Investigation in the cannabigerol derivative VCE-003.2 as a disease-modifying agent in a mouse model of experimental synucleinopathy

Abstract

Background: The cannabigerol derivative VCE-003.2, which has activity at the peroxisome proliferator-activated receptor-γ has afforded neuroprotection in experimental models of Parkinson's disease (PD) based on mitochondrial dysfunction (6-hydroxydopamine-lesioned mice) and neuroinflammation (LPS-lesioned mice). Now, we aim to explore VCE-003.2 neuroprotective properties in a PD model that also involves protein dysregulation, other key event in PD pathogenesis.

Methods: To this end, an adeno-associated viral vector serotype 9 coding for a mutated form of the α-synuclein gene (AAV9-SynA53T) was unilaterally delivered in the substantia nigra pars compacta (SNpc) of mice. This model leads to motor impairment and progressive loss of tyrosine hydroxylase-labelled neurons in the SNpc.

Results: Oral administration of VCE-003.2 at 20 mg/kg for 14 days improved the performance of mice injected with AAV9-SynA53T in various motor tests, correlating with the preservation of tyrosine hydroxylase-labelled neurons in the SNpc. VCE-003.2 also reduced reactive microgliosis and astrogliosis in the SNpc. Furthermore, we conducted a transcriptomic analysis in the striatum of mice injected with AAV9-SynA53T and treated with either VCE-003.2 or vehicle, as well as control animals. This analysis aimed to identify gene families specifically altered by the pathology and/or VCE-003.2 treatment. Our data revealed pathology-induced changes in genes related to mitochondrial function, lysosomal cell pathways, immune responses, and lipid metabolism. In contrast, VCE-003.2 treatment predominantly affected the immune response through interferon signaling.

Conclusion: Our study broadens the neuroprotective potential of VCE-003.2, previously described against mitochondrial dysfunction, oxidative stress, glial reactivity and neuroinflammation in PD. We now demonstrate its efficacy against another key pathogenic event in PD as α-synuclein dysregulation. Furthermore, our investigation sheds light on the molecular mechanisms underlying VCE-003.2 revealing its role in regulating interferon signaling. These findings, together with a favorable ADMET profile, enhance the preclinical interest of VCE-003.2 towards its future clinical development in PD.

Keywords: Cannabinoids; PPAR-γ; Parkinson’s disease; VCE-003.2; alpha-synuclein.

Fig. 1

Representative scheme of the experimental protocol used for the induction of α-synuclein-based PD model used in this study and the treatment with VCE-003.2, with indication of timelines for treatments, behavioral analysis and animal euthanasia

Fig. 2

Behavioural analysis in the Cylinder Rearing Test (CRT) and in the Elevated-Body Swing Test (EBST) of vehicle-treated control (sham-operated) adult male mice and unilaterally AAV9-SynA53T-injected adult male mice treated with vehicle or VCE-00.2 (20 mg/kg) given orally. Treatments were daily and prolonged for 2 weeks. Data (preference score (left) and total number of movements (right)) corresponded to 24 h after the last dose and were expressed as means ± SEM of more than 4 animals per group. They were analyzed by one-way ANOVA followed by the Tukey test (**p < 0.01 versus vehicle-treated sham-operated mice; #p < 0.05, ##p < 0.01 versus vehicle-treated AAV9-SynA53T-injected mice)

Fig. 3

Intensity of the immunostaining for TH measured in a selected area of the SNpc of vehicle-treated control (sham-operated) adult male mice and unilaterally AAV9-SynA53T-injected adult male mice treated with vehicle or VCE-00.2 (20 mg/kg) given orally. Treatments were daily and prolonged for 2 weeks. Data corresponded to 24 h after the last dose and were expressed as means ± SEM of more than 5 animals per group. They were analyzed by one-way ANOVA followed by the Tukey test (***p < 0.005 versus vehicle-treated sham-operated mice; ###p < 0.005 versus vehicle-treated AAV9-SynA53T-injected mice). Representative immunostaining images for each experimental group, with indication of the approximate area quantified, are shown in right panels (scale bar = 200 μm)

Fig. 4

Intensity of the immunostaining for CD68 and GFAP measured in a selected area of the SN of vehicle-treated control (sham-operated) adult male mice and unilaterally AAV9-SynA53T-injected adult male mice treated with vehicle or VCE-00.2 (20 mg/kg) given orally. Treatments were daily and prolonged for 2 weeks. Data corresponded to 24 h after the last dose and were expressed as means ± SEM of more than 5 animals per group. They were analyzed by one-way ANOVA followed by the Tukey test (**p < 0.01, ***p < 0.005 versus vehicle-treated sham-operated mice; ##p < 0.01 versus vehicle-treated AAV9-SynA53T-injected mice). Representative CD68 immunostaining images for each experimental group are shown in right panels (scale bar = 200 μm)

Fig. 5

Volcano plots showing differentially expressed genes (DEGs). X-axis shows the logFC and Y-axis shows -log(P-value). Every dot represents a specific gene (black are genes at FDR > 0.05 and red are genes at FDR < 0.05). Left panel: Volcano plot of the DEGs of AAV9-SynA53T-injected mice versus sham-operated mice, both treated with vehicle. Right panel: Volcano plot of the DEGs of VCE-003.2-treated AAV9-SynA53T-injected mice versus those treated with vehicle. DEGs were observed at FDR < 0.05

Fig. 6

Pathway enrichment analysis using Hallmark Pathways from GO using pre-ranked GSEA. Pathways represented are significant at q-value < 0.05. X-axis shows the directionality and Y-axis the biological pathway, with the green color tone reflecting the levels of significance. The significant enrichment for the comparison of AAV9-SynA53T-injected mice versus sham-operated mice, both treated with vehicle, is presented on the left, whereas the one for the comparison of VCE-003.2-treated AAV9-SynA53T-injected mice versus those treated with vehicle is presented on the right

Fig. 7

Transcription factor enrichment analysis using DoRothEA showing the altered regulons that were significant at q-value < 0.05. X-axis shows the directionality and Y-axis the different regulons, with the green color tone reflecting the levels of significance. The significant enrichment for the comparison of AAV9-SynA53T-injected mice versus sham-operated mice, both treated with vehicle, is presented on the left, whereas the one for the comparison of VCE-003.2-treated AAV9-SynA53T-injected mice versus those treated with vehicle is presented on the right