disappear, regardless of how much estrogen is given exogenously [67].
These reports suggest that estrogen can only be neuroprotective if sufficient estrogen levels are present in the brain before morphological changes, such as Aβ plaque formation, occur in AD. Therefore, estrogen treatment could prevent or slow down the development of AD.
Once the brain has been deprived of estrogen for an extended time, the protective effect no longer occurs, and subsequent hormone treatment might even be detrimental to cognition [66,67].

Seladin-1 and Brain Physiology and Pathology
Studies to investigate the causes of AD have resulted in the discovery of a new protein approximately 20 years ago by Greeve [68] using a differential mRNA display approach.
This approach identified a novel gene, named seladin-1, the abbreviation for Selective Alzheimer's Disease indicator-1, which was differentially expressed in selective vulnerable brain regions of AD patients, such as the hippocampus, amygdala, inferior temporal cortex, and the entorhinal cortex.
A later study showed that this protein is actually a well-known enzyme, 3-betahydroxysterole delta-24-reductase, that is encoded by the DHCR24 gene and was identified as a human homolog of the DIMINUTO/DWARF1 gene described previously in plants [73]. Seladin-1/DHCR24 is highly conserved and expressed in the CNS, especially in neurons under basal conditions [70]. It catalyzes the last step in the biosynthesis of cholesterol from desmosterol [74]. The deficiency of seladin-1/DHCR24 has been shown to decrease cholesterol levels in the plasma membrane and subsequently, reduce the formation and stability of lipid rafts [75].
Thus, as lipid rafts are important for mediating cell function [76] downstream cellular signaling is also affected [68]. Seladin-1/DHCR24 has also been reported to play a role in cellular responses against oxidative and oncogenic stress and inflammation [77][78][79][80][81][82]. Independent from its enzymatic activity, seladin-1 protein has been shown to protect neurons exposed to Aβ and oxidative stress from apoptotic cell death [70] and inhibit caspase-3 activity responsible for apoptosis [83]. These studies also indicated that seladin-1 is a downstream mediator of the neuroprotective activity of estrogen [69]. In our study, the aromatase inhibitor, letrozole, increased seladin-1 protein levels significantly in human neuroblastoma cells (SH-SY5Y). It is known that a decrease in brain E 2 levels is perceived as stress by neurons, and that brain aromatase levels increase as a protective/ compensatory mechanism [19]. For this reason, we hypothesized that after aromatase inhibition, seladin-1 levels are also increased as a protective mechanism to compensate for the decline in E 2 synthesis. To test this hypothesis, we measured the E 2 level in SH-SY5Y cells and observed that it was significantly decreased in the letrozole-treated group. Thus, increased seladin-1 can trigger an incremental change in cholesterol, the precursor for E 2 synthesis [84] ( Fig. 3).
When seladin-1 is silenced, the integrity of the membrane is disturbed, resulting in the formation of Ca 2+ -permeable pores on the membrane and subsequent cytotoxicity due to Ca 2+ hyperexcitation [85]. Seladin-1 overexpression has been shown to prevent this damage by preserving membrane integrity [15,68,75,76]. When seladin-1 is knocked out, the integrity of the membrane deteriorates, making easier for β-secretases (BACE) to break down amyloid precursor protein (APP) into Aβ, thereby inhibiting plasmin, the enzyme that degrades Abs, and causing Aβ accumulation [15,68,75,76].
In seladin-1 knock-out (SelKO) mouse models, homozygous mice were found to be born with severe dermopathies and die shortly after birth, so experiments are performed using heterozygous mice. AD mice were crossed with SelKO mice (AD/SelKO) in order to mimic the decreased expression of seladin-1 in AD; the membrane and intracellular cholesterol levels were decreased, and desmosterol levels were increased in the AD/SelKO mice. Significant Ab 1-40 and Aβ 1-42 increases were also observed in AD/SelKO mice compared to that in the AD mice [75].
In individuals with desmosterolosis, a rare congenital anomaly in humans, there is a significant decrease in seladin-1 enzyme activity due to gene mutations, and serious neurophysiological changes and developmental anomalies are seen [86].
Seladin-1 also plays an important role in nerve protection, similar to aromatase, via its essential enzymatic activity [71,72]. It is not a coincidence that expression of both aromatase and seladin-1 enzymes, especially in brain regions sensitive to AD, are found to be reduced. Another common feature of these enzymes is an increase in their expression as an acute response to neurodegenerative injury.

