The neuroprotective effect of GPR4 inhibition through attenuation of caspase mediated apoptotic cell death in MPTP induced mouse model of Parkinson’s disease

GPR4, a member of proton activated GPCRs group. Previously we have reported that GPR4 is constitutively active at physiological pH and knockout of GPR4 has shown to protect dopaminergic neuronal cells from caspase-dependent mitochondrial apoptotic cell death. In this study we have investigated the role of GPR4 in 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) treated mice model of Parkinson’s disease. Subchronic administration of MPTP in mice produces oxidative stress induced apoptotic cell death of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and motor deficits. Treatment with NE52-QQ57, a selective antagonist of GPR4 reduced dopaminergic neuronal loss MPTP-intoxicated C57BL6/J mice and improved motor deficit and memory impairment. Co-treatment with NE52-QQ57 significantly decreases the protein level of the proapoptotic marker (Bax), and increases the antiapoptotic marker (Bcl-2) in the SNpc and striatum tissue collected from the brain of MPTP inflicted mice. Further, MPTP-induced activation of caspase 3 and cleavage of poly (ADP-ribose) polymerase (PARP) was significantly decreased in the SNpc and striatum tissue of NE52-QQ57 cotreated mice. Further mice receiving both MPTP and NE52-QQ57 mice showed significantly higher TH positive cells in the SNpc and striatum than MPTP treated mice alone. Moreover, NE52-QQ57 cotreatment improved the motor activity in the rotarod test and pole test and also improved spatial memory in Y maze test. Our findings suggest GPR4 as a potential therapeutic target for PD whereas the activation GPR4 is involved in the caspase mediated apoptotic cell death in SNpc and striatum of MPTP-intoxicated mice.

angiogenesis etc. [15][16][17]. Previously, pharmacological inhibition of GPR4 has been reported to remediate myocardial infarction [18] and genetic deletion of GPR4 improved cardiac function through lowering blood pressure [19], and inhibited apoptosis in renal ischemia reperfusion injury [20] intestinal inflammation [21]. However, there was no study on the role of GPR4 on the apoptosis cell death neurodegenerative disease before we have investigated the effect of GPR4 knockout and overexpression in the dopaminergic neuronal cells [22]. Our previous study showed, GPR4 knockout or pharmacological inhibition improves the neurotoxin induced caspase 3 dependent apoptotic cell death. Here in this study, we have used sub chronically administered MPTP-induced Parkinsonism in C57BL/6J mice to further elaborate the role of GPR4 in the classical mitochondrial apoptotic cell death. We have used orally active GPR4 selective antagonist to evaluate how pharmacological inhibition of GPR4 protects neural cell loss in the SNpc through increasing the level of tyrosine hydroxylase (TH) and attenuation of caspase mediated apoptotic cell death. The dose of NE52-QQ57 (30 mg/kg/, p.o.) was adopted from Hosford et al., 2018 and MPTP (30 mg/kg, i.p.) for 5 days has been adopted from Kim et al., 2015 [11, 23]. Mice were cotreated with NE52-QQ57 (30 mg/kg/, p.o.) and MPTP (30 mg/kg, i.p.) for 5 days but the NE52-QQ57 (30 mg/kg/, p.o.) treatment was continued till the day before sacrifice. After three days of last MPTP injection mice were sacrificed after behavioral studies and tissue of substantia nigra and striatum of mice brain were collected (Figure 1a).

Expression of GPR4 is upregulated in MPTP-induced PD mouse model
It is acknowledged that the activation of GPR4 differs based on the cell types of the and number of GPR4 expressed in that cell. Different regions of brains have very distinct type of cell types which shows different level of GPR4 activities [11]. We have investigated the expression of the GPR4 receptor different regions of mice brain using immunoblot analysis (supplementary figure 1). We have found that the expression level of GPR4 in SNpc and striatum after subchronic administration of MPTP (30 mg/kg, i.p.) for 5 days was highest after the three day of last MPTP injection.   The effect of GPR4 antagonist, NE52-QQ57, on the TH protein expressions in MPTP-treated mice. MPTP was treated (30mg/kg/day) for 5 days. Mice were sacrificed and substantia nigra and striatum tissue were collected after three days of last MPTP administration. (a). TH protein expression SNpc region of mice brain (n=3) and densitometric analysis. (b). TH protein expression striatum region of mice brain (n=3) and densitometric analysis. β-Actin was utilized as an internal control. Data represented as mean ± SEM.
