Abstract Title

NADPH oxidase (NOX1) mediates testosterone-induced neurodegeneration

Presenter Name

Mavis Tenkorang

RAD Assignment Number

1715

Abstract

Background: Parkinson’s disease (PD) has been recorded as the second most common neurological disease. Oxidative stress (OS) plays a key role in the pathogenesis of PD. Several studies have established that Parkinson’s disease (PD) is sex biased, affecting more men than women. Testosterone, a primary male sex hormone and a known oxidative stressor, has been implicated in PD. Previous studies in our lab have shown that testosterone via a non-genomic mechanism exacerbates OS damage in dopaminergic neurons. However, the mechanism by which testosterone increases OS is unknown. We found that testosterone acts through a membrane associated androgen receptor (mAR) variant – AR45 leading to the activation of proinflammatory mediators; NF-κB and COX2. NADPH Oxidase 1 (NOX 1) is a major OS generator in cells, hence a potential contributor to the pathogenesis of neurological diseases. It is possible that NOX 1 complexes with the mAR to mediate this destructive process.

Purpose: The primary objective of this study is to determine the underlying mechanism by which testosterone increases OS in dopaminergic neurons. We therefore hypothesize that in dopaminergic cells, testosterone increases oxidative stress by activating NOX 1. Ultimately, our goal is to identify pathways regulated by testosterone in dopaminergic neurons in order to provide effective pharmacological targets to enhance the treatment of PD.

Methods: We used a dopaminergic cell line (N27 cells). For an oxidative stressor, we used tert-butyl-hydrogen peroxide (H2O2) to induce 20% cell loss prior to testosterone (100nm) administration. NOX 1 inhibitor, Apocynin was administered before H2O2 exposure. To examine membrane associated androgen receptor and not the classical androgen receptor, we used cell impermeable DHT-BSA (500 nM) to confirm that NOX 1’s effect is through a non-genomic mechanism. Cell viability and OS were quantified using the MTT and Reduced Thiols assays respectively. To determine if NOX 1 interacts with a mAR, we immunoprecipitated the mAR and probed for NOX 1.

Results: Apocynin alone had no effect on cell viability and OS. Further, Apocynin alone, did not alter H2O2-induced cell loss, indicating that H2O2 increases OS via a non-NOX 1 mechanism. However, Apocynin blocked testosterone’s induced cell loss and OS generation suggesting that NOX 1 mediates testosterone’s damaging effects in an OS environment. Inhibition of NOX 1 also blocked DHT-BSA’s damaging effects on cell viability in an OS environment. NOX 1 protein also complexes with the mAR.

Conclusion: Testosterone-induced cell loss is mediated by a NOX1/mAR complex, indicating that NOX 1 is involved in testosterone-induced OS generation. The findings of these experiments provide a better insight into testosterone’s role in neurodegeneration and its underlying mechanism.

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Research Area

Neuroscience

Presentation Type

Poster

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NADPH oxidase (NOX1) mediates testosterone-induced neurodegeneration

Background: Parkinson’s disease (PD) has been recorded as the second most common neurological disease. Oxidative stress (OS) plays a key role in the pathogenesis of PD. Several studies have established that Parkinson’s disease (PD) is sex biased, affecting more men than women. Testosterone, a primary male sex hormone and a known oxidative stressor, has been implicated in PD. Previous studies in our lab have shown that testosterone via a non-genomic mechanism exacerbates OS damage in dopaminergic neurons. However, the mechanism by which testosterone increases OS is unknown. We found that testosterone acts through a membrane associated androgen receptor (mAR) variant – AR45 leading to the activation of proinflammatory mediators; NF-κB and COX2. NADPH Oxidase 1 (NOX 1) is a major OS generator in cells, hence a potential contributor to the pathogenesis of neurological diseases. It is possible that NOX 1 complexes with the mAR to mediate this destructive process.

Purpose: The primary objective of this study is to determine the underlying mechanism by which testosterone increases OS in dopaminergic neurons. We therefore hypothesize that in dopaminergic cells, testosterone increases oxidative stress by activating NOX 1. Ultimately, our goal is to identify pathways regulated by testosterone in dopaminergic neurons in order to provide effective pharmacological targets to enhance the treatment of PD.

Methods: We used a dopaminergic cell line (N27 cells). For an oxidative stressor, we used tert-butyl-hydrogen peroxide (H2O2) to induce 20% cell loss prior to testosterone (100nm) administration. NOX 1 inhibitor, Apocynin was administered before H2O2 exposure. To examine membrane associated androgen receptor and not the classical androgen receptor, we used cell impermeable DHT-BSA (500 nM) to confirm that NOX 1’s effect is through a non-genomic mechanism. Cell viability and OS were quantified using the MTT and Reduced Thiols assays respectively. To determine if NOX 1 interacts with a mAR, we immunoprecipitated the mAR and probed for NOX 1.

Results: Apocynin alone had no effect on cell viability and OS. Further, Apocynin alone, did not alter H2O2-induced cell loss, indicating that H2O2 increases OS via a non-NOX 1 mechanism. However, Apocynin blocked testosterone’s induced cell loss and OS generation suggesting that NOX 1 mediates testosterone’s damaging effects in an OS environment. Inhibition of NOX 1 also blocked DHT-BSA’s damaging effects on cell viability in an OS environment. NOX 1 protein also complexes with the mAR.

Conclusion: Testosterone-induced cell loss is mediated by a NOX1/mAR complex, indicating that NOX 1 is involved in testosterone-induced OS generation. The findings of these experiments provide a better insight into testosterone’s role in neurodegeneration and its underlying mechanism.