*2.7. Knockout of GPR4 Increases the Mitochondrial Membrane Potential (MMP) in Neurotoxin-Stimulated SH-SY5Y Cells*

Excess intracellular ROS leads to swelling of the mitochondrial matrix and rupture of the outer membrane, which opens up the mitochondrial permeability transition pores (mPTPs). As a result, the mitochondrial membrane potential (MMP) is disrupted and mitochondrial oxidative stress-mediated apoptosis is initiated [4].

In this study, to measure the MMP, 24 h serum-starved SH-SY5Y cells were treated with MPP<sup>+</sup> (1 mM) for 24 h in serum-free culture media. MMP was then determined through a JC-10 fluorescence quantitative assay. Similarly, in a separate experiment in a 6-well plate, cells were utilised for JC-10 fluorescence microscopy to visualise the red and green fluorescence.

Aggregated JC-10 is an indicator of MMP; the greater the ratio of red/green fluorescence, the higher the level of MMP. In a quantitative JC-10 fluorescence microplate assay, the MPP+-treated SHSY-5Y cells presented a lower red/green fluorescence ratio (44.75 ± 0.82%) than the untreated SH-SY5Y cells (Figure 7A). The MPP+-treated GPR4-OE cells, meanwhile, displayed the lowest red/green fluorescence ratio of all the samples (39.44 ± 0.39%), indicating a loss of MMP. In contrast, GPR4 knockout prevented the loss of MMP for the MPP+-treated GPR4-KO cells, as indicated by a higher red/green fluorescence ratio (65.44 <sup>±</sup> 0.99%). Therefore, the MPP+-stimulated GPR4-KO cells demonstrated a higher level of MMP than either the MPP+-stimulated SH-SY5Y (44.75 <sup>±</sup> 0.82%) or the GPR-OE (39.44 <sup>±</sup> 0.39%) cells.

In this study, the aggregated JC-10 created red fluorescence in the polarised mitochondrial membrane. When the MMP collapsed in apoptotic cells, the JC-10 retained its monomeric form, which is characterised by green fluorescence. An increase in the red/green fluorescence intensity ratio indicated intact mitochondria. In a separate experiment, to visualise the JC-10 fluorescence dye in the MPP+-treated cells, JC-10 fluorescence microscopy was employed. In the control SHSY-5Y cells, both red and green fluorescence was observed, with a high level of red fluorescence and low level of green fluorescence. In contrast, the level of green fluorescence was higher and the red fluorescence remarkably lower in both the MPP+-treated SHSY-5Y and GPR4-OE cells, when compared with the control SHSY-5Y cells. In the MPP+-treated GPR4-KO cells, the red fluorescence was restored close to that of the control SHSY-5Y cells, while the level of green fluorescence was decreased (Figure 7B). These results suggest that GPR4 knockout restores the MPP+-induced a loss of MMP in dopaminergic neurons.

#### *2.8. Knockout of GPR4 Decreases the Intracellular Calcium in Neurotoxin-Stimulated SH-SY5Y Cells*

Increases in intracellular Ca2<sup>+</sup> in association with MPP<sup>+</sup>- or H2O2-mediated apoptotic cell death have been previously reported [32]. Several studies have suggested that an increase in the intracellular Ca2<sup>+</sup> released from the ER store by the inositol trisphosphate receptor (IP3R) is directly responsible for mitochondrial Ca2<sup>+</sup> overload [33,34]. However, the exact mechanism by which MPP<sup>+</sup> or H2O2 stimulation increases the intracellular calcium is not clearly understood. Interestingly, several studies have demonstrated that H2O2-/MPP+-mediated mitochondrial oxidative stress is associated with an intracellular Ca2<sup>+</sup> spike, which increases the Bax/Bcl-2 ratio, the release of cytochrome C, mitochondrial depolarisation, and the Caspase-3 activity in neuronal cells [19,32]. Previous reports have suggested that many G protein coupled-receptors (GPCRs), such as GPR4, which releases Gβγ and activates Gi, are capable of Ca<sup>2</sup><sup>+</sup> signalling. Few GPCRs, however, harness Gβγ-dependent activation of PLC<sup>β</sup> to release ER-stored Ca2<sup>+</sup> into the cytoplasm through PIP2 degradation [35,36]. In this study, the MPP+-treated GPR4-OE cells demonstrated an increased proteolytic degradation of PIP2, in comparison with the SH-SY5Y cells treated with MPP<sup>+</sup>. Contrastingly, the MPP<sup>+</sup>-stimulated GPR4-KO cells presented a particularly low degradation of PIP2 compared with both the MPP+-stimulated SH-SY5Y and GPR4-OE cells (Figure 8A).

