Oxidative stress in transgenic mice with oligodendroglial alpha-synuclein overexpression replicates the characteristic neuropathology of multiple system atrophy

Abstract

Multiple system atrophy (MSA) is a progressive neurodegenerative disorder characterized by parkinsonism unresponsive to dopaminergic therapy, cerebellar ataxia, and dysautonomia. Neuropathology shows a characteristic neuronal multisystem degeneration that is associated with widespread oligodendroglial alpha-synuclein (alpha-SYN) inclusions. Presently no animal model completely replicates the specific neuropathology of MSA. Here we investigated the behavioral and pathological features resulting from oligodendroglial alpha-SYN overexpression in transgenic mice exposed to mitochondrial inhibition by 3-nitropropionic acid. In transgenic mice 3-nitropropionic acid induced or augmented motor deficits that were associated with MSA-like pathology including striatonigral degeneration and olivopontocerebellar atrophy. Widespread astrogliosis and microglial activation were also observed in the presence of alpha-SYN in oligodendrocytes. Our results indicate that combined mitochondrial inhibition and overexpression of oligodendroglial alpha-SYN generates a novel model of MSA that may be useful for evaluating both pathogenesis and treatment strategies.

Figure 1

Behavioral motor impairment. a: Mean daily motor impairment scores increased substantially after day 8 in control and Tg mice receiving high-dose 3-NP. In contrast, low-dose 3-NP induced significant behavioral impairment in Tg, but not in control mice at day 15. b: Significant dose-dependent effect of 3-NP treatment on the total motor impairment score was observed (total for 15 days). c: Open-field horizontal activity was significantly impaired both by low- and high-dose 3-NP without dose dependency. d: However, when analyzing rearing as one of the open-field activity measures 3-NP showed a clear dose-response effect that was more marked in Tg mice exposed to low-dose 3-NP. T_turn (e) and T_total (f) of the pole test showed similar results suggesting a combined overall genotype and 3-NP effect. Forelimb (g) and hindlimb (h) stride length decreases confirmed 3-NP and overall genotype effects. Further, even without treatment a significantly shorter hindlimb stride length was observed in Tg mice. Significant changes are indicated as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001 for 3-NP treatment effects within the control or Tg group; °, P < 0.05; °°, P < 0.01 for comparisons of control and Tg mice. For all tests, n > 6 per group. Flex field activity test and pole test were performed on day 13. Stride length measurements were performed on day 14.

Figure 2

Striatonigral neuropathology. a and b: DARPP-32 immunohistochemistry of striatum in a control saline-treated mouse and in a Tg mouse after treatment with high-dose 3-NP. Arrows point to the lesion in the dorsolateral striatum. Insets represent higher magnification of the striatum. In Tg + 3NP mice the center of the lesion is deprived of cells and the periphery of the lesion shows a few remaining degenerating cell bodies. c: Neuronal cell counts in the striatum revealed dose-dependent loss of DARPP-32-labeled neurons in C57BL/6 (control) and Tg mice that was more pronounced in the latter when exposed to low-dose 3-NP. d: Analysis of striatal volume showed a similar 3-NP and genotype effect. e: The density of TH immunoreactivity in the striatum of control and Tg mice was significantly decreased after 3-NP intoxication. There was also a significant overall group effect of Tg versus control mice (P < 0.01). f: There was a reduction of TH-positive neurons in SNc in untreated Tg versus control mice. After 3-NP administration neuronal loss in SNc increased dose dependently with greater loss in Tg mice receiving low-dose 3-NP. g and h: Photomicrographs showing TH-immunopositive dopaminergic neurons in SNc of control (saline-treated) and 3-NP-treated Tg mice. Significant changes are indicated as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001 for 3-NP treatment effects within the control or Tg group; °, P < 0.05; °°, P < 0.01; °°°, P < 0.001 for comparisons of control and Tg mice. Scale bars: 1 mm (a); 50 μm (insets in a, b); 100 μm (g, h).

Figure 3

Cerebellar, pontine, and olivary neuropathology. a: Cresyl violet staining demonstrated Purkinje cell loss in 3-NP-treated Tg but not in control mice. b: Morphometric analysis confirmed significant loss of Purkinje cells and of cerebellar volume in Tg mice treated with high-dose 3-NP. In contrast, deep cerebellar nuclei were preserved. c: Representative photomicrographs of TH-immunopositive neurons in locus ceruleus (LC) in control and Tg mice, without or with 3-NP treatment. d: Morphometric analysis showed neuronal loss of LC in untreated Tg compared to control mice. 3-NP induced dose-dependent cell loss in both control and Tg mice, with a significant difference between both groups. Cell counts revealed loss of neurons in the pontine nuclei and inferior olivary complex of Tg but not control mice exposed to low- and high-dose 3-NP. Significant changes are indicated as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001 for 3-NP treatment effects within the control or Tg group; °, P < 0.05; °°, P < 0.01; °°°, P < 0.001 for comparisons of control and Tg mice. Scale bar, 100 μm (c).

Figure 4

Astrogliosis. GFAP immunohistochemistry demonstrating widespread astrogliosis in Tg mice after 3-NP intoxication including substantia nigra (SN), cerebellum, pontine nuclei (PN), and inferior olive (IO). In contrast, 3-NP-treated control C57BL/6 mice show weak astroglial activation in SN but not in the other brain structures presented. Scale bars: 50 μm (SN, cerebellum, PN); 0.2 mm (IO).

Figure 5

Microglial activation. CD68 immunohistochemistry demonstrated microglial activation in several brain regions. Untreated Tg mice showed diffuse microglial infiltration in white matter tracts [capsula interna (delineated by arrows), cerebellum] that was not observed in untreated control mice. In Tg mice, intoxication with 3-NP augmented microglial activation in the sites mentioned above but remained absent in control mice. Scale bars, 0.2 mm.

Figure 6

Oligodendroglial pathology. a: Immunohistochemistry for α-SYN with 15G7 antibody, demonstrating the density of α-SYN-positive profiles in oligodendrocytes in the striatum of a Tg mouse. b: Immunohistochemistry for α-SYN with 15G7 antibody, demonstrating a single oligodendrocyte of a Tg mouse with a typical polar accumulation of α-SYN reminiscent of a GCI (arrow). c: Semiquantitative analysis of the density of oligodendrocytes in the striatum of control C57BL/6 and Tg mice showed loss of oligodendrocytes in response to 3-NP intoxication regardless of the mouse genotype.