Mitochondrial inhibitor 3-nitroproprionic acid enhances oxidative modification of alpha-synuclein in a transgenic mouse model of multiple system atrophy

Abstract

Multiple system atrophy (MSA) is a progressive neurodegenerative disease characterized by autonomic failure, parkinsonism, cerebellar ataxia, and oligodendrocytic accumulation of alpha-synuclein (alphasyn). Oxidative stress has been linked to neuronal death in MSA and the mitochondrial toxin 3-nitropropionic acid (3NP) is known to enhance the motor deficits and neurodegeneration in transgenic mice models of MSA. However, the effect of 3NP administration on alphasyn itself has not been studied. In this context, we examined the neuropathological effects of 3NP administration in alphasyn transgenic mice expressing human alphasyn (halphasyn) under the control of the myelin basic protein (MBP) promoter and the effect of this administration on posttranslational modifications of alphasyn, on levels of total alphasyn, and on its solubility. We demonstrate that 3NP administration altered levels of nitrated and oxidized alphasyn in the MBP-halphasyn tg while not affecting global levels of phosphorylated or total alphasyn. 3NP administration also exaggerated neurological deficits in the MBP-halphasyn tg mice, resulting in widespread neuronal degeneration and behavioral impairment.

Fig. 1

3NP-induced αsyn posttranslational modifications in MBP-hαsyn mice. Immunoblot analysis was carried out on homogenized brain samples from vehicle- and 3NP-treated NTg and MBP-hαsyn mice processed to obtain soluble (TBS), detergent-soluble (SDS), and detergent-insoluble (urea) fractions (A). Quantitative analysis of the blots was conducted to examine levels of nitrated αsyn in TBS (B), SDS (C), and urea (D) fractions, oxidized αsyn in TBS (E), SDS (F), and urea (G) fractions, phosphorylated αsyn in TBS (H), SDS (I), and urea (J) fractions, and total αsyn in TBS (K), SDS (L), and urea (M) fractions. Actin was used as a loading control in each case. *Significant difference (P < 0.05, one-way ANOVA and post hoc Fisher).

Fig. 2

3NP-induced shift in solubility of posttranslationally modified αsyn in MBP-hasyn mice. Composite bar graphs of the levels of nitrated (A), oxidized (B), phosphorylated (C), and total (D) αsyn in the TBS, SDS, and urea fractions.

Fig. 3

Immunohistochemical analysis of the effect of 3NP treatment on nitrated αsyn. Immunohistochemical analysis was conducted to examine the levels of nitrated αsyn in the basal ganglia of vehicle-treated NTg mice (A), 3NP-treated NTg mice (B), vehicle-treated MBP-hαsyn mice (C), and 3NP-treated MBP-hαsyn mice (D). Quantitative analysis of basal ganglia levels of nitrated αsyn (E). Immunohistochemical analysis was also conducted to examine the levels of nitrated αsyn in the frontal cortex of vehicle-treated NTg mice (F), 3NP-treated NTg mice (G), vehicle-treated MBP-hαsyn mice (H), and 3NP-treated MBP-hαsyn mice (I). Quantitative analysis of frontal cortical levels of nitrated αsyn (J). Scale bar = 50 μM. *Significant difference (P < 0.05, one-way ANOVA and post hoc Fisher). [Color figure can be viewed in the online issue, which is available at www.interscience. wiley.com.]

Fig. 4

Immunohistochemical analysis of the effect of 3NP treatment on phosphorylated αsyn. Immunohistochemical analysis was conducted to examine the levels of phosphorylated αsyn in the basal ganglia of vehicle-treated NTg mice (A), 3NP-treated NTg mice (B), vehicle-treated MBP-hαsyn mice (C), and 3NP-treated MBP-hαsyn mice (D). Quantitative analysis of basal ganglia levels of phosphorylated αsyn (E). Immunohistochemical analysis was also conducted to examine the levels of phosphorylated αsyn in the frontal cortex of vehicle-treated NTg mice (F), 3NP-treated NTg mice (G), vehicle-treated MBP-hαsyn mice (H), and 3NP-treated MBP-hαsyn mice (I). Quantitative analysis of frontal cortical levels of phosphorylated αsyn (J). Scale bar = 50 μM. *Significant difference (P < 0.05, one-way ANOVA and post hoc Fisher). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Fig. 5

