NADPH Oxidase Mediates Membrane Androgen Receptor-Induced Neurodegeneration

Abstract

Oxidative stress (OS) is a common characteristic of several neurodegenerative disorders, including Parkinson disease (PD). PD is more prevalent in men than in women, indicating the possible involvement of androgens. Androgens can have either neuroprotective or neurodamaging effects, depending on the presence of OS. Specifically, in an OS environment, androgens via a membrane-associated androgen receptor (mAR) exacerbate OS-induced damage. To investigate the role of androgens on OS signaling and neurodegeneration, the effects of testosterone and androgen receptor activation on the major OS signaling cascades, the reduced form of NAD phosphate (NADPH) oxidase (NOX)1 and NOX2 and the Gαq/inositol trisphosphate receptor (InsP3R), were examined. To create an OS environment, an immortalized neuronal cell line was exposed to H2O2 prior to cell-permeable/cell-impermeable androgens. Different inhibitors were used to examine the role of G proteins, mAR, InsP3R, and NOX1/2 on OS generation and cell viability. Both testosterone and DHT/3-O-carboxymethyloxime (DHT)-BSA increased H2O2-induced OS and cell death, indicating the involvement of an mAR. Furthermore, classical AR antagonists did not block testosterone's negative effects in an OS environment. Because there are no known antagonists specific for mARs, an AR protein degrader, ASC-J9, was used to block mAR action. ASC-J9 blocked testosterone's negative effects. To determine OS-related signaling mediated by mAR, this study examined NOX1, NOX2, Gαq. NOX1, NOX2, and the Gαq complex with mAR. Only NOX inhibition blocked testosterone-induced cell loss and OS. No effects of blocking either Gαq or G protein activation were observed on testosterone's negative effects. These results indicate that androgen-induced OS is via the mAR-NOX complex and not the mAR-Gαq complex.

Figure 1.

Experimental design. N27 cells were plated in 96-well cell culture plates and incubated at 37°C in 5% CO₂ for 24 h. Two-hour pretreatment was conducted for all inhibitors and AR degrader. At 80% confluency, cells were exposed to the OS or, H₂O₂, for 4 h followed by testosterone treatment.

Figure 2.

Coimmunoprecipitation of AR45, NOX1, and NOX2 to determine the protein complex. NOX1, NOX2, and AR45 proteins were precipitated using specific antibodies and then probed with respective antibodies for NOX1, NOX2, and AR45 to determine protein–protein interactions. NOX1, NOX2, and AR45 interact to form a protein complex. (A) Gα_q couples with AR45 but does not interact with either NOX1 or NOX2. (B) Testosterone can bind to the mAR, AR45, and activate multiple signaling pathways via its protein interactions with NOX1, NOX2, and Gα_q in a lipid raft. Caveolin (purple), flotillin (blue), and phospholipids (orange) are shown. Co-IP, coimmunoprecipitation; WB, Western blot.

Figure 3.

Testosterone’s detrimental effects are not mediated through the classical genomic pathway. Testosterone alone does not affect cell viability. H₂O₂ induced ∼20% cell loss, which was exacerbated by testosterone. (A) The AR antagonist bicalutamide did not block testosterone’s negative effects in an OS environment. (B) Testosterone did not alter AR45 expression. The AR degrader, ASC J9, significantly decreased the expression of AR45, irrespective of the presence of testosterone. (C) ASC J9 blocked testosterone-induced cell loss in an OS environment, but it did not influence H₂O₂-induced cell loss. Results are reported as mean ± SEM. Results were determined by ANOVA followed by a Fisher least significant difference post hoc test. *P ≤ 0.05 vs all groups; ^#P ≤ 0.05 vs control; ⁺P ≤ 0.05 vs HT groups. B, bicalutamide; C, vehicle control; H, H₂O₂; HT, posttreatment T; J9, ASC J9; T, 100 nM testosterone.