Brain aromatase and seladin-1 expression and interactions in AD
AD, the major cause of dementia, is a progressive neurodegenerative disorder that is characterized by memory loss and cognitive deficits with both genetic and environmental components [23,70,87]. Reduced serum estrogen in postmenopausal women has been widely reported to increase the risk of AD and is correlated with AD-related neuropathological changes [88][89][90].
It has been shown that estrogen therapy, HRT, could protect at least some postmenopausal women against cognitive impairment, dementia and AD, if the hormone treatment commenced soon after menopause; a later start of HRT may be detrimental [107][108][109][110][111]. This would concur with the hypothesis of the "healthy cell bias of estrogen action" [112].
Another important factor that could define subgroups of women for potential estrogen therapy is genetic. Polymorphisms related to gonadal steroid synthesis and metabolism could affect the outcomes of HRT. Therefore it is likely that certain variants of the aromatase gene may be associated with the risk of AD in women. It has also been shown that CYP19 polymorphisms affect the risk of AD in women.
Indeed, CYP19 gene variants could potentially affect the risk for AD by reducing or increasing the conversion of androgens into estrogens, resulting in altered protection against neuronal injury or neurodegeneration through multiple mechanisms [108]. As a result, alterations to plasma estrogen levels cannot always reflect the changes occurring in the CNS, and, despite its protective effect in vitro, exogenous estrogen treatment may not have a therapeutic effect [19,99,108]. Therefore, the protective effect of local estrogen may be dominant in the brain, rather than plasma estrogen, and the importance of local brain aromatase expression and activity is emphasized as the source of E 2 in the brain [18,42,89,106,109].
In humans, sex differences in the development of AD are frequently discussed; for example, the prevalence and severity of AD and the rate of decline is higher and cognitive Figure 3. Schematic of the interaction between aromatase and seladin-1. Decrease in brain E 2 levels is perceived as stress by neurons, and brain aromatase levels increase as a protective/compensatory mechanism [19]. After aromatase inhibition, seladin-1 levels increased as a protective mechanism to compensate for the decline in E 2 synthesis. Then, E 2 binds the estrogen response element (ERE) on the aromatase gene and regulates the aromatase promoter for neuroprotective activity of estrogen [69]. Figure 4. Schematic for the increased aromatase in the DG region of male SelKO/AD mice. Aromatase immunoreactivity in neurons increased significantly in the dentate gyrus (DG) region of male SelKO/AD mice compared to Sel + /AD (control) mice. Thus, we speculated that aromatase levels may increase either to improve neurogenesis in the DG or as a result of neurogenesis after seladin-1 gene expression downregulation in AD deterioration is faster and more pronounced in women than in men [110,111]. In general, the strong decline of circulating E 2 due to menopause is assumed to be a major risk factor for AD in women [63]. Neuroprotective effects of estrogen, an increased risk of dementia after menopause, and a 19-29% higher prevalence of AD in women compared to men stimulated an investigation into the relationship between estrogen, the estrogen-synthesizing enzyme aromatase, and AD [64,65,108,112,113].
In 3-month-old female 5XFAD mice, a significant decrease was observed in total aromatase expression, which was accompanied by reduced aromatase protein level in the CA1 and CA3 regions. It is possible that the increased production of Aβ inhibits aromatase expression in the young mice, which, in turn, results in the loss of neuroprotection by E 2 [91].
This finding indicates that the function of brainderived aromatase differs between male and female animal models as well.
Furthermore, a transgenic mouse model for AD (APP23 mice), in which the animals develop Aβ plaques and other pathological changes observable in the brains of AD patients [114,115], was crossbred with aromatase-KO (Ar −/− ) mice to test the influence of E 2 on the formation of Aβ plaques. The resulting female progeny, which is Ar +/− and therefore E 2 -haploinsufficient, demonstrated faster and more severe Aβ plaque formation and less effective Aβ clearance than did aromatase-expressing APP23 mice [89]. Ovariectomy of APP23 females, i.e., the elimination of their major source of systemic E 2 , did not mimic the effects of genetically-induced aromatase deficiency that affected all aromatase-expressing tissues, including the brain. These results suggest that brain-derived E 2 , rather than ovary-derived E 2 , counteracts Aβ plaque formation and is, therefore, neuroprotective in female mice.
