Preclinical Investigation in Neuroprotective Effects of the GPR55 Ligand VCE-006.1 in Experimental Models of Parkinson's Disease and Amyotrophic Lateral Sclerosis

Abstract

Cannabinoids act as pleiotropic compounds exerting, among others, a broad-spectrum of neuroprotective effects. These effects have been investigated in the last years in different preclinical models of neurodegeneration, with the cannabinoid type-1 (CB₁) and type-2 (CB₂) receptors concentrating an important part of this research. However, the issue has also been extended to additional targets that are also active for cannabinoids, such as the orphan G-protein receptor 55 (GPR55). In the present study, we investigated the neuroprotective potential of VCE-006.1, a chromenopyrazole derivative with biased orthosteric and positive allosteric modulator activity at GPR55, in murine models of two neurodegenerative diseases. First, we proved that VCE-006.1 alone could induce ERK1/2 activation and calcium mobilization, as well as increase cAMP response but only in the presence of lysophosphatidyl inositol. Next, we investigated this compound administered chronically in two neurotoxin-based models of Parkinson's disease (PD), as well as in some cell-based models. VCE-006.1 was active in reversing the motor defects caused by 6-hydroxydopamine (6-OHDA) in the pole and the cylinder rearing tests, as well as the losses in tyrosine hydroxylase-containing neurons and the elevated glial reactivity detected in the substantia nigra. Similar cytoprotective effects were found in vitro in SH-SY5Y cells exposed to 6-OHDA. We also investigated VCE-006.1 in LPS-lesioned mice with similar beneficial effects, except against glial reactivity and associated inflammatory events, which remained unaltered, a fact confirmed in BV2 cells treated with LPS and VCE-006.1. We also analyzed GPR55 in these in vivo models with no changes in its gene expression, although GPR55 was down-regulated in BV2 cells treated with LPS, which may explain the lack of efficacy of VCE-006.1 in such an assay. Furthermore, we investigated VCE-006.1 in two genetic models of amyotrophic lateral sclerosis (ALS), mutant SOD1, or TDP-43 transgenic mice. Neither the neurological decline nor the deteriorated rotarod performance were prevented with this compound, and the same happened with the elevated microglial and astroglial reactivities, albeit modest spinal motor neuron preservation was achieved in both models. We also analyzed GPR55 in these in vivo models and found no changes in both TDP-43 transgenic and mSOD1 mice. Therefore, our findings support the view that targeting the GPR55 may afford neuroprotection in experimental PD, but not in ALS, thus stressing the specificities for the development of cannabinoid-based therapies in the different neurodegenerative disorders.

Keywords: 6-hydroxydopamine; GPR55 receptors; Parkinson’s disease; TDP-43 transgenic mice; VCE-006.1; amyotrophic lateral sclerosis; cannabinoids; chromenopyrazole; lipopolysaccharide; mSOD1 mice.

Figure 1

(A) Chemical structure of VCE-006.1. (B) VCE-006.1 and LPI induces ERK1/2 activation in DU145 cells. The cells were stimulated as indicated and the expression of phospho-ERK1/2 and total ERK1/2 determined by immunoblots. (C) VCE-006.1 and LPI induces [Ca²⁺] immobilization in U937 cells. U937 cells were loaded with Indo1-AM, treated with the compounds, and the calcium mobilization was measured by ratiometric fluorescence as indicated under Materials and Methods. (D) GPR55 activity of VCE-006.1 at different concentrations (1, 5, and 10 µM) in the absence or the presence of 10 µM LPI on HEK293T-GPR55-CRE-luc cells. Results are expressed as the fold induction of GPR55 activity and represent means ± SEM of data generated in 6 independent experiments, each conducted in triplicates. Statistical significance was determined by one-way ANOVA followed by the Tukey test (* p < 0.05, ** p < 0.01, *** p < 0.005 vs. control (basal) and VCE-006.1 alone; ^# p < 0.05 vs. LPI and VCE-006.1 (1 µM) + LPI).

