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. 2009 Oct 16;150(12):5539–5548. doi: 10.1210/en.2009-0640

Androgens Induce Dopaminergic Neurotoxicity via Caspase-3-Dependent Activation of Protein Kinase Cδ

Rebecca L Cunningham 1, Andrea Giuffrida 1, James L Roberts 1
PMCID: PMC2795716  PMID: 19837873

Abstract

Aged men have a greater incidence of Parkinson’s disease (PD) than women. PD is a neurodegenerative condition associated with the loss of dopamine neurons in the nigrostriatal pathway. This study examined the neurotoxic effects of androgens in a dopaminergic cell line (N27 cells) and the downstream signaling pathways activated by androgens. Treatment of N27 cells with testosterone- and dihydrotestosterone-induced mitochondrial dysfunction, protein kinase C (PKC)-δ cleavage, and apoptosis in dopaminergic neuronal cells. Inhibition of caspase-3 prevented the cleavage of PKCδ from the full-length element to the catalytic fragment and apoptosis in N27 cells, suggesting that androgen-induced apoptosis is mediated by caspase-3-dependent activation of PKCδ. Androgen-induced apoptosis may be specific to dopamine neurons as evidenced by a lack of testosterone-induced apoptosis in GnRH neurons. These results support a neurotoxic consequence of testosterone on dopaminergic neurons and may provide insight into the gender bias found in PD.

Androgens induce apoptosis in a dopaminergic cell line via caspase-3-dependent activation of PKCδ.

Recent studies report sex differences concerning the prevalence of certain neurodegenerative disorders (1,2), such as Parkinson’s disease (PD). Aged men have a greater incidence of PD than aged women (3,4,5,6,7). Clinical characteristics of PD include motor disturbances, such as tremor rigidity and bradykinesia (8,9,10,11). The primary pathological feature of PD is the loss of dopamine neurons within the substantia nigra pars compacta of the midbrain (8,9,11,12,13).

Oxidative stress is involved in mediating this loss of dopamine neurons in PD (6,7,11,14,15). Furthermore, animal models of PD have shown that oxidative stress leads to initiation of the apoptotic pathway (16,17,18,19). Loss of dopamine receptors in the midbrain has been found in both animal models of PD (20) and animals treated with androgens (21). Therefore, it is possible that testosterone may play a role in the progression of PD. Previous reports have shown that testosterone can increase cellular apoptosis (22,23,24). However, the role of testosterone in dopamine neuron death occurring in PD has not been examined.

Apoptosis is a form of controlled cell death (25,26), characterized by cell shrinkage, DNA fragmentation, and chromatin condensation (27,28,29). Apoptosis is associated with the cellular activation of the caspase family of cysteine proteases (25,26). Caspase-3 mediates the execution phase of the apoptosis cycle (28,30,31,32,33). Upon caspase-3 activation, protein kinase C (PKC)-δ is proteolytically cleaved, resulting in its activation within the cytosol and subsequent cell death (32,34,35). Several studies report that PKCδ positively feeds back on caspase-3 activation, thus inducing further apoptosis (36,37,38). PKCδ has also been shown to be a mediator of apoptosis in PD animal models (36,39). Several studies report that PKCδ is responsive to both oxidative stress and testosterone (37,38,40,41). Increased apoptosis via caspase-3 activation in response to oxidative stressors, such as hydrogen peroxide, has been reported in the dopaminergic N27 cell line (7). Testosterone levels in the LNCaP prostate cells were positively correlated with PKCδ levels (41). The likelihood that testosterone directly regulates PKCδ expression is further supported by the existence of a hormone response element for testosterone located 4.7 kb upstream of the transcription start site of the PKCδ promoter region (41).

It is important to note that androgens and its effects on oxidative stress have not been studied within a dopaminergic cell line. Therefore, the purpose of this study was to investigate the role of androgens in PD, using N27 cells as a model to test the neurotoxic effects of these hormones on dopamine neurons. Furthermore, to determine the specificity of androgen-induced neurotoxicity, a nondopaminergic cell line, GT1-7, was included.

