Low-dose 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine causes inflammatory activation of astrocytes in nuclear factor-κB reporter mice prior to loss of dopaminergic neurons

Abstract

Neuroinflammation is implicated in the progression of numerous disease states of the CNS, but early inflammatory signaling events in glial cells that may predispose neurons to injury are not easily characterized in vivo. To address this question, we exposed transgenic mice expressing a nuclear factor-κB (NF-κB)-driven enhanced green fluorescent protein (EGFP) reporter construct to low doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and examined inflammatory activation of astrocytes in relation to neurobehavioral and neuropathological outcomes. The highest dose of MPTP (60 mg/kg total dose) caused a decrease in locomotor activity and a reduction in stride length. No significant loss of dopaminergic neurons in the substantia nigra was apparent at any dose. In contrast, expression of tyrosine hydroxylase in striatal fibers was reduced at 60 mg/kg MPTP, as were levels of dopamine and DOPAC. Colocalized expression of EGFP and inducible nitric oxide synthase (NOS2) occurred in astrocytes at 30 and 60 mg/kg MPTP and was associated with increased protein nitration in nigral dopaminergic neurons. Inhibition of NF-κB in primary astrocytes by expression of mutant IκBα suppressed expression of NOS2 and protected cocultured neurons from astrocyte-mediated apoptosis. These data indicate that inflammatory activation of astrocytes and enhanced nitrosative stress occurs at low doses of MPTP prior to loss of dopaminergic neurons. NF-κB-mediated expression of NOS2 appears to be a sensitive indicator of neuroinflammation that correlates with MPTP-induced neurochemical and neurobehavioral deficits prior to loss of dopaminergic neurons in the subtantia nigra.

Fig. 1

Low-dose MPTP induces changes in locomotor activity. A: Locomotor activity over the 7-day treatment period as measured by open-field activity. B: Locomotor activity data for day 1 posttreatment. The highest dose of 60 mg/kg MPTP resulted in significant reduction in three of the four locomotor parameters tested 24 hr after dosing, which completely recovered by 6 days posttreatment. Data are expressed as mean ± SEM (n = 10). C: Stride length measurements 6 days posttreatment expressed as percentage change compared with baseline (day 0).

Fig. 2

MPTP induces dose-dependent neuropathological changes in the nigrostriatal system. A: Representative 10× confocal montage images of tyrosine hydroxylase (TH; red) expression in the substantia nigra pars compacta of saline control and 60 mg/kg MPTP-treated animals counterstained with DAPI (blue). Representative 60× confocal images of TH-positive neurons in the SNpc of saline control and 60 mg/kg MPTP-treated animals (inset). B: Estimated number of total TH⁺ neurons in the SNpc as determined by stereological counting (n = 5). C: Representative 10× confocal montage images of TH immunostaining in the striatum of saline control and 60 mg/kg MPTP-treated animals counterstained with DAPI. D: Quantitation of TH expression in the striatum. Data expressed as mean fluorescence intensity in relative fluorescence units ± SEM (n = 5). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Fig. 3

Low-dose MPTP exposure results in decreased striatal dopamine levels. Dopamine (A) and DOPAC (B) concentrations and DOPAC/dopamine ratio (C) as determined by HPLC analysis. Catecholamine concentrations are expressed as ng/mg protein ± SEM (n = 3).

Fig. 4

Neuroinflammatory activation of astrocytes occurs in NF-κB reporter mice prior to loss of dopaminergic neurons. A: Representative 40× images of astrocytes (GFAP; purple) expressing intrinsic GFP fluorescence (green), inducible nitric oxide synthase (NOS2; red), and counterstained with DAPI (blue). B: Quantification of the number of GFAP⁺astrocytes expressing intrinsic GFP. C: Quantification of the number of GFAP⁺astrocytes expressing NOS2. D: Quantification of the number of GFAP⁺astrocytes expressing both GFP and NOS2. Data are expressed as percentage ± SEM (n = 9). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Fig. 5

MPTP exposure increased global protein nitration in dopmainergic neurons in the substantia nigra. A: Representative 40× images of tyrosine hydroxylase (TH; green) and 3-nitrotyrosine (3-NT; red) expression in the SNpc of saline control and 30 mg/kg MPTP-treated animals counterstained with DAPI. B: Quantification of 3-NT mean fluorescence intensity (n = 6 individual animals per group). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Fig. 6

Suppression of NF-κB signaling in astrocytes prior to inflammatory activation with MPTP protects cocultured striatal neurons. A: Real-time RT-PCR quantification of NOS2 mRNA expression following treatment with 10 µM MPTP, 1 pg/ml TNF-α, and 1 ng/ml IFNγ in the presence of mutant IκBα or empty adenoviral vector (EV). B: Representative 40× images of caspase activity in primary striatal neurons after 6 hr of incubation with stimulated astrocytes expressing mutant IκBα or a control vector. C: Quantitation of caspase activity. Data expressed as mean fluorescence intensity in relative fluorescence units ± SEM (n = 3 independent experiments, with images taken from a minimum of eight microscopic fields per coverslip). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]