Blockade of nociceptin/orphanin FQ transmission attenuates symptoms and neurodegeneration associated with Parkinson's disease

Abstract

The opioid-like neuropeptide nociceptin/orphanin FQ (N/OFQ) and its receptor (NOP) are expressed in the substantia nigra (SN), a brain area containing dopamine neurons that degenerate in Parkinson's disease. Endogenous N/OFQ facilitates nigral glutamate release and inhibits nigrostriatal dopamine transmission and motor behavior. Here, we present evidence suggesting that endogenous N/OFQ may contribute to Parkinson's disease. Pharmacological blockade of the SN N/OFQ-NOP receptor system attenuated parkinsonian-like akinesia/hypokinesia in 6-hydroxydopamine hemilesioned or haloperidol-treated rats, whereas deletion of the NOP receptor gene conferred mice partial protection from haloperidol-induced motor depression. The antiparkinsonian action of NOP receptor antagonists was associated with reduction of glutamate release in the SN. In 6-hydroxydopamine hemilesioned rats, enhancement of N/OFQ expression and release was detected in the lesioned compared with the unlesioned SN, indicating that parkinsonism may be associated with overactivation of the N/OFQ-NOP receptor system in the SN. Finally, deletion of the N/OFQ gene conferred mice partial protection against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced loss of SN dopamine neurons. Based on these data, we propose that NOP receptor antagonists may represent a novel approach for combined (symptomatic and neuroprotective) therapy of Parkinson's disease.

Figure 1.

Characterization of motor activity in vehicle-injected and 6-OHDA-injected (hemiparkinsonian) rats. A-C, Hemiparkinsonian rats displayed overall reduction in motor activity and motor asymmetry compared with vehicle-injected rats, as shown by an increase in the total time spent on the blocks in the bar test (the contralateral forepaw being more severely affected; A), reduction in the number of steps made by the contralateral forepaw in the drag test (B), and reduced rotarod performance (C). C, The rotarod performance of naive rats has also been reported. Data are mean ± SEM of 10-33 determinations obtained from 26 vehicle-injected, 33 hemiparkinsonian, and 18 naive rats. °°p < 0.01 versus the ipsilateral forepaw. ^##p < 0.01 versus vehicle-injected and naive rats (ANOVA followed by Newman-Keuls post hoc test). Error bars represent SEM.

Figure 2.

UFP-101 relieved akinesia/hypokinesia in hemiparkinsonian rats. A-E, Injection of UFP-101 (0.1-30 nmol in 0.5 μl) in the SNr reduced the time spent on the blocks in the bar test (in seconds; A), increased the number of steps of the contralateral forepaw in the drag test (B), improved overall motor performance (calculated as percentage of the control session) in the rotarod test (C, D), and induced contralateral rotations (E). A-D, The bar and drag tests (A, B) were performed 10 min after injection; the rotarod test was performed 10 min (C) and 60 min (D) after injection. Motor asymmetry was evaluated by separately measuring activity of the paws ipsilateral and contralateral (parkinsonian) to the lesioned side. E, Turning behavior was assessed by counting the number of rotations in the direction opposite to the injection side (i.e., contralateral) in 90 min. A-E, Data are mean ± SEM of 7-12 determinations obtained from 27 hemiparkinsonian and 30 vehicle-injected rats (A-D) or 36 hemiparkinsonian and 42 vehicle-injected rats (E). ^*p < 0.05; ^**p < 0.01 versus saline-treated rats. °p < 0.05; °°p < 0.01 versus the ipsilateral forepaw. ^#p < 0.05; ^##p < 0.01 versus vehicle-injected rats (ANOVA followed by Newman-Keuls post hoc test). Error bars represent SEM.

Figure 3.