Figure 4.

The role of G protein and InsP₃R receptor in testosterone-induced neurodegeneration. (A and B) GDPβS trilithium, a GDP analog, and BIM-46187, a Gα_q inhibitor, did not protect the cells from testosterone’s detrimental effects in an OS environment. (C) The InsP₃R inhibitor 2-APB was able to block testosterone’s damaging effects in an OS environment. Results are reported as mean ± SEM. Results were determined by ANOVA followed by a Fisher least significant difference post hoc test. *P ≤ 0.05 vs all groups; ^#P ≤ 0.05 vs control; ⁺P ≤ 0.05 vs HT groups. C, vehicle control; G, GDPβS trilithium; Gq, BIM-46187; H, H₂O₂; HT, posttreatment T; I, 2-APB; T, 100 nM testosterone.

Figure 5.

The effects of nonspecific NOX inhibitor on OS generation and cell loss. (A) DPI was toxic to N27 cells, regardless of the concentration used. Apocynin did not show toxicity. (B) Testosterone alone increased OS generation, as evidenced by decreased reduced thiols. Apocynin blocked testosterone’s effects on OS generation. (C) Testosterone alone had no effect on cell viability. Apocynin did not protect cells from H₂O₂’s effects. Apocynin blocked testosterone-induced cell loss in an OS environment. (D) Similarly, apocynin blocked DHT-BSA exacerbation of H₂O₂-induced cell loss. Results are reported as mean ± SEM. Results were determined by ANOVA followed by a Fisher least significant difference post hoc test. *P ≤ 0.05 vs all groups; ^#P ≤ 0.05 vs control; ⁺P ≤ 0.05 vs HT groups. A, apocynin; C, vehicle control; H, H₂O₂; HT, posttreatment testosterone; T, 100 nM testosterone, 500 nm DHT-BSA.

Figure 6.

The effects of selective NOX inhibitors on testosterone-induced cell loss. (A) The selective NOX1 inhibitor, ML171, had no effect on cell viability. H₂O₂-induced cell loss was not blocked by ML171. ML171 partially blocked testosterone-induced cell loss in the presence of OS. (B) The selective NOX2 inhibitor, GSK2795039, had no effect on cell viability. H₂O₂-induced cell loss was not blocked by GSK2795039. GSK2795039 partially blocked testosterone-induced cell loss in the presence of OS. Results are reported as mean ± SEM. Results were determined by ANOVA followed by a Fisher least significant difference post hoc test. *P ≤ 0.05 vs all groups; ^#P ≤ 0.05 vs control; ⁺P ≤ 0.05 vs HT groups. C, vehicle control; H, H₂O₂; HT, posttreatment T; N, ML 171; N2, GSK2795039; T, 100 nM testosterone, 500 nm DHT-BSA.

Figure 7.

The effects of AR degradation on NOX1 and NOX2 protein expression. (A and B) Degrading the AR45, using ASC J9, did not affect NOX1 or NOX2 expression. Data are expressed as a normalized ratio of protein band density of (A) NOX1 and (B) NOX2 against GAPDH and presented as mean ± SD. Results were determined by ANOVA followed by a Fisher least significant difference post hoc test. C, vehicle control; H, H₂O₂; HT, posttreatment T; J9, ASC J9; T, 100 nM testosterone.

Figure 8.

Working model. Membrane AR (i.e., AR45) resides in a lipid raft within the plasma membrane and complexes with NOX1, NOX2, and Gα_q. Testosterone can bind and activate AR45, which in turn stimulates NOX1 and NOX2, resulting in OS generation. Alternatively, the AR45–NOX complex can upregulate InsP₃R activity to increase intracellular calcium release, resulting in increased OS. Dysregulation of this pathway can lead to neurotoxicity, such as during PD. The AR45–Gα_q pathway is not involved in testosterone-induced cell loss in OS environments. PLC, Phospholipase C.