Beneficial effects of the phytocannabinoid Δ9-THCV in L-DOPA-induced dyskinesia in Parkinson's disease.

The antioxidant and CB2 receptor agonist properties of Δ9-tetrahydrocannabivarin (Δ9-THCV) afforded neuroprotection in experimental Parkinson's disease (PD), whereas its CB1 receptor antagonist profile at doses lower than 5 mg/kg caused anti-hypokinetic effects. In the present study, we investigated the anti-dyskinetic potential of Δ9-THCV (administered i.p. at 2 mg/kg for two weeks), which had not been investigated before. This objective was investigated after inducing dyskinesia by repeated administration of L-DOPA (i.p. at 10 mg/kg) in a genetic model of dopaminergic deficiency, Pitx3ak mutant mice, which serves as a useful model for testing anti-dyskinetic agents. The daily treatment of these mice with L-DOPA for two weeks progressively increased the time spent in abnormal involuntary movements (AIMs) and elevated their horizontal and vertical activities (as measured in a computer-aided actimeter), signs that reflected the dyskinetic state of these mice. Interestingly, when combined with L-DOPA from the first injection, Δ9-THCV delayed the appearance of all these signs and decreased their intensity, with a reduction in the levels of FosB protein and the histone pAcH3 (measured by immunohistochemistry), which had previously been found to be elevated in the basal ganglia in L-DOPA-induced dyskinesia. In addition to the anti-dyskinetic effects of Δ9-THCV when administered at the onset of L-DOPA treatment, Δ9-THCV was also effective in attenuating the intensity of dyskinesia when administered for three consecutive days once these signs were already present (two weeks after the onset of L-DOPA treatment). In summary, our data support the anti-dyskinetic potential of Δ9-THCV, both to delay the occurrence and to attenuate the magnitude of dyskinetic signs. Although further studies are clearly required to determine the clinical significance of these data in humans, the results nevertheless situate Δ9-THCV in a promising position for developing a cannabinoid-based therapy for patients with PD.

Benefits of VCE-003.2, a cannabigerol quinone derivative, against inflammation-driven neuronal deterioration in experimental Parkinson's disease: possible involvement of different binding sites at the PPARγ receptor.

BACKGROUND: Neuroprotection with cannabinoids in Parkinson's disease (PD) has been afforded predominantly with antioxidant or anti-inflammatory cannabinoids. In the present study, we investigated the anti-inflammatory and neuroprotective properties of VCE-003.2, a quinone derivative of the non-psychotrophic phytocannabinoid cannabigerol (CBG), which may derive its activity at the peroxisome proliferator-activated receptor-γ (PPARγ). The compound is also an antioxidant. METHODS: We evaluated VCE-003.2 in an in vivo [mice subjected to unilateral intrastriatal injections of lipopolysaccharide (LPS)] model of PD, as well as in in vitro (LPS-exposed BV2 cells and M-213 cells treated with conditioned media generated from LPS-exposed BV2 cells) cellular models. The type of interaction of VCE-003.2 at the PPARγ receptor was furtherly investigated in bone marrow-derived human mesenchymal stem cells (MSCs) and sustained with transcriptional assays and in silico docking studies. RESULTS: VCE-003.2 has no activity at the cannabinoid receptors, a fact that we confirmed in this study using competition studies. The administration of VCE-003.2 to LPS-lesioned mice attenuated the loss of tyrosine hydroxylase (TH)-containing nigrostriatal neurons and, in particular, the intense microgliosis provoked by LPS in the substantia nigra, measured by Iba-1/Cd68 immunostaining. The analysis by qPCR of proinflammatory mediators such as tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and inducible nitric oxide synthase (iNOS) in the striatum showed they were markedly elevated by the LPS lesion and strongly reduced by the treatment with VCE-003.2. The effects of VCE-003.2 in LPS-lesioned mice implied the activation of PPARγ receptors, as they were attenuated when VCE-003.2 was co-administered with the PPARγ inhibitor T0070907. We then moved to some in vitro approaches, first to confirm the anti-inflammatory profile of VCE-003.2 in cultured BV2 cells exposed to LPS. VCE-003.2 was able to attenuate the synthesis and release of TNF-α and IL-1ß, as well as the induction of iNOS and cyclooxygenase-2 (COX-2) elicited by LPS in these cells. However, we found such effects were not reversed by GW9662, another classic PPARγ antagonist. Next, we investigated the neuroprotective effects of VCE-003.2 in cultured M-213 neuronal cells exposed to conditioned media generated from LPS-exposed cultured BV2 cells. VCE-003.2 reduced M-213 cell death, but again, such effects were not reversed by T0070907. Using docking analysis, we detected that VCE-003.2 binds both the canonical and the alternative binding sites in the PPARγ ligand-binding pocket (LBP). Functional assays further showed that T0070907 almost abolished PPARγ transcriptional activity induced by rosiglitazone (RGZ), but it did not affect the activity of VCE-003.2 in a Gal4-Luc system. However, T0070907 inhibited the effects of RGZ and VCE-003.2 on the expression of PPARγ-dependent genes upregulated in MSCs. CONCLUSIONS: We have demonstrated that VCE-003.2 is neuroprotective against inflammation-driven neuronal damage in an in vivo model of PD and in in vitro cellular models of neuroinflammation. Such effects might involve PPARγ receptors, although in silico and in vitro experiments strongly suggest that VCE-003.2 targets PPARγ by acting through two binding sites at the LBP, one that is sensitive to T0070907 (canonical binding site) and other that is not affected by this PPARγ antagonist (alternative binding site).