Materials and Methods

Reagents

RPMI 1640 medium was purchased from Life Technologies, Inc. (Gaithersburg, MD), and fetal bovine serum was from American Type Culture Collection (Manassas, VA). Testosterone propionate, dihydrotestosterone (DHT), estradiol, and flutamide were from Sigma (St. Louis, MO). Caspase-3/CPP32 inhibitor (Z-DEVD-FMK) was from BioVision Inc. (Mountain View, CA). The Vybrant apoptosis assay kit was from Invitrogen (Eugene, OR), the fluorescent thiol detection kit was from Cell Technology (Mountain View, CA), and the QuantiChrom peroxide assay kit was from Bioassay Systems (Hayward, CA). N-acetyl-cysteine (NAC; A9165) was from Sigma. The rabbit polyclonal PKCδ (sc-213), rabbit polyclonal androgen receptor (sc-815; C19), horseradish peroxidase-conjugated antimouse IgG secondary antibody, and antirabbit IgG secondary antibody were from Santa Cruz Biotechnology (Santa Cruz, CA). The monoclonal mouse β-actin (A5441) antibody was from Sigma-Aldrich. The polyclonal rabbit peroxiredoxin-3 antiserum (ab16830) was from Abcam Inc. (Cambridge, MA).

Cell cultures

Two cell lines were used in this study: N27 and the GT1-7. Both the GT1-7 (42,43,44,45) and the N27 cell lines (Fig. 1A) express androgen receptors. The N27 cell line (a gift from Dr. Curt Freed, University of Colorado Health Science Center, Denver, CO) comprises a homogenous population of tyrosine hydroxylase immortalized cells derived from mesencephalic tissue of 12-d-old rat fetuses, which produce dopamine and its metabolites. These cells express the dopamine transporter, PKC isoforms, and adrenoreceptors (46). However, they do not express dopamine D1 receptors (46), which is consistent with mature substantia nigra dopamine neurons (47,48). Furthermore, grafted N27 cells have been shown to significantly reduce the rate of methamphetamine-induced rotations in unilateral 6-hydroxydopamine lesioned rats (46). This cell line has previously been used as a model to study neurotoxicity (36,49,50). Cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 μg/ml); grown at 37 C in a 5% CO2 incubator; and subcultured weekly. All experiments were performed between passages 12 and 19 and at approximately 80% confluency in RPMI 1640 serum-free media to avoid confounding variables (hormonal content within serum). Removal of serum does not induce a fasting or deprivation state due to the medium being heavy in glucose, amino acids, and necessary nutrients (51). No significant differences in the percentage of dead cells were found between 1 and 48 h exposure to serum-free media (0.163 ± 0.083 and 0.121 ± 0.113%, respectively) using propidium iodide fluorescence, a measure of cell death. These cells continue to proliferate in a serum-free environment (Cunningham, R. L., unpublished observations).

Figure 1.

Figure 1

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Androgen-induced apoptosis in N27 cells via the classical androgen receptor. N27 cells express androgen receptors. A testis homogenate was used as a positive control (A). Apoptosis was examined using Hoechst 33342. N27 cells were treated with vehicle control (n = 5), testosterone (100 nm, n = 4), flutamide (500 nm, n = 3), flutamide + testosterone (n = 3), DHT (100 nm, n = 4), or T-BSA (100 nm; n = 3). Nuclear staining was visualized by fluorescent microscopy. Apoptotic cells were distinguished by the presence of bright blue nuclei (B). Apoptotic nuclei were quantified from 10 random fields per chamber from each of the independent experiments. The data are expressed as the mean percent of apoptotic cells ± sem (C). Both testosterone and DHT significantly increased apoptosis in N27 cells compared with control. *, P < 0.05. T-BSA did not increase apoptosis. The androgen receptor antagonist, flutamide, inhibited testosterone-induced apoptosis. ANOVA was followed by Student’s PLSD post hoc test. Scale bar, 200 μm. Arrow indicates an apoptotic cell.

The hypothalamic GnRH GT1-7 cell line was a gift from Dr. Xin-Yun Lu (University of Texas Health Science Center, San Antonio, TX). This cell line was maintained in DMEM/F12 media supplemented with 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 μg/ml) grown at 37 C in a 5% CO2 incubator and subcultured weekly. Experiments were performed in serum-free medium.