Surprisingly, compared with APP23/Ar +/+ mice, Aβ plaque production is reduced in male APP23/Ar +/− mice, suggesting that endogenous testosterone may protect against AD in males and that the neuroprotective role of brainderived E 2 may be sex-dependent [116].
Moreover, in order to investigate interactions between aromatase and seladin-1, we used 5XFAD mice crossed with ArKO or SelKO mice.
Using immunohistochemical analysis, we observed that aromatase immunoreactivity in neurons increased significantly in the dentate gyrus (DG) region of male SelKO/ AD mice compared to Sel + /AD (control) mice. Furthermore, we also observed that E 2 levels increased in this group, whereas a significant decrease was observed in the ArKO group.
Aromatase increases in the DG of 14-16-weekold SelKO/AD male mice, during the earlier stages of AD [84]. In AD, the aromatase level may increase at earlier stages as a protective mechanism against neurodegeneration. Similar results were observed in vitro when serum, the cholesterol source, was removed from cell culture media [42], and in vivo as a compensatory response to acute neurotoxin administration [117][118][119]. It is tempting to assume that an ADrelated increase in aromatase expression, and, therefore, a potential increase in E 2 synthesis, represents a protective reaction of the tissue to early stages of the developing disease [91].
Furthermore, the alteration of aromatase levels, especially in the DG, can be associated with the role of the DG in neurogenesis [120]. Thus, we speculated that aromatase levels may increase either to improve neurogenesis in the DG or as a result of neurogenesis after seladin-1 gene expression downregulation in AD (Fig. 4). The reason why aromatase increase did not occur in female SelKO/AD mice was not apparent, in contrast to male mice, but may be due to the compensatory effect of higher peripheral estrogen against decreased brain estrogen in these non-ovariectomized young female mice. Brains of SelKO/AD male mice may need more local aromatase and estrogen expression to compensate for less peripheral estrogen, caused by the deprivation of the protective effects of gonadal estrogen relative to females, and the lack of the neuroprotective effects of seladin-1. We suggested that the CNS response to neurodegeneration is reflected as an increase in the local aromatase level. Therefore, the aromatase level does not increase in SelKO/ AD female mice because the neuroprotective effect of peripheral estrogen compensates for the changes in local estrogen expression [84].
In women, decreases in gonadal estrogen and local aromatase expression may help to explain their vulnerability to neurodegenerative events and the increase of AD diagnoses after menopause. The sex differences found in the mouse model are in agreement with previous findings, which indicate that the neuroprotective role of brain-derived E 2 may be more important in females than in males [89,113,116], and may help to explain why women are more prone to AD than men [91,110].
Cerebral ischemia or "ischemic stroke" is caused by advanced age, hypertension, previous history of stroke or a transient ischemic attack, cardiac arrest, traumatic brain injury, diabetes, cigarette smoking, atrial fibrillation and high cholesterol [128], and results in neuronal death predominantly in brain regions that are most intrinsically vulnerable, such as the CA1 region of the hippocampus [129].

Estrogens display neuroprotective
properties and promote neural regeneration after traumatic brain injury and cerebral ischemia by decreasing apoptotic signaling, neuroinflammation, and oxidative stress and by normalizing glutamate concentrations [130].
Aromatase expression increases following brain trauma, suggesting that aromatase plays an important role in neuroprotection by increasing local estrogen levels [23]. These neuroprotective effects of estrogen during brain ischemia have been well-established in ovariectomized rodents and result in a significant decrease in the lesion size and infarct volume [13,130,131].
Aromatase plays an important role in endogenous E 2 -mediated protective mechanisms and presents a novel target for neuroprotective therapy in ischemic pathology [19]. Female ArKO mice have significantly increased ischemic damage induced by reversible MCAO in all areas of the examined brain compared to wild-type littermates.
The same result was observed when E 2 production was pharmacologically inhibited by an aromatase inhibitor, fadrozole [19].
Aromatase protein increased after MCAO, and studies evaluating the peri-infarct location and astrocytic localization implicate the potential for aromatase to promote the survival of cells in the penumbra after experimental stroke by local synthesis of estrogens [41]. Peterson et al. (2001) have shown that aromatase mRNA and protein are rapidly and locally upregulated in RGCs, and that these cells migrate to injury sites following neural damage in zebra finch brains.
