Neuroscience
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Browsing Neuroscience by Author "Holmes, Shaletha S."
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Item INDUCTION OF APOPTOSIS VIA TESTOSTERONE IN OXIDATIVELY DAMAGED DOPAMINERGIC CELLS(2014-03) Simmons, Olivia C.; Holmes, Shaletha S.; Cunningham, RebeccaFollowing 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.Item THE EFFECTS OF ANDROGENS ON CASPASE-1 MEDIATED SIGNALING IN OXIDATIVE STRESSED DOPAMINE NEURONS(2014-03) Holmes, Shaletha S.; Su, Chang; Singh, Meharvan; Cunningham, RebeccaParkinson’s disease (PD), a neurodegenerative disorder characterized by oxidative stress and the loss of dopamine neurons in the midbrain, affects the aging population. In fact, males have a higher risk for PD than females. While the mechanisms remain elusive, one possibility may be that androgens, such as testosterone, play a potential role. Our studies suggest that androgens can increase the expression of Caspase-1, an enzyme whose activity increases with oxidative stress and can result in mitochondrial collapse, ubiquitination, alpha-synuclein–positive lewy body accumulation, inflammation and apoptosis. Therefore, we hypothesize that in oxidative stress conditions, androgens suppress KLF4, a negative regulator of caspase-1, resulting in the increase of Caspase-1 to promote toxic protein accumulation, inflammation, and apoptotic neuronal cell death. This study is related to the role of testosterone in high levels of oxidative stress associated with aging. Purpose (a): Oxidative stress and an extensive loss of dopamine neurons in the nigrostriatal pathway are hallmarks of Parkinson’s disease (PD), a neurodegenerative disorder affecting millions of people. Males have a higher risk for PD than females. While the mechanisms remain elusive, one possibility may be that androgens, such as testosterone, play a potential role. Our studies suggest that androgens can increase the expression of Caspase-1, an enzyme whose activity increases with oxidative stress and can result in mitochondrial collapse, ubiquitination, alpha-synuclein–positive lewy body accumulation, inflammation and apoptosis. We hypothesize that in oxidative stress conditions, androgens suppress KLF4, a negative regulator of caspase-1, resulting in overexpression of Caspase-1 leading to toxic protein accumulation, inflammation, and apoptosis. Methods (b): We exposed a dopaminergic cell line (N27 cells) to a sublethal concentration of the pro-oxidant, tert-butyl hydrogen peroxide (H2O2) for 24 hrs and assessed cell viability in the presence or absence of testosterone. Results (c): Physiologically relevant concentrations of testosterone (0, 1, 10, 100 nM) failed to compromise cell viability in non-oxidatively stressed cells1. In contrast, testosterone did promote cell death in the H2O2 pre-treated cells. In H202 treated cells, testosterone increased caspase-1 expression and activation, as evidenced by an increase in cleaved caspase-1. In addition, KLF4 expression was decreased by testosterone in H2O2 treated cells. The role of KLF4 as a negative regulator of caspase-1 was confirmed in experiments showing that siRNA-mediated knockdown of KLF4 increased caspase-1 levels in H2O2 treated cells. Testosterone increased H202 mediated expression of COX2 signaling, a protein associated with inflammation. Also, testosterone decreased H2O2-induced ubiquitin expression resulting in the accumulation of toxic proteins. Further, testosterone increased H202 induced in apoptosis. Conclusions (d): Overall, these results indicate that androgens such as testosterone exert negative effects under oxidative stress conditions through the suppression of KLF4 and activation of caspase-1 signaling pathways leading to cell death. Thus, supporting a role for androgens for the gender bias observed in PD.