Genetic and pharmacological evidence that endogenous nociceptin/orphanin FQ contributes to dopamine cell loss in Parkinson's disease

Abstract

To investigate whether the endogenous neuropeptide nociceptin/orphanin FQ (N/OFQ) contributes to the death of dopamine neurons in Parkinson's disease, we undertook a genetic and a pharmacological approach using NOP receptor knockout (NOP(-/-)) mice, and the selective and potent small molecule NOP receptor antagonist (-)-cis-1-methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1-yl]methyl]-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol (SB-612111). Stereological unbiased methods were used to estimate the total number of dopamine neurons in the substantia nigra of i) NOP(-/-) mice acutely treated with the parkinsonian neurotoxin 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP), ii) naïve mice subacutely treated with MPTP, alone or in combination with SB-612111, iii) rats injected with a recombinant adeno-associated viral (AAV) vector overexpressing human mutant p.A53T α-synuclein, treated with vehicle or SB-612111. NOP(-/-) mice showed a 50% greater amount of nigral dopamine neurons spared in response to acute MPTP compared to controls, which was associated with a milder motor impairment. SB-612111, given 4 days after MPTP treatment to mimic the clinical condition, prevented the loss of nigral dopamine neurons and striatal dopaminergic terminals caused by subacute MPTP. SB-612111, administered a week after the AAV injections in a clinically-driven protocol, also increased by 50% both the number of spared nigral dopamine neurons and striatal dopamine terminals, and prevented accompanying motor deficits induced by α-synuclein. We conclude that endogenous N/OFQ contributes to dopamine neuron loss in pathogenic and etiologic models of Parkinson's disease through NOP receptor-mediated mechanisms. NOP receptor antagonists might prove effective as disease-modifying agents in Parkinson's disease, through the rescue of degenerating nigral dopamine neurons and/or the protection of the healthy ones.

Keywords: Adeno associated viral vectors; MPP(+); MPTP; NOP receptor; Neuroprotection; Nociceptin/orphanin FQ; Parkinson's disease; SB-612111; α-Synuclein.

Fig 1

NOP^−/− mice are less susceptible than NOP^+/+ mice to develop hypokinesia in response to MPTP. Motor activity was measured using the bar (A), drag (B), and rotarod (C) tests, before (day 0) and for up to 6 days after systemic administration of MPTP (20 × 4 mg/kg, i.p.). Data are mean ± SEM of 6–9 mice per group. Statistical analysis was performed on the average performance of the last three days of observation (D4-D6) *p < 0.05, **p < 0.01 different from saline-treated mice of the same genotype; #p < 0.05, different from MPTP-treated NOP^−/− mice (one-way ANOVA followed by Newman–Keuls test for multiple comparisons).

Fig 2

NOP^−/− mice are more resistant than NOP^+/+ mice to MPTP-induced dopamine cell loss. Representative microphotographs (left) of tyrosine hydroxylase (TH) positive neurons in substantia nigra compacta (SNc) of NOP^+/+ and NOP^−/− mice, at 7 days after saline or MPTP (20 × 4 mg/kg, i.p.) administration. Stereological quantification of dopamine neurons in SNc (right). Data are mean ± SEM of 6–9 mice per group. **p < 0.01, different from saline-treated mice of the same genotype; ##p < 0.01, different from MPTP-treated NOP^+/+ mice (one-way ANOVA followed by the Newman–Keuls test for multiple comparisons).

Fig 3

SB-612111 attenuates subacute MPTP-induced neurotoxicity. Representative microphotographs (left) of tyrosine hydroxylase (TH) positive fibers in striatum. Stereological count of TH positive neurons in substantia nigra compacta (A) and quantification of TH positive terminals in striatum (B) of mice treated with MPTP (25 mg/kg, once daily for 7 days, i.p.) and/or SB-612111 (10 mg/kg, twice daily for 10 days, i.p.). Immunohistochemistry was performed 8 days after the last MPTP injection. Data are expressed as number of positive neurons (A) or mean gray level of TH striatal immunoreactivity (B), and are mean ± SEM of n = 7 mice per group. p** < 0.01 different from Saline/vehicle; #p < 0.05, ##p < 0.01 different from MPTP/vehicle (one-way ANOVA followed by the Newman–Keuls test for multiple comparisons).

Fig 4

SB-612111 attenuates motor deficits induced by AAV2/9 A53T human α-syn (hα-syn) injection in rats. SB-612111 (1 mg/kg, s.c., twice daily) or its vehicle, were administered for 8 weeks starting one week after AAV2/9-hα-syn injection, and motor activity assessed using the stepping test once a week. Data are expressed as number of backhand steps and are mean ± SEM of n = 7 (Vehicle) and n = 8 (SB-612111) rats per group. ##p < 0.01 (two-way ANOVA followed by Bonferroni test for multiple comparisons).

Fig 5

SB-612111 attenuates neurodegeneration induced by AAV2/9 A53T human α-syn (hα-syn) injection in rats. Stereological count of TH positive DA neurons in the substantia nigra compacta (A) and quantification of TH positive terminals in the striatum (B) of rats injected with AAV2/9-hα-syn and treated with SB-612111 (1 mg/kg, twice daily for 8 weeks, s.c.) or vehicle. Data are expressed as number of TH positive neurons (A) or mean gray level of TH immunoreactivity (B), and are mean±SEM of n= 7(Vehicle) and n = 8(SB-612111) rats per group. ##p < 0.01 (Student t-test).

Fig 6

SB-612111 does not change the load of hα-syn (A) and phosphorylated-α-syn (p-α-syn) (B) in the SNc of AAV A53T human α-syn injected rats. Data are expressed as immunopositive surface percentage of total nigral surface, and are mean ± SEM of n = 7 (Vehicle) and n = 8 (SB-612111) rats per group.