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Neuroprotective and neurotoxic outcomes of androgens and estrogens in an oxidative stress environment
Biology of Sex Differences volume 11, Article number: 12 (2020)
The role of sex hormones on cellular function is unclear. Studies show androgens and estrogens are protective in the CNS, whereas other studies found no effects or damaging effects. Furthermore, sex differences have been observed in multiple oxidative stress-associated CNS disorders, such as Alzheimer’s disease, depression, and Parkinson’s disease. The goal of this study is to examine the relationship between sex hormones (i.e., androgens and estrogens) and oxidative stress on cell viability.
N27 and PC12 neuronal and C6 glial phenotypic cell lines were used. N27 cells are female rat derived, whereas PC12 cells and C6 cells are male rat derived. These cells express estrogen receptors and the membrane-associated androgen receptor variant, AR45, but not the full-length androgen receptor. N27, PC12, and C6 cells were exposed to sex hormones either before or after an oxidative stressor to examine neuroprotective and neurotoxic properties, respectively. Estrogen receptor and androgen receptor inhibitors were used to determine the mechanisms mediating hormone-oxidative stress interactions on cell viability. Since the presence of AR45 in the human brain tissue was unknown, we examined the postmortem brain tissue from men and women for AR45 protein expression.
Neither androgens nor estrogens were protective against subsequent oxidative stress insults in glial cells. However, these hormones exhibited neuroprotective properties in neuronal N27 and PC12 cells via the estrogen receptor. Interestingly, a window of opportunity exists for sex hormone neuroprotection, wherein temporary hormone deprivation blocked neuroprotection by sex hormones. However, if sex hormones are applied following an oxidative stressor, they exacerbated oxidative stress-induced cell loss in neuronal and glial cells.
Sex hormone action on cell viability is dependent on the cellular environment. In healthy neuronal cells, sex hormones are protective against oxidative stress insults via the estrogen receptor, regardless of sex chromosome complement (XX, XY). However, in unhealthy (e.g., high oxidative stress) cells, sex hormones exacerbated oxidative stress-induced cell loss, regardless of cell type or sex chromosome complement. The non-genomic AR45 receptor, which is present in humans, mediated androgen’s damaging effects, but it is unknown which receptor mediated estrogen’s damaging effects. These differential effects of sex hormones that are dependent on the cellular environment, receptor profile, and cell type may mediate the observed sex differences in oxidative stress-associated CNS disorders.
Sex differences have been of interest as far back as 1871 with Charles Darwin’s publication titled “The descent of man, and selection in relation to sex” . Unfortunately, the number of studies on sex differences is sparse; leading to the National Institutes of Health (NIH) 2015 requirement of sex to be examined in NIH-funded studies. Further knowledge of sex differences is necessary as medicine is moving toward individualized precision medicine .
Numerous CNS disorders exhibit sex differences, which may result in the need for sex-specific standard of care. Men have an increased risk for Parkinson’s disease , autism , and schizophrenia . Conversely, Alzheimer’s disease , major depression [7, 8], and stress disorders [9, 10] are more prevalent in women than men. Menopause in women also influences the prevalence of CNS disorders, such as Alzheimer’s disease , stroke , Parkinson’s disease [13,14,15], depression , anxiety disorders , and schizophrenia [18, 19]. During menopause, estradiol levels abruptly decline from 15–350 pg/ml (depending on menstrual cycle stage) to less than 10 pg/ml, which is below circulating estradiol levels in men (10–40 pg/ml) . Interestingly, testosterone levels (8–60 ng/dL) are maintained in women during menopause [21,22,23]; these testosterone levels are 27–30 fold less than the levels observed in healthy young adult men (240–950 ng/dL) and middle-aged men (219–929 ng/dL) [24, 25].
Both androgens (i.e., testosterone and dihydrotestosterone, DHT) and estrogens (i.e., 17β-estradiol) along with their cognate receptors can influence the CNS by altering both structure and function [26, 27]. Within the past 25 years, studies have examined sex differences in androgen and estrogen receptor expression in the brain, such as the hypothalamus, cortex, and hippocampus (Table 1). Generally, sex hormones have protective effects in the CNS [36, 37]. However, recent findings indicate that the effects of sex hormones may depend on the cell type, cellular environment, and receptor expression profile [38,39,40,41]. Further, most studies only examined full-length androgen receptors, using antibodies (i.e., PG-21, Santa Cruz N-20, and Chemicon Ab561) that target amino acid sequences in the N terminus region of the androgen receptor (Table 1). These antibodies are unable to indicate the presence of an androgen receptor variant (i.e., AR45) that is missing the regulatory N terminus domain .
The AR45 localizes to plasma membrane lipid rafts in multiple brain regions, such as the entorhinal cortex, the hippocampus, and the substantia nigra , and is present in the human postmortem brain tissue (Fig. 1c). This androgen receptor variant is unresponsive to classical androgen receptor antagonists and involved in non-genomic actions of androgens . Specifically, AR45 interacts with G proteins and the NADPH oxidase (NOX) signaling pathways, which can lead to increased oxidative stress and cell loss [39, 42].
Oxidative stress results from the dysregulation of free radical homeostasis, which can damage lipids, proteins, and DNA. Free radicals are molecules that contain unpaired electrons and play important roles in cellular function (e.g., signal transduction and gene transcription) . The most common free radicals are hydroxyls, superoxides, and nitric oxide, which can produce hydrogen peroxide and peroxynitrate. Further, the most common reactive oxygen species (ROS) that produce free radicals are hydrogen peroxide and peroxynitrate. These free radicals and ROS are primarily generated via mitochondrial aerobic metabolism to create energy (ATP production) .
The brain is the highest consumer of energy in the body, in which it uses 20% of available energy for cellular communication and housekeeping functions . Under normal physiological conditions, ~ 2% of oxygen used to generate ATP is converted to ROS. In unhealthy or aged brains, more oxygen is converted to ROS , increasing the susceptibility of the brain to oxidative stress and damage. Oxidative damage can result in chronic diseases and has been shown to be associated with CNS diseases, such as Parkinson’s disease , autism [48, 49], schizophrenia , Alzheimer’s disease , stroke , major depression [53, 54], and anxiety disorders [55, 56]. Since sex differences are observed in these oxidative stress-associated CNS disorders and it is unclear what impact androgens and estrogens have on oxidative stress signaling, it is important to examine the relationship between sex hormones and oxidative stress.
In these studies, we focused on N27 neuronal-derived female rat cells, PC12 neuronal phenotype male rat cells, and C6 glial-derived male rat cells. These cell lines express estrogen receptors α/β and AR45, but do not express the full-length androgen receptor (Fig. 1a, b). These cell lines will allow further investigation of the effects of the novel androgen receptor variant, AR45, on cell survival. We chose to examine AR45 over the full-length androgen receptor as our prior studies found no effects of androgen receptor antagonists on oxidative stress endpoints, such as cell viability [39, 41].
Materials and methods
Fetal bovine serum (FBS, 35-010-CV), Dulbecco’s modified Eagle’s medium (DMEM, 10-017-CV), and l-glutamine (25-005-CI) were purchased from Corning. Charcoal/dextran-stripped fetal bovine serum (CS-FBS, S11650) was purchased from Atlanta Biologicals. DMSO (D128), SuperSignal West Femto Substrate (34096), Pierce BCA Protein Assay Kit (23225), tris-buffered saline (TBS, BP2471), and Tween-20 (BP337) were purchased from Thermo Fisher Scientific. Penicillin-streptomycin solution (PS, 15140-122), phosphate-buffered saline (PBS, 10010-031), and TrypLE Select LE 10X (A12177-01) were purchased from Gibco. Androgen receptor degrader ASC-J9 (J9, HY-15194) was purchased from MedChem Express. ICI 182,780 (ICI, 1047) and Androgen R/NR3C4 (MAB5876) were purchased from R&D Systems. Actin (ADI-CSA-400) was purchased from Enzo Life Sciences. tert-Butyl hydroperoxide (H2O2, A13926) and thiazolyl blue tetrazolium bromide solution (MTT, L11939) were purchased from Alfa Aesar. RPMI-1640 medium (SH30027.02) and RPMI-1640 Phenol Red and l-glutamine-free medium (SH30605.01) were purchased from Hyclone. 4-Androsten-17β-ol-3-one (testosterone, A6950-000) and dihydrotestosterone 3-CMO: BSA (DHT-BSA, A2574-050) were purchased from Steraloids. 17β-Estradiol (E-8875) and NGF (N0513) were purchased from Millipore Sigma. The antibodies AR-C19 (SC-815), AR-N20 (SC-816), ERα (SC-542), and ERβ (SC-8974) were purchased from Santa Cruz Biotechnology. GAPDH (GTX627408) was purchased from GeneTex, and goat anti-rabbit (31460) and goat anti-mouse (31430) secondary antibodies were purchased from Invitrogen. NP40 lysis buffer (J619-500) was purchased from Ameresco. RIPA lysis buffer (N653) was purchased from VWR. 15-well/15 uL any KD mini protean gels (456-9036) and Immun-Blot PVDF membranes (162-0177) were purchased from Bio-Rad.