J-113397 and levodopa relieved akinesia/hypokinesia and attenuated motor asymmetry in hemiparkinsonian rats. A-D, Systemic (intraperitoneal) injection of J-113397 (0.1-3 mg/kg) or levodopa (1 mg/kg plus 15 mg/kg benserazide) reduced the time spent on the blocks (in seconds) and attenuated motor asymmetry in the bar test (A), increased the number of steps of the contralateral forepaw in the drag test (B), and improved overall motor performance (calculated as percentage of the control session) in the rotarod test (C, D). Motor asymmetry was evaluated by separate measures at the paws ipsilateral and contralateral (parkinsonian) to the lesioned side. The bar and drag tests (A, B) were performed 20 min after injection; the rotarod test was performed 20 min (C) and 70 min (D) after injection. A-D, Data are mean ± SEM of six to eight determinations obtained from 45 hemiparkinsonian and 35 vehicle-injected rats. ^*p < 0.05; ^**p < 0.01 versus saline-treated rats. °p < 0.05; °°p < 0.01 versus the ipsilateral paw. ^#p < 0.05; ^##p < 0.01 versus vehicle-injected rats (ANOVA followed by Newman-Keuls post hoc test). Error bars represent SEM.

Figure 4.

Mice lacking the NOP receptor (NOP^-/-) are more resistant to haloperidol-induced akinesia than wild-type (NOP^+/+) mice. A-F, Mice were tested before (control) and after 0.3 mg/kg (A, C, E) or 0.8 mg/kg (B, D, F) haloperidol (intraperitoneal). A-F, Akinesia was measured for up to 8 h by using the bar (A, B), drag (C, D), and rotarod (E, F) tests. NOP^-/- mice having received haloperidol were less akinetic than NOP^+/+ mice. Data are expressed as means ± SEM of 7-11 determinations obtained from 22 NOP^-/- and 22 NOP^+/+ mice. ^**p < 0.01 versus control. °p < 0.05; °°p < 0.01 versus NOP^+/+ mice (ANOVA followed by Newman-Keuls post hoc test). Error bars represent SEM.

Figure 5.

J-113397 normalized GLU release in the rat SNr and reversed haloperidol-induced akinesia. Rats were made akinetic with haloperidol (0.8 mg/kg, i.p.) and treated with J-113397 (1 mg/kg, i.p.) or saline when akinesia was fully instated (i.e., 75 min after haloperidol). A, B, In the same animals, J-113397 reduced SNr GLU release elevated previously by haloperidol (A) and attenuated akinesia, simultaneously evaluated by using the bar test (B). Two-way ANOVA revealed a significant haloperidol by J-113397 interaction. Data are expressed as means ± SEM of eight to nine determinations. Basal GLU (in nanomolar concentration) and AUC (arbitrary units; 75-180 min window) values were, respectively, as follows: 107.6 ± 14.5, 8830 ± 257 (saline n = 8); 110.7 ± 7.1, 8548 ± 252 (J-113397; n = 7); 102.6 ± 14.2, 17349 ± 1038 (haloperidol, n = 9); and 99.1 ± 11.0, 12128 ± 888 (haloperidol plus J-113397; n = 8). A significant correlation (r² = 0.584; p < 0.0001) was found between reduction of immobility time and nigral GLU release. Error bars represent SEM.

Figure 6.

J-113397 and UFP-101 reduce GLU release in the SNr of hemiparkinsonian rats. A-D, Reverse dialysis of UFP-101 (1-10 μm; 60 min; A, B) or systemic administration of J-113397 (0.1-3 mg/kg, i.p.; C, D) reduced GLU extracellular levels in both the lesioned and unlesioned SNr of awake hemiparkinsonian rats. Data (means of 5-12 experiments) are expressed as percentage ± SEM of basal pretreatment levels (calculated as the mean of the two sample before the treatment; A, D) or AUC (arbitrary units in the 0-90 min window; E, F) values. Comparison between effects in the lesioned and unlesioned SNr has been made on AUC values. The probe location is shown in supplemental Fig. 2S (available at www.jneurosci.org as supplemental material). Basal GLU (in nanomolar concentration) values in the unlesioned and lesioned SNr were, respectively, as follows: E, 114.6 ± 16.5 (n = 7) and 107.9 ± 12.9 (n = 10; washout); 94.3 ± 10.1 (n = 8) and 95.3 ± 11.1 (n = 8; 1 μm UFP-101); 114.9 ± 17.6 (n = 5) and 103.6 ± 5.5 (n = 5; 3 μm UFP-101); 118.5 ± 10.7 (n = 12) and 103.2 ± 7.9 (n = 9; 10 μm UFP-101); F, 102.3 ± 7.4 (n = 6) and 89.7 ± 6.5 (n = 5; saline); 87.8 ± 7.6 (n = 6) and 100.6 ± 8.6 (n = 6; 0.1 mg/kg J-113397); 112.4 ± 12.4 (n = 11) and 92.7 ± 7.1 (n = 12; 1 mg/kg J-113397), 95.4 ± 14.0 (n = 6) and 101.4 ± 10.0 (n = 6; 3 mg/kg J-113397). ^*p < 0.05; ^**p < 0.01 versus saline-treated rats. °p < 0.05; °°p < 0.01 versus unlesioned SNr (ANOVA followed by Newman-Keuls post hoc test). Error bars represent SEM.