Targeting the cannabinoid CB2 receptor to attenuate the progression of motor deficits in LRRK2-transgenic mice.

Most of cases of Parkinson's disease (PD) have a sporadic origin, with their causes mostly unknown, although overexposure to some environmental factors has been found to occur in some cases. Other forms of parkinsonism are the consequence of dominant or recessive mutations in specific genes, e.g. α-synuclein, parkin and, more recently, leucine-rich repeat kinase 2 (LRRK2), whose G2019S mutation represents the most prevalent form of late-onset, autosomal dominant familial PD. A transgenic mouse model expressing the G2019S mutation of LRRK2 is already available and apparently may represent a valuable experimental model for investigating PD pathogenesis and novel treatments. We designed a long-term study with these animals aimed at: (i) elucidating the changes experienced by the endocannabinoid signaling system in the basal ganglia during the progression of the disease in these mice, paying emphasis in the CB2 receptor, which has emerged as a promising target in PD, and (ii) evaluating the potential of compounds selectively activating this CB2 receptor, as disease-modifying agents in these mice. Our results unequivocally demonstrate that LRRK2 transgenic mice develop motor impairment consisting of small anomalies in rotarod performance (presumably reflecting a deficit in motor coordination and dystonia) and a strong deficiency in the hanging-wire test (reflecting muscle weakness), rather than hypokinesia which was difficult to be demonstrated in the actimeter. These behavioral responses occurred in absence of any evidence of reactive gliosis and neuronal losses, as well as synaptic deterioration in the basal ganglia, except an apparent impairment in autophagy reflected by elevated LAMP-1 immunolabelling in the striatum and substantia nigra. Furthermore, there were no changes in the status of the CB2 receptor, as well as in other elements of the endocannabinoid signaling, in the basal ganglia, but, paradoxically, the selective activation of this receptor partially reversed the deficits in the hanging-wire test of LRRK2 transgenic mice. This was accompanied by normalization in LAMP-1 immunolabelling in the basal ganglia, although it is possible that other CNS structures, remaining to be identified, are involved in the behavioral improvement. In summary, our data support the interest of the CB2 receptor as a potential pharmacological target in LRRK2 transgenic mice, although the neuronal substrates underlying these benefits might be not completely related to the basal ganglia and to the presumed parkinsonian features of these mice.

Potential of the cannabinoid CB(2) receptor as a pharmacological target against inflammation in Parkinson's disease.

Inflammation is an important pathogenic factor in Parkinson's disease (PD), so that it can contribute to kill dopaminergic neurons of the substantia nigra and to enhance the dopaminergic denervation of the striatum. The cannabinoid type-2 (CB2) receptor has been investigated as a potential anti-inflammatory and neuroprotective target in different neurodegenerative disorders, but still limited evidence has been collected in PD. Here, we show for the first time that CB2 receptors are elevated in microglial cells recruited and activated at lesioned sites in the substantia nigra of PD patients compared to control subjects. Parkinsonian inflammation can be reproduced experimentally in rodents by intrastriatal injections of lipopolysaccharide (LPS) which, through an intense activation of glial elements and peripheral infiltration, provokes a rapid deterioration of the striatum that may extend to the substantia nigra too. Using this experimental model, we recently described a much more intense deterioration of tyrosine hydroxylase (TH)-containing nigral neurons in CB2 receptor-deficient mice compared to wild-type animals, supporting a potential neuroprotective role for this receptor. In the present study, we further explored this issue. First, we found elevated levels of the CB2 receptor measured by qRT-PCR in the striatum and substantia nigra of LPS-lesioned mice, as well as an increase in the immunostaining for this receptor in the LPS-lesioned striatum. Second, we found a significant increase in CD68 immunostaining, which serve to identify activated microglia and also infiltrated peripheral macrophages, in these brain structures in response to LPS insult, which was much more intense in CB2 receptor-deficient mice in the case of the substantia nigra. Next, we observed that the activation of CB2 receptors with a selective agonist (HU-308) reversed LPS-induced elevation of CD68 immunostaining in the striatum and the parallel reduction in TH immunostaining. Lastly, we found that LPS elevated the gene expression of different pro-inflammatory mediators in both the striatum and the substantia nigra, whereas the selective activation of CB2 receptors reduced a part of these mediators, e.g. inducible nitric oxide synthase, although exclusively in the striatum. In conclusion, we have provided the first evidence on the up-regulation of CB2 receptors in glial elements in postmortem tissues of PD patients, which has been confirmed in an inflammatory model of this disease. In addition, we have provided evidence on the benefits derived from their activation in relation with the activation of microglial cells, the infiltration of macrophages and also certain capability of these cells to generate proinflammatory factors.