Treatments

N27 cells were exposed to physiologically relevant concentrations of androgens (10 or 100 nm), representing the low and high end of the normal range in human males (52,53,54,55,56). Cells were analyzed after 48 h of androgen exposure. The androgens used were the aromatizable androgen, testosterone propionate, the nonaromatizable androgen, DHT, and the nonmembrane permeable androgen, testosterone conjugated to BSA (T-BSA). Androgens were applied either individually or in combination with the androgen receptor antagonist flutamide (500 nm), the caspase-3/CPP32 inhibitor Z-DEVD-FMK (4 μm), or the antioxidant NAC (1 mm). The steroid hormones, testosterone and dihydrotestosterone, along with flutamide were from stock solutions made in ethanol (final concentration of ethanol <0.01%). T-BSA and Z-DEVD-FMK were dissolved in culture media. In this study we mainly used testosterone because this hormone is the most clinically relevant androgen measured in human male sera (57). In addition, aging has been shown to impact testosterone levels in males (58), without affecting DHT levels significantly (59,60). The same treatment paradigm was used with the hypothalamic cell line GT1-7 to determine whether androgens had similar effects in nondopaminergic neurons.

Apoptosis assay

Apoptosis was quantified using the Vybrant apoptosis assay kit no. 5 (Invitrogen) according to the manufacturer’s instructions. Briefly, equal volumes of Hoechst 33342 solution and propidium iodide (1:1000) were added to cells cultured in four-chamber slides, and the mixture was incubated for 30 min on ice. Fluorescence was measured under a fluorescence microscope with a ×20 objective, using filters for 4′,6′-diamino-2-phenylindole (DAPI) and Texas Red dye. The blue fluorescent dye Hoechst 33342 stains chromatin of apoptotic nuclei more vividly than nonapoptotic nuclei, whereas the red fluorescent dye propidium iodide is permeable only to the nuclei of dead cells that have lost plasma membrane integrity. Apoptotic and dead cells were quantified from 10 random fields per chamber in at least three independent experiments. Results are expressed as percentage of apoptotic cells (100 × Hoechst-positive cells/number of cells per field) and dead cells (100 × propidium iodide-positive cells/number of cells per field).

Detection of reduced thiols

The level of reduced thiols in cell lysate (1 × 105 cells) was determined by using the fluorescent thiol detection kit (Cell Technology) according to the manufacturer’s instructions in five independent experiments. This assay uses 2,4-dinitrobenzenesulfonyl fluorescein (dye) as a fluorescent probe for general reduced thiols. Fluorescence was excited at 405 nm and emitted at 525 nm.

Cell lysates

Cells were lysed in lysis buffer [50 mm Tris-HCL (pH 7.5), 100 mm NaCl, 0.1% Triton X-100, 0.1% sodium dodecyl sulfate, 1 mm EDTA] containing protease and phosphatase inhibitor cocktail [1:100 (P8340); Sigma] on ice, and the lysates were clarified by centrifugation at 4 C for 20 min at 13,000 rpm (16,000 × g) in a refrigerated microcentrifuge. Protein concentrations were determined by using the micro-BCA assay (Pierce, Rockford IL) according to the manufacturer’s instructions.

Peroxide assay

Quantichrom peroxide assay kit DIOX-250 (Bioassay System) was used to determine peroxide concentration according to manufacturer’s instructions. Equal amounts (45 μg) of protein were incubated at room temperature with 100 μl of either the working reagent (ammonium iron sulfate/sulfuric acid and xylenol orange) or the control reagent (sulfuric acid and xylenol orange) for 30 min in a 96-well plate. OD was measured using a microplate reader at 585 nm in at least three independent experiments.

Western blot analysis

Equal amounts (20 μg) of protein were separated in a 4–20% polyacrylamide gel, transferred to nitrocellulose membranes (0.2 μm pore size) at 4 C, and blocked for 1 h at room temperature with Tris-buffered saline containing 0.05% Tween 20 [TBST (pH 7.4)] and 5% nonfat dried milk. After washing with TBST, membranes were incubated with specific antibodies at 1:500 concentration overnight at 4 C. Membranes were then washed again with TBST and incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies at 1:2000 concentration for 1 h at room temperature. The protein bands were visualized with an enhanced chemiluminescence’s detection kit (GE Healthcare, Piscataway, NJ) according to the manufacturer’sinstructions. The intensity of the bands was quantified by densitometry using the National Institutes of Health Image J program (Bethesda, MD) in three independent experiments.

Statistical analysis

Statistical analysis was computed by the StatView 5.0 software (SAS Institute, Cary, NC). All data were expressed as mean ± sem and analyzed by a Student’s unpaired t test or an ANOVA for studies with more than two groups followed by Fisher’s protected least significant difference (PLSD) post hoc test. P ≤ 0.05 was designated as significant.

