Mechanisms Underlying Membrane Androgen Receptor-Induced Neurodegeneration

Date
2019-03-05
Authors
Duong, Phong
Tenkorang, Mavis
Wilson, Elizabeth
Cunningham, Rebecca
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Purpose: A common characteristic of several neurodegenerative disorders is oxidative stress (OS). Many neurodegenerative disorders are more prevalent in men and postmenopausal women. Our lab has shown testosterone via a non-genomic mechanism exacerbates OS damage in neurons. Indeed, our lab was the first to discover the presence of the androgen receptor (AR) splice variant, AR45, in the brain. We found testosterone can initiate signaling cascades via this membrane associated AR (mAR), leading to increased OS. However, the mechanism for OS generation is unknown. NADPH Oxidase 1 and 2 (NOX 1/2) are major OS generators, and potential targets for androgen-induced OS and cell death. Based on our studies showing protein-protein interactions between NOX1/2, AR45 and Gαq, we hypothesize testosterone increases OS by activating mAR complexed with NOX 1/2, initiating IP3 signaling. Method: Using an immortalized neuronal cell line (N27 cells), we exposed cells to hydrogen peroxide (H2O2) prior to testosterone (100 nM) or DHT-BSA (500nM). Inhibitors were used to examine AR, IP3 and NOX1/2 signaling. Cell viability and OS were quantified. In addition to in vitro experiments, we examined the effects of NOX 1/2 on DHT exacerbation of chronic intermittent hypoxia, CIH (AHI=10) induced OS by treating adult male Long Evans rats with the NOX1/2 inhibitor, apocynin (4mg/kg). Results: Classical AR antagonists did not block testosterone’s negative effects, indicating classical AR does not mediate these effects. Since AR antagonists do not block mAR, we used an AR protein degrader, ASC-J9 (5uM). ASC-J9 blocked testosterone’s negative effects. Next, we examined signaling cascades associated with proteins complexed with mAR-AR45. To block NOX actions, we used apocynin (10 uM). Apocynin did not alter H2O2-induced cell loss, indicating H2O2 increases OS via a non-NOX mechanism. However, apocynin completely blocked testosterone induced cell loss and OS, suggesting the involvement of NOX1/2. Consistent with our in vitro data, apocynin also decreased OS generation in DHT-treated rats exposed to CIH, during sleep phase for 7 days. Inhibition of IP3 receptor blocked testosterone’s negative effects, indicating that testosterone may activate IP3 signaling via the mAR-NOX complex. Conclusion: NOX and IP3 play a crucial role in mAR-induced neurodegeneration. Future studies will examine the mAR-NOX complex as a therapeutic target for neurodegenerative diseases.

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