GPR4 Knockout Improves the Neurotoxin-Induced, Caspase-Dependent Mitochondrial Apoptosis of the Dopaminergic Neuronal Cell

Abstract

In Parkinson’s disease, mitochondrial oxidative stress-mediated apoptosis is a major cause of dopaminergic neuronal loss in the substantia nigra (SN). G protein-coupled receptor 4 (GPR4), previously recognised as an orphan G protein coupled-receptor (GPCR), has recently been claimed as a member of the group of proton-activated GPCRs. Its activity in neuronal apoptosis, however, remains undefined. In this study, we investigated the role of GPR4 in the 1-methyl-4-phenylpyridinium ion (MPP+) and hydrogen peroxide (H2O2)-treated apoptotic cell death of stably GPR4-overexpressing and stably GPR4-knockout human neuroblastoma SH-SY5Y cells. In GPR4-OE cells, MPP+ and H2O2 were found to significantly increase the expression levels of both mRNA and proteins of the pro-apoptotic Bcl-2-associated X protein (Bax) genes, while they decreased the anti-apoptotic B-cell lymphoma 2 (Bcl-2) genes. In addition, MPP+ treatment activated Caspase-3, leading to the cleavage of poly (ADP-ribose) polymerase (PARP) and decreasing the mitochondrial membrane potential (ΔΨm) in GPR4-OE cells. In contrast, H2O2 treatment significantly increased the intracellular calcium ions (Ca2+) and reactive oxygen species (ROS) in GPR4-OE cells. Further, chemical inhibition by NE52-QQ57, a selective antagonist of GPR4, and knockout of GPR4 by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 decreased the Bax/Bcl-2 ratio and ROS generation, and stabilised the ΔΨm, thus protecting the SH-SY5Y cells from MPP+- or H2O2-induced apoptotic cell death. Moreover, the knockout of GPR4 decreased the proteolytic degradation of phosphatidylinositol biphosphate (PIP2) and subsequent release of the endoplasmic reticulum (ER)-stored Ca2+ in the cytosol. Our results suggest that the pharmacological inhibition or genetic deletion of GPR4 improves the neurotoxin-induced caspase-dependent mitochondrial apoptotic pathway, possibly through the modulation of PIP2 degradation-mediated calcium signalling. Therefore, GPR4 presents a potential therapeutic target for neurodegenerative disorders such as Parkinson’s disease.

The Neuroprotective Effect of GPR4 Inhibition through Attenuation of Caspase Mediated Apoptotic Cell Death in MPTP Induced Mouse Model of Parkinson’s Disease

“…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].…”

“…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.…”

“…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.…”

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

“…These findings demonstrate that the pharmacological inhibition of GPR4 prevented MPTP-induced mitochondrial oxidative stress and prevented the initiation of the caspase 3-dependent apoptotic cell death pathway. This result aligns with our previously published in vitro data that examined both the genetic and pharmacological inhibition of GPR4 in a human dopaminergic cell line [22].…”

“…However, no study has examined the role played by GPR4 in the apoptotic neuronal cell death associated with neurodegenerative disease. We were the first to investigate the effects of GPR4 knockout and overexpression in dopaminergic neuronal cells [ 22 ]. Our previous study showed that GPR4 knockout or pharmacological inhibition reduced neurotoxin-induced caspase 3-dependent apoptotic cell death.…”