Results

Testosterone increases apoptosis through the classical intracellular androgen receptor

To determine whether androgen exposure elicits apoptosis in dopaminergic N27 neurons, these cells were chronically (48 h) treated with testosterone, the nonaromatizable androgen DHT, the androgen receptor antagonist flutamide, flutamide + testosterone, the nonmembrane permeable androgen T-BSA, or vehicle (control). After androgen treatment, an apoptosis assay that uses the dye Hoechst 33342 as a marker for apoptosis was performed (Fig. 1B). Both testosterone and DHT (100 nm) induced apoptosis, as evidenced by an increase in bright blue fluorescence-labeled cells compared controls (Fig. 1B), whereas testosterone at 10 nm had no effect (data not shown). On the other hand, T-BSA, an androgen that binds only to membrane androgen receptors (61,62), had no effect on apoptosis when used at the same concentration (100 nm) as testosterone (Fig. 1, B and C), as well as at 10 nm or 1 μm (data not shown). Androgen-induced apoptosis was blocked by flutamide (Fig. 1, B and C). Cell death, as expressed as the number of propidium iodide-positive cells per total number of cells per field, was significantly increased by testosterone or DHT (7.502 ± 0.668 and 8.238 ± 1.490%, respectively) compared with controls (0.986 ± 0.461%). The testosterone-induced cell death was reversed by flutamide (1.083 ± 0.142%), which had no effect on cell death when given alone (1.084 ± 0.308%). T-BSA also had no effect (0.473 ± 0.031%).

Testosterone increases mitochondrial dysfunction in N27 cells

Previous studies suggested that mitochondrial dysfunction promotes neurotoxicity in dopamine cells (63,64,65). Mitochondrial dysfunction was examined by measuring reduced thiol levels and peroxide levels. Testosterone exposure significantly increased peroxide levels (Fig. 2A) and significantly decreased the amount of reduced thiols (Fig. 2B) in dopaminergic neurons compared with vehicle control. Protein expression of peroxiredoxin-3, a mitochondrial antioxidant specific for peroxides, was also significantly increased by testosterone exposure (Fig. 2C). The androgen receptor antagonist flutamide and the antioxidant NAC blocked androgen-induced oxidative stress (Fig. 2B).

Figure 2.

Figure 2

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Testosterone-induced mitochondrial dysfunction. The levels of reduced thiols were assayed by using a fluorescent probe for general reduced thiols, and peroxides were assayed with a colorimetric kit in five independent experiments. N27 cells were treated with either testosterone or vehicle control for 48 h. Testosterone (100 nm) significantly increased peroxide concentration (*, P < 0.05), whereas 10 nm testosterone had no effect (A). Testosterone (100 nm) significantly decreased reduced thiols compared with control (*, P < 0.05). The antioxidant NAC significantly blocked testosterone’s effect on reduced thiols, as did the androgen receptor antagonist flutamide (B). To localize the increased peroxide radicals, Western blot analysis of the protein peroxiredoxin-3 (PRDX3), a mitochondrial antioxidant specific for peroxide free radicals, was analyzed in three independent experiments to localize the increased peroxide radicals. Testosterone significantly increased PRDX3 expression (*, P < 0.05) (C). ANOVA was followed by Student’s PLSD post hoc test.

Testosterone activates the PKCδ apoptotic signaling pathway

To determine the mechanism of androgen-induced apoptosis in N27 cells, proteolytic activation of PKCδ was measured at 12, 24, and 48 h after testosterone exposure. Testosterone treatment induced cleavage of the full-length PKCδ (72–74 kDa) into the active catalytic fragments (42 and 38 kDa) at 48 h after treatment (Fig. 3A). Because previous studies have shown that dopaminergic apoptosis is caspase-3 dependent (36,38), cells were cotreated with testosterone and the caspase-3-specific inhibitor Z-DEVD-FMK (4 μm). The androgen-induced PKCδ cleavage was significantly inhibited by the Z-DEVD-FMK (Fig. 3B). As shown in Fig. 4, A and B, caspase-3 inhibition also blocked androgen-induced apoptosis in dopamine neurons. Significantly fewer bright blue fluorescent-positive cells were found in the inhibitor + testosterone group compared with the testosterone group, whereas similar levels of blue fluorescent-labeled cells were observed in the control and inhibitor-only groups. Androgen-induced cell death, as measured by propidium iodide fluorescence, was suppressed by Z-DEVD-FMK (0.897 ± 0.227%) compared with controls (0.986 ± 0.461%).

