Oral administration of the cannabigerol derivative VCE-003.2 promotes subventricular zone neurogenesis and protects against mutant huntingtin-induced neurodegeneration

Abstract

Background: The administration of certain cannabinoids provides neuroprotection in models of neurodegenerative diseases by acting through various cellular and molecular mechanisms. Many cannabinoid actions in the nervous system are mediated by CB₁ receptors, which can elicit psychotropic effects, but other targets devoid of psychotropic activity, including CB₂ and nuclear PPARγ receptors, can also be the target of specific cannabinoids.

Methods: We investigated the pro-neurogenic potential of the synthetic cannabigerol derivative, VCE-003.2, in striatal neurodegeneration by using adeno-associated viral expression of mutant huntingtin in vivo and mouse embryonic stem cell differentiation in vitro.

Results: Oral administration of VCE-003.2 protected striatal medium spiny neurons from mutant huntingtin-induced damage, attenuated neuroinflammation and improved motor performance. VCE-003.2 bioavailability was characterized and the potential undesired side effects were evaluated by analyzing hepatotoxicity after chronic treatment. VCE-003.2 promoted subventricular zone progenitor mobilization, increased doublecortin-positive migrating neuroblasts towards the injured area, and enhanced effective neurogenesis. Moreover, we demonstrated the proneurogenic activity of VCE-003.2 in embryonic stem cells. VCE-003.2 was able to increase neuroblast formation and striatal-like CTIP2-mediated neurogenesis.

Conclusions: The cannabigerol derivative VCE-003.2 improves subventricular zone-derived neurogenesis in response to mutant huntingtin-induced neurodegeneration, and is neuroprotective by oral administration.

Keywords: Cannabinoid; Huntingtin; Neurodegeneration; Neurogenesis; PPAR.

Fig. 1

Experimental design for pharmacological manipulation and characterization of in vivo model of Huntington’s disease based on adeno-associated viral expression of mutant huntingtin exon1

Fig. 2

VCE-003.2 exerts a pro-neurogenic effect in vitro. a-b Mouse embryonic stem cells (R1 line) were treated with Vehicle, VCE-003.2 (500 nM) during neural differentiation for 21 days. Representative immunofluorescence images and quantification of CTIP2- and doublecortin-positive cells is shown (n = 3). c Luciferase reporter assay of the A4-MARS sequence of the CTIP2 locus was performed 24 h after P19 cell transfection under neuronal differentiation conditions and subject to pharmacological regulation as above. d P19 neurosphere formation assay was performed by culturing the cells for 24 h in the presence of Vehicle or VCE-003.2. Statistics: Unpaired t-test vs Vehicle a * p < 0.05; t = 2.82; df = 4; 95% Confidence interval (CI) = 0.3149 to 36.85; R² = 0.66. b * p < 0.05; t = 2.91; df = 4; 95% CI = 0.02 to 1.03; R² = 0.67. c * p < 0.05; t = 2.87; df = 5; 95% CI = 0.09 to 1.72; R² = 0.62. d ** < 0.01 t = 5.91; df = 362; 95% CI = 66.20 to 132.20; R2 = 0.08. Scale bar, 50 μm

Fig. 3

Oral administration of VCE-003.2 attenuates microglial activation and motor impairment induced by mutant-huntingtin expression. Wild type C57Bl/6 N mice were injected bilaterally with mutant huntingtin expressing AAV-htt94Q and AAV-htt16Q as control. VCE-003.2 or vehicle were administered orally daily (10 mg/kg) and mice analyzed at 2 after lesion. a Motor function was assessed in the RotaRod test and mean latency to fold quantified. b Representative confocal microscopy images of huntingtin-CFP and immunoreactivity with an antibody for microglia (Iba1). Iba1 immunoreactivity was quantified in the indicated experimental groups. AAV-htt16Q Vehicle and VCE-003.2 (n = 3 and 6, respectively), AAV-htt94Q Vehicle and VCE-003.2 (n = 3 and 5, respectively). Statistics: One-way ANOVA. a F = 5.94; R² = 0.29. **p < 0.01; q = 4.88 AAV-htt16Q Veh vs. Mut-htt94q Veh and #p < 0.05; q = 3.90 vs AAV-htt94Q Veh vs. AAV-htt94Q VCE-003.2. b F = 5.87; R² = 0.91. #p < 0.05; q = 5.32 vs AAV-htt16Q VCE-003.2 vs. AAV-htt94Q Veh and ##p < 0.01; q = 3.49 vs AAV-htt94Q VCE-003.2 vs. AAV-htt94Q Veh. **p < 0.01; q = 9.30 AAV-htt94Q Veh vs. AAV-htt16Q Veh. Scale bar, 100 μm