Frozen hippocampal postmortem tissue from male and female donors, aged 66–93, Caucasian, was obtained from the Institute for Healthy Aging’s Brain Bank at the University of North Texas Health Science Center. Tissue storage period was less than 9 years. All cases exhibited Alzheimer’s disease-associated pathology.
This study used only rat-based cell lines in our experimental procedures. 1RB3AN27 (N27) dopaminergic cells (kind gift from Randy Strong, Ph.D., at University of Texas Health Science Center; RRID: CVCL_D584), PC12 dopaminergic adherent cells (ATCC CRL-1721.1; RRID: CVCL_F659), and C6 glial cells (ATCC CCL-107; RRID: CVCL_0194) were used in experimental paradigms. N27 cells are derived from SV40 T antigen-transformed neurons originally from embryonic female rat mesencephalic cells . PC12 neuroblastic cells are derived from a pheochromocytoma from a male rat [58, 59]. C6 glioma cells were derived from the male rats [60, 61]. N27 and PC12 cells were grown in RPMI-1640 media supplemented with 10% FBS and 1% PS (culture media). Although the adherent PC12 cells do not require NGF differentiation for adherence, dopaminergic neuronal phenotype, and response to oxidative stressors , we added NGF (100 ng/ml) to the PC12 media at time of plating . C6 cells were originally propagated in DMEM supplemented with 10% FBS and 1% PS. Following 48 h of growth, C6 cells were switched to the RPMI culture media. There were no significant differences observed in function or morphology following the medium switch in C6 cells (unpublished observation). Cells were maintained in a sterile environment at 37 oC with 5% CO2 and sub-cultured every 2–3 days.
To ensure the quality and integrity of the different cell lines, all experiments were conducted between passages 16–21 (undifferentiated N27), 8–14 (undifferentiated C6), and 5–10 (differentiated PC12). We also characterized these cells based on their morphology, doubling time, and a well-characterized response to tert-butyl hydrogen peroxide (H2O2) and testosterone [39,40,41]. Cell lines were switched to RPMI 1640 serum-free media supplemented with 10% CS-FBS and 1% PS (experimental media) prior to induction of experimental compounds to avoid hormonal content found within regular FBS [39,40,41]. CS-FBS does not contain steroid hormones (i.e., estradiol, testosterone, thyroid hormones). Testosterone, estradiol, and DHT-BSA were from stock solutions made in DMSO (final DMSO concentration < 0.001%).
Cell culture treatments
Reported LC-MS/MS brain hormone levels in male rats are (1) 5–24 nM testosterone [64,65,66,67], (2) 2.3–3.2 nM DHT [64, 67], and 0.2–0.9 nM estradiol [64, 67]. Since little to no albumin is present in healthy brains [68, 69], brain hormones are not protein bound and considered free. In order to compensate for the albumin (2.1 g/dl) in the CS-FBS, we used the Vermeulen calculation to determine the appropriate hormone dosage to attain physiological brain hormone levels [70, 71]. Therefore, in CS-FBS 100 nM, testosterone is 8 nM calculated free testosterone, 1 nM estradiol is 0.07 nM calculated free estradiol, and 500 nM DHT-BSA is 24 nM calculated free DHT-BSA. The higher DHT-BSA dosage was used to compensate for decreased hormone binding due to the 20 DHT molecules per 1 BSA molecule composition of DHT-BSA . Based on this, the levels of hormones used in this study are a reasonable approximation of brain hormone levels.
N27 and C6 cells were plated onto 96-well plates at a density of 1.5–2.0 × 106 cells/mL with culture media, whereas PC12 cells were plated at 6.0 × 104 cells/mL in culture media. All cells were left to proliferate overnight, except for PC12 cells that required 48 h. For treatments under the neuroprotective paradigm, cells were exposed to testosterone , 17β-estradiol, and DHT-BSA in experimental media (i.e., CS-FBS) for 2 h. Following the hormone exposure, N27 cells were treated with 15–20 uM of H2O2 in experimental media, PC12 cells with 30 uM of H2O2 in experimental media, and C6 cells with 50 uM of H2O2 in experimental media for 18 h to induce 20–30% cell loss. Cells under the neurotoxic paradigm underwent 10–20 uM (N27), 30 uM (PC12), or 50 uM (C6) of H2O2 for 2 h to induce 20–30% cell loss before hormone exposure for an additional 18 h. Cell viability was determined following each treatment paradigm.
Hormone receptor inhibitors
The estrogen receptor α/β inhibitor, ICI 182, 780 (ICI), and the androgen receptor degrader, ASC-J9 (J9), were used for this study. Inhibitor dose was chosen based on the IC50 data. Inhibitors and the degrader were made from stock solutions in DMSO (final DMSO concentration < 0.001%). For paradigms involving hormone receptor inhibitors, cells were exposed to ICI (300 pM) in experimental media for 1 h prior to induction of any hormones or oxidative stressor. For the androgen receptor degrader, J9 (5 uM), cells were exposed to the degrader for 30 min followed by either hormones or oxidative stressor for 1 h. Cells were then treated with either hormones or oxidative stressor for an additional 2 h.
Cell viability was determined by MTT assay. Media was aspirated from all wells, replenished with 100 uL of RPMI-1640 phenol red-free medium, and supplemented with 10% CS-FBS, 1% PS, and 1% l-glutamine. This was followed by the addition of 20 uL of 5 mg/mL of MTT solution to each well. Experimental plates were then covered in foil to block additional light and incubated at 37 oC with 5% CO2 for 3 h. Following incubation, plates were read at an absorbance of 595 nm. The colorimetric intensity is directly proportional to the number of viable cells in each well. Readings from respective treatment groups were then normalized to the vehicle control group to determine cell viability [39,40,41]. Three independent experiments, using different cell cultures, were conducted.
Western blot analysis
In order to confirm hormone receptor expression (Fig. 1), untreated N27, PC12, and C6 cells were collected and homogenized. We included Jurkat whole cell lysate (Abcam ab7899) as a positive control for the full-length androgen receptor and estrogen receptors α/β, and the MCF7 breast cancer cell line (ATCC HTB-22; RRID: CVCL_0031) was used as a positive control for estrogen receptors α/β. Additionally, we examined androgen receptor expression in human hippocampal tissues (25–50 ug). Tissues were homogenized using a RIPA lysis buffer mixture supplemented with protease inhibitor cocktail, 1 uM DTT, and 1 mM EDTA. Following homogenization and separation into whole cell lysate and membrane fractions , protein concentrations were determined using the Pierce BCA Protein Assay Kit per manufacturer’s instructions. Equal amounts of protein (20 ug) were separated in a Bio-Rad Any KD polyacrylamide gel at 25 mA for approximately 1 h and transferred onto a PVDF membrane at 50 V at 4 oC for 2–3 h. Following transfer, membrane blots were blocked using 5% non-fat milk in TBST for 30 min at room temperature. After blocking, membranes were incubated with constant agitation in primary antibodies (ERα 1:1000, ERβ 1:1000, ARC19 1:1000 for cell lines, Androgen R/NR3C4 1:1000 and ARN20 1:1000 for human tissue, Actin 1:1000, and GAPDH 1:10000) in 1% TBST non-fat milk either 2 h at room temperature or overnight at 4 oC. Membranes were then washed with 10% TBST twice for 10 min each before being incubated with secondary antibodies (Goat Anti-Rb HRP 1:1000 and Goat Anti-Ms HRP 1:10000) in 1% TBST non-fat milk for 30 min at room temperature. Afterwards, membranes were washed with 10% TBST two times for 10 min each. Visualization of bands was performed using SuperSignal West Femto Maximum Sensitivity Substrate and imaged for 30–90 s. Band intensity was then quantified by densitometry using the National Institutes of Health ImageJ program and normalized to GAPDH or actin levels. Three independent experiments were used.
