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

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.

Figure 1. NAC increases striatal area occupied by TH-positive terminals in SNCA-overexpressing mice at 12 months of age.

A–D. Representative images of TH-immunostained 30 µm sections of alanine or NAC-treated wild-type or transgenic mouse striatum. A. Wild-type, alanine treated; B. Wild-type, NAC treated; C. Transgenic, alanine treated; D. Transgenic, NAC treated. E. Density of TH-positive terminals in the striatum of alanine or NAC-treated wild-type or transgenic mouse striatum. F. Density of DAT-positive terminals in the striatum of alanine or NAC-treated wild-type or transgenic mouse striatum. Data in E and F were analyzed using a 2-tailed Student's t-test. All relevant statistically significant comparisons are indicated on the graph. WT ALA N = 4, WT NAC  = 6, TG ALA  = 4, TG NAC  = 3.

Figure 2. Treatment with NAC Reduces Levels of SNCA in PDGFb-SNCA Transgenic Mice.

A–D. Representative images of human SNCA-immunostained 30 µm sections of alanine or NAC treated wild-type or transgenic mouse cortex. A. Wild-type, alanine treated; B. Wild-type, NAC treated; C. Transgenic, alanine treated; D. Transgenic, NAC treated. E. Human SNCA staining intensity (integrated density) of the cortex of wild-type and transgenic mice treated with either alanine or NAC normalized to intensity of staining of the corpus callosum on the same brain section. WT data is shown to demonstrate the level of non-specific background staining. F. Quantification of the number of intensely stained cells per cortical region of interest. G. Human SNCA staining intensity (integrated density) of the striatum of wild-type and transgenic mice treated with either alanine or NAC normalized to intensity of staining data from the corpus callosum of the same brain section. H. Quantification of the average number of intensely stained cells per striatal region of interest. Because of low numbers of SNCA-positive cells in the striatum, ROI data from three separate striatal sections were averaged. Data in E–H were analyzed using a 2-tailed Student's t-test. All relevant statistically significant comparisons are indicated on the graph. All groups N = 4.

Figure 3. SNCA-Overexpressing Mice Treated with NAC for 5–7 Weeks Exhibit Increased Glutathione in the Substantia Nigra but not in Cortex.

A. Levels of total glutathione in the Substantia substantia nigra (SN) B. Levels of total glutathione in the cortex (Ctx) of SNCA overexpressing mice (TG) versus wild-type littermate controls (WT) exposed to drinking water supplemented with NAC or control drinking water supplemented with alanine (ALA). Data were analyzed using a 2-tailed Student's t-test. All relevant statistically significant comparisons are indicated on the graph. WT ALA N = 6, WT NAC N = 6, TG ALA N = 5, TG NAC N = 6.

Figure 4. Early increases in SN glutathione after NAC treatment are not seen after 12 months of treatment.

Levels of total glutathione in the striatum (Str), substantia nigra (SN) and frontal cortex (Ctx) of SNCA overexpressing mice (TG) versus wild-type littermate controls (WT) exposed to drinking water supplemented with NAC or control drinking water supplemented with alanine (ALA) from weaning through 1 year of age. Though levels of glutathione are consistently lower in the SN compared to Str or Ctx at 1 year of age, there are no significant differences between WT versus TG mice, or between ALA versus NAC treated mice. WT ALA N = 4, WT NAC N = 5, TG ALA N = 3, TG NAC N = 3.

Figure 5. 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.

Figure 6. Performance on the accelerating Rotarod was unaffected by SNCA-overexpression or by chronic NAC treatment.

Wild-type (WT) and SNCA-overexpressing (TG) mice treated with either Alanine (ALA) or NAC were tested at 1 year of age on a Rotarod (Ugo Basile) accelerating from 2–40 rpm over a period of 240 seconds. There were no significant differences between the groups tested. WT ALA N = 13, WT NAC N = 14, TG ALA N = 12, TG NAC N = 16.

Figure 7. Performance on the Pole Test was unaffected by SNCA-overexpression or NAC treatment.

A. Time for the mouse to turn on the pole and face downwards (T_turn). B. Time for the mouse to reach the bottom of the pole (T_LA). There were no significant differences between the groups tested. WT ALA N = 12, WT NAC N-12, TG ALA N = 13, TG NAC N = 16.