Figure 2

Response in the cylinder rearing test (A) and in the pole test (B) of male mice subjected to unilateral 6-OHDA lesions or sham-operated and daily treated with VCE-006.1 (20 mg/kg, i.p.) for 2 weeks. Values are means ± SEM of more than 6 animals per group. Data were assessed by one-way ANOVA followed by the Tukey test (*** p < 0.005 vs. the two sham-operated groups; ^# p < 0.05, ^## p < 0.01 vs. the vehicle-treated 6-OHDA lesioned mice).

Figure 3

Quantification of TH (B) and LAMP-1 (C) immunoreactivities, including representative images (A) (TH; scale bar = 100 µm) and (D) (LAMP-1; scale bar = 50 µm)), measured in a selected area of the substantia nigra pars compacta of male mice subjected to unilateral 6-OHDA lesions or sham-operated and daily treated with VCE-006.1 (20 mg/kg, i.p.) for 2 weeks. Values correspond to % of the ipsilateral lesioned side vs. contralateral non-lesioned side and are expressed as means ± SEM of more than 6 animals per group. Data were assessed by one-way ANOVA followed by the Tukey test (** p < 0.01, *** p < 0.005 vs. the two sham-operated groups; ^# p < 0.05 vs. the vehicle-treated 6-OHDA lesioned mice).

Figure 4

Quantification of Cd68 (B) and GFAP (C) immunoreactivities, including representative images (A) (Cd68; scale bar = 100 µm) and (D) (GFAP; scale bar = 50 µm)), measured in a selected area of the substantia nigra pars compacta of male mice subjected to unilateral 6-OHDA lesions or sham-operated and daily treated with VCE-006.1 (20 mg/kg, i.p.) for 2 weeks. Values correspond to % of the ipsilateral lesioned side vs. contralateral non-lesioned side and are expressed as means ± SEM of more than 6 animals per group. Data were assessed by one-way ANOVA followed by the Tukey test (* p < 0.05, *** p < 0.005 vs. the two sham-operated groups; ^## p < 0.01 vs. the vehicle-treated 6-OHDA lesioned mice).

Figure 5

Cell viability measured with the MTT assay in cultured SH-SY5Y cells at 24 h to be treated with different concentrations of VCE-006.1 (0.5, 1, 2, 5, 10, and 20 µM) against 6-OHDA (200 µM). In all cases, a group with cells exposed to vehicle was also included to determine the 100% of cell viability. Values are means ± SEM of at least 4 independent experiments, each performed in triplicate. Data were assessed by the one-way ANOVA followed by the Tukey (*** p < 0.005 vs. control cells; ^### p < 0.005 vs. cells exposed to 6-OHDA + vehicle; ^@ p < 0.05 vs. cells treated with the other VCE-006.1 concentrations).

Figure 6

Response in the cylinder rearing test (A) and in the pole test (B) of male mice subjected to unilateral LPS lesions or sham-operated and daily treated with VCE-006.1 (20 mg/kg, i.p.) for 2 weeks. Values are means ± SEM of more than 6 animals per group. Data were assessed by one-way ANOVA followed by the Tukey test (* p < 0.05, ** p < 0.01, *** p < 0.005 vs. the two sham-operated groups).

Figure 7

Quantification of TH (B), LAMP-1 (C), Cd68 (D), and GFAP (E) immunoreactivities, including representative images for TH immunostaining ((A); scale bar = 100 µm), measured in a selected area of the substantia nigra pars compacta of male mice subjected to unilateral LPS lesions or sham-operated and daily treated with VCE-006.1 (20 mg/kg, i.p.) for 2 weeks. Values correspond to % of the ipsilateral lesioned side vs. contralateral non-lesioned side and were expressed as means ± SEM of more than 5 animals per group. Data were assessed by one-way ANOVA followed by the Tukey test (* p < 0.05, *** p < 0.005 vs. the two sham-operated groups).

Figure 8

mRNA levels for TNF-α (A), IL-1β (B), iNOS (C), COX-2 (D), CB₁ receptor (E), CB₂ receptor (F), and PPAR-γ (G) measured by qPCR in the striatum of male mice subjected to unilateral LPS lesions or sham-operated and daily treated with VCE-006.1 (20 mg/kg, i.p.) for 2 weeks. GAPDH was used as an endogenous reference gene for data normalization. Values correspond to fold of change vs. sham-operated controls and are expressed as means ± SEM of more than 5 animals per group. Data were assessed by one-way ANOVA followed by the Tukey test (* p < 0.05, ** p < 0.01, *** p < 0.005 vs. the two sham-operated groups).

