INDUCTION OF APOPTOSIS VIA TESTOSTERONE IN OXIDATIVELY DAMAGED DOPAMINERGIC CELLS

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2014-03

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Simmons, Olivia C.
Cunningham, Rebecca

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Abstract

Following an episode of stroke, reduction of blood supply to brain cells can lead to conditions of oxidative stress (OS) in brain cells, or neurons, specifically in the dopaminergic neurons. The loss of dopaminergic neurons manifests itself as Parkinson’s disease (PD). Classically, men have a higher incidence of developing PD post-stroke than females. This suggests a role of testosterone (T) in the development of PD after induction of OS in neurons. We postulate that T in OS-induced states will activate the pathway of apoptosis, or programmed cell death, via an enzyme called caspase to induce death of dopaminergic brain cells, and thus symptoms of PD. To test our hypothesis, we first pre-treated dopaminergic cell lines with hydrogen peroxide (H2O2) to simulate stroke-induced OS. The cells were then treated with differing concentrations of T (0, 1, 10, 100 nM), representing the physiologic ranges of T in humans. Expressions of pro-caspase-3 and pro-caspase-9, the uncleaved precursors to caspase-3 & caspase-9, respectively, in the cells were quantified using Western Blot analysis. Statistical significance of our findings was reported using ANOVA and Fisher’s post hoc analysis with SAS software. Our experiments showed a trend of decreased expression of pro-caspase-9, and a significant decrease in pro-caspase-3 expression in the H+T treatment conditions as compared to the control conditions. These results point to the apoptotic cell death pathway via caspase-3 and caspase-9 as the mechanism by which increased T levels lead to PD in stroke patients. Purpose (a): Following ischemic stroke, reduction of blood supply to brain cells can lead to conditions of oxidative stress (OS) in neurons, specifically in the dopaminergic neurons of the substantia nigra (SN). The loss of dopaminergic neurons manifests itself as Parkinson’s disease (PD). Classically, men have a higher incidence of developing PD post-stroke than females. This suggests a role of testosterone (T) in the development of PD after induction of OS in neurons. We postulate that T in OS-induced states will activate the caspase pathway of apoptosis to induce dopaminergic cell death, and thus symptoms of PD. Methods (b): To test our hypothesis, we first pre-treated N27 dopaminergic cell lines with hydrogen peroxide (H2O2) to simulate stroke-induced OS. The cells were then treated with differing concentrations of T (0, 1, 10, 100 nM), representing the physiologic ranges of T in humans. Expressions of pro-caspase-3 and pro-caspase-9, the uncleaved precursors to caspase-3 & caspase-9, respectively, in the cells were quantified using Western Blot analysis. Statistical significance of our findings was reported using ANOVA and Fisher’s post hoc analysis with SAS software and p <0.05 as significant. Results (c): Our experiments showed a trend of decreased expression of pro-caspase-9, and a significant decrease in pro-caspase-3 expression in the H+T treatment conditions as compared to the control conditions. Conclusions (d): These results point to the apoptotic pathway via caspase-3 and caspase-9 as the mechanism by which increased T levels lead to PD in stroke patients.

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Research Appreciation Day Award Winner - 2014 Medical Student Government Association - 3rd Place Research Award

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