Fig. 4

Oral administration of VCE-003.2 is neuroprotective from mutant-huntingtin-induced neurodegeneration. Wild type mice were injected bilaterally with mutant huntingtin expressing AAV-htt94Q (mtHtt) and AAV-htt16Q (wtHtt) as control. VCE-003.2 or vehicle were administered orally daily (10 mg/kg) and mice analyzed 30 days after lesion. Confocal microscopy characterization of huntingtin-CFP and using an antibody for the MSN marker DARPP32. Quantification of MSN survival after lesion for the indicated experimental groups. AAV-htt16Q (Vehicle and VCE-003.2, n = 5 each) and AAV-htt94Q (Vehicle and VCE-003.2, n = 7 each). Statistics: One-way ANOVA. a F = 31.56; R² = 0.80. ##p < 0.01; q = 5.28 AAV-htt94Q VCE-003.2 vs. AAV-htt94Q Veh. **p < 0.01; q = 10.63 vs AAV-htt94Q Veh vs. AAV-htt94Q Veh vs. AAV-htt16Q Veh and **p < 0.01; q = 5.56 vs AAV-htt94Q VCE-003.2 vs. AAV-htt16Q Veh. b F = 27.15; R² = 0.78. ##p < 0.01; q = 10.74 AAV-htt94Q VCE-003.2 vs. AAV-htt94Q Veh and **p < 0.01; q = 10.85 vs AAV-htt94Q Veh vs. AAV-htt16Q Veh. Scale bar, 100 μm

Fig. 5

Subventricular zone neural progenitor mobilization is increased by oral administration of VCE-003.2. Mice were analyzed 4 weeks after lesion induced by AAV-htt16Q and AAV-htt94Q bilateral injection and daily treatment with vehicle or VCE-003.2 (10 mg/kg). Confocal microscopy characterization of the SVZ was performed with GFAP, htt and Ki-67-specific antibodies in the indicated experimental groups. Proliferating SVZ-neural stem cells were quantified based on GFAP and Ki-67 immunofluorescence colocalization. AAV-htt16Q (Veh and VCE-003.2, n = 7 and 6, respectively) and AAV-htt94Q (Veh and VCE-003.2, n = 7 and 8, respectively). Statistics: One-way ANOVA. F = 5.78; R² = 0.42. ##p < 0.01; q = 5.29 AAV-htt94Q VCE-003.2 vs. AAV-htt94Q Veh. Scale bar, 100 μm

Fig. 6

Subventricular zone neural progenitor mobilization is increased by oral administration of VCE-003.2. Mice were analyzed 4 weeks after lesion induced by AAV-htt16Q and AAV-htt94Q bilateral injection and daily treatment with vehicle or VCE-003.2 (10 mg/kg). Confocal microscopy characterization of the SVZ was performed with Ascl1-specific antibody in the indicated experimental groups. Quantification of transit amplifying progenitors labelled with Ascl1. AAV-htt-16Q (Vehicle and VCE-003.2, n = 5 each) and AAV-htt94Q (Vehicle and VCE-003.2, n = 5 each). Statistics: One-way ANOVA. F = 10.12; R² = 0.65. ##p < 0.01; q = 4.25 AAV-htt94Q VCE-003.2 vs. AAV-htt94Q Veh and **p < 0.01; q = 7.12 AAV-htt94Q VCE-003.2 vs. AAV-htt16Q Veh. Scale bar, 100 μm

Fig. 7

Oral administration of VCE-003.2 exerts a pro-neurogenic action. Mice were analyzed 4 weeks after lesion induced by AAV-htt16Q and AAV-htt94Q bilateral injection and administered daily with vehicle or VCE-003.2 (10 mg/kg). a Confocal microscopy characterization of migrating neuroblasts identified with doublecortin antibody and quantification in the striatum of the indicated mice groups. b Effective neurogenesis was determined by quantification of double positive cells labelled with BrdU and NeuN antibodies. AAV-htt16Q Vehicle and VCE-003.2 (n = 7 and 5, respectively); AAV-htt94Q Vehicle and VCE-003.2 (n = 9 and 11, respectively). Statistics: One-way ANOVA. a F = 14.43; R² = 0.69. ##p < 0.01; q = 6.95 AAV-htt94Q VCE-003.2 vs. AAV-htt94Q Veh and **p < 0.01; q = 7.59 AAV-htt94Q VCE-003.2 vs. AAV-htt16Q Veh. b F = 13.99; R² = 0.59. ##p < 0.01; q = 6.50 AAV-htt94Q VCE-003.2 vs. AAV-htt94Q Veh and **p < 0.01; q = 7.59 AAV-htt94Q VCE-003.2 vs. AAV-htt16Q Veh. Scale bar, 100 μm