**Figure 7.** The measurement of mitochondrial membrane potential (MMP) in MPP+-treated SH-SY5Y cells that were stably GPR4-OE or GPR4-GPR4-KO. 24 h serum-starved SH-SY5Y cells were treated with MPP<sup>+</sup> (1 mM) for 24 h in serum-free culture media; a JC-10 fluorescence quantitative assay (according to the manufacturer's instructions) and fluorescence microscopy were then employed to measure their MMP. (**A**) The percentage ratios of J-aggregates (red) and J-monomers (green). (**B**) MMP changes were monitored with a JC-10 dye that is detectable through fluorescence microscopy. Mean ± SEM (*n* = 3) was employed to express the data. Tukey's multiple comparison test was performed using a one-way ANOVA. Each \* *p* < 0.05 refers to the sample concentration compared with the same group of non-treated cells.

To evaluate whether GPR4 overexpression increased intracellular calcium through Gβγ modulation of the PLCβ-PIP2 pathway, SH-SY5Y cells were treated with MPP<sup>+</sup> (1 mM) for 24 h in serum-free media. Cell lysates were analysed through western blotting to determine the degradation of PIP2. The SH-SY5Y cells were treated with H2O2 (200 μM) for 2 h 30 min to determine their relative intracellular Ca2+, utilising a Fluo-4 AM calcium indicator in a fluorescence microplate assay. Similarly, in a separate 6-well plate, cells stained with a Fluo-4 AM calcium indicator were observed under a fluorescence microscope.

The quantitative analysis of intracellular Ca2<sup>+</sup>, as indicated by the Fluo-4 AM microplate assay, found levels of intracellular Ca2<sup>+</sup> for the H2O2-treated GPR4-OE cells that were 369.58 <sup>±</sup> 24.75% higher than those for the non-treated GPR4-OE cells. In contrast, the H2O2-treated GPR4-KO cells presented significantly lower levels of intracellular Ca2<sup>+</sup> (181.28 <sup>±</sup> 0.85%), in comparison with the H2O2-treated SH-SY5Y (243.25 ± 7.81%) and H2O2-treated GPR4-OE cells (369.58 ± 24.75%; Figure 8B).

In a separate experiment to visualise intracellular Ca2<sup>+</sup> levels in the H2O2-treated cells, Fluo-4 AM fluorescence microscopy was employed. This round of microscopy demonstrated similar results to those obtained from the quantitative microplate assay. H2O2-treated GPR4-OE cells displayed the highest levels of green Fluo-4 AM fluorescence, while H2O2-treated GPR4-KO cells produced lower levels of green Fluo-4 AM fluorescence than both the H2O2-treated SH-SY5Y and GPR4-KO cells (Figure 8C). Overall, these data suggest that the increase in intracellular calcium associated with H2O2- or MPP<sup>+</sup>-mediated mitochondrial oxidative stress is exaggerated by GPR4 overexpression, whereas GPR4 knockout prevents an increase in intracellular Ca2<sup>+</sup> through the decrease of PIP2 degradation, and thus restricts the release of Ca2<sup>+</sup> from the ER by preventing the degradation of

PIP2. Therefore, GPR4-PLCβ-PIP2 signalling may act as a key factor through which GPR4 increases intracellular calcium and potentiates mitochondrial oxidative stress-mediated apoptosis.

**Figure 8.** Phosphatidylinositol biphosphate (PIP2) calcium signalling and intracellular calcium levels in MPP+-treated SH-SY5Y cells that were stably GPR4-OE or GPR4-KO. 24 h serum-starved SH-SY5Y cells were treated with MPP<sup>+</sup> (1 mM) for 24 h in serum-free culture media for the purpose of immunoblotting. The SH-SY5Y cells were then treated with H2O2 (200 μM) for 2 h 30 min in serum-free culture media and subjected to a Fluo-4 AM fluorescence assay and fluorescence microscopy. Detection of the Fluo-4 AM fluorescence intensity and related imaging were carried out according to the manufacturer's instructions. Cells were counter-stained with Hoechst dye. (**A**) An immunoblot of the PIP2 and β-Actin. (**B**) A quantitative analysis of the Fluo-4 AM fluorescence intensity in H2O2- (200 μM) treated cells. (**C**) Fluo-4 AM calcium imaging of the intracellular calcium level. Mean ± SEM (*n* = 3) was employed to express the data. Tukey's multiple comparison test was performed using a one-way ANOVA. Each \* *p* < 0.05 refers to the sample concentration compared with the same group of non-treated cells.

