The expression of cannabinoid type 1 receptor and 2-arachidonoyl glycerol synthesizing/degrading enzymes is altered in basal ganglia during the active phase of levodopa-induced dyskinesia

Highlights

• During the active phase of LID, the expression of CB₁ receptor increases in the putamen, GPe and STN.

• 2-AG metabolism would be increased in pallidal-STN projections of dyskinetic animals OFF L-dopa.

• AEA synthesizing/degrading enzymes are altered in the GPe of untreated parkinsonian monkeys.

• CB₁ receptor and 2-AG metabolic pathway may be considered therapeutic targets for the treatment of dyskinesia.

Abstract

Management of levodopa-induced dyskinesias (LID) is one of the main challenges in the treatment of Parkinson's disease patients. Mechanisms involved in the appearance of these involuntary movements are not well known but modifications in the activity of different neurotransmitter pathways seem to play an important role. The objective of this study was to determine differences in the expression levels of the endocannabinoid system (ECS) elements that would support a role in LID. The basal ganglia nuclei, putamen, external segment of the globus pallidus (GPe), internal segment of the globus pallidus (GPi), subthalamic nucleus (STN) and substantia nigra (SN) were dissected out from cryostat sections obtained from two groups of parkinsonian monkeys treated with levodopa to induce dyskinesias. One group of dyskinetic animals was sacrificed under the effect of levodopa, during the active phase of LID, and the other group 24 h after the last levodopa dose (OFF levodopa). Biochemical analysis by real-time PCR for ECS elements was performed. CB₁ receptor expression was upregulated in the putamen, GPe and STN during the active phase of dyskinesia and downregulated in the same nuclei and in the SN when dyskinetic animals were OFF levodopa. Changes in the 2-arachidonoyl glycerol (2-AG) synthesizing/degrading enzymes affecting the pallidal-subthalamic projections in dyskinetic animals OFF levodopa would suggest that 2-AG may play a role in LID. Anandamide (AEA) synthesizing/degrading enzymes were altered specifically in the GPe of untreated parkinsonian monkeys, suggesting that increased AEA levels may be a compensatory mechanism. These results indicate that the expression of the ECS elements is influenced by alterations in dopaminergic neurotransmission. On one hand, changes in CB₁ receptor expression and in the 2-AG synthesizing/degrading enzymes suggest that they could be a therapeutic target for the active phase of LID. On the other hand, AEA metabolism could provide a non-dopaminergic target for symptomatic relief. However, further research is needed to unravel the mechanism of action of the ECS and how they could be modulated for a therapeutic purpose.

Introduction

Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by the loss of dopaminergic neurons projecting to the striatum, a brain region involved in the execution and planning of motor behavior (Redgrave et al., 2010). The dopamine precursor l-3,4-dihydroxyphenylalanine (l-dopa or levodopa) remains the most effective and best tolerated treatment for PD motor symptoms (Cotzias et al., 1967)(Fox et al., 2011). However, long-term l-dopa administration is associated with the appearance of motor fluctuations and disabling abnormal involuntary movements (AIMs) known as l-dopa-induced dyskinesias (LID) (Bastide et al., 2015). Approximately, 80% of patients experience LID within 5 years of treatment affecting profoundly their quality of life (Bastide et al., 2015). LID is associated with the activation of dopamine receptors, downstream changes in proteins and genes and with abnormalities in non-dopaminergic systems all of which combine to produce alterations in the neuronal firing pattern between the basal ganglia and the cortex (Bastide et al., 2015). LID remains as the major challenge in the management of PD patients due to the lack of effective pharmacological treatments.