These findings suggest that injury-dependent upregulation of aromatase may be a conserved characteristic of the vertebrate brain and an important component of the initial response of neural tissue to injury [40].
In addition to aromatase expression, transcription of the C/EBPβ protein, a well-known mediator of injury and inflammatory responses in peripheral tissues [132] and potentially, in the brain [133,134], is increased in ischemic hippocampi and decreased after treatment with the aromatase inhibitor, megestrol acetate. We speculate that, because aromatase is a gene associated with regeneration, it is likely to be a direct transcriptional target of the C/EBP family [135].
Moreover, altered lipid metabolism is also believed to be a key event that contributes to CNS injuries, such as stroke [136,137].
SREBP-1 is a transcription factor best known for regulating lipid and cholesterol metabolism.
The active fragment of SREBP is transported to the nucleus where it binds to the promoters of SREBP target genes, most of which are involved in the synthesis and metabolism of lipids [138].
A recent study reported that, while the detailed mechanisms by which SREBP-1 activation leads to neuronal cell death remain to be established, the researchers discovered a method to inhibit SREBP-1 and, thereby, significantly reduce cell death [139]. In addition, another study showed that the removal of fetal bovine serum, a source of cholesterol, from cell medium results in increased astrocytic aromatase expression and activity [140]. We also observed an increase in aromatase expression when the serum was Inactivation of the SREBP-1 pathway was shown to reduce neuronal damage after stroke in mice [139]. Moreover, another report suggests that the excitatory receptor-dependent activation of SREBP-1 promotes neuronal cell death [142].
Our conclusions from these studies support the central hypothesis that the upregulation of aromatase in ischemic hippocampi and its downregulation in megestrol acetate-treated and indinavir-treated tissues may partly depend on transcription factors, such as C/EBPβ [135] and SREBP-1 [141], respectively. Our findings indicate that ischemia, as well as chronic neurodegenerative processes, lead to increased cytoplasmic aromatase and nuclear C/EBPβ and SREBP-1. Thus, it is possible to hypothesize an interaction between this enzyme and transcription factors [135,141].
Interestingly, clinical and experimental findings after stroke are abundant and highlight important sex differences [143,144]. Clinical studies showed that aging women display worse outcomes following an ischemic stroke than men, and that women also have higher mortality after hemorrhagic strokes [145]. In addition, rodents ischemic stroke models demonstrate that young females have smaller infarcted area than young males [146] and that they exhibit less severe stroke consequences during proestrus (high 17β-E 2 concentration) than during metestrus (low 17β-E 2 concentration) [147].
During aging, mortality is higher in females than in males, and males display greater bleeding and mortality during hemorrhagic strokes [145].
The study also showed that estrogen treatments improve outcomes in young females and males after ischemic and hemorrhagic strokes, although their effects in aging females during ischemia are controversial [145]. Other clinical and experimental findings also document the impact of sex and sex steroids in other CNS insults [148,149].  [152][153][154] and significantly decreased Ca +2 levels [155]. This decrease was strongly correlated with reduced Aβ 42 and Aβ 40 levels in the ipsilateral thalamus of MCAO rats. Seladin-1 decreased at both mRNA and protein levels after MCAO; conversely, bepridil treatment restored seladin-1 expression in the ipsilateral thalamus of MCAO rats and was associated with the improved neuronal survival [156]. The seladin-1 protein is encoded by the DHCR24 gene and is a potential neuroprotective factor. This notion stems from previous studies, which showed that seladin-1 plays a cytoprotective role in oxidative stressinduced apoptosis by scavenging reactive oxygen species [83]. Moreover, seladin-1 interacts with the p53 tumor suppressor protein [77], a redox-sensitive transcription factor involved in the pathogenesis of brain ischemia and AD. Importantly, seladin-1 expression is downregulated in large pyramidal neurons in specific regions in AD brain and suggests that seladin-1 is associated with selective neuronal vulnerability [68][69][70][71][72].

Seladin-1 in Stroke
From this evidence, it seems reasonable to speculate that neuronal damage accompanied by a reduction in seladin-1, similar to reports in certain AD brains, will have important consequences in the CNS response to ischemia.
