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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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Rotenone-induced Bax activation is reduced in 14-3-3θ-overexpressing cells.a) Less Bax translocated to mitochondria in 14-3-3θ cells in response to rotenone. After treatment with 5 µM rotenone for 24 hours, vector control and 14-3-3θ cell lysates were subfractionated into cytosolic and mitochondrial fractions and immunoblotted with a polyclonal rabbit antibody against Bax. For each fraction, lanes for vector control and 14-3-3θ cells are from the same gel and exposure time but are separated for clarity with regard to quantification. Bax levels were normalized to tubulin for the cytosolic fraction or cyclophilin D for the mitochondrial fraction. Bax levels for rotenone-treated cells are shown as the relative percentage of the corresponding untreated cells. Densitometric quantification included seven separate experiments. Error bars reflect SEM. *p<0.05, **p<0.01 (one sample t-test). b) Total Bax levels were unchanged with rotenone treatment in either cell line. After treatment with 5 µM rotenone for 24 hours, whole cell lysates were immunoblotted with an anti-Bax antibody. c) Fewer 14-3-3θ cells were positive for activated Bax upon rotenone treatment. After treatment without (i-iv) or with rotenone (v-viii) for 16 hours, vector control and 14-3-3θ cells were fixed in 2% paraformaldehyde and immunostained with a monoclonal mouse antibody against the active Bax conformation (6A7) and a goat Alexa 488-conjugated anti-mouse secondary antibody (i, ii, v, vi). Nuclei were stained with Hoechst 33342 (iii, iv, vii, viii). The number of 6A7-positive cells was quantitated with rater blind to experimental conditions. Error bars reflect SEM. **p<0.01, ***p<0.001 (Bonferroni's multiple comparison test). Scale bar  = 50 µm. d) Rotenone-induced Bax oligomerization was reduced in 14-3-3θ cells. Vector control and 14-3-3θ stable cells were treated with 5 µM rotenone for 24 hours. Mitochondrially-enriched fractions were crosslinked and immunoblotted for oligomers with an anti-Bax antibody. Cyclophilin D served as loading control. Densitometric quantification includes three independent experiments. Error bars reflect SEM. ***p<0.001 (Bonferroni's multiple comparison test). n.s.  =  non-significant.
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pone-0021720-g002: Rotenone-induced Bax activation is reduced in 14-3-3θ-overexpressing cells.a) Less Bax translocated to mitochondria in 14-3-3θ cells in response to rotenone. After treatment with 5 µM rotenone for 24 hours, vector control and 14-3-3θ cell lysates were subfractionated into cytosolic and mitochondrial fractions and immunoblotted with a polyclonal rabbit antibody against Bax. For each fraction, lanes for vector control and 14-3-3θ cells are from the same gel and exposure time but are separated for clarity with regard to quantification. Bax levels were normalized to tubulin for the cytosolic fraction or cyclophilin D for the mitochondrial fraction. Bax levels for rotenone-treated cells are shown as the relative percentage of the corresponding untreated cells. Densitometric quantification included seven separate experiments. Error bars reflect SEM. *p<0.05, **p<0.01 (one sample t-test). b) Total Bax levels were unchanged with rotenone treatment in either cell line. After treatment with 5 µM rotenone for 24 hours, whole cell lysates were immunoblotted with an anti-Bax antibody. c) Fewer 14-3-3θ cells were positive for activated Bax upon rotenone treatment. After treatment without (i-iv) or with rotenone (v-viii) for 16 hours, vector control and 14-3-3θ cells were fixed in 2% paraformaldehyde and immunostained with a monoclonal mouse antibody against the active Bax conformation (6A7) and a goat Alexa 488-conjugated anti-mouse secondary antibody (i, ii, v, vi). Nuclei were stained with Hoechst 33342 (iii, iv, vii, viii). The number of 6A7-positive cells was quantitated with rater blind to experimental conditions. Error bars reflect SEM. **p<0.01, ***p<0.001 (Bonferroni's multiple comparison test). Scale bar  = 50 µm. d) Rotenone-induced Bax oligomerization was reduced in 14-3-3θ cells. Vector control and 14-3-3θ stable cells were treated with 5 µM rotenone for 24 hours. Mitochondrially-enriched fractions were crosslinked and immunoblotted for oligomers with an anti-Bax antibody. Cyclophilin D served as loading control. Densitometric quantification includes three independent experiments. Error bars reflect SEM. ***p<0.001 (Bonferroni's multiple comparison test). n.s.  =  non-significant.