Analyses were performed using IBM SPSS Statistics version 21 software. Statistical comparisons were made by two or three-way ANOVA using oxidative stressor (H2O2), hormones (testosterone, 17β-estradiol, DHT-BSA), and inhibitors as independent factors. This was followed by Fisher’s LSD post hoc analysis to evaluate differences between groups. Results are expressed as mean± SEM, and p value less than or equal to 0.05 (p≤ 0.05) indicates statistically significant differences. Each experiment was replicated at least three times using different cell cultures.
Testosterone and 17β-estradiol are protective in N27 and PC12 cells but not C6 cells
In vitro testosterone-mediated neuroprotection has been observed in several neuronal cells, including N27 cells [40, 73]. Similarly, in this study, we observed significant effects of the oxidative stressor H2O2 (F1, 8 = 475.2, η2 = 0.94, p < 0.05) and the hormone testosterone (F1, 8 = 13.8, η2 = 0.03, p < 0.05) on cell viability, along with an interaction between H2O2 and testosterone (F1, 8 = 8.7, η2 = 0.02, p < 0.05). Two-hour pretreatment of N27 cells with testosterone (100 nM) prior to oxidative stress, protected the cells by attenuating H2O2-induced cell loss (Fig. 2a). Consistent with our prior studies, testosterone alone did not have any effect on cell viability [39,40,41].
To examine if 17β-estradiol protects against oxidative stress damage, N27 cells were pretreated with 17β-estradiol, followed by H2O2. Significant effects of oxidative stressor H2O2 (F1, 8 = 56.2, η2 = 0.64, p < 0.05) and 17β-estradiol (F1, 8 = 11.2, η2 = 0.13, p < 0.05), along with an interaction between H2O2 and estradiol (F1, 8 = 12.5, η2 = 0.14, p < 0.05) on cell viability, were observed (Fig. 2b).
Unlike testosterone and 17β-estradiol, DHT-BSA did not alter H2O2-induced cell loss in N27 cells (Fig. 2c). DHT and 17β-estradiol are testosterone metabolites, which are ligands for the androgen receptor and the estrogen receptor, respectively. The addition of the BSA molecule to DHT restricts its activity to membrane androgen receptors. These results indicate that the putative membrane-associated androgen receptor does not mediate steroid hormone neuroprotection against oxidative stress insults, suggesting that the estrogen receptor mediates testosterone- and estrogen-mediated neuroprotection.
To determine if this effect is specific to neuronal-derived cells, we examined the neuroprotective properties of testosterone, 17β-estradiol, and DHT-BSA in C6 glial-derived cells. Similar to N27 cells, this cell line does not contain full-length androgen receptors but does express the androgen receptor variant, AR45 (Fig. 1). Unlike the N27 cells, neither testosterone (Fig. 2d) nor estradiol (Fig. 2e) was protective against H2O2-induced cell loss in C6 cells. Likewise, DHT-BSA did not protect the cells from H2O2-induced cell loss (Fig. 2f).
Since the N27 cells are a female-derived cell line, we conducted similar experiments in the PC12 male-derived cell line to examine if sex chromosome complement may underlie this protective effect in neuronal-like cells (Fig. 3). We observed significant effects of the oxidative stressor H2O2 (F1, 24 = 73.64, η2 = 0.68, p < 0.05) on cell viability, along with an interaction between H2O2 and hormones (F2,24 = 3.418, η2 = 0.06, p < 0.05). Two-hour pretreatment of PC12 cells with testosterone (100 nM) prior to oxidative stress protected the cells by attenuating H2O2-induced cell loss. In contrast, DHT-BSA did not alter H2O2-induced cell loss in PC12 cells. These findings are similar to results in the female-derived N27 cells.
Interestingly, two different concentrations of H2O2 were necessary to induce 20% cell loss in the cell lines. The N27 and PC12 cells were more sensitive to H2O2 (20–30 uM) than C6 cells to H2O2 (50 uM). This sensitivity to H2O2 may be due to dopamine metabolism of N27 and PC12 cells, which increases the oxidative stress burden and possibly sensitizes the cells to subsequent oxidative stressors [40, 74].
Estrogen receptor mediates neuroprotection
Since protection was not observed in C6 cells, we focused on N27 cells for further investigation into the mechanisms underlying hormone-mediated protection. Both testosterone and estradiol were protective against a mild oxidative stress insult that caused 20% cell loss (Fig. 2a, b), and thus, we wanted to ensure that these hormones would be protective against harsher oxidative stress insults. Therefore, we increased the H2O2 concentration to 50 uM to induce 80% cell loss (Fig. 4a, b). 17β-estradiol pretreatment significantly protected the cells from oxidative stress-induced cell death (Fig. 4a), as evidenced by significant effects of H2O2 (F1, 8 = 1176.8, η2 = 0.93, p < 0.05), estradiol (F1, 8 = 44.3, η2 = 0.03, p < 0.05), and an interaction between H2O2 and estradiol (F1, 8 = 42.2, η2 = 0.03, p < 0.05) on cell viability. Similarly, testosterone pretreatment protected N27 cells from H2O2-induced cell loss (Fig. 4b), in which significant effects of H2O2 (F1, 8 = 877.7, η2 = 0.93, p < 0.05) and testosterone (F1, 8 = 37.7, η2 = 0.04, p < 0.05), along with an interaction between these two factors (F1, 8 = 27.5, η2 = 0.03, p < 0.05), were observed.
To determine if the estrogen receptor is mediating neuroprotection, we blocked the estrogen receptor with ICI that inhibits estrogen α/β receptors . We observed significant effects of H2O2 (F1, 16 = 210.7, η2 = 0.86, p < 0.05), estradiol (F1, 16 = 15.3, η2 = 0.06, p < 0.05), and an interaction between H2O2 and estradiol (F1, 16 = 11, η2 = 0.04, p < 0.05) on cell viability (Fig. 4c). No effects of 17β-estradiol alone were observed. Approximately 20% cell loss was induced by H2O2. As expected, 17β-estradiol protected the cells from H2O2’s neurotoxic effects. This neuroprotective effect of estradiol on cell viability was blocked by the co-application of ICI (300 pM, IC50 concentration) with 17β-estradiol prior to H2O2 (F1, 16 = 7.2, η2 = 0.03, p < 0.05), confirming that 17β-estradiol’s neuroprotective effect is through the estrogen α/β receptor (Fig. 4c).
Hormone neuroprotection lost when cells are temporarily hormone deficient
Even though the FBS media (~ 27.5 pg/ml estradiol) was replaced with CS-FBS media in the prior experiments, cells were always exposed to hormones due to exogenous hormone application (e.g. testosterone, 17β-estradiol) in CS-FBS media. In this set of experiments, N27 cells were incubated in CS-FBS media in the absence of hormones 1 h prior to exogenous hormone (17β-estradiol and testosterone) treatment for 2 h (Fig. 5). After hormone treatment, cells were exposed to H2O2. Interestingly, neither 17β-estradiol nor testosterone-protected N27 cells from H2O2-induced cells loss (Fig. 5). These results highlight a “window of opportunity” for hormone action, in which hormone deprivation for at least 1 h was sufficient to ameliorate the neuroprotective effects of 17β-estradiol and testosterone against a subsequent oxidative stressor.
