The Neuroprotective Effects of GPR4 Inhibition through the Attenuation of Caspase Mediated Apoptotic Cell Death in an MPTP Induced Mouse Model of Parkinson's Disease

Abstract

The proton-activated G protein-coupled receptor (GPCR) 4 (GPR4) is constitutively active at physiological pH, and GPR4 knockout protected dopaminergic neurons from caspase-dependent mitochondria-associated apoptosis. This study explored the role of GPR4 in a 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-treated mouse model of Parkinson's disease (PD). In mice, subchronic MPTP administration causes oxidative stress-induced apoptosis in the dopaminergic neurons of the substantia nigra pars compacta (SNpc), resulting in motor deficits. NE52-QQ57, a selective GPR4 antagonist, reduced dopaminergic neuronal loss in MPTP-treated mice, improving motor and memory functions. MPTP and NE52-QQ57 co-treatment in mice significantly decreased pro-apoptotic marker Bax protein levels and increased anti-apoptotic marker Bcl-2 protein levels in the SNpc and striatum. MPTP-induced caspase 3 activation and poly (ADP-ribose) polymerase (PARP) cleavage significantly decreased in the SNpc and striatum of mice co-treated with NE52-QQ57. MPTP and NE52-QQ57 co-treatment significantly increased tyrosine hydroxylase (TH)-positive cell numbers in the SNpc and striatum compared with MPTP alone. NE52-QQ57 and MPTP co-treatment improved rotarod and pole test-assessed motor performance and improved Y-maze test-assessed spatial memory. Our findings suggest GPR4 may represent a potential therapeutic target for PD, and GPR4 activation is involved in caspase-mediated neuronal apoptosis in the SNpc and striatum of MPTP-treated mice.

Keywords: GPR4 receptor; MPTP; PARP; Parkinson’s disease; apoptosis; caspase 3; neurodegeneration.

Figure 1

The effects of subchronic MPTP administration on the GPR4 protein expression levels in the substantia nigra and striatum of the mouse brain. (a) Schematic representation of the treatment and behavioral study schedule for animal experiments. GPR4 protein expression and its densitometric analysis in (b) the substantia nigra pars compacta (SNpc) and (c) the striatum measured in tissue samples collected 3 days after the final MPTP administration. β-Actin expression levels used as an internal control. Data are presented as the mean ± SEM. One-way ANOVA, followed by Tukey’s multiple comparison test, was used to compare differences between groups. # p < 0.05 when the MPTP-treated group was compared with the vehicle-only group; * p < 0.05 when other treated groups were compared with the MPTP-treated group. p.o.: oral administration; i.p.: intraperitoneal; q.d.: once a day.

Figure 2

The effects of the GPR4 antagonist NE52-QQ57 on TH protein expression levels in MPTP-treated mice. MPTP was administered (30 mg/kg/day) for 5 days. Mice were sacrificed, and the substantia nigra and striatum tissues were collected 3 days after the final MPTP administration. (a) TH protein expression in the SNpc region of the mouse brain (n = 3) and densitometric analysis. (b) TH protein expression in the striatum region of the mouse brain (n = 3) and densitometric analysis. β-Actin was utilized as an internal control. Data represented the mean ± SEM. One-way ANOVA, followed by Tukey’s multiple comparison test, was used to compare differences between groups. # p < 0.05 when the MPTP-treated group was compared with the vehicle-only group; * p < 0.05 when other treated groups were compared with the MPTP-treated group.

Figure 3

The effects of the GPR4 antagonist NE52-QQ57 on pro-apoptotic Bax and anti-apoptotic Bcl-2 protein expressions in MPTP-treated mice. MPTP was administered (30 mg/kg/day) for 5 days. Mice were sacrificed 3 days after the final MPTP injection, and the substantia nigra and striatum tissues were collected. (a) Bax and Bcl-2 protein expression in the SNpc region of the mouse brain (n = 3) and densitometric analysis. (b) Bax and Bcl-2 protein expression in the striatum region of the mouse brain (n = 3) and densitometric analysis. β-Actin was utilized as an internal control. Data are presented as the mean ± SEM. One-way ANOVA, followed by Tukey’s multiple comparison test, was used. # p < 0.05 when the MPTP-treated group was compared with the vehicle-only group; * p < 0.05 when other treated groups were compared with the MPTP-treated group.

Figure 4

The effects of the GPR4 antagonist NE52-QQ57 on the expression of pro-apoptotic proteins, cleaved PARP and cleaved caspase 3, and caspase 3 activity in MPTP-treated mice. MPTP was administered (30 mg/kg/day) for 5 days. Mice were sacrificed, and the substantia nigra and striatum tissue was collected 3 days after the final MPTP administration. (a) Cleaved PARP-1 and cleaved caspase 3 protein expression and caspase 3 activity assay in the mouse SNpc (n = 3) and densitometric analysis. (b) Cleaved PARP-1 and cleaved caspase 3 protein expression and caspase 3 activity assay in the mouse striatum (n = 3) and densitometric analysis. β-Actin was utilized as an internal control. Data are presented as the mean ± SEM. One-way ANOVA, followed by Tukey’s multiple comparison test, was used to compare differences between groups. # p < 0.05 when the MPTP-treated group was compared with the vehicle-only group; * p < 0.05 when the other treated groups were compared with the MPTP-treated group.

Figure 5

GPR4 inhibition attenuates the depletion of TH-positive cells in the striatum and SNpc of a subchronic MPTP-treated mouse model of PD. MPTP was administered (30 mg/kg/day) for 5 days. After performing behavioral experiments, the mice were anaesthetized for the immunohistochemical study three days after the final MPTP administration. Mice were sacrificed, and the whole brain was isolated to collect sections from the SNpc and striatum regions. (a). Representative images of TH-positive fibers immunoreactivity (IR) in sections of the striatum, with densitometric analysis of optical density of TH positive fibers (n = 3–4). (b). Representative images of TH-positive cell IR in the SNpc sections, with densitometric analysis for TH positive cells (n = 3–4). Data are presented as mean ± SEM. One-way ANOVA, followed by Tukey’s multiple comparison test, was used to determine significant differences between groups. # p < 0.05 when the MPTP-treated group was compared with the vehicle-only group; * p < 0.05 when the other treated groups were compared with the MPTP-treated group.

Figure 6

GPR4 inhibition attenuates motor deficits in a subchronic MPTP-treated mouse model of PD. MPTP was administered (30 mg/kg/day) for 5 days. Behavioral experiments were performed three days after the final MPTP administration (a) PD-like bradykinesia was measured using a pole test. (b) Motor coordination function was measured using a Rotarod test. Both the fall latency and the distance travelled were measured. (c) Spatial memory measured using Y-maze. Each dot represents the average of five individual trials, and the thick bar represents the mean ± standard deviation. Data represented as mean ± SEM (n = 5 of triplicates). One-way ANOVA, followed by Tukey’s multiple comparison test, was used to compare differences between groups. # p < 0.05 when the MPTP-treated group is compared with the vehicle-only group; * p < 0.05 other treated groups compared with the MPTP-treated group.