Mentions: Since 14-3-3θ interacts with Bax as demonstrated by immunoprecipitation, we hypothesized that increased binding of Bax in the presence of higher 14-3-3θ levels could reduce Bax activation. In response to an apoptotic trigger, Bax undergoes conformational alterations and translocates to the mitochondrial outer membrane, where it undergoes oligomerization that leads to pore formation [28], [29]. We first tested whether 14-3-3θ overexpression altered the translocation of Bax into the mitochondria in response to rotenone. Control and 14-3-3θ stable cells were treated with 5 µM rotenone for approximately 24 hours, and then cell lysates were fractionated into cytosolic and mitochondrial fractions. For vector control cells treated with rotenone, immunoblotting of these fractions showed that Bax levels decreased to 48% of untreated cells in the cytosolic fraction (p<0.01, one sample t-test) and increased to 194% of untreated cells in the mitochondrial fraction (p<0.05, one sample t-test); this suggests that Bax translocates to the mitochondria upon rotenone treatment (Fig. 2a). In contrast, incubation of 14-3-3θ cells with rotenone did not result in significant changes in the cytosolic or mitochondrial Bax levels as determined by Western blotting (Fig. 2a). Levels of total Bax protein were similar in control and 14-3-3θ cell lines and were not affected by rotenone treatment in both cell lines, as determined by Western blotting of whole cell lysates (Fig. 2b).


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)

Rotenone-induced Bax activation is reduced in 14-3-3θ-overexpressing cells.a) Less Bax translocated to mitochondria in 14-3-3θ cells in response to rotenone. After treatment with 5 µM rotenone for 24 hours, vector control and 14-3-3θ cell lysates were subfractionated into cytosolic and mitochondrial fractions and immunoblotted with a polyclonal rabbit antibody against Bax. For each fraction, lanes for vector control and 14-3-3θ cells are from the same gel and exposure time but are separated for clarity with regard to quantification. Bax levels were normalized to tubulin for the cytosolic fraction or cyclophilin D for the mitochondrial fraction. Bax levels for rotenone-treated cells are shown as the relative percentage of the corresponding untreated cells. Densitometric quantification included seven separate experiments. Error bars reflect SEM. *p<0.05, **p<0.01 (one sample t-test). b) Total Bax levels were unchanged with rotenone treatment in either cell line. After treatment with 5 µM rotenone for 24 hours, whole cell lysates were immunoblotted with an anti-Bax antibody. c) Fewer 14-3-3θ cells were positive for activated Bax upon rotenone treatment. After treatment without (i-iv) or with rotenone (v-viii) for 16 hours, vector control and 14-3-3θ cells were fixed in 2% paraformaldehyde and immunostained with a monoclonal mouse antibody against the active Bax conformation (6A7) and a goat Alexa 488-conjugated anti-mouse secondary antibody (i, ii, v, vi). Nuclei were stained with Hoechst 33342 (iii, iv, vii, viii). The number of 6A7-positive cells was quantitated with rater blind to experimental conditions. Error bars reflect SEM. **p<0.01, ***p<0.001 (Bonferroni's multiple comparison test). Scale bar  = 50 µm. d) Rotenone-induced Bax oligomerization was reduced in 14-3-3θ cells. Vector control and 14-3-3θ stable cells were treated with 5 µM rotenone for 24 hours. Mitochondrially-enriched fractions were crosslinked and immunoblotted for oligomers with an anti-Bax antibody. Cyclophilin D served as loading control. Densitometric quantification includes three independent experiments. Error bars reflect SEM. ***p<0.001 (Bonferroni's multiple comparison test). n.s.  =  non-significant.
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Related In: Results  -  Collection