Figure 7.

Plasticity of the N/OFQ-NOP receptor system after 6-OHDA lesion. A, B, Relative optical density values of ppN/OFQ (A) and NOP (B) mRNA hybridization to riboprobes 7 weeks after injection of vehicle (vehicle-injected; open bars; n = 5) or 6-OHDA in the right SN (hemiparkinsonian; solid bars; n = 6) of rats. The results are expressed as mean ± SEM percentage of control signal (in the noninjected side) over SNc and SNr. Representative images of ppN/OFQ mRNA (below A) and NOP receptor mRNA (below B) expression in the SNc (arrow) and SNr (arrowheads) in hemiparkinsonian rats are presented. C, Extracellular levels of N/OFQ were measured by microdialysis in the lesioned and unlesioned SNr of nine hemiparkinsonian rats, 7 weeks after 6-OHDA. Microdialysis was performed at rest and under two 30 min rotarod sessions performed (15 rpm) at the beginning of the third and fourth collection periods. Data (mean ± SEM of 9 determinations) are expressed as N/OFQ-LI in femtomoles. ^**p < 0.01 versus pretreatment values. °°p < 0.01 versus unlesioned SNr. ^##p < 0.01 versus vehicle-injected rats (ANOVA followed by Newman-Keuls post hoc test). Error bars represent SEM.

Figure 8.

Deletion of the ppN/OFQ gene attenuated the MPTP-induced loss of TH/NeuN-immunoreactive neurons in the SN. A, Total number of TH/NeuN-immunoreactive neurons in the SN after MPTP treatment in wild-type (ppN/OFQ^+/+) and ppN/OFQ knock-out (ppN/OFQ^-/-) mice. MPTP treatment produced a significantly greater decrease in the number of TH-positive cells in the ppN/OFQ^+/+ mice (n = 8) than in ppN/OFQ^-/- mice (n=7). B, Total TH-positive neurons throughout the rostral- caudal axis of the SN. ^*p < 0.05 versus saline-treated mice. ^#p < 0.05 versus ppN/OFQ^+/+ mice (ANOVA followed by Newman-Keuls post hoc test). Error bars represent SEM.

Figure 9.

Deletion of the ppN/OFQ gene attenuates the MPTP-induced loss of TH immunoreactivity in the dorsolateral CP. Density of TH immunoreactivity in the rostral (1.10 mm from bregma) CP. No significant difference was detected between saline-treated wild-type (+/+) or knock-out (-/-) mice (2.39 ± 0.17 and 2.75 ± 0.30, respectively). For comparison purposes, all saline-treated animals were combined and termed control. A, B, MPTP produced a significant loss of TH staining in the dorsolateral (A), but not ventral (B), portion of the CP. MPTP significantly decreased TH staining in the dorsolateral CP of ppN/OFQ^+/+ (n = 8), but not ppN/OFQ^-/- (n = 7), mice. Representative images of the CP sections from saline- and MPTP-treated mice are also presented. ^*p < 0.05 versus control (ANOVA followed by Newman-Keuls post hoc test). Error bars represent SEM.