Mechanisms Underlying Membrane Androgen Receptor-Induced Neurodegeneration

A common characteristic of several neurodegenerative disorders is oxidative stress (OS). Many neurodegenerative disorders are more prevalent in men and postmenopausal women compared to premenopausal women, indicating the possible involvement of androgens (men > postmenopausal women > premenopausal women) in neurodegeneration. Our lab found testosterone can have either neuroprotective or neurodamaging effects depending on the presence of OS in the cellular environment. We have 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. Subcellular localization of AR45 is in the lipid rafts of the plasma membrane in several brain regions affected by neurodegenerative disorders (eg. substantia nigra, hippocampus). 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 Gα q, we hypothesize testosterone increases OS by activating mAR complexed with NOX 1/2, initiating IP 3 signaling. Using an immortalized neuronal cell line (N27 cells), we exposed cells to hydrogen peroxide (H 2 O 2 ) prior to testosterone (100 nM) or DHT‐BSA (500nM). Inhibitors were used to examine G protein, androgen receptor, IP 3 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). Classical AR antagonists did not block testosterone's negative effects, indicating the classical AR does not mediate these effects. Since AR antagonists do not block mAR, we used an AR protein degrader, ASC‐J9 (5uM). Unlike AR antagonists, the AR degrader blocked testosterone's negative effects. Next, we examined signaling cascades associated with proteins complexed with mAR‐AR45, such as NOX1/2 and Gα q . To block NOX actions, we used apocynin (10 uM), a nonspecific NOX inhibitor. Apocynin did not alter H 2 O 2 ‐induced cell loss, indicating H 2 O 2 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 the oxidative stressor, CIH, during sleep phase for 7 days. Inhibition of Gα q or G protein activation did not alter testosterone's negative effects on cell viability. However, inhibition of IP 3 receptor blocked these effects. Interestingly, NOX can influence IP 3 receptor mediated signaling, indicating that testosterone may activate IP 3 signaling via the mAR‐NOX complex and not the mAR‐ Gα q complex localized in membrane lipid raft. Future studies will examine the mAR‐NOX complex as a therapeutic target for neurodegenerative diseases. Support or Funding Information NIH/NINDS R01 NS088514 to RLC This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .