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Oral N-acetyl-cysteine attenuates loss of dopaminergic terminals in alpha-synuclein overexpressing mice.

Clark J, Clore EL, Zheng K, Adame A, Masliah E, Simon DK - PLoS ONE (2010)

Bottom Line: Despite the transient nature of the impact of NAC on brain glutathione, the loss of dopaminergic terminals at 1 year associated with SNCA overexpression was significantly attenuated by NAC supplementation, as measured by immunoreactivity for tyrosine hydroxylase in the striatum (p = 0.007; unpaired, two-tailed t-test), with a similar but nonsignificant trend for dopamine transporter (DAT) immunoreactivity.This was associated with a decrease in nuclear NFkappaB localization and an increase in cytoplasmic localization of NFkappaB in the NAC-treated transgenics.Overall, these results indicate that oral NAC supplementation decreases SNCA levels in brain and partially protects against loss of dopaminergic terminals associated with overexpression of alpha-synuclein in this model.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Beth Israel Deaconess Medical Center, Boston, Massachusetts, United States of America.

ABSTRACT
Levels of glutathione are lower in the substantia nigra (SN) early in Parkinson's disease (PD) and this may contribute to mitochondrial dysfunction and oxidative stress. Oxidative stress may increase the accumulation of toxic forms of alpha-synuclein (SNCA). We hypothesized that supplementation with n-acetylcysteine (NAC), a source of cysteine--the limiting amino acid in glutathione synthesis, would protect against alpha-synuclein toxicity. Transgenic mice overexpressing wild-type human alpha-synuclein drank water supplemented with NAC or control water supplemented with alanine from ages 6 weeks to 1 year. NAC increased SN levels of glutathione within 5-7 weeks of treatment; however, this increase was not sustained at 1 year. Despite the transient nature of the impact of NAC on brain glutathione, the loss of dopaminergic terminals at 1 year associated with SNCA overexpression was significantly attenuated by NAC supplementation, as measured by immunoreactivity for tyrosine hydroxylase in the striatum (p = 0.007; unpaired, two-tailed t-test), with a similar but nonsignificant trend for dopamine transporter (DAT) immunoreactivity. NAC significantly decreased the levels of human SNCA in the brains of PDGFb-SNCA transgenic mice compared to alanine treated transgenics. This was associated with a decrease in nuclear NFkappaB localization and an increase in cytoplasmic localization of NFkappaB in the NAC-treated transgenics. Overall, these results indicate that oral NAC supplementation decreases SNCA levels in brain and partially protects against loss of dopaminergic terminals associated with overexpression of alpha-synuclein in this model.

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Long-term NAC treatment affects sub-cellular localization of NFκB.A. 2.5 µg of cytoplasmic protein lysate from the cortex of 3 alanine- and 3 NAC-treated PDGFb-SNCA mice was run on a 10-well 4–15% SDS-PAGE gel (Bio-Rad). After transfer, the membrane was cut around the 50 kD marker and probed with anti- NFκB p65 antibody (Santa Cruz) and anti-β-actin antibody (Santa Cruz). 2.5 µg of nuclear protein lysate was run on a separate 10-well 4–15% SDS-PAGE gel and the treated in the manner outlined above. In both cases a single 65 kDa band was observed for NFκB and a single 43 kDa band was observed for β-actin. B. Band quantification of cytoplasmic NFκB normalized to β-actin levels. C. Band quantification of nuclear NFκB normalized to Histone H3 levels. Data were analyzed using a 2-tailed Student's t-test. All relevant statistically significant comparisons are indicated on the graphs. The experiment was repeated three times; representative results from a 1 minute exposure of both blots are shown.
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pone-0012333-g005: Long-term NAC treatment affects sub-cellular localization of NFκB.A. 2.5 µg of cytoplasmic protein lysate from the cortex of 3 alanine- and 3 NAC-treated PDGFb-SNCA mice was run on a 10-well 4–15% SDS-PAGE gel (Bio-Rad). After transfer, the membrane was cut around the 50 kD marker and probed with anti- NFκB p65 antibody (Santa Cruz) and anti-β-actin antibody (Santa Cruz). 2.5 µg of nuclear protein lysate was run on a separate 10-well 4–15% SDS-PAGE gel and the treated in the manner outlined above. In both cases a single 65 kDa band was observed for NFκB and a single 43 kDa band was observed for β-actin. B. Band quantification of cytoplasmic NFκB normalized to β-actin levels. C. Band quantification of nuclear NFκB normalized to Histone H3 levels. Data were analyzed using a 2-tailed Student's t-test. All relevant statistically significant comparisons are indicated on the graphs. The experiment was repeated three times; representative results from a 1 minute exposure of both blots are shown.

Mentions: NAC can influence the activity of several transcription factors, including inhibition of NFκB [25], increased activation of NFκB has been observed in Parkinson's disease models [39]–[41], and inhibition of NFκB in glial cells has been proposed as a promising neuroprotective strategy [42]. Nuclear and cytoplasmic fractions were isolated from cortical tissues of wild-type and PDGFb-SNCA mice. Proteins were separated by SDS-PAGE and probed with anti- NFκB p65 antibody (Santa Cruz). An increased amount of NFκB was seen in the cytoplasm of NAC-treated PDGFb-SNCA cortical tissue compared to alanine-treated transgenics (Fig. 5A & 5B); accordingly, the amount of nuclear NFκB was decreased in these animals (Fig. 5A & 5C). Total NFκB normalized to nuclear and cytoplasmic loading controls did not vary significantly between PDGFb-SNCA transgenic mice treated with either alanine or NAC (data not shown).