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pone-0021720-g002: Rotenone-induced Bax activation is reduced in 14-3-3θ-overexpressing cells.a) Less Bax translocated to mitochondria in 14-3-3θ cells in response to rotenone. After treatment with 5 µM rotenone for 24 hours, vector control and 14-3-3θ cell lysates were subfractionated into cytosolic and mitochondrial fractions and immunoblotted with a polyclonal rabbit antibody against Bax. For each fraction, lanes for vector control and 14-3-3θ cells are from the same gel and exposure time but are separated for clarity with regard to quantification. Bax levels were normalized to tubulin for the cytosolic fraction or cyclophilin D for the mitochondrial fraction. Bax levels for rotenone-treated cells are shown as the relative percentage of the corresponding untreated cells. Densitometric quantification included seven separate experiments. Error bars reflect SEM. *p<0.05, **p<0.01 (one sample t-test). b) Total Bax levels were unchanged with rotenone treatment in either cell line. After treatment with 5 µM rotenone for 24 hours, whole cell lysates were immunoblotted with an anti-Bax antibody. c) Fewer 14-3-3θ cells were positive for activated Bax upon rotenone treatment. After treatment without (i-iv) or with rotenone (v-viii) for 16 hours, vector control and 14-3-3θ cells were fixed in 2% paraformaldehyde and immunostained with a monoclonal mouse antibody against the active Bax conformation (6A7) and a goat Alexa 488-conjugated anti-mouse secondary antibody (i, ii, v, vi). Nuclei were stained with Hoechst 33342 (iii, iv, vii, viii). The number of 6A7-positive cells was quantitated with rater blind to experimental conditions. Error bars reflect SEM. **p<0.01, ***p<0.001 (Bonferroni's multiple comparison test). Scale bar  = 50 µm. d) Rotenone-induced Bax oligomerization was reduced in 14-3-3θ cells. Vector control and 14-3-3θ stable cells were treated with 5 µM rotenone for 24 hours. Mitochondrially-enriched fractions were crosslinked and immunoblotted for oligomers with an anti-Bax antibody. Cyclophilin D served as loading control. Densitometric quantification includes three independent experiments. Error bars reflect SEM. ***p<0.001 (Bonferroni's multiple comparison test). n.s.  =  non-significant.
Mentions: Since 14-3-3θ interacts with Bax as demonstrated by immunoprecipitation, we hypothesized that increased binding of Bax in the presence of higher 14-3-3θ levels could reduce Bax activation. In response to an apoptotic trigger, Bax undergoes conformational alterations and translocates to the mitochondrial outer membrane, where it undergoes oligomerization that leads to pore formation [28], [29]. We first tested whether 14-3-3θ overexpression altered the translocation of Bax into the mitochondria in response to rotenone. Control and 14-3-3θ stable cells were treated with 5 µM rotenone for approximately 24 hours, and then cell lysates were fractionated into cytosolic and mitochondrial fractions. For vector control cells treated with rotenone, immunoblotting of these fractions showed that Bax levels decreased to 48% of untreated cells in the cytosolic fraction (p<0.01, one sample t-test) and increased to 194% of untreated cells in the mitochondrial fraction (p<0.05, one sample t-test); this suggests that Bax translocates to the mitochondria upon rotenone treatment (Fig. 2a). In contrast, incubation of 14-3-3θ cells with rotenone did not result in significant changes in the cytosolic or mitochondrial Bax levels as determined by Western blotting (Fig. 2a). Levels of total Bax protein were similar in control and 14-3-3θ cell lines and were not affected by rotenone treatment in both cell lines, as determined by Western blotting of whole cell lysates (Fig. 2b).

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