Androgens and estrogens are damaging in an oxidative stress environment
We previously published that testosterone in an oxidative stress environment is damaging in N27 cells [39,40,41]. In this study, testosterone, 17β-estradiol, and DHT-BSA further decreased cell viability in the presence of oxidative stress in both N27 and C6 cells (Fig. 6). Regardless of cell line, hormone alone did not have any effect on cell viability. In N27 cells, significant effects were observed with H2O2 (F1, 8 = 222.6, η2 = 0.78, p < 0.05), testosterone (F1, 8 = 22.4, η2 = 0.08, p < 0.05), and an interaction between H2O2 and testosterone (F1, 8 = 34, η2 = 0.12, p < 0.05) on cell viability (Fig. 6a). Estradiol had similar effects as testosterone on N27 cell viability in an oxidative stress environment (Fig. 6b), as evidenced by significant effects with H2O2 (F1, 8 = 129.4, η2 = 0.67, p < 0.05), 17β-estradiol (F1, 8 = 31.9, η2 = 0.16, p < 0.05), and an interaction between H2O2 and 17β-estradiol (F1, 8 = 26.8, η2 = 0.14, p < 0.05) on cell viability. Consistent with our prior studies , the membrane androgen receptor agonist, DHT-BSA, exacerbated H2O2-induced cell loss (Fig. 6c), as shown by significant effects with H2O2 (F1, 8 = 287.2, η2 = 0.70, p < 0.05), DHT-BSA (F1, 8 = 56.2, η2 = 0.14, p < 0.05), and an interaction between H2O2 and DHT-BSA (F1, 8 = 58.1, η2 = 0.14, p < 0.05). Similar to N27 cells, we observed damaging effects of hormones in an oxidative stress environment in PC12 cells. Significant effects were observed with H2O2 (F1, 12 = 88.064, η2 = 0.7, p < 0.05), DHT-BSA (F1, 12 = 11.027, η2 = 0.09, p < 0.05), and an interaction between H2O2 and DHT-BSA (F1, 12 = 14.198, η2 = 0.11, p < 0.05) on cell viability (Fig. 7).
Unlike our results that showed only neuroprotection in the N27 cell line, androgens and estrogens exacerbated H2O2-induced cell loss in the C6 cells. Specifically, testosterone (F1, 8 = 383.3, η2 = 0.17, p < 0.05), H2O2 (F1, 8 = 1488.2, η2 = 0.66, p < 0.05), and an interaction between H2O2 and testosterone (F1, 8 = 364.1, η2 = 0.16, p < 0.05) were observed in C6 cell viability (Fig. 6d). We observed the same responses with 17β-estradiol (F1, 8 = 77.7, η2 = 0.15, p < 0.05), H2O2 (F1, 8 = 341.9, η2 = 0.68, p < 0.05), and an interaction between the two variables (F1, 8 = 77.6, η2 = 0.15, p < 0.05) on cell viability (Fig. 6e). Figure 6f shows the same response with the membrane androgen receptor agonist, DHT-BSA, in which there were significant effects of H2O2 (F1, 8 = 109.2, η2 = 0.69, p < 0.05), DHT-BSA (F1, 8 = 23.2, η2 = 0.15, p < 0.05), and an interaction between oxidative stressor and DHT-BSA (F1, 8 = 19, η2 = 0.12, p < 0.05) on cell viability.
Cytosolic estrogen and androgen receptors do not mediate hormone toxicity
Our prior studies show that inhibiting cytosolic androgen receptors with flutamide, enzalutamide, or bicalutamide does not block androgen’s damaging effects in an oxidative stress environment [39, 40]. It is unknown what role estrogen receptors play in the damaging effects of testosterone or estradiol. Since estrogen α/β receptors are involved in neuroprotection (Fig. 4c), it is possible that these receptors may also mediate their damaging effects. Using N27 cells, ICI did not block testosterone’s negative effects in an oxidative stress environment (Fig. 8a), as evidence by significant effects of H2O2 (F1, 16 = 864.1, η2 = 0.67, p < 0.05), testosterone (F1, 16 = 224.4, η2 = 0.17, p < 0.05), and an interaction between oxidative stressor and testosterone (F1, 16 = 179.8, η2 = 0.14, p < 0.05) on cell viability but no effects of ICI (F1, 16 = 0.152, η2 = 0.0001, p > 0.05). A similar lack of response was observed with ICI and estradiol in an oxidative stress environment (Fig. 8b), wherein H2O2 (F1, 16 = 347, η2 = 0.66, p < 0.05), 17β-estradiol (F1, 16 = 96.7, η2 = 0.18, p < 0.05), and an interaction between H2O2 and 17β-estradiol (F1, 16 = 66.9, η2 = 0.13, p < 0.05) had significant effects on N27 cell viability but not ICI (F1, 16 = 0.030, η2 = 0.00006, p > 0.05).
Likewise, in C6 cells ICI did not block testosterone or estradiol exacerbation of H2O2-induced cell loss. In Fig. 8c, C6 cell viability was significantly impacted by H2O2 (F1, 16 = 2162.1, η2 = 0.63, p < 0.05), testosterone (F1, 16 = 657.5, η2 = 0.19, p < 0.05), and an interaction between oxidative stressor and testosterone (F1, 16 = 610.5, η2 = 0.18, p < 0.05), but no effects of ICI (F1, 16 = 1.589, η2 = 0.0005, p > 0.05). Similar effects were found using estradiol in Fig. 8d, in which we observed significant effects of H2O2 (F1, 16 = 1235.5, η2 = 0.70, p < 0.05), 17β-estradiol (F1, 16 = 256.1, η2 = 0.15, p < 0.05), and an interaction between oxidative stressor and 17β-estradiol (F1, 16 = 246.5, η2 = 0.14, p < 0.05), but no effects of ICI (F1, 16 = 0.006, η2 = 0.00, p > 0.05) in C6 cells.
Non-genomic mechanisms underlie hormone toxicity
Since neither estrogen receptor α/β nor cytosolic androgen receptors mediate hormone toxicity, we focused on the AR45 androgen receptor variant that is expressed in plasma membrane lipid rafts in the CNS . We previously published that ASC-J9 (J9), an androgen receptor degrader, was able to protect the N27 cells from testosterone’s damaging effects in an oxidative stress environment . However, it is unknown if AR45 mediates androgen toxicity in C6 cells.
Similar to our prior studies, degradation of the AR45 via J9 protected N27 cells from testosterone-induced cell loss in the presence of H2O2 (Fig. 9a). We observed significant effects of H2O2 (F1, 16 = 86.2, η2 = 0.67, p < 0.05), testosterone (F1, 16 = 7.5, η2 = 0.06, p < 0.05), and a significant interaction between H2O2 and testosterone (F1, 16 = 10.9, η2 = 0.08, p < 0.05), along with a significant interaction between H2O2, testosterone, and AR degrader (F1, 16 = 8.8, η2 = 0.07, p < 0.05). Notably, J9 did not affect H2O2-induced cell loss, indicating that it does not have off-target scavenging effects at the current concentration in N27 cells.
We observed different results using C6 cells. Contrary to the observable neuroprotective actions by J9 in N27 cells, J9 acts as a scavenger in C6 cells. Specifically, J9 attenuated H2O2-induced cell loss (F1, 16 = 90, η2 = 0.25, p < 0.05), and thus, its effect on testosterone could not be determined (Fig. 9b). A lower dose (1 uM) of J9 was used. However, it was ineffective and did not impact C6 cell viability (data not shown).
The role of sex hormones, such as androgens and estrogens, on cellular function is unclear. For example, some studies show androgens and estrogens are protective in the CNS. However, other studies found either no effects or damaging effects, especially in oxidative stress environments. Interestingly, sex differences have been observed in multiple oxidative stress-associated CNS disorders, such as Parkinson’s disease , autism [48, 49], schizophrenia , Alzheimer’s disease , stroke , major depression [53, 54], and anxiety disorders [55, 56], indicating a role for sex hormones. Since it is unclear what impact androgens and estrogens have on oxidative stress signaling, the current study examined the relationship between sex hormones and oxidative stress on cell viability.
