WIN55,212-2, a cannabinoid receptor agonist, protects against nigrostriatal cell loss in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson's disease

Abstract

Parkinson's disease (PD) is characterized by the progressive loss of nigrostriatal dopamine neurons leading to motor disturbances and cognitive impairment. Current pharmacotherapies relieve PD symptoms temporarily but fail to prevent or slow down the disease progression. In this study, we investigated the molecular mechanisms by which the non-selective cannabinoid receptor agonist WIN55,212-2 (WIN) protects mouse nigrostriatal neurons from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity and neuroinflammation. Stereological analyses showed that chronic treatment with WIN (4 mg/kg, intraperitoneal), initiated 24 h after MPTP administration, protected against MPTP-induced loss of tyrosine hydroxylase-positive neurons in the substantia nigra pars compacta independently of CB1 cannabinoid receptor activation. The neuroprotective effect of WIN was accompanied by increased dopamine and 3,4-dihydroxyphenylacetic acid levels in the substantia nigra pars compacta and dorsal striatum of MPTP-treated mice. At 3 days post-MPTP, we found significant microglial activation and up-regulation of CB2 cannabinoid receptors in the ventral midbrain. Treatment with WIN or the CB2 receptor agonist JWH015 (4 mg/kg, intraperitoneal) reduced MPTP-induced microglial activation, whereas genetic ablation of CB2 receptors exacerbated MPTP systemic toxicity. Furthermore, chronic WIN reversed MPTP-associated motor deficits, as revealed by the analysis of forepaw step width and percentage of faults using the inverted grid test. In conclusion, our data indicate that agonism at CB2 cannabinoid receptors protects against MPTP-induced nigrostriatal degeneration by inhibiting microglial activation/infiltration and suggest that CB2 receptors represent a new therapeutic target to slow the degenerative process occurring in PD.

Fig. 1

Effect of WIN on MPTP-induced loss of TH⁺ neurons in the SNc. Saline or MPTP (4x 20 mg/kg, i.p.) were administered every 2 hr on day 0 to CB₁^+/+ mice. Vehicle or WIN (1 mg/kg, i.p. or 4 mg/kg, i.p.) were administered every day over a 5 day period, starting 24 hr after the last injection of saline or MPTP. Animals were sacrificed on day 7. (A) Representative microimages of midbrain immunostained for TH (scale bar = 100 μm). (B) Unbiased stereological quantification of TH⁺ neurons in the SNc. ^###p<0.001 compared to saline-treated control; *p<0.05 as shown (ANOVA followed by Bonferroni’s multiple comparisons post-test; n=5 per treatment).

Fig. 2

Measurement of DA and DOPAC levels and DA turnover (DOPAC/DA) in the dorsal striatum (A) and ventral midbrain (B). Saline or MPTP (4x 20 mg/kg, i.p.) were administered every 2 hr on day 0. Vehicle or WIN (4 mg/kg, i.p.) were administered every day over a 5 day period, starting 24 hr after the last injection of MPTP. Animals were sacrificed on day 7 post-MPTP treatment. *p<0.05 and **p<0.01 compared to saline+vehicle; ^#p<0.05 compared to MPTP+vehicle (ANOVA followed by Newman-Keuls’ multiple comparisons post-test; n=4 per treatment).

Fig. 3

Effect of genetic ablation of CB₁ on WIN-mediated protection against MPTP-induced loss of TH⁺ neurons. CB₁^+/+ or CB₁^−/− mice received 4 injections of MPTP (20 mg/kg, i.p.) every 2 hr on day 0. Vehicle or WIN (4 mg/kg, i.p.) were administered every day over a 5 day period, starting 24 hr after the last injection of MPTP. Animals were sacrificed on day 7 and TH⁺ neurons were quantified via unbiased stereology. ^###p<0.001 compared to saline-treated CB₁^+/+ (dashed line); *p<0.05 and ***p<0.001 as shown (ANOVA followed by Bonferroni’s multiple-comparisons post-test; n=5 per treatment).

Fig. 4

Effects of MPTP on brain CB₂ receptor expression. Saline or MPTP (4x 20 mg/kg, i.p.) were administered every 2 hr on day 0 and animals were sacrificed on day 3. (A) Western blot analysis of CB₂ receptor expression in ventral midbrain and dorsal striatum. Bands were quantified by densitometry. **p<0.01 compared to saline-treated control (unpaired Student’s t-test; n=4 per treatment). (B) Double-label immunofluorescence confirms that CB₂ receptors (green) and MAC-1 (red) co-localize in the SNc (scale bars = 10 μm). (C) Immunofluorescence confirms that CB₂ receptors are present in spleen from CB₂^+/+ but not CB₂^−/− mice (scale bars = 10 μm).

Fig. 5

Effects of MPTP and WIN on microglial activation/infiltration. Saline or MPTP (4x 20 mg/kg, i.p.) were administered every 2 hr on day 0 and animals were sacrificed on day 3. Vehicle or cannabinoids were administered on days 1–3. (A) Representative microimages of the ventral midbrain immunostained for MAC-1 (scale bar = 100 μm). (B) Higher magnification image of the SNc immunostained for MAC-1 3 days post-MPTP treatment (scale bar = 25 μm). (C) Densitometry of MAC-1 expression in ventral midbrain following western blotting. *p<0.05 and **p<0.01 compared to saline-treated control (dashed line); ***p<0.001 compared to MPTP+vehicle (ANOVA followed by Bonferroni’s multiple comparisons post-test; n=3 per treatment).

Fig. 6

WIN reverses MPTP-induced motor deficits. Forepaw step length (A) and percentage of forepaw step faults (B) were measured in mice receiving saline or MPTP and post-treated with vehicle or WIN (4 mg/kg, i.p.) as previously described. *p<0.05, **p<0.01 and ***p<0.001 as shown (ANOVA followed by Newman-Keuls’ post-test; n=8 per treatment).