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.
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).
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-1β (IL-1β), 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-1β, 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).