The major findings of this study are (1) the first evidence of AR45 protein expression in the human brain, (2) testosterone and estrogen are protective against subsequent oxidative stress insults in neuronal-derived cells but not in glial-derived cells, (3) the estrogen receptor α/β mediates sex hormone neuroprotection, (4) a 1-h window of opportunity exists for sex hormone neuroprotection, (5) sex hormone administration following oxidative stress exacerbates oxidative stress damage in neuronal- and glial-derived cells, (6) the estrogen receptor is not involved in sex hormone-mediated toxicity, and (7) AR45 mediates androgen exacerbation of oxidative stress-induced cell loss. Since no differences were observed due to sex chromosome complement (N27 female-derived and PC12 male-derived neuronal phenotypic cells, C6 male-derived glial cells), the observed findings indicate that sex hormones’ cellular effects are not dependent on genotype (XX, XY) but rather are more specific to cell type, receptor profile, and the environmental status of the cell (e.g., oxidative stress load).
Although it is known that the full-length androgen receptor is expressed in the hippocampus [76, 77], our data showed lack of full-length androgen receptor expression in frozen hippocampal postmortem tissue from individuals diagnosed with Alzheimer’s disease (Fig. 1c). This result is not unexpected, as the full-length androgen receptor protein is known to degrade into fragments (e.g., 70 kDa fragments) under conditions such as freezing [42, 78, 79]. Further, the presence of 70 kDa androgen receptor fragment increases with age . Interestingly, aging is associated with increased oxidative stress [81, 82]. Thus, it is a possible oxidative stress may play a role in androgen receptor degradation, as oxidative stress has a bidirectional relationship with calcium-dependent calpain proteases [83,84,85] that can cleave full-length androgen receptors into 70 kDa fragments [86,87,88,89]. Future studies will examine if aging and neurodegenerative disorders are associated with increased expression of androgen receptor fragments.
The first study on the characterization and distribution of AR45 in humans by Ahrens-Fath 2005 failed to observe AR45 expression in the human brain tissue . The Ahrens-Fath study used the whole brain tissue, whereas we used a specific region of the brain tissue (i.e., hippocampus). The Ahrens-Fath study failed to show full-length androgen receptor transcript expression (positive control) in the human brain , which is widely known to be present in the human brain [32, 77]. Therefore, the lack of full-length androgen receptor and AR45 transcript expression in the human brain in the Ahrens-Fath study could be a false negative result. Since our data shows that AR45 protein is present in the human brain tissue and does not respond to classical androgen receptor antagonists along with data from Hu  showing AR45 mRNA expression in the aged human brain tissue, this protein may be an important pharmacological therapeutic target for neurodegenerative conditions.
Neuronal- and glial-derived cells responded differently to the oxidative stressor, H2O2. The neuronal phenotypic N27 and PC12 cell lines were more sensitive to oxidative stress than the glial-derived C6 cells. This result is consistent with several reported studies [92,93,94]. Glia cell (e.g., astrocytes and microglia) functions are diverse. They range from maintaining the brain environment , energy storage, and synaptic maintenance by modulating the neurotransmitter release and uptake (e.g., glutamate and GABA) [96,97,98,99,100,101], regulating the action potentials via potassium modulation [102, 103], and synthesizing and releasing the neurotrophic factors and neurosteroids [104,105,106,107]. These glial cell functions may underlie their resistance to oxidative stress insults.
The role of glial cells in neuronal degeneration is of increasing interest. The glia to neuron ratio (GNR) may play a role in the observed sex differences in brain regions linked with oxidative stress-associated diseases. In this study, we reviewed the literature for GNR in various brain regions associated with Alzheimer’s disease, Parkinson’s disease, major depression, anxiety disorders, schizophrenia, and autism spectrum disorders, as these disorders exhibit sex differences in prevalence (Table 2). We generally observed fewer glial cells per neuron in brain regions linked with more male-biased oxidative stress CNS disorders. For example, in the striatum, substantia nigra pars compacta, spinal cord, and cerebellum, neuronal cells far outnumbered glial cells. The presence of fewer glial cells could increase the susceptibility of these brain regions to oxidative stress damage due to the loss of glial supportive mechanisms from oxidative stress damage. Interestingly, testosterone itself is an oxidative stressor [39,40,41, 129]. Our results show that under conditions of oxidative stress, testosterone can exacerbate oxidative stress damage via a membrane-associated androgen receptor (AR45) [39,40,41, 129]. Therefore, androgens could be involved in the observed sex differences in these brain regions.
Testosterone’s effects are state dependent. Under low oxidative stress conditions, testosterone and its metabolite 17β-estradiol are neuroprotective via the estrogen receptor. We did not observe protective effects in C6 glial-derived cells. Interestingly, our data showed a window of opportunity for neuroprotection by sex hormones. If neuronal-derived cells were hormone-deficient at least 1 h, neither testosterone nor estrogen protected cells from subsequent oxidative stress insults. These results are consistent with findings from the Women’s Health Initiative concerning the loss of estrogen-mediated protection in menopausal women. Specifically, estrogen was protective in women less than 10 years from menopause . Estrogens are associated with decreased homocysteine, a marker of oxidative stress , in women within 10 years from menopause [130, 132]. However, homocysteine levels greater than 8 umol/L in postmenopausal women were associated with negative effects of estrogen . Further, homocysteine levels greater than 14 umol/L in individuals over 60 years of age were linked with Alzheimer’s disease risk [134, 135], which is more prevalent in postmenopausal women . Since our results show that estrogens were not protective in an oxidative stress environment, homocysteine levels may be useful as a biomarker for the “window of opportunity” for estrogen protection.
The classical cytosolic androgen receptor did not mediate androgen’s effects on neuroprotection, nor did classical androgen receptor antagonists affect androgen-mediated toxicity. Based on these results, medical use of androgen receptor antagonists is unlikely to interfere with androgen’s neuroprotective or damaging effects in neuronal and glial cells. Currently, androgen receptor antagonists are used to treat benign prostatic hyperplasia, prostate cancer, alopecia, hypersexuality, precocious puberty, and transgender transition in men, whereas in women, these drugs are used to treat acne, hirsutism, hyperandrogenism, and amenorrhea.
In contrast, the use of estrogen receptor antagonists could have a significant adverse effect by blocking testosterone- and 17β-estradiol-mediated neuroprotection. Currently, estrogen receptor antagonists are used in men and women to treat multiple conditions. Estrogen receptor antagonists are used to treat gynecomastia, breast cancer, and hypogonadism in men; breast cancer, ovulation induction, and transgender transition in women. Notably, the use of estrogen receptor antagonists (i.e., tamoxifen) are associated with increased Parkinson’s disease risk in women [136,137,138]. However, the role of estrogen receptor antagonists in Alzheimer’s disease risk in women is less clear [139,140,141,142,143]. No studies have examined the impact of estrogen receptor antagonists on CNS conditions in men.
Perspectives and significance
The effects of androgens and estrogens on neuronal and glial cell viability are dependent on the cellular environment. In healthy neuronal cells, androgens and estrogens are protective against oxidative stress insults via the estrogen receptor. However, in unhealthy (e.g., high oxidative stress) neuronal and glial cells, sex hormones have negative effects on cell viability by exacerbating oxidative stress-induced cell loss. Additionally, the non-genomic AR45 receptor is involved in androgen’s damaging effects, but it is unknown which receptor mediates estrogen’s damaging effects. These state-dependent effects of sex hormones may mediate the observed sex differences in oxidative stress-associated CNS disorders.
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.
Darwin C. The descent of man, and selection in relation to sex. London, UK: John Murray; 1871.
Collins FS, Varmus H. A new initiative on precision medicine. N Engl J Med. 2015;372(9):793–5.
Baldereschi M, et al. Parkinson’s disease and parkinsonism in a longitudinal study: two-fold higher incidence in men. ILSA Working Group. Italian Longitudinal Study on Aging. Neurology. 2000;55(9):1358–63.
Baron-Cohen S, et al. Why are autism spectrum conditions more prevalent in males? PLoS biology. 2011;9(6):e1001081.
Aleman A, Kahn RS, Selten JP. Sex differences in the risk of schizophrenia: evidence from meta-analysis. Arch Gen Psychiatry. 2003;60(6):565–71.
Mazure CM, Swendsen J. Sex differences in Alzheimer’s disease and other dementias. Lancet Neurol. 2016;15(5):451–2.