Thus, paucity in seladin-1 could affect the amount of damage and time of recovery from ischemia, as it is highly dependent on the amount of stress to the affected area [82]. Inflammation related mediators, such as COX-2, iNOS, TNF-α, and IL-10, were increased after ischemia in Sel+/− mice, compared with WT counterparts [82].

Brain aromatase and seladin-1 in epilepsy.
Epilepsy is the most common serious neurological disease and is defined as "a disease of the brain characterized by an enduring predisposition to generate epileptic seizures" [157]. Epilepsy, antiepileptic drugs (AEDs), and the reproductive system exhibit complex mechanisms of action. Reproductive endocrine and sexual dysfunction are more common in patients with partial epilepsy than in those with generalized epilepsy [157][158][159][160][161][162][163][164][165][166][167], particularly in temporal lobe epilepsy that affects the limbic system, because this area is extensively interconnected with the hypothalamic nuclei that regulate gonadal function [168][169][170]. This region controls gonadal hormones, including estrogen, which is converted from testosterone by aromatase. Patients with temporal lobe epilepsy have higher aromatase activity in the cerebral cortex than in subcortical areas.

Inhibition of cerebral aromatization results
in lower focal availability of estrogen and, theoretically, improves seizure control [171].
While the increase in aromatase expression, and, thus, the synthesis of estrogen, in head trauma, ischemia and AD are neuroprotective, the effects of estrogen on epilepsy are complex.
A recent study established that estrogens are epileptogenic [172] and have proconvulsive effects [173]. This effect is due to the ability of estrogens to lower the seizure threshold, thereby increase seizure discharges [172,174], and to increase neural membrane excitability [173,175]. However, there have also been some studies that hint at a potential anticonvulsant role of estrogen [175], highlighting not only the complex role of gonadal hormones in the body but also the complex etiology of epilepsy itself.
Thus, E 2 has mixed effects, ranging from no effect, mild anticonvulsant to proconvulsant effects, on seizures that depend on whether physiological or supraphysiological doses have been used [176]. In that sense, aromatase enzyme inhibitors may have great potential for use as an antiepileptic treatment [177]. In some clinical trials, aromatase inhibitors have been used to treat prostate hypertrophy; some success was reported in men with complex partial epilepsy treated using this drug and other aromatase inhibitors [178][179][180]. In fact, letrozole, approved for the treatment of breast cancer by the Food and Drug Administration (FDA) [181], has been clinically successful in treating epilepsy in men [178]. In a case study, a 61-year-old man with temporal lobe epilepsy and sexual dysfunction due to low testosterone levels used letrozole to normalize his testosterone level and improve his sexual function and seizure control [178,180]. Therefore, testosterone supplementation, concomitant aromatase inhibitors [182], and  [186], increase hepatic synthesis of sex hormone-binding globulin (SHBG) [187], and increase serum E 2 levels, either in absolute concentrations or relative to bioavailable testosterone (BAT), and are associated with hyposexuality and hypogonadism [188,189].
Therefore, a small increase in E 2 level, presumably as a result of an AED-induced aromatase activity increase, could have a disproportionately large negative feedback effect and contribute to hypogonadism [190].
The use of aminoglutethimide, a firstgeneration aromatase inhibitor, has been attempted as an antiepileptic drug in combination with other standard drugs [194].
Letrozole is a third-generation, reversible, nonsteroidal aromatase inhibitor, that was approved by FDA for the treatment of postmenopausal women with hormone receptor-positive or hormone receptorunknown locally advanced and metastatic breast cancer [195]. A recent preclinical study demonstrated the protective effect of letrozole in preventing kindling induced a number of antiepileptic compounds with a high potential for aromatase inhibition [177].

Conclusion
The identification of the brain-specific aromatase promoter and the activity of local estrogen through ERα and its downstream regulator, We also thank the Technology Transfer Center of Hacettepe University for supporting the advance editing of the manuscript.
Moreover, letrozole was previously reported to inhibit the testosterone-induced increase in PTZ seizure activity in mice [197]. Letrozole administration prior to KA significantly increased the latency to onset of seizures and reduced seizure occurrence in mice. However, the drug demonstrated no discernible effects on KA-mediated neurotoxicity [6].
It is interesting to note that letrozole is easily transported across the blood-brain barrier after systemic application and exerts an inhibitory influence on hippocampal estrogen synthesis, as it does in other regions of the body in mice [198].