**1. Introduction**

Parkinson's disease (PD) is a neurodegenerative disorder characterised by dopamine deficiency. An important pathological basis of PD is the loss of dopaminergic neurons due to apoptotic cell death in the substantia nigra (SN) of the brain [1]. An array of evidence suggests that reactive oxygen species (ROS)-induced oxidative stress is a major cause of the dopaminergic neuronal loss in the SN [2]. Mitochondria are key players in apoptosis during this neurodegeneration. In a cell undergoing apoptosis, mitochondria increase the production of oxyradicals and open the pores of its membranes,

leading to the depolarisation of its transmembrane potential (Δψm) and the release of cytochrome C [3,4]. The B-cell lymphoma 2 (Bcl-2) family of proteins, which includes anti-apoptotic Bcl-2 and pro-apoptotic Bcl-2-associated X protein (Bax) members, plays a critical role in the initiation of the apoptotic pathway. When anti-apoptotic Bcl-2 localises in the mitochondrial inner membrane from the outer membrane, the intermembrane space protein, cytochrome C, is released. This binds to apoptotic peptidase activating factor 1 (APAF1) and forms a heptameric structure, known as the apoptosome. Apoptosomes recruit caspase 9 and activate a series of events, eventually activating Caspase-3 as a result [5]. Surprisingly, poly (ADP-ribose) polymerase (PARP), a nuclear protein involved in DNA repair that is specifically cleaved to a signature 89-kDa fragment, has been implicated as an early marker for apoptotic cell death in neurons [6]. The characteristic hallmarks of the mitochondrial oxidative damage-induced apoptosis pathway can be considered to be the opening of the mitochondrial permeability transition pore (mPTP), the collapse of the mitochondrial membrane potential (ΔΨm), the release of cytochrome C, the activation of Caspase-3, and proteolytic degradation of PARP [7,8]. Surprisingly, the endoplasmic reticulum (ER) acts as a reservoir of calcium ions (Ca2<sup>+</sup>). The ER can release Ca2<sup>+</sup> through either its ryanodine (RYR) or inositol trisphosphate (IP3) receptors. Ca2+, once released from the ER, is taken up by the mitochondrial Ca2<sup>+</sup> uniporter (MCU) located in the mitochondrial inner membrane [9]. An increase in mitochondrial Ca2<sup>+</sup> is associated with an increase of the mPTP opening and ROS generation, a decrease in ΔΨm, and release of cytochrome C, as well as excitotoxicity and apoptosis [10–12]. Recent findings suggest that an increase in the Ca2<sup>+</sup> released from the ER can increase the mitochondrial oxidant stress of the substantia nigra pars compacta (SNc) dopaminergic (DA) neurons [10,13]. Alteration of the intracellular calcium homeostasis PD cybrids has also been reported [14].

1-methyl-4-phenylpyridinium ion (MPP<sup>+</sup>) and hydrogen peroxide (H2O2) are the most widely employed neurotoxins, due to their ability to mimic a PD-like syndrome with apoptotic cell death through mitochondrial oxidative damage, in both cellular and animal models of PD [15–17]. MPP+, the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), generates 5–7 times more ROS by selectively inhibiting mitochondrial complex I, thus initiating pro-apoptotic Bax/Bcl-2-dependent apoptotic cell death [15]. Besides, MPP<sup>+</sup>- and H2O2-mediated oxidative stress have also been reported to cause mitochondrial oxidative stress-mediated apoptotic cell death [18]. Moreover, MPP<sup>+</sup>- and H2O2-mediated cell death are associated with the alteration of intracellular Ca2<sup>+</sup> homeostasis. An increase in intracellular Ca2<sup>+</sup> due to ER stress also potentiates a decrease in ΔΨm, the release of cytochrome C, and the induction of apoptosis [12,19–21]. Therefore, the inhibition of pro-apoptotic signalling, or the decrease of intracellular ROS and Ca2+, may be beneficial for the protection of dopaminergic neuronal loss in PD.

G protein-coupled receptor 4 (GPR4), a proton-sensing receptor, is highly sensitive to the alteration of extracellular proton concentration [22]. It belongs to a small G protein-coupled proton-sensing receptor family that includes ovarian cancer G protein-coupled receptor 1 (OGR1), also referred to as GPR68, G2A, also termed GPR132, and T-cell death-associated gene 8 (TDAG8), also known as GPR65. These receptors signal through either to phosphoinositide mediated increase in intracellular Ca2<sup>+</sup> or through modulating adenylate cyclase activity [23,24]. Information regarding the distribution and biology of GPR4 in the brain of an individual with PD is limited. An abundant level of GPR4 expression was observed in the retro-trapezoidal nucleus locus coeruleus, the cerebrovascular endothelium, the neurons of the dorsal raphe, and the lateral septum of a GPR4-knock-in mouse model [25]. Recently, the role of GPR4 in inflammation during ER stress pathway-mediated apoptotic cell death has been reported. In human umbilical vein endothelial cells (HUVEC) and other disease models, such as that of myocardial ischemic mice, knocking out GPR4 has been found to reduce cardiomyocyte apoptosis and improve cardiac function [26–28]. However, no study has yet to elucidate the role of GPR4 on apoptotic cell death in neurodegenerative disorders. Therefore, this work aims to forge an understanding of the role of GPR4 in neurotoxin-induced, mitochondrial oxidative stress-mediated

apoptosis in a PD model. In particular, through our study of the overexpression and genetic deletion of GPR4, we investigate the role of GPR4 in the mitochondrial apoptosis pathway.

### **2. Results**