Rutter M, Caspi A, Moffitt TE. Using sex differences in psychopathology to study causal mechanisms: unifying issues and research strategies. J Child Psychol Psychiatry. 2003;44(8):1092–115.
Gobinath AR, Choleris E, Galea LA. Sex, hormones, and genotype interact to influence psychiatric disease, treatment, and behavioral research. J Neurosci Res. 2017;95(1-2):50–64.
Maeng LY, Milad MR. Sex differences in anxiety disorders: interactions between fear, stress, and gonadal hormones. Horm Behav. 2015;76:106–17.
Yonkers KA, et al. Chronicity, relapse, and illness--course of panic disorder, social phobia, and generalized anxiety disorder: findings in men and women from 8 years of follow-up. Depress Anxiety. 2003;17(3):173–9.
Brinton RD, et al. Perimenopause as a neurological transition state. Nat Rev Endocrinol. 2015;11(7):393–405.
Lisabeth LD, et al. Age at natural menopause and risk of ischemic stroke: the Framingham heart study. Stroke. 2009;40(4):1044–9.
Haaxma CA, et al. Gender differences in Parkinson’s disease. J Neurol Neurosurg Psychiatry. 2007;78(8):819–24.
Cereda E, et al. Reproductive factors and clinical features of Parkinson’s disease. Parkinsonism Relat Disord. 2013;19(12):1094–9.
Ragonese P, et al. Age at menopause predicts age at onset of Parkinson’s disease. Mov Disord. 2006;21(12):2211–4.
Clayton, A.H. and P.T. Ninan, Depression or menopause? Presentation and management of major depressive disorder in perimenopausal and postmenopausal women. Prim Care Companion J Clin Psychiatry, 2010. 12(1): p. PCC.08r00747.
Hu LY, et al. Risk of psychiatric disorders following symptomatic menopausal transition: a nationwide population-based retrospective cohort study. Medicine (Baltimore). 2016;95(6):e2800.
Gupta R, Assalman I, Bottlender R. Menopause and schizophrenia. Menopause Int. 2012;18(1):10–4.
Aloysi A, Van Dyk K, Sano M. Women’s cognitive and affective health and neuropsychiatry. Mt Sinai J Med. 2006;73(7):967–75.
Mayo Clinic Laboratories. Test ID: ESTF. Estrogens, Estrone (E1) and Estradiol (E2), Fractionated, Serum. 2019 [cited 2019 6/26/2019]; Available from: https://www.mayocliniclabs.com/test-catalog/Clinical+and + Interpretive/84230.
Christensen A, Pike CJ. Menopause, obesity and inflammation: interactive risk factors for Alzheimer's disease. Front Aging Neurosci. 2015;7:130.
Santoro N, Randolph JF Jr. Reproductive hormones and the menopause transition. Obstet Gynecol Clin North Am. 2011;38(3):455–66.
Ala-Fossi SL, et al. Ovarian testosterone secretion during perimenopause. Maturitas. 1998;29(3):239–45.
Mayo Clinic Laboratories. Test ID: TTFB testosterone, total, bioavailable, and free, serum. 2019 [cited 2019 6/26/2019]; Available from: https://www.mayocliniclabs.com/test-catalog/Clinical+and + Interpretive/83686.
Travison TG, et al. Harmonized reference ranges for circulating testosterone levels in men of four cohort studies in the United States and Europe. The Journal of Clinical Endocrinology & Metabolism. 2017;102(4):1161–73.
Nugent BM, Schwarz JM, McCarthy MM. Hormonally mediated epigenetic changes to steroid receptors in the developing brain: implications for sexual differentiation. Horm Behav. 2011;59(3):338–44.
Zhang JM, et al. Impact of sex and hormones on new cells in the developing rat hippocampus: a novel source of sex dimorphism? Eur J Neurosci. 2008;27(4):791–800.
Herbison AE. Sexually dimorphic expression of androgen receptor immunoreactivity by somatostatin neurones in rat hypothalamic periventricular nucleus and bed nucleus of the stria terminalis. J Neuroendocrinol. 1995;7(7):543–53.
Keil KP, et al. In vivo and in vitro sex differences in the dendritic morphology of developing murine hippocampal and cortical neurons. Sci Rep. 2017;7(1):8486.
Tsai H-W, et al. Age- and sex-dependent changes in androgen receptor expression in the developing mouse cortex and hippocampus. Neuroscience Journal. 2015;2015:11.
Lu SF, et al. Androgen receptor in mouse brain: sex differences and similarities in autoregulation. Endocrinology. 1998;139(4):1594–601.
Kruijver FP, et al. Sex differences in androgen receptors of the human mamillary bodies are related to endocrine status rather than to sexual orientation or transsexuality. J Clin Endocrinol Metab. 2001;86(2):818–27.
Lumbroso S, et al. Immunohistochemical localization and immunoblotting of androgen receptor in spinal neurons of male and female rats. Eur J Endocrinol. 1996;134(5):626–32.
Ravizza T, et al. Sex differences in androgen and estrogen receptor expression in rat substantia nigra during development: an immunohistochemical study. Neuroscience. 2002;115(3):685–96.
Karlsson, S.A., et al., Neural androgen receptors modulate gene expression and social recognition but not social investigation. Frontiers in Behavioral Neuroscience, 2016. 10(41).
Pike CJ. Testosterone attenuates beta-amyloid toxicity in cultured hippocampal neurons. Brain Res. 2001;919(1):160–5.
Zárate, S., T. Stevnsner, and R. Gredilla, Role of estrogen and other sex hormones in brain aging. Neuroprotection and DNA Repair. Frontiers in aging neuroscience, 2017. 9: p. 430-430.
Brinton RD. The healthy cell bias of estrogen action: mitochondrial bioenergetics and neurological implications. Trends Neurosci. 2008;31(10):529–37.
Tenkorang MAA, Duong P, Cunningham RL. NADPH oxidase mediates membrane androgen receptor-induced neurodegeneration. Endocrinology. 2019;160(4):947–63.
Holmes S, et al. Oxidative stress defines the neuroprotective or neurotoxic properties of androgens in immortalized female rat dopaminergic neuronal cells. Endocrinology. 2013;154(11):4281–92.
Holmes S, et al. Effects of oxidative stress and testosterone on pro-inflammatory signaling in a female rat dopaminergic neuronal cell line. Endocrinology. 2016;157(7):2824–35.
Garza-Contreras, J., et al., Presence of androgen receptor variant in neuronal lipid rafts. eNeuro, 2017. 4(4).
McCord JM. The evolution of free radicals and oxidative stress. Am J Med. 2000;108(8):652–9.
Halliwell B. Free radicals, antioxidants, and human disease: curiosity, cause, or consequence? Lancet. 1994;344(8924):721–4.
Du F, et al. Tightly coupled brain activity and cerebral ATP metabolic rate. Proceedings of the National Academy of Sciences. 2008;105(17):6409.
Lepoivre M, et al. Quenching of the tyrosyl free radical of ribonucleotide reductase by nitric oxide. Relationship to cytostasis induced in tumor cells by cytotoxic macrophages. J Biol Chem. 1994;269(34):21891–7.
Wei Z, et al. Oxidative stress in Parkinson’s disease: a systematic review and meta-analysis. Front Mol Neurosci. 2018;11:236.
Rossignol DA, Frye RE. Evidence linking oxidative stress, mitochondrial dysfunction, and inflammation in the brain of individuals with autism. Front Physiol. 2014;5:150.
Chauhan, A. and V. Chauhan, Oxidative stress in autism. Pathophysiology: the official journal of the International Society for Pathophysiology/ISP, 2006. 13(3): p. 171-181.
Emiliani FE, Sedlak TW, Sawa A. Oxidative stress and schizophrenia: recent breakthroughs from an old story. Current opinion in psychiatry. 2014;27(3):185–90.
Butterfield DA, Halliwell B. Oxidative stress, dysfunctional glucose metabolism and Alzheimer disease. Nature Reviews Neuroscience. 2019;20(3):148–60.
Li W, Yang S. Targeting oxidative stress for the treatment of ischemic stroke: upstream and downstream therapeutic strategies. Brain Circulation. 2016;2(4):153–63.
Michel TM, Pulschen D, Thome J. The role of oxidative stress in depressive disorders. Curr Pharm Des. 2012;18(36):5890–9.