Endocannabinoids, their synthesizing/degrading enzymes and receptors constitute the endocannabinoid system (ECS) which plays a crucial role in controlling neuronal excitability and synaptic transmission (Castillo et al., 2012). The most relevant endogenous cannabinoids in the brain are the 2-arachidonoyl glycerol (2-AG) (Mechoulam et al., 1995)(Sugiura et al., 1995)(Stella et al., 1997) and the arachidonoyl ethanolamide or anandamide (AEA) (Devane et al., 1992) which act through paracrine or autocrine cannabinoid receptors (Castillo et al., 2012). Diacylglycerol lipase α (DAGLα) is localized in postsynaptic neurons (Katona et al., 2006)(Lafourcade et al., 2007) and it is required for Ca²⁺-dependent 2-AG production and for endocannabinoid-mediated synaptic plasticity (Gao et al., 2010)(Tanimura et al., 2010). Monoacylglycerol lipase (MAGL), located presynaptically (Gulyas et al., 2004), is the main degradative enzyme for 2-AG in the brain (Blankman et al., 2007) and the point of control of 2-AG signaling (Hashimotodani et al., 2007). Postsynaptic depolarization and intracellular Ca²⁺ influx promote AEA production. One of the main enzymes for AEA synthesis in the brain is N-acyl-phosphatidylethanolamine-hydrolyzing phospholipase D (NAPE-PLD) (Okamoto et al., 2007) that can be expressed post- and presynaptically (Cristino et al., 2008)(Egertová et al., 2008). Biological actions of AEA are terminated by enzymatic hydrolysis by fatty acid amide hydrolase (FAAH) which is located in intracellular compartments (Cravatt et al., 1996)(Gulyas et al., 2004). 2-AG is a full agonist at cannabinoid type 1 (CB₁) and type 2 (CB₂) receptors (Freund et al., 2003). AEA is a partial agonist of the classic CB₁ and CB₂ receptors and also binds to peroxisome proliferator-activated (PPAR) and transient receptor potential vanilloid type 1 (TRPV-1) receptors (Pertwee et al., 2010). Several lines of evidence support the involvement of the ECS in the regulation of motor functions and in movement disorders. The different elements of the ECS are particularly abundant in basal ganglia (Fernández-Ruiz, 2009), plant-derived and synthetic cannabinoids have an inhibitory effect in motor activity (Sañudo-Peña et al., 1999) and the ECS is altered in experimental models and human forms of motor diseases (Fernández-Ruiz, 2009). Interestingly, the ECS exerts a major modulatory action in basal ganglia by its ability to modulate GABAergic and glutamatergic synapsis, and dopamine neurotransmission (Sañudo-Peña et al., 1999)(Gerdeman et al., 2002)(Kreitzer and Malenka, 2007)(Adermark et al., 2009)(García et al., 2016).

Studies related to the ECS and LID have been focused mainly in the CB₁ receptor. In the 6-OHDA rat model, CB₁ mRNA receptor expression did not change following 6-OHDA lesion but increased with l-dopa therapy (Zeng et al., 1999). Animals were sacrificed 3 h after the last dose of l-dopa, although evidence of a dyskinetic behavior was not provided (Zeng et al., 1999). CB₁ receptor binding and CB₁ receptor agonist WIN55,212–2-stimulated [³⁵S]GTPγS binding were increased in the striatum of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) marmosets and PD patients and returned to control levels upon l-dopa therapy (Lastres-Becker et al., 2001). However, a decrease of CB₁ mRNA receptor was reported in the striatum and in the external segment of the globus pallidus (GPe) of PD patients under l-dopa therapy (Hurley et al., 2003). These studies suggest a relationship between CB₁ receptor and dyskinesia but remained inconclusive since the time elapsed between the last dose of l-dopa and the sacrifice of the MPTP marmosets was not mentioned (Lastres-Becker et al., 2001), neither the presence of dyskinesias in PD patients (Lastres-Becker et al., 2001)(Hurley et al., 2003). 2-AG levels were elevated in the striatum and decreased in the GPe of MPTP-lesioned primates sacrificed during the active phase of dyskinesia but how 2-AG levels would lead to dyskinesia remains unknown (van der Stelt et al., 2005). Pharmacological studies in 6-hidroxydopamine rodent models of LID showed that WIN55,212-2 attenuated non-locomotive AIMs through CB₁ receptor (Morgese et al., 2007). In contrast, elevation of AEA levels with the FAAH inhibitor URB597 eliminated all types of AIMs when co-administered with the TRPV-1 antagonist capsazepine (Morgese et al., 2007), this effect was mediated by the activation of PPARγ (Martinez et al., 2015). Stimulation of TRPV-1 with oleoylethanolamide, a N-acylethanolamide that do not bind cannabinoid receptors, also had anti-dyskinetic properties (González-Aparicio and Moratalla, 2014). Cannabidiol, a non-psychoactive phytocannabinoid, reduced AIMs when it was administrated with a TRPV-1 antagonist by acting on CB₁ and PPARγ (dos-Santos-Pereira et al., 2016).

Pharmacological studies in MPTP primates and humans support the role of the ECS in LID with apparently paradoxical outcomes. Both, CB₁ receptor agonists and antagonists show anti-dyskinetic properties (van der Stelt et al., 2005)(Fox et al., 2002)(Sieradzan et al., 2001). Further research is needed to determine the role of the ECS in LID. We hypothesize that the elements of the ECS would change differentially in basal ganglia nuclei during the active phase of dyskinesia. Thus, the aim of this study was to determine differences in the expression levels of the ECS elements that might support a role for the ECS in the neuronal plasticity present during the occurrence of LID and/or when the l-dopa effect has gone.