Tukey's multiple comparison test of One-way ANOVA-was used. #p < 0.05 when the MPTP treated group is compared with the control group; *p < 0.05 other treated groups compared with the MPTP treated group.
2.3. Inhibition of GPR4 decreases MPTP-induced increase of proapoptotic Bax/Bcl-2 ratio in SNpc and striatum Figure 3: The effect of GPR4 antagonist, NE52-QQ57, on the pro-apoptotic Bax and Bcl-2 protein expressions in MPTP-treated mice. MPTP was treated (30mg/kg/day) for 5 days. Mice were sacrificed and Substantia nigra and Striatum tissue was collected after three days of last MPTP administration. (a). Bax and Bcl-2 protein expression SNPc region of mice brain (n=3) and densitometric analysis. (b). Bax and Bcl-2 protein expression Striatum region of mice brain (n=3) and densitometric analysis. β-Actin was utilised as an internal control. Data represented as mean ± SEM. Tukey's multiple comparison test of One-way ANOVA-was used. #p < 0.05 when the MPTP treated group is compared with the control group; *p < 0.05 other treated groups compared with the MPTP treated group.
The Bcl-2 family proteins Bax and Bcl-2 play a role of central regulator of mitochondrial apoptotic pathway. Bcl-2 protein's activity is dependent on its counteracting twin Bax. Activation of BAX result is an initiation step of apoptosis, while Bcl-2 protects from the program cell death and promote cells survival. Activation of Bax initiates the release of cytochrome C from mitochondrial intermembrane space to the cytosol which activates the proteolytic caspases [24]. To investigate the effect of GPR4 inhibition on Bax and Bcl-2 protein expression mice were cotreated with NE52-QQ57 (30 mg/kg/, p.o.) and MPTP (30 mg/kg, i.p.) for 5 days and continued till the day before sacrifice.
Three day after the last MPTP injection brain tissue from SNpc and striatum were collected and processed for immunoblot.
The level of Bax protein expression in SNpc of mice treated with MPTP led to a significantly (p < 0.05) increase, more than three folds (3.13±0.28 fold) and reduced the Bcl-2 protein expression to almost half (0.57±0.15 fold) (p < 0.05) comparison with vehicle only group (Figure 3a). The ratio of Bax/Bcl-2 shows more than seven-fold increase (7.55±2.57 fold) than the vehicle only group. In the tissue collected from striatum of MPTP treated group of mice, the expression level of Bax was significantly (p < 0.05) increased (1.39±0.16 fold) and the level of Bcl-2 expression was almost half (0.52±0.09 fold) fold than the vehicle treated group. Whereas, the Bax/Bcl-2 ratio was significantly (p < 0.05) higher (3.22±0.82 fold) than the vehicle treated group (Figure 3b). However, in NE52-QQ57 -MPTP cotreated group significant (p < 0.05) decrease of the Bax expression (0.89±0.024 fold) and less depletion of Bcl-2 protein expression (1.11±0.11 fold) was observed than the MPTP treated group.
Moreover, the Bax/Bcl-2 ratio in NE52-QQ57 -MPTP cotreated group was also significantly (p < 0.05) lower (0.82±0.06 fold) than the MPTP treated group (Figure 3a). These data suggest that sub chronic administration of MPTP causes the imbalance of mitochondrial Bax/Bcl-2 protein ratio and that may initiate the mitochondrial caspase mediated apoptotic pathway. Treatment with selective GPR4 antagonist NE52-QQ57 prevents the increase of proapoptotic protein Bax and also prevent the depletion of antiapoptotic protein Bcl-2 which indicates the anti-apoptotic effect of NE52-QQ57.  activity assay of SNPc tissue region of mice brain (n=3) and densitometric analysis. (b). Cleaved PARP-1 and Cleaved Caspase 3 protein expression and Caspase 3 activity assay of striatum region of mice brain (n=3) and densitometric analysis. β-Actin was utilized as an internal control. Data represented as mean ± SEM. Tukey's multiple comparison test of One-way ANOVA-was used. #p < 0.05 when the MPTP treated group is compared with the control group; *p < 0.05 other treated groups compared with the MPTP treated group.
