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14-3-3theta protects against neurotoxicity in a cellular Parkinson's disease model through inhibition of the apoptotic factor Bax.

Slone SR, Lesort M, Yacoubian TA - PLoS ONE (2011)

Bottom Line: We found that 14-3-3θ overexpression reduced Bax activation and downstream signaling events, including cytochrome C release and caspase 3 activation.A 14-3-3θ mutant incapable of binding Bax failed to protect against rotenone.These data suggest that 14-3-3θ's neuroprotective effects against rotenone are at least partially mediated by Bax inhibition and point to a potential therapeutic role of 14-3-3s in Parkinson's disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Center for Neurodegeneration and Experimental Therapeutics, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.

ABSTRACT
Disruption of 14-3-3 function by alpha-synuclein has been implicated in Parkinson's disease. As 14-3-3s are important regulators of cell death pathways, disruption of 14-3-3s could result in the release of pro-apoptotic factors, such as Bax. We have previously shown that overexpression of 14-3-3θ reduces cell loss in response to rotenone and MPP(+) in dopaminergic cell culture and reduces cell loss in transgenic C. elegans that overexpress alpha-synuclein. In this study, we investigate the mechanism for 14-3-3θ's neuroprotection against rotenone toxicity. While 14-3-3s can inhibit many pro-apoptotic factors, we demonstrate that inhibition of one factor in particular, Bax, is important to 14-3-3s' protection against rotenone toxicity in dopaminergic cells. We found that 14-3-3θ overexpression reduced Bax activation and downstream signaling events, including cytochrome C release and caspase 3 activation. Pharmacological inhibition or shRNA knockdown of Bax provided protection against rotenone, comparable to 14-3-3θ's neuroprotective effects. A 14-3-3θ mutant incapable of binding Bax failed to protect against rotenone. These data suggest that 14-3-3θ's neuroprotective effects against rotenone are at least partially mediated by Bax inhibition and point to a potential therapeutic role of 14-3-3s in Parkinson's disease.

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14-3-3θ overexpression reduces rotenone-induced disruption of mitochondrial membrane potential.Vector control and 14-3-3θ cells were treated with varying doses of rotenone or 10 µM carbonyl cyanide 3-chlorophenylhydrazone (CCCP), a mitochondrial toxin, for 24 hours. Mitochondrial membrane potential was assayed by the JC-1 assay. Ratio of aggregated JC-1 (red) to monomer JC-1 (green) for each condition was normalized to that ratio for the corresponding untreated cells. Results reflect three independent experiments with three replicates per experiment. Error bars reflect SEM. **p<0.01, ***p<0.001 (Bonferroni's multiple comparison test).
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pone-0021720-g006: 14-3-3θ overexpression reduces rotenone-induced disruption of mitochondrial membrane potential.Vector control and 14-3-3θ cells were treated with varying doses of rotenone or 10 µM carbonyl cyanide 3-chlorophenylhydrazone (CCCP), a mitochondrial toxin, for 24 hours. Mitochondrial membrane potential was assayed by the JC-1 assay. Ratio of aggregated JC-1 (red) to monomer JC-1 (green) for each condition was normalized to that ratio for the corresponding untreated cells. Results reflect three independent experiments with three replicates per experiment. Error bars reflect SEM. **p<0.01, ***p<0.001 (Bonferroni's multiple comparison test).

Mentions: Our Bax knockdown experiments implicate additional mechanisms besides Bax inhibition for 14-3-3θ's neuroprotective effects. Since rotenone can directly inhibit Complex I to disrupt the mitochondrial membrane potential (Δψm), 14-3-3θ could modulate this effect in a manner independent of Bax inhibition. We measured the effect of 14-3-3θ overexpression on Δψm. Changes in Δψm were determined by using the ratiometric fluorescent dye JC-1. As expected, Δψm was reduced with increasing doses of rotenone in vector control cells. The rotenone-mediated decrease in Δψm was significantly attenuated in 14-3-3θ cells (Fig. 6). The mitochondrial respiratory chain uncoupler, carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was used as a positive control for disruption of Δψm (Fig. 6).