Figure 3.

Figure 3

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Testosterone-induced proteolytic cleavage of PKCδ via the caspase-3 pathway. Activation of PKCδ was assayed in N27 cells after testosterone treatment at 12, 24, and 48 h. Western blot analysis was used to determine the catalytic fragments of PKCδ. Testosterone induced PKCδ cleavage at 48 h after treatment (A). Inhibition of caspase-3 by Z-DEVD-FMK (4 μm) significantly blocked testosterone-induced PKCδ proteolysis (#, P < 0.05) (B). ANOVA was followed by Student’s PLSD post hoc test.

Figure 4.

Figure 4

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Inhibition of testosterone-induced apoptosis by caspase-3 inhibition in N27 cells. Apoptosis was assessed by using Hoechst 33342. N27 cells were treated with vehicle control (n = 5), testosterone (100 nm, n = 4), the caspase-3 inhibitor Z-DEVD-FMK (4 μm, n = 3), or Z-DEVD-FMK + testosterone (n = 3). Nuclear staining was visualized by fluorescent microscopy. Apoptotic cells were identified by the presence of bright blue-labeled cells. Testosterone significantly increased apoptosis compared with control (*, P < 0.05) (A). Inhibition of caspase-3 blocked testosterone-induced apoptosis (B). ANOVA was followed by Student’s PLSD post hoc test. Scale bar, 200 μm. Arrow indicates an apoptotic cell.

Androgen-induced apoptosis is not a generalized neuronal response

To ascertain whether androgens induce apoptosis in other neuronal cell types, the nondopaminergic cell line GT1-7 was exposed to chronic (48 h) androgen treatment using the same paradigm as described for the N27 cells. Fig. 5A illustrates fluorescent micrographs of Hoechst-33342-labeled GT1-7 neurons. The testosterone and control groups had similar levels of bright blue fluorescent-labeled cells (Fig. 5B), and no differences were found with respect to cell death (testosterone, 1.738 ± 0.141%; control, 2.176 ± 0.107%). GT1-7 cells (2.111 ± 0.782) had lower basal levels of oxidative stress than N27 cells (5.447 ± 0.240) as measured by a peroxide assay. Nevertheless, testosterone did not increase oxidative stress (Fig. 5C).

Figure 5.

Figure 5

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Testosterone does not induce apoptosis in the GT1-7 cells line. Androgen-induced apoptosis was examined in a nondopaminergic cell line (GT1-7) using Hoechst 33342 in four independent experiments. GT1-7 cells were treated with either vehicle control or testosterone (100 nm). No differences in bright blue fluorescent-labeled apoptotic cells were found between the testosterone and control groups (A and B). Testosterone did not increase peroxide levels in GT1-7 cells (C). Student’s unpaired t test. Scale bar, 200 μm. Arrow indicates an apoptotic cell.

Discussion

This study shows that physiologically relevant levels of androgens induce neurotoxicity in dopaminergic neurons. The key findings include: 1) testosterone increases apoptosis and cell death in N27 cells via the classical intracellular androgen receptor, 2) androgens increase oxidative stress-induced mitochondrial dysfunction, 3) testosterone induces caspase-3-dependent proteolytic activation of PKCδ, and 4) androgen-induced apoptosis is not a generalized neuronal event. These findings suggest that androgens may contribute to the gender disparities concerning the prevalence of PD (1,2).

A model for androgen-induced apoptosis in dopaminergic neurons is presented in Fig. 6. This model proposes that androgens may promote dopaminergic cell damage through a cascade that involves compromised mitochondrial function and increased oxidative stress, leading to caspase-3-dependent cleavage (and thus activation) of PKCδ. Upon PKCδ cleavage, DNA fragmentation and apoptosis would result (66). This model suggests that an androgen receptor antagonist can reverse the apoptotic events triggered by androgens. On the other hand, pharmacological inhibition of caspase-3 activity can block the apoptotic effects of PKCδ activation.

Figure 6.

Figure 6

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Putative model of androgen-induced apoptosis in N27 cells. Androgens enter the cell, bind to the classical intracellular androgen receptor (AR), and induce oxidative stress leading to mitochondrial dysfunction. Release of cytochrome c from the mitochondria activates the apoptotic caspase cascade, promoting PKCδ activation. Pharmacological inhibition of the androgen receptor by flutamide or of caspase-3 activity by Z-DEVD-FMK blocks androgen-induced apoptosis.