In response to oxidative stress and poly(ADP-ribose) polymerase (PARP), is the most abundant nuclear enzyme. The cleavage of PARP is a reliable marker of apoptosis which is done by DEVD-ase caspases, a family of proteases like caspase 3 activated during apoptosis [25,26]. To further access the effect of GPR4 inhibition by NE52-QQ57 on apoptosis of dopaminergic neurons, we have measured the level of cleaved PARP, its proteolytic enzyme caspase 3 protein and caspase activity of SNpc and striatum tissue collected from MPTP inflicted mice.
After treatment with MPTP (30 mg/kg, i.p.) for 5 days the level of cleaved PARP protein expression in SNpc of mice led to a significant (p < 0.05) increase of cleaved PARP (3.78±0.6 fold) and cleaved caspase 3 protein to almost two and a half (2.32±0.06 fold) (p < 0.05) comparison with vehicle only group ( Figure 4a). Additionally, in a separate quantitative caspase 3 activity assay MPTP treated group showed more than double caspase 3 activity (194.923±25.86%) than the vehicle treated group.
However, in the striatum MPTP treated mice group significantly (#p < 0.05) increased cleaved PARP (1.84±0.11 fold) and cleaved caspase 3 level (1.46±0.26 fold). Also, the quantitative analysis of caspase 3 activity showed a significant (#p < 0.05) increase of (133.03±4.87%) caspase 3 activity in the MPTP treated mice. Whereas, cotreatment with NE52-QQ57 and MPTP prevents the increase of cleaved PARP (0.85±0.14 fold), cleaved caspase 3 level (0.97±0.23 fold) and significantly (*p < 0.05) prevented the increase of caspase 3 activity (98.87±9.02%) ( Figure 4b). Overall, the data suggest that inhibition of GPR4 has a strong effect on the inhibition of apoptotic cell deaths through inhibition of mitochondrial caspase 3 mediated cleavage of PARP. However, of GPR4 inhibition prevented the MPTP mediated increase of caspase 3 activity in both SNpc and striatum tissues but the protective effect of NE52-QQ57 was higher in SNpc than the striatum.   Tyrosine hydroxylase (TH), an enzyme that converts L-tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA). The conversion of L-tyrosine to DOPA is the rate-limiting and initial step in the dopamine biosynthesis pathway in dopaminergic neurons [27].   and distance travelled (

Discussion
In this study, we have investigated the effect of pharmacological inhibition of GPR4 in the mitochondrial oxidative stress-induced apoptotic cell death in PD. Activation of pH has been reported in many different pH ranges in different cells and animal model. Hosford and his team have also reported the expression of GPR4 in cerebrovascular endothelium and neurons of retrotrapezoidal nucleus locus coeruleus, dorsal raphe, and lateral septum of mice at physiological pH.
We have shown the GPR4 is activate in the physiological pH (supplement data S1) and capable of potentiating the effect of MPP + , an active metabolite of MPTP [22]. Here we intended to find out if MPTP treatment has any effect on GPR4 expression level over time in cerebellum, cerebral cortex, substantia nigra, hippocampus, pons medulla, mid brain and striatum of mice brain after 1, 3 and 7 days of last MPTP injection. In cerebellum and cerebral cortex no significant change in the expression of GPR4 were observed. Whereas, significant increased in the GPR4 level in the SNpc, hippocampus, on the neuronal loss, deficits in memory and motor performance Parkinson's disease model we have used selective GPR4 antagonist NE52-QQ57 on MPTP induced Parkinson's disease model. TH, an essential enzyme in dopamine biosynthesis converts tyrosine to dopamine by using tetrahydrobiopterin and molecular oxygen [27]. The decrease of TH activity is positively correlated with the dopaminergic neuronal cells. Moreover, behavioral deficit is also correlated with the number of TH-positive neurons in SNpc and striatum [23]. We found that GPR4 inhibition protects from the loss of TH in the striatum and SNpc in response to MPTP intoxication. Indicating that GPR4 inhibition may improve the dopaminergic loss associated motor activity and memory deficit.
To further investigate how selective GPR4 inhibition prevents the MPTP intoxicated neuronal loss through the mitochondrial oxidative stress mediated apoptotic pathway we investigated the ratio of Bax and Bcl-2 protein as early indication of the early phases of apoptotic cascade [28,29].
