Neuroprotective effect of long-term NDI1 gene expression in a chronic mouse model of Parkinson disorder

Abstract

Previously, we showed that the internal rotenone-insensitive nicotinamide adenine dinucleotide (NADH)-quinone oxidoreductase (NDI1) gene from Saccharomyces cerevisiae (baker's yeast) can be successfully inserted into the mitochondria of mice and rats and the expressed enzyme was found to be fully functional. In this study, we investigated the ability of the Ndi1 enzyme to protect the dopaminergic neurons in a chronic mouse model of Parkinson disorder. After expression of the NDI1 gene in the unilateral substantia nigra of male C57BL/6 mice for 8 months, a chronic Parkinsonian model was created by administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) with probenecid and evaluated using neurochemical and behavioral responses 1-4 weeks post-MPTP/probenecid injection. We showed that expression of Ndi1 was able to significantly prevent the loss of dopamine and tyrosine hydroxylase as well as the dopaminergic transporters in the striatum of the chronic Parkinsonian mice. Behavioral assessment based on a methamphetamine-induced rotation test and spontaneous swing test further supported neurological preservation in the NDI1-treated Parkinsonian mice. The data presented in this study demonstrate a protective effect of the NDI1 gene in dopaminergic neurons, suggesting its therapeutic potential for Parkinson-like disorders.

FIG. 1.

Effect of probenecid on striatal DA system in mice. A control study was performed to evaluate the effect of probenecid alone on the levels of DA, DOPAC, and 5-HT (a) and TH immunoreactivity (b). Animals (N = 25) were separated into five groups and injected with various concentrations of probenecid, i.p., twice a week for 5 weeks. Following an additional 4 weeks, one-half of the animals from each group were sacrificed for HPLC analysis and the other half were used for immunohistochemical analysis. (a) Compared to saline-injected controls, probenecid (0, 75, 150, 250 mg/kg/injection) did not alter the striatal contents of DA, DOPAC, and 5-HT. At 0 mg/kg level, only DMSO (10 mL/kg), the vehicle in which probenecid was dissolved, was injected. The 100% values (in ng/mg tissue) were 6.37 (DA), 0.53 (DOPAC), and 0.72 (5-HT). (b) A representative photomicrograph showing that chronic probenecid (250 mg/kg per injection) did not change the density of TH-immunoreactivity in the neostriatum. Probenecid at concentrations lower than 250 mg/kg per injection and DMSO alone also produced no effect on striatal TH-immunoreactivity (pictures not shown). Data are stated as mean ± SEM. DA, Dopamine; DOPAC, 3,4-dihydroxyphenylacetic acid; 5-HT, 5-hydroxytryptamine; HPLC, high-performance liquid chromatography; DMSO, dimethylsulfoxide; TH, tyrosine hydroxylase; SEM, standard error of the mean.

FIG. 2.

Effect of NDI1 treatment on animal behaviors in chronic MPTP/probenecid-treated mice. Mice were tested for behavioral deficits at 1 and 4 weeks after chronic MPTP/probenecid treatment. (a) For the methamphetamine-induced rotation test, animals were injected with 1.5 mg/kg methamphetamine and behavior was recorded for 40 min, following a 15-min acclimation period. There was no preference in turn direction for the control (N = 9) or chronic MPTP/probenecid (N = 6) group. However, a preferential contralateral body turning was found in chronic MPTP/probenecid-treated mice 4 weeks after treatment, which had been pretreated with NDI1 gene for 8 months (right hemisphere) (N = 8). (b) For the elevated body swing test, the number of body swings to the left or right, greater than 30° from the vertical position were counted. Similar observations were obtained with the elevated body swing test as that of the methamphetamine-induced rotation test. Data are presented as the mean percentage ± SEM. ***p < 0.001; **p < 0.01. MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Prob., probenecid.

FIG. 3.

Effect of NDI1 on biogenic amine levels in the striatum of chronic MPTP/probenecid-treated mice. The levels of DA(a), the major metabolite of DA, DOPAC (b), and serotonin or 5-HT (c) were measured in each side of neostriatum from control (N = 5) and chronic MPTP/probenecid-treated mice 5 weeks after treatment, which had been pretreated without (N = 5) or with (N = 7) NDI1 gene for 8 months. Bilateral loss of over 90% of DA and DOPAC was detected in the striatum of chronic parkinsonian mice 5 weeks after systemic injections with MPTP/probenecid (a and b). The striatal content of 5-HT was not affected by the chronic MPTP/probenecid treatment (c). A partial prevention of DA and DOPAC loss was detected in the right striatum of the chronic parkinsonian mice, which had been pretreated (right hemisphere) ipsilaterally with rAAV-Ndi1 8 months before the chronic MPTP/probenecid treatment (a and b). The striatal content of 5-HT was not affected by the rAAV-NDI1 pretreatment in chronic parkinsonian mice (c). Results are expressed as mean ± SEM. ***p < 0.001; **p < 0.01. MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; DA, Dopamine; DOPAC, 3,4-dihydroxyphenylacetic acid; 5-HT, 5-hydroxytryptamine; SEM, standard error of the mean; LH, left hemisphere; RH, right hemisphere; Prob., probenecid.

FIG. 4.

Effect of Ndi1 treatment on striatal dopaminergic proteins and Ndi1 in the chronic parkinsonian mice. The expression of several dopaminergic functional proteins was analyzed in each side of neostriatum from control (N = 8) and chronic MPTP/probenecid-treated mice 5 weeks after treatment, which had been pretreated without (N = 6) or with (N = 8) NDI1 gene for 8 months. The protein expression of VMAT2, DAT, and TH on the left (LH) and right hemispheres (RH) in the chronic parkinsonian mice 5 weeks after systemic injections with MPTP/probenecid was markedly reduced (a and b). Marked Ndi1 expression could be detected in the injected side of the striatum from the chronic parkinsonian mice pretreated with NDI1 (a and b). Sustained expression of VMAT2, DAT, and TH proteins was observed in the right striatum of the chronic parkinsonian mice, which had been pretreated ipsilaterally with rAAV-NDI1 8 months the chronic MPTP/probenecid treatment (a and b). Results are expressed as mean ± SEM. ***p 0.001; **p < 0.01; *p < 0.05. MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; VMAT2, vesicular monoamine transporter 2; DAT, dopamine transporter; TH, tyrosine hydroxylase; SEM, standard error of the mean; Prob., probenecid.

FIG. 5.

Effect of Ndi1 treatment on the TH-positive cells in the substantia nigra of the chronic parkinsonian mice. Representative photomicrographs of TH staining of brain sections illustrate the density of TH-positive cells in the left and right substantia nigra between the control and chronic MPTP/probenecid-treated mice without or with 8-month rAAV-NDI1 pretreatment (right hemisphere, shown by arrow). Scale bar, 500 μm. The results are compared in the histograms for the control group (N = 6), the chronic MPTP/probenecid-treated group (N = 8), and the rAAV-NDI1-treated group (N = 10). Chronic systemic MPTP/probenecid treatment resulted in a substantial loss of TH-immunoreactivity bilaterally in the substantia nigra 5 weeks posttreatment when compared with the control animals. When mice were preinjected with rAAV-NDI1 in the right substantia nigra (arrow) 8 months prior to MPTP/probenecid treatment, near-normal TH-immunoreactivity was observed in the Ndi1-pretreated side of substantia nigra 5 weeks after the induction of chronic Parkinsonism. ***p < 0.001. TH, Tyrosine hydroxylase; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Prob., probenecid.