Black CN, et al. Is depression associated with increased oxidative stress? A systematic review and meta-analysis. Psychoneuroendocrinology. 2015;51:164–75.
Salim S. Oxidative stress and psychological disorders. Current neuropharmacology. 2014;12(2):140–7.
Fedoce ADG, et al. The role of oxidative stress in anxiety disorder: cause or consequence? Free Radic Res. 2018;52(7):737–50.
Adams FS, et al. Characterization and transplantation of two neuronal cell lines with dopaminergic properties. Neurochem Res. 1996;21(5):619–27.
American Type Culture Collection. PC-12 Adh (ATCC® CRL-1721.1™). 2014, July 1; Available from: https://www.atcc.org/products/all/CRL-1721.1.aspx#documentation.
Greene LA, Tischler AS. Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proceedings of the National Academy of Sciences of the United States of America. 1976;73(7):2424–8.
Benda P, et al. Differentiated rat glial cell strain in tissue culture. Science. 1968;161(3839):370–1.
Hubner S, et al. Protective effects of fetal zone steroids are comparable to estradiol in hyperoxia-induced cell death of immature glia. Endocrinology. 2017;158(5):1419–35.
Baumann A, et al. Tyrosine hydroxylase binding to phospholipid membranes prompts its amyloid aggregation and compromises bilayer integrity. Scientific reports. 2016;6:39488.
Grau, C.M. and L.A. Greene, Use of PC12 cells and rat superior cervical ganglion sympathetic neurons as models for neuroprotective assays relevant to Parkinson’s disease. Methods in molecular biology (Clifton, N.J.), 2012. 846: p. 201-211.
Okamoto M, et al. Mild exercise increases dihydrotestosterone in hippocampus providing evidence for androgenic mediation of neurogenesis. Proc Natl Acad Sci U S A. 2012;109(32):13100–5.
Higashi T, et al. Studies on neurosteroids XVIII LC-MS analysis of changes in rat brain and serum testosterone levels induced by immobilization stress and ethanol administration. Steroids. 2006;71(7):609–17.
Tobiansky DJ, et al. Testosterone and corticosterone in the mesocorticolimbic system of male rats: effects of gonadectomy and caloric restriction. Endocrinology. 2018;159(1):450–64.
Caruso D, et al. Comparison of plasma and cerebrospinal fluid levels of neuroactive steroids with their brain, spinal cord and peripheral nerve levels in male and female rats. Psychoneuroendocrinology. 2013;38(10):2278–90.
LeVine SM. Albumin and multiple sclerosis. BMC Neurology. 2016;16(1):47.
Roos KL. Principles of neurologic infectious diseases. 1st ed. New York: McGraw-Hill Medical Pub. Division; 2005.
Mazer NA. A novel spreadsheet method for calculating the free serum concentrations of testosterone, dihydrotestosterone, estradiol, estrone and cortisol: with illustrative examples from male and female populations. Steroids. 2009;74(6):512–9.
Vermeulen A, Verdonck L, Kaufman JM. A critical evaluation of simple methods for the estimation of free testosterone in serum. The Journal of clinical endocrinology and metabolism. 1999;84(10):3666–72.
Estrada M, Varshney A, Ehrlich BE. Elevated testosterone induces apoptosis in neuronal cells. J Biol Chem. 2006;281(35):25492–501.
Ahlbom E, Prins GS, Ceccatelli S. Testosterone protects cerebellar granule cells from oxidative stress-induced cell death through a receptor mediated mechanism. Brain Res. 2001;892(2):255–62.
Lotharius J, et al. Effect of mutant alpha-synuclein on dopamine homeostasis in a new human mesencephalic cell line. J Biol Chem. 2002;277(41):38884–94.
Nicholson RI, et al. Responses to pure antiestrogens (ICI 164384, ICI 182780) in estrogen-sensitive and -resistant experimental and clinical breast cancer. Ann N Y Acad Sci. 1995;761:148–63.
Simerly RB, et al. Distribution of androgen and estrogen receptor mRNA-containing cells in the rat brain: an in situ hybridization study. J Comp Neurol. 1990;294(1):76–95.
Dart DA, et al. Visualising androgen receptor activity in male and female mice. PloS one. 2013;8(8):–e71694.
Kemppainen JA, et al. Androgen receptor phosphorylation, turnover, nuclear transport, and transcriptional activation. Specificity for steroids and antihormones. J. Biol. Chem. 1992;267(2):968–74.
Gregory CW, He B, Wilson EM. The putative androgen receptor-A form results from in vitro proteolysis. J Mol Endocrinol. 2001;27(3):309–19.
Tsai HW, et al. Age- and sex-dependent changes in androgen receptor expression in the developing mouse cortex and hippocampus. Neurosci J. 2015;2015:525369.
Cunningham RL, et al. Oxidative stress, testosterone, and cognition among Caucasian and Mexican-American men with and without Alzheimer’s disease. Journal of Alzheimer’s disease : JAD. 2014;40:563–73.
Tenkorang MA, Snyder B, Cunningham RL. Sex-related differences in oxidative stress and neurodegeneration. Steroids. 2018;133:21–7.
Ray SK, et al. Oxidative stress and Ca2+ influx upregulate calpain and induce apoptosis in PC12 cells. Brain Res. 2000;852(2):326–34.
Chen B, et al. Inhibition of calpain reduces oxidative stress and attenuates endothelial dysfunction in diabetes. Cardiovasc Diabetol. 2014;13:88.
Páramo B, et al. Calpain activation induced by glucose deprivation is mediated by oxidative stress and contributes to neuronal damage. The International Journal of Biochemistry & Cell Biology. 2013;45(11):2596–604.
Pelley RP, et al. Calmodulin-androgen receptor (AR) interaction: calcium-dependent, calpain-mediated breakdown of AR in LNCaP prostate cancer cells. Cancer Res. 2006;66(24):11754–62.
Mudryj M, Tepper CG. On the origins of the androgen receptor low molecular weight species. Hormones & cancer. 2013;4(5):259–69.
Libertini SJ, et al. Evidence for calpain-mediated androgen receptor cleavage as a mechanism for androgen independence. Cancer Res. 2007;67(19):9001–5.
Irvine RA, et al. Inhibition of p160-mediated coactivation with increasing androgen receptor polyglutamine length. Human Molecular Genetics. 2000;9(2):267–74.
Ahrens-Fath I, et al. Androgen receptor function is modulated by the tissue-specific AR45 variant. FEBS J. 2005;272(1):74–84.
Hu DG, et al. Identification of androgen receptor splice variant transcripts in breast cancer cell lines and human tissues. Horm Cancer. 2014;5(2):61–71.
Iwata-Ichikawa E, et al. Glial cells protect neurons against oxidative stress via transcriptional up-regulation of the glutathione synthesis. J Neurochem. 1999;72(6):2334–44.
Bhatia, T.N., et al., Astrocytes do not forfeit their neuroprotective roles after surviving intense oxidative stress. Frontiers in Molecular Neuroscience, 2019. 12(87).
Lassmann H, van Horssen J. Oxidative stress and its impact on neurons and glia in multiple sclerosis lesions. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease. 2016;1862(3):506–10.
Fetler L, Amigorena S. Neuroscience. Brain under surveillance: the microglia patrol. Science. 2005;309(5733):392–3.
Kimelberg HK, Katz DM. High-affinity uptake of serotonin into immunocytochemically identified astrocytes. Science. 1985;228(4701):889–91.
Kimelberg HK, Norenberg MD. Astrocytes. Sci Am. 1989;260(4):66 -72, 74, 76.
Schousboe A, et al. Role of astrocytic transport processes in glutamatergic and GABAergic neurotransmission. Neurochem Int. 2004;45(4):521–7.
Boucsein C, et al. Purinergic receptors on microglial cells: functional expression in acute brain slices and modulation of microglial activation in vitro. Eur J Neurosci. 2003;17(11):2267–76.
Light AR, et al. Purinergic receptors activating rapid intracellular Ca increases in microglia. Neuron Glia Biol. 2006;2(2):125–38.