Cannabinoid-dopamine interactions in the physiology and physiopathology of the basal ganglia.

UNLABELLED: Endocannabinoids and their receptors play a modulatory role in the control of dopamine transmission in the basal ganglia. However, this influence is generally indirect and exerted through the modulation of GABA and glutamate inputs received by nigrostriatal dopaminergic neurons, which lack cannabinoid CB1 receptors although they may produce endocannabinoids. Additional evidence suggests that CB2 receptors may be located in nigrostriatal dopaminergic neurons, and that certain eicosanoid-related cannabinoids may directly activate TRPV1 receptors, which have been found in nigrostriatal dopaminergic neurons, thus allowing in both cases a direct regulation of dopamine transmission by specific cannabinoids. In addition, CB1 receptors form heteromers with dopaminergic receptors which provide another pathway to direct interactions between both systems, in this case at the postsynaptic level. Through these direct mechanisms or through indirect mechanisms involving GABA or glutamate neurons, cannabinoids may interact with dopaminergic transmission in the basal ganglia and this is likely to have important effects on dopamine-related functions in these structures (i.e. control of movement) and, particularly, on different pathologies affecting these processes, in particular, Parkinson's disease, but also dyskinesia, dystonia and other pathological conditions. The present review will address the current literature supporting these cannabinoid-dopamine interactions at the basal ganglia, with emphasis on aspects dealing with the physiopathological consequences of these interactions. LINKED ARTICLES: This article is part of a themed section on Updating Neuropathology and Neuropharmacology of Monoaminergic Systems. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v173.13/issuetoc.

Identification of CB2 receptors in human nigral neurons that degenerate in Parkinson's disease.

It is well-demonstrated that cannabinoid CB2 receptors located in glial cells are up-regulated in neurodegenerative disorders serving as a target to control glial influences to neurons. Recent evidence indicates that CB2 receptors may be also located in certain neuronal subpopulations and serve as a marker of neuronal losses. We investigated this possibility in the post-mortem substantia nigra of Parkinson's disease (PD) patients and controls. Immunostaining for the CB2 receptor was found in tyrosine hydroxylase-positive neurons in the substantia nigra, a fact confirmed with double-staining analyses. The signal was found in controls but also in PD patients, in which CB2 receptor labelling was significantly lower, in parallel to the losses of these neurons experienced in the disease. These data show for the first time that CB2 receptors are located in tyrosine hydroxylase-containing neurons in the substantia nigra at levels significantly lower in PD patients compared to controls.

Prospects for cannabinoid therapies in basal ganglia disorders.

Cannabinoids are promising medicines to slow down disease progression in neurodegenerative disorders including Parkinson's disease (PD) and Huntington's disease (HD), two of the most important disorders affecting the basal ganglia. Two pharmacological profiles have been proposed for cannabinoids being effective in these disorders. On the one hand, cannabinoids like Δ(9) -tetrahydrocannabinol or cannabidiol protect nigral or striatal neurons in experimental models of both disorders, in which oxidative injury is a prominent cytotoxic mechanism. This effect could be exerted, at least in part, through mechanisms independent of CB(1) and CB(2) receptors and involving the control of endogenous antioxidant defences. On the other hand, the activation of CB(2) receptors leads to a slower progression of neurodegeneration in both disorders. This effect would be exerted by limiting the toxicity of microglial cells for neurons and, in particular, by reducing the generation of proinflammatory factors. It is important to mention that CB(2) receptors have been identified in the healthy brain, mainly in glial elements and, to a lesser extent, in certain subpopulations of neurons, and that they are dramatically up-regulated in response to damaging stimuli, which supports the idea that the cannabinoid system behaves as an endogenous neuroprotective system. This CB(2) receptor up-regulation has been found in many neurodegenerative disorders including HD and PD, which supports the beneficial effects found for CB(2) receptor agonists in both disorders. In conclusion, the evidence reported so far supports that those cannabinoids having antioxidant properties and/or capability to activate CB(2) receptors may represent promising therapeutic agents in HD and PD, thus deserving a prompt clinical evaluation.