Oxidative stress has been described as an integral component of the pathogenesis of PD (67,68,69). In this study we examined the effects of chronic exposure to androgens on oxidative stress by measuring the levels of reduced thiols and peroxide free-radical species in N27 cells. Reduced thiols can be used as an indirect measure (or sensor) for reactive oxygenated species (ROS). Indeed, one cellular mechanism to counteract increased ROS is the production of the thiol-reducing glutathione and thioredoxin, which maintain the reduction-oxidation homeostasis of the cell (70,71). Thus, decreased reduced thiols are indicative of increased ROS in the cell. Our data indicate that testosterone decreased reduced thiol levels in N27 cells, suggestive of a testosterone-induced increase of ROS. This effect was blocked by the antioxidant NAC. Furthermore, testosterone increased peroxide free-radical species (Fig. 2), which are indicative of increased mitochondrial oxidative stress. These results have led to the formulation of our working model (Fig. 6), suggesting that chronic exposure to testosterone promotes mitochondrial dysfunction, thus further increasing the oxidative stress load of dopamine neurons. Such a cascade of events is consistent with prior studies implicating oxidative stress in the pathogenesis of PD (67,72).

The effects of physiological levels of androgens on dopamine neuron apoptosis have not been examined at time periods longer than 24 h, and only data using supraphysiological levels of testosterone are available (22). Previous studies have shown that 24 h exposure to testosterone at 10 and 100 nm had no effect on dopaminergic SH-SY5Y cell viability (22). These results are in agreement with our study, showing no effect of 24 h androgen exposure in N27 cells. However, longer (48 h) incubation induced apoptosis, suggesting that cell viability is dependent on the length of androgen exposure.

Androgens have been shown to have both neuroprotective (73,74,75) and toxic effects (41), depending on the system and concentration of androgens being studied (73,74). Furthermore, androgens can activate different intracellular pathways, such as the ERK/MAPK pathway that have been implicated in neuroprotection (73,75). On the other hand, physiological levels of androgens have been shown to increase apoptosis in prostate cells (41) and mixed cortical cells in the presence of an aromatase inhibitor (76). Different androgen metabolites or activation of either a putative membrane or the classical intracellular androgen receptor might explain these diverging effects. Testosterone can be converted to estradiol via aromatase, or reduced to DHT through the enzyme, 5α-reductase (77,78,79). Estrogen has consistently been shown to be neuroprotective (80,81,82), and increased dopaminergic apoptosis has been reported in aromatase knockout mice (83). These studies suggest that androgenic mechanisms contribute to dopaminergic apoptosis and estrogenic mechanisms mediate dopaminergic neuroprotection.

A recent study in primary cortical astrocytes showed that apoptosis as a result of androgen exposure might be linked to activation of membrane androgen receptors (23). Interestingly, the apoptotic effects of T-BSA, which does not permeate the plasma membrane, have been shown to decrease Akt phosphorylation in primary cortical astrocytes (signaling pathway involved in cellular protection) and to increase caspase activation (23). Thus, it is possible that the subtype or the location (i.e. membrane vs. nuclear) of the androgen receptor is crucial in determining the protective or toxic properties of testosterone.

Because membrane androgen receptors mediate apoptosis in glial cells, we examined the impact of these receptors on apoptosis in N27 cells. Interestingly, whereas testosterone promoted apoptosis, T-BSA did not, even when applied at equimolar concentrations. On the other hand, the nonaromatizable androgen, DHT, also increased apoptosis, supporting an androgen-mediated mechanism. Lastly, the androgen receptor antagonist, flutamide, blocked the effects of testosterone and DHT. Flutamide has been shown to preferentially block the classical intracellular androgen receptor rather than the membrane androgen receptor and further distinguishes the two receptor mechanisms (23). Therefore, these results show for the first time that androgen-induced apoptosis in dopaminergic neuronal cells occurs via the classical intracellular androgen receptor, rather than a putative membrane androgen receptor that is believed to be pharmacologically distinct.