Taylor DL, et al. Stimulation of microglial metabotropic glutamate receptor mGlu2 triggers tumor necrosis factor alpha-induced neurotoxicity in concert with microglial-derived Fas ligand. J Neurosci. 2005;25(11):2952–64.
Ransom BR, Sontheimer H. The neurophysiology of glial cells. J Clin Neurophysiol. 1992;9(2):224–51.
Newman E, Reichenbach A. The Muller cell: a functional element of the retina. Trends Neurosci. 1996;19(8):307–12.
Fawcett JW, Asher RA. The glial scar and central nervous system repair. Brain Res Bull. 1999;49(6):377–91.
Silver J, Miller JH. Regeneration beyond the glial scar. Nat Rev Neurosci. 2004;5(2):146–56.
Do Rego JL, et al. Neurosteroid biosynthesis: enzymatic pathways and neuroendocrine regulation by neurotransmitters and neuropeptides. Front Neuroendocrinol. 2009;30(3):259–301.
Gatson JW, et al. Aromatase is increased in astrocytes in the presence of elevated pressure. Endocrinology. 2011;152(1):207–13.
Dombrowski SM, Hilgetag CC, Barbas H. Quantitative architecture distinguishes prefrontal cortical systems in the rhesus monkey. Cerebral Cortex. 2001;11(10):975–88.
Smith AD. Imaging the progression of Alzheimer pathology through the brain. Proceedings of the National Academy of Sciences. 2002;99(7):4135–7.
Caligiore D, et al. Parkinson’s disease as a system-level disorder. NPJ Parkinsons Dis. 2016;2:16025.
Pandya M, et al. Where in the brain is depression? Current psychiatry reports. 2012;14(6):634–42.
Martin EI, et al. The neurobiology of anxiety disorders: brain imaging, genetics, and psychoneuroendocrinology. The Psychiatric clinics of North America. 2009;32(3):549–75.
Harrison PJ. The neuropathology of schizophrenia: a critical review of the data and their interpretation. Brain. 1999;122(4):593–624.
Ha S, et al. Characteristics of brains in autism spectrum disorder: structure, function and connectivity across the lifespan. Experimental neurobiology. 2015;24(4):273–84.
Herculano-Houzel S. The human brain in numbers: a linearly scaled-up primate brain. Frontiers in human neuroscience. 2009;3:31.
Herculano-Houzel S, Dos Santos SE. You do not mess with the glia. Neuroglia. 2018;1(1):193–219.
Keller, D., C. Erö, and H. Markram, Cell densities in the mouse brain: a systematic review. Frontiers in Neuroanatomy, 2018. 12(83).
von Bartheld CS, Bahney J, Herculano-Houzel S. The search for true numbers of neurons and glial cells in the human brain: a review of 150 years of cell counting. The Journal of comparative neurology. 2016;524(18):3865–95.
Smeyne M, et al. Glia cell number modulates sensitivity to MPTP in mice. Glia. 2005;52(2):144–52.
Perez-Costas E, Melendez-Ferro M, Roberts RC. Basal ganglia pathology in schizophrenia: dopamine connections and anomalies. Journal of neurochemistry. 2010;113(2):287–302.
Pakkenberg B, Gundersen HJ. Total number of neurons and glial cells in human brain nuclei estimated by the disector and the fractionator. J Microsc. 1988;150(Pt 1):1–20.
Halliday GM. Thalamic changes in Parkinson’s disease. Parkinsonism & Related Disorders. 2009;15:S152–5.
Hoogendijk WJ, et al. Image analyser-assisted morphometry of the locus coeruleus in Alzheimer’s disease, Parkinson’s disease and amyotrophic lateral sclerosis. Brain. 1995;118(Pt 1):131–43.
Giorgi FS, et al. The neuroanatomy of the reticular nucleus locus coeruleus in Alzheimer’s disease. Frontiers in neuroanatomy. 2017;11:80.
Prakash KG, et al. Neuroanatomical changes in Parkinson’s disease in relation to cognition: an update. Journal of advanced pharmaceutical technology & research. 2016;7(4):123–6.
Rubinow MJ, Juraska JM. Neuron and glia numbers in the basolateral nucleus of the amygdala from preweaning through old age in male and female rats: a stereological study. The Journal of comparative neurology. 2009;512(6):717–25.
Poulin SP, et al. Amygdala atrophy is prominent in early Alzheimer’s disease and relates to symptom severity. Psychiatry research. 2011;194(1):7–13.
Bjugn R, Gundersen HJ. Estimate of the total number of neurons and glial and endothelial cells in the rat spinal cord by means of the optical disector. J Comp Neurol. 1993;328(3):406–14.
Wilson EN, et al. Chronic intermittent hypoxia induces hormonal and male sexual behavioral changes: hypoxia as an advancer of aging. Physiol Behav. 2018;189:64–73.
Lakryc, E.M., et al., What is the influence of hormone therapy on homocysteine and crp levels in postmenopausal women? Clinics (Sao Paulo, Brazil), 2015. 70(2): p. 107-113.
Perna AF, Ingrosso D, De Santo NG. Homocysteine and oxidative stress. Amino Acids. 2003;25(3-4):409–17.
Christodoulakos GE, et al. Endogenous sex steroids and circulating homocysteine in healthy Greek postmenopausal women. Hormones (Athens). 2006;5(1):35–41.
Bruschi F, et al. Age, menopausal status and homocysteine levels in women around menopause. Eur J Obstet Gynecol Reprod Biol. 2005;120(2):195–7.
Seshadri S, et al. Plasma homocysteine as a risk factor for dementia and Alzheimer’s disease. The New England journal of medicine. 2002;346(7):476–83.
Kang SS, Wong PW, Malinow MR. Hyperhomocyst(e)inemia as a risk factor for occlusive vascular disease. Annu Rev Nutr. 1992;12:279–98.
Gradus JL, et al. The association between adjustment disorder diagnosed at psychiatric treatment facilities and completed suicide. Clin Epidemiol. 2010;2:23–8.
Hong C-T, et al. Tamoxifen and the risk of Parkinson’s disease in female patients with breast cancer in Asian people: a nationwide population-based study. Journal of breast cancer. 2017;20(4):356–60.
Lin HF, et al. Tamoxifen usage correlates with increased risk of Parkinson’s disease in older women with breast cancer: a case-control study in Taiwan. Eur J Clin Pharmacol. 2018;74(1):99–107.
Liao, K.-F., C.-L. Lin, and S.-W. Lai, Nationwide case-control study examining the association between tamoxifen use and Alzheimer’s disease in aged women with breast cancer in Taiwan. Frontiers in Pharmacology, 2017. 8(612).
Ording AG, et al. Null Association between tamoxifen use and dementia in danish breast cancer patients. Cancer Epidemiology Biomarkers &. Prevention. 2013;22(5):993–6.
Henderson VW, et al. Raloxifene for women with Alzheimer disease: a randomized controlled pilot trial. Neurology. 2015;85(22):1937–44.
Yaffe K, et al. Effect of raloxifene on prevention of dementia and cognitive impairment in older women: the Multiple Outcomes of Raloxifene Evaluation (MORE) randomized trial. Am J Psychiatry. 2005;162(4):683–90.
Sun LM, et al. Long-term use of tamoxifen reduces the risk of dementia: a nationwide population-based cohort study. QJM. 2016;109(2):103–9.
Postmortem tissue was obtained from the Institute for Healthy Aging’s Brain Bank at the University of North Texas Health Science Center. The authors would like to thank Drs. Rosalie Uht and Shaohua Yang of the Institute for Healthy Aging’s Brain Bank for their assistance.
This study was supported by the NIH R01 NS0091359 to RLC.
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This study was performed under a human subject protocol approved by the University of North Texas Health Science Center Institutional Review Board.
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The authors declare that they have no competing interests.
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Duong, P., Tenkorang, M.A.A., Trieu, J. et al. Neuroprotective and neurotoxic outcomes of androgens and estrogens in an oxidative stress environment. Biol Sex Differ 11, 12 (2020). https://doi.org/10.1186/s13293-020-0283-1
- Membrane androgen receptor
- Window of opportunity
- Estrogen receptors
- Oxidative stress
- Human hippocampus
- Sex differences