Oral N-acetyl-cysteine attenuates loss of dopaminergic terminals in alpha-synuclein overexpressing mice.

Clark J, Clore EL, Zheng K, Adame A, Masliah E, Simon DK - PLoS ONE (2010)

Long-term NAC treatment affects sub-cellular localization of NFκB.A. 2.5 µg of cytoplasmic protein lysate from the cortex of 3 alanine- and 3 NAC-treated PDGFb-SNCA mice was run on a 10-well 4–15% SDS-PAGE gel (Bio-Rad). After transfer, the membrane was cut around the 50 kD marker and probed with anti- NFκB p65 antibody (Santa Cruz) and anti-β-actin antibody (Santa Cruz). 2.5 µg of nuclear protein lysate was run on a separate 10-well 4–15% SDS-PAGE gel and the treated in the manner outlined above. In both cases a single 65 kDa band was observed for NFκB and a single 43 kDa band was observed for β-actin. B. Band quantification of cytoplasmic NFκB normalized to β-actin levels. C. Band quantification of nuclear NFκB normalized to Histone H3 levels. Data were analyzed using a 2-tailed Student's t-test. All relevant statistically significant comparisons are indicated on the graphs. The experiment was repeated three times; representative results from a 1 minute exposure of both blots are shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2925900&req=5

pone-0012333-g005: Long-term NAC treatment affects sub-cellular localization of NFκB.A. 2.5 µg of cytoplasmic protein lysate from the cortex of 3 alanine- and 3 NAC-treated PDGFb-SNCA mice was run on a 10-well 4–15% SDS-PAGE gel (Bio-Rad). After transfer, the membrane was cut around the 50 kD marker and probed with anti- NFκB p65 antibody (Santa Cruz) and anti-β-actin antibody (Santa Cruz). 2.5 µg of nuclear protein lysate was run on a separate 10-well 4–15% SDS-PAGE gel and the treated in the manner outlined above. In both cases a single 65 kDa band was observed for NFκB and a single 43 kDa band was observed for β-actin. B. Band quantification of cytoplasmic NFκB normalized to β-actin levels. C. Band quantification of nuclear NFκB normalized to Histone H3 levels. Data were analyzed using a 2-tailed Student's t-test. All relevant statistically significant comparisons are indicated on the graphs. The experiment was repeated three times; representative results from a 1 minute exposure of both blots are shown.
Mentions: NAC can influence the activity of several transcription factors, including inhibition of NFκB [25], increased activation of NFκB has been observed in Parkinson's disease models [39]–[41], and inhibition of NFκB in glial cells has been proposed as a promising neuroprotective strategy [42]. Nuclear and cytoplasmic fractions were isolated from cortical tissues of wild-type and PDGFb-SNCA mice. Proteins were separated by SDS-PAGE and probed with anti- NFκB p65 antibody (Santa Cruz). An increased amount of NFκB was seen in the cytoplasm of NAC-treated PDGFb-SNCA cortical tissue compared to alanine-treated transgenics (Fig. 5A & 5B); accordingly, the amount of nuclear NFκB was decreased in these animals (Fig. 5A & 5C). Total NFκB normalized to nuclear and cytoplasmic loading controls did not vary significantly between PDGFb-SNCA transgenic mice treated with either alanine or NAC (data not shown).

Bottom Line: Despite the transient nature of the impact of NAC on brain glutathione, the loss of dopaminergic terminals at 1 year associated with SNCA overexpression was significantly attenuated by NAC supplementation, as measured by immunoreactivity for tyrosine hydroxylase in the striatum (p = 0.007; unpaired, two-tailed t-test), with a similar but nonsignificant trend for dopamine transporter (DAT) immunoreactivity.This was associated with a decrease in nuclear NFkappaB localization and an increase in cytoplasmic localization of NFkappaB in the NAC-treated transgenics.Overall, these results indicate that oral NAC supplementation decreases SNCA levels in brain and partially protects against loss of dopaminergic terminals associated with overexpression of alpha-synuclein in this model.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Beth Israel Deaconess Medical Center, Boston, Massachusetts, United States of America.

ABSTRACT
Levels of glutathione are lower in the substantia nigra (SN) early in Parkinson's disease (PD) and this may contribute to mitochondrial dysfunction and oxidative stress. Oxidative stress may increase the accumulation of toxic forms of alpha-synuclein (SNCA). We hypothesized that supplementation with n-acetylcysteine (NAC), a source of cysteine--the limiting amino acid in glutathione synthesis, would protect against alpha-synuclein toxicity. Transgenic mice overexpressing wild-type human alpha-synuclein drank water supplemented with NAC or control water supplemented with alanine from ages 6 weeks to 1 year. NAC increased SN levels of glutathione within 5-7 weeks of treatment; however, this increase was not sustained at 1 year. Despite the transient nature of the impact of NAC on brain glutathione, the loss of dopaminergic terminals at 1 year associated with SNCA overexpression was significantly attenuated by NAC supplementation, as measured by immunoreactivity for tyrosine hydroxylase in the striatum (p = 0.007; unpaired, two-tailed t-test), with a similar but nonsignificant trend for dopamine transporter (DAT) immunoreactivity. NAC significantly decreased the levels of human SNCA in the brains of PDGFb-SNCA transgenic mice compared to alanine treated transgenics. This was associated with a decrease in nuclear NFkappaB localization and an increase in cytoplasmic localization of NFkappaB in the NAC-treated transgenics. Overall, these results indicate that oral NAC supplementation decreases SNCA levels in brain and partially protects against loss of dopaminergic terminals associated with overexpression of alpha-synuclein in this model.

Show MeSH
Related in: MedlinePlus