PKCδ has been shown to be a mediator of apoptosis in animal models of PD (36,39). Previous studies showed that PKCδ is highly expressed in the striatum and substantia nigra (SN) of the brain (32,84). Dopamine neuron loss in the striatum and SN are one of the hallmark features of PD (6,7,11,14,15,20). Also, PKCδ expression increases with age (85), which is a risk factor for PD (3,4,5). Proteolytic activation of PKCδ is involved in the regulation of gene transcription in apoptosis (66). In keeping with these findings, we observed increased PKCδ cleavage in N27 cells after 48 h testosterone exposure, suggesting that the observed apoptosis is PKCδ-mediated.

PKCδ activation can occur through three different mechanisms: 1) membrane translocation of PKCδ, 2) tyrosine phosphorylation, and 3) caspase-3-dependent proteolytic activation (32). Mitochondrial dysfunction resulting from free radicals, increased ROS production, and subsequent cytochrome c release, can activate the caspase-3 pathway (86,87,88). In this study, we showed that testosterone increased oxidative stress and free-radical production and confirmed the involvement of the caspase-3 signaling pathway in these responses. Inhibition of caspase-3 by Z-DEVD-FMK blocked the testosterone-induced PKCδ cleavage and apoptosis in N27 cells, suggesting that androgen-induced apoptosis in these neurons occurs through caspase-3-dependent proteolytic activation of PKCδ. These results are consistent with previous reports of PKCδ-mediated apoptosis in animal models of PD (36,39).

The observation that testosterone did not induce apoptosis in the nondopaminergic GT1-7 cell line suggests that dopaminergic N27 cells are more sensitive to the neurotoxic effects of androgens. One inherent property of dopamine cells is a high degree of ROS formation and oxidative stress due to the catabolism of dopamine itself (89,90,91). Androgen-induced apoptosis in N27 cells may result from an additive effect of testosterone further increasing oxidative stress and free-radical species in a system that already possesses a high oxidative stress load.

Clinical studies show that aged males have a higher incidence of PD (1,2). The onset of clinical symptoms of PD occurs after approximately 60% loss of dopamine neurons within the SN pars compacta (SNc) (8). In rat SN, androgen receptors are mainly expressed in dopaminergic neurons of the SNc and are not expressed in the SN pars reticulata region (92), suggesting a possible role for androgens in rendering these cells more vulnerable than other subpopulations of dopamine neurons (such as those in the ventral tegmental area). Changes in androgen receptor levels along with reciprocal changes in testosterone levels have been found in the aged brain (cortex, hypothalamus, and hippocampus) (93,94,95), although no studies have systematically examined the impact of age and hormone levels on androgen receptor expression in dopamine neurons within the SNc. Nevertheless, age and gender have been shown to alter androgen receptor phosphorylation, thus affecting its function (96). Indeed, the phosphorylation levels of mouse cortical androgen receptors in response to testosterone are higher in aged males than young males and aged or young females (96). If similar changes occur in other brain regions, such as the SNc, phosphorylation (activation) of androgen receptors could contribute to further oxidative stress in dopamine neurons. Conversely, testosterone levels decrease with aging (95,97,98,99,100) and PD (101,102), but testosterone levels continue to remain higher in aged males than females (103). Thus, this persistent difference in testosterone levels could also result in higher oxidative stress in dopamine neurons, which over time, may contribute to the increased risk of PD in males. Further studies are required to confirm this hypothesis and determine whether the relationship between androgen receptor function and testosterone levels contributes to the pathogenesis of PD.

In conclusion, these results demonstrate that androgens increase apoptosis in dopaminergic neurons by inducing mitochondrial dysfunction through increased production of free-radical species and oxidative stress, leading to PKCδ activation.

Acknowledgments

We thank Dr. Meharvan Singh for his insightful comments and technical advice on the manuscript.

Footnotes

This work was supported by National Institutes of Health (NIH) Grant AG-08538 and American Parkinson’s Disease Association (APDA) grant Roger Duvoisin Award (to J.L.R.), NIH GrantNS050401-05A1 (to A.G.), and NIH Grant F32NS061417-01 (to R.L.C.).

Disclosure Summary: A.G. and J.L.R. have nothing to declare. R.L.C. received grant support from 2008 to 2011 from the National Institutes of Health.

First Published Online October 16, 2009

Abbreviations: DHT, Dihydrotestosterone; NAC, N-acetyl-cysteine; PD, Parkinson’s disease; PKC, protein kinase C; PLSD, protected least significant difference; ROS, reactive oxygenated species; SN, substantia nigra; SNc, SN pars compacta; T-BSA, testosterone conjugated to BSA; TBST, Tris-buffered saline containing Tween 20.

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