Figure 9

mRNA levels for TNF-α (A), IL-1β (B), and GPR55 (C) measured by qPCR in BV2 cells exposed to LPS and/or VCE-006.1 (1 µM) for 20 h, and mRNA levels for GPR55 measured by qPCR in the striatum of male mice subjected to unilateral 6-OHDA (D) or LPS (E) lesions or sham-operated and daily treated with VCE-006.1 (20 mg/kg, i.p.) for 2 weeks. In all cases, GAPDH was used as an endogenous reference gene for data normalization, and values correspond to fold change vs. controls and are expressed as means ± SEM of more than 5 animals per group. Data were assessed by one-way ANOVA followed by the Tukey test (*** p < 0.005 vs. the control group).

Figure 10

Hanging wire response (A), rotarod performance (B), and neurological score (C), analyzed mSOD1 transgenic and wild-type male mice at specific weeks during a chronic treatment from 63 day-old to 125 day-old with VCE-006.1 (20 mg/kg, daily and i.p.) or vehicle, and quantification of the number of Nissl-stained motor neurons (E), including representative images ((D); scale bar = 100 µm), in the lumbar ventral horn (marked with a dotted line) of the spinal cord in all experimental groups after the chronic treatment. Values are means ± SEM of more than 6 animals per group. Behavioral data were assessed by two-way ANOVA (with repeated measures), whereas Nissl staining data were assessed by one-way ANOVA, in both cases followed by the Tukey test (* p < 0.05, ** p < 0.01, *** p < 0.005 vs. wild-type mice; ^## p < 0.01 vs. mSOD1 mice treated with vehicle).

Figure 11

Quantification of GFAP (B) and Iba-1 (D) immunoreactivities, including representative images ((A) and (C), respectively; scale bar = 100 µm), in the lumbar ventral horn (marked with a dotted line) of the spinal cord in wild-type and mSOD1 transgenic mice after a chronic treatment from 63 day-old to 125 day-old with VCE-006.1 (20 mg/kg, daily and i.p.) or vehicle. Values are means ± SEM of more than 6 animals per group. Data were assessed by one-way ANOVA followed by the Tukey test (*** p < 0.005 vs. wild-type mice).

Figure 12

Clasping response (A) and rotarod performance (B) analyzed TDP-43 transgenic and wild-type male mice at specific weeks during a chronic treatment of 30 days with VCE-006.1 (20 mg/kg, daily and i.p.) or vehicle, and quantification of the number of Nissl-stained motor neurons (D), including representative images ((C); scale bar = 100 µm), in the lumbar ventral horn (marked with a dotted line) of the spinal cord in all experimental groups after the chronic treatment. Values are means ± SEM of more than 6 animals per group. Behavioral data were assessed by two-way ANOVA (with repeated measures), whereas Nissl staining data were assessed by one-way ANOVA, in both cases followed by the Tukey test (* p < 0.05, ** p < 0.01, *** p < 0.005 vs. wild-type mice; ^# p < 0.05 vs. TDP-43 mice treated with vehicle).

Figure 13

Quantification of GFAP (B) and Iba-1 (D) immunoreactivity, including representative images ((A,C), respectively; scale bar = 100 µm), in the lumbar ventral horn (marked with a dotted line) of the spinal cord in wild-type and TDP-43 transgenic mice after chronic treatment of 30 days with VCE-006.1 (20 mg/kg, daily and i.p.) or vehicle. Values are means ± SEM of more than 6 animals per group. Data were assessed by one-way ANOVA followed by the Tukey test (* p < 0.05, ** p < 0.01, *** p < 0.005 vs. wildtype mice).

Figure 14

mRNA levels for GPR55 measured by qPCR in the spinal cord of male mSOD1 (at 123 days of age; (A)) or TDP-43 (at 65 (B) and 105 (C) days of age) transgenic mice, and their corresponding wild-type mice. GAPDH was used as an endogenous reference gene for data normalization. Values correspond to fold change vs. controls and are expressed as means ± SEM of more than 5 animals per group. Data were assessed by the unpaired Student’s t-test.