14-3-3theta protects against neurotoxicity in a cellular Parkinson's disease model through inhibition of the apoptotic factor Bax.

Slone SR, Lesort M, Yacoubian TA - PLoS ONE (2011)

14-3-3θ overexpression reduces rotenone-induced disruption of mitochondrial membrane potential.Vector control and 14-3-3θ cells were treated with varying doses of rotenone or 10 µM carbonyl cyanide 3-chlorophenylhydrazone (CCCP), a mitochondrial toxin, for 24 hours. Mitochondrial membrane potential was assayed by the JC-1 assay. Ratio of aggregated JC-1 (red) to monomer JC-1 (green) for each condition was normalized to that ratio for the corresponding untreated cells. Results reflect three independent experiments with three replicates per experiment. Error bars reflect SEM. **p<0.01, ***p<0.001 (Bonferroni's multiple comparison test).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3140482&req=5

pone-0021720-g006: 14-3-3θ overexpression reduces rotenone-induced disruption of mitochondrial membrane potential.Vector control and 14-3-3θ cells were treated with varying doses of rotenone or 10 µM carbonyl cyanide 3-chlorophenylhydrazone (CCCP), a mitochondrial toxin, for 24 hours. Mitochondrial membrane potential was assayed by the JC-1 assay. Ratio of aggregated JC-1 (red) to monomer JC-1 (green) for each condition was normalized to that ratio for the corresponding untreated cells. Results reflect three independent experiments with three replicates per experiment. Error bars reflect SEM. **p<0.01, ***p<0.001 (Bonferroni's multiple comparison test).
Mentions: Our Bax knockdown experiments implicate additional mechanisms besides Bax inhibition for 14-3-3θ's neuroprotective effects. Since rotenone can directly inhibit Complex I to disrupt the mitochondrial membrane potential (Δψm), 14-3-3θ could modulate this effect in a manner independent of Bax inhibition. We measured the effect of 14-3-3θ overexpression on Δψm. Changes in Δψm were determined by using the ratiometric fluorescent dye JC-1. As expected, Δψm was reduced with increasing doses of rotenone in vector control cells. The rotenone-mediated decrease in Δψm was significantly attenuated in 14-3-3θ cells (Fig. 6). The mitochondrial respiratory chain uncoupler, carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was used as a positive control for disruption of Δψm (Fig. 6).

Bottom Line: We found that 14-3-3θ overexpression reduced Bax activation and downstream signaling events, including cytochrome C release and caspase 3 activation.A 14-3-3θ mutant incapable of binding Bax failed to protect against rotenone.These data suggest that 14-3-3θ's neuroprotective effects against rotenone are at least partially mediated by Bax inhibition and point to a potential therapeutic role of 14-3-3s in Parkinson's disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Center for Neurodegeneration and Experimental Therapeutics, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.

ABSTRACT
Disruption of 14-3-3 function by alpha-synuclein has been implicated in Parkinson's disease. As 14-3-3s are important regulators of cell death pathways, disruption of 14-3-3s could result in the release of pro-apoptotic factors, such as Bax. We have previously shown that overexpression of 14-3-3θ reduces cell loss in response to rotenone and MPP(+) in dopaminergic cell culture and reduces cell loss in transgenic C. elegans that overexpress alpha-synuclein. In this study, we investigate the mechanism for 14-3-3θ's neuroprotection against rotenone toxicity. While 14-3-3s can inhibit many pro-apoptotic factors, we demonstrate that inhibition of one factor in particular, Bax, is important to 14-3-3s' protection against rotenone toxicity in dopaminergic cells. We found that 14-3-3θ overexpression reduced Bax activation and downstream signaling events, including cytochrome C release and caspase 3 activation. Pharmacological inhibition or shRNA knockdown of Bax provided protection against rotenone, comparable to 14-3-3θ's neuroprotective effects. A 14-3-3θ mutant incapable of binding Bax failed to protect against rotenone. These data suggest that 14-3-3θ's neuroprotective effects against rotenone are at least partially mediated by Bax inhibition and point to a potential therapeutic role of 14-3-3s in Parkinson's disease.

Show MeSH
Related in: MedlinePlus