Immunohistochemical analysis of the effect of 3NP treatment on human αsyn. Immunohistochemical analysis was conducted to examine the levels of human αsyn in the basal ganglia of vehicle-treated NTg mice (A), 3NP-treated NTg mice (B), vehicle-treated MBP-hαsyn mice (C), and 3NP-treated MBP-hαsyn mice (D). Quantitative analysis of basal ganglia levels of human αsyn was also performed (E). Immunohistochemical analysis was also conducted to examine the levels of human αsyn in the frontal cortex of vehicle-treated NTg mice (F), 3NP-treated NTg mice (G), vehicle-treated MBP-hαsyn mice (H), and 3NP-treated MBP-hαsyn mice (I). Quantitative analysis of frontal cortical levels of human αsyn was also performed (J). Scale bar = 50 μm. *Significant difference (P < 0.05, one-way ANOVA and post hoc Fisher). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Fig. 6

3NP treatment induced colocalization of αsyn and caspase activation in oligodendrocytes in MBP-hαsyn mice. Active caspase 3 immunoreactivity in vehicle-treated NTg mice (A), 3NP-treated NTg mice (D), vehicle-treated MBP-hαsyn mice (G), and 3NP-treated MBP-hαsyn mice (J). αsyn immunoreactivity in vehicle-treated NTg mice (B), 3NP-treated NTg mice (E), vehicle-treated MBP-hαsyn mice (H), and 3NP-treated MBP-hαsyn mice (K). Colocalization of active caspase 3 and αsyn signal in vehicle-treated NTg mice (C), 3NP-treated NTg mice (F), vehicle-treated MBP-hαsyn mice (I), and 3NP-treated MBP-hαsyn mice (L). Quantitative analysis of the levels of activated caspase 3 immunoreactive neurons and oligodendroglia in vehicle- and 3NP-treated NTg and MBP-hasyn mice (M). Scale bar = 50 μM.

Fig. 7

3NP treatment exacerbates behavioral deficits in MBP-hαsyn mice. Motor behavior was assessed by the pole test (A). Mice were placed at the top of a vertical pole, and total time to descend (T-Total) was measured. Mice received 2 days of training, five trials each day, and were analyzed on the third day. Peak grip strength (g) was assessed (B) in three consecutive trials by allowing the mice to grasp a grid connected to an isometric dynamometer; mice were slowly moved backward until they released the bar. *Significant difference (P < 0.05, one-way ANOVA and post hoc Fisher).

Fig. 8

Widespread neuropathology as a result of 3NP treatment. Immunohistochemical analysis was performed to examine the number of TH-positive cells in the substantia nigra of vehicle- and 3NP-treated NTg and vehicle- and 3NP-treated MBP-hαsyn mice (A–D, respectively, and analyzed in E). Neuronal density, as determined by NeuN immunoreactivity, was analyzed the striatum of vehicle- and 3NP-treated NTg and vehicle- and 3NP-treated MBP-hasyn mice (F–I, respectively, and analyzed in J). Neuronal density was also examined in the frontoparietal cortex of vehicle- and 3NP-treated NTg and vehicle- and 3NP-treated MBP-hαsyn mice (K–N, respectively, and analyzed in O). Scale bar = 50 μM in A–D. Scale bar = 200 μM in F–I, K–N. *Significant difference (P < 0.05, one-way ANOVA and post hoc Fisher). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Fig. 9

Oligodendroglial pathology as a result of 3NP treatment. In order to examine the effects of 3NP treatment on oligodendrocytes, levels of APC, an oligodendrocytic marker, were examined in the corpus callosum of vehicle- and 3NP-treated NTg and vehicle- and 3NP-treated MBP-hasyn mice (A–D, respectively, and analyzed in E). APC levels were also examined in the cerebellar white matter of vehicle- and 3NP-treated NTg and vehicle- and 3NP-treated MBP-hasyn mice (F–I, respectively, and analyzed in J). Scale bar = 200 μM. *Significant difference (P < 0.05, one-way ANOVA and post hoc Fisher). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]