MPTP inflicted apoptotic cell deaths bear the characteristic hallmarks of an increase in the Bax/Bcl-2 ratio, the release of cytochrome-C, and the Caspase-3 activation, which cleaves PARP and induces apoptotic cell death [30]. We further investigated the cleavage of caspase 3, caspase 3 activity and cleavage of PARP. Cleavage of PARP is a major substrate of caspases, which is also a valuable marker of apoptosis. In MPTP induced mitochondrial oxidative stress a dramatic increase of caspase 3 activity and PARP cleavage has been reported in neuronal cell death in brain, where caspase-3 has been reported to play as the final effector of caspase-dependent apoptosis [35,36]. Our results show that MPTP dramatically increases the protein level of both cleaved caspase 3 and cleaved PARP in both SNpc and striatum.
Additionally, the caspase 3 activity was also significantly increased in both SNpc and striatum tissues.
Although the fold of cleaved caspase 3 and PARP proteins and caspase 3 activity was not same in both tissues, NE 52-QQ57 cotreatment significantly prevented the cleavage of both proteins and caspase3 activity in SNpc and striatum. Which demonstrate the pharmacological inhibition of GPR4 prevents the MPTP induced mitochondrial oxidative stress and prevent the caspase 3 dependent apoptotic cell death. This result is equivocal with our previously published in vitro data using both genetic and pharmacological inhibition of GPR4 in the human dopaminergic neuronal cell [22]. striatum. Moreover, in this study, the level of depletion of TH positive cells in the MPTP treated mice is similar to the level of TH positive cell depletion in the subchronic model of PD reported previously [37]. Our finding shows similarity with previously studies where demonstrating that TH activity is correlated with the behavioral deficit in toxin induced animal model of PD [23,28].
Further, we investigate if the pharmacological inhibition of GPR4 has any effect on the memory impairment and behavioral deficit we performed, Rotarod, Y-maze and Pole tests. The pole test is used as a behavioral test to assess the bradykinesia in PD mouse models [38]. Pharmacological inhibition of GPR4 receptor improved the MPTP induced bradykinesia of mice. To evaluates the motor activity we have used the rotarod test which is commonly used in the mice model of PD [39].
The rotarod results show GPR4 antagonist significantly prevented MPTP induced motor deficit in mice. Cognitive impairment of spatial memory is reported after subchronic MPTP injection in different studies [40][41][42]. To assess the impairment of spatial memory we have used Y maze test which is widely performed in MPTP treated subchronic model of PD. Here our study shows that inhibition of GPR4 activity significantly improved the impaired spontaneous alteration in MPTP treated mice.
Interestingly our immunoblot data suggest the higher level of GPR4 expression in the hippocampus region also suggest that GPR4 activity in may be connected with the memory deficit in neurodegenerative disorders like PD which provides a platform for further investigation of the involvement of GPR4 in memory deficit.  and 50 ±5% humidity) and were allowed food and water ad libitum. The room's lights were on   Pole test: Pole test to investigate bradykinesia in mice was conducted based on our previous work with slight modification [23]. At the top of a rough surfaced wooden pole mice were placed upwardly which was 8 mm in diameter and 55 cm tall. The total time to descend and time to turn was measured.

Reagents and
The time until the mouse arrived at the floor is referred as Time to descend. Delay or extension or of the time which usually takes to complete the test was considered as a reflection of bradykinesia. Each mouse was subjected to performed the test for five times successively.
Y-Maze test: To assess the spontaneous alternation performances of mice Y maze test was performed using a similar procedure described previously in [23]. Spontaneous alternation performances were assessed by recording animal behavior during a single session in a Y-maze. In the Y maze apparatus, each arm was 40 cm long, 12 cm wide and 30 cm in height. Each mouse being naïve or previously unintroduced to the maze, was placed at the end of one arm and allowed to move freely through every arm of the maze. During an 8 min session total entry to arms and spontaneous alteration of arms entries were recorded visually. Arm entry was considered to be accomplished when the hind paws of a mouse was completely placed in any arm. Whereas, alternation was considered when a mouse enters into all the three arms on overlapping triplet sets. The spontaneous alternation performances was calculated as percentage alternation based on the formula: percent alternation = [(number of alternations)/(total number of arm entries − 2)] × 100%.

Statistical Analyses
GraphPad Prism software version 5 (GraphPad, La Jolla, CA, USA) is used to perform statistical analyses. Data are represented as means ± standard error (SEM) of three to five independent experiments. One-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test was performed to determine the significant differences between the nontreated and treated groups. A P-values < 0.05 were considered statistically significant.  T-Cell Death-Associated Gene 8 OGR1 The Ovarian Cancer G Protein-Coupled Receptor 1