### **3. Discussion**

In this study, we investigated the roles of GPR4 overexpression, pharmacological inhibition, and genetic knockout in the mitochondrial oxidative stress-induced apoptotic cell death that is associated with PD. Although many studies have reported the activation of GPR4 at the physiological pH range (7.0–7.4), overexpression of GPR4 showed relatively high GPR4 activity at neutral pH 7.4 [37]. In transiently GPR4-overexpressing HEK293 cells, GPR4 is inactive at pHs higher than 8.0, whereas it is highly active at the physiological pH, 7.4, and substantially less active at pHs down to 6.8 (plausible in the range of physiological acidification) [38]. The pH sensitivity of GPR4 has been reported to vary for different cells, though potentially due to the methods employed in different laboratories [25]. In the natively GPR4-expressing cell, HUVEC, pHs from 7.4 to 7.0 have been shown to result in a 1.5-fold activation of GPR4 [25]. In this study, we found an increase in GPR4 mRNA expression at pH 7.4 in both SH-SY5Y and stably GPR4-OE cells in serum-starved media (data not added). A very slight increase in the expression of GPR4 was observed at pH 6.4. Therefore, to maintain consistency, we conducted all the experiments at a pH ~7.4. This was also the pH of the culture media that we employed.

Human-derived neuroblastoma SH-SY5Y cells are widely used in neuroscientific research as an in vitro model for the investigation of neuronal differentiation and neuroprotective events. Stimulation with several neurotoxins, such as MPP<sup>+</sup>, MPTP, rotenone, 6-OHDA, and H2O2, has been utilised to induce oxidative stress-mediated apoptotic death, thereby mimicking neurodegenerative diseases, including PD and aging [39–41]. To determine the final concentration of H2O2 and MPP+, SH-SY5Y cells were treated with H2O2 at different concentrations, ranging from 75 μM to 125 μM, for 24 h, as well

as with MPP+, ranging from 250 μM to 1 mM. H2O2 and MPP<sup>+</sup> both decreased the cell viability in a concentration-dependent manner, with optimum cytotoxicity being observed at concentrations of 125 μM for H2O2 and 1 mM for MPP+; these concentrations were selected for further experiments to determine their cytotoxicity in the serum-free SH-SY5Y cell line. In our study, GPR4 mRNA and protein expressions were increased in a time-dependent manner for 24 h in both MPP<sup>+</sup>- and H2O2-treated SH-SY5Y cells. Hence, GPR4 is directly linked with MPP<sup>+</sup>- and H2O2-induced apoptotic cell death.

Both the pro-apoptotic protein, Bax, and the anti-apoptotic protein, Bcl-2, are members of the Bcl-2 family and are directly involved in apoptotic cell death. The balance between these two proteins of the Bcl-2 family, or an increase in the Bax/Bcl-2 ratio, indicates the early phases of an apoptotic cascade [29,30]. Significant increases in ROS, or the Bax/Bcl-2 ratio, result in the collapse of the mitochondrial membrane potential, the release of cytochrome C, the activation of Caspase-3, the cleavage of PARP, and, subsequently, apoptotic cell death [6,7]. Both MPP<sup>+</sup>- and H2O2-induced apoptotic cell deaths bear the characteristic hallmarks of an increase in the Bax/Bcl-2 ratio, the release of cytochrome-C, and the activation of the proteolytic enzyme, Caspase-3, which cleaves PARP and induces apoptotic cell death [7,8]. In our study, the overexpression of GPR4 in SH-SY5Y cells significantly increased the effect of either MPP<sup>+</sup> or H2O2 and increased the Bax/Bcl-2 ratio, as was seen in both the immunoblot and RT-PCR. As a result, this significantly increased the protein level of the cleaved Caspase-3, the Caspase-3 mediated cleavage of PARP, and the Caspase-3 activity. On the contrary, the CRISPR/Cas9 knockout of GPR4 was found to result in a lesser increase in the Bax/Bcl-2 mRNA and protein ratio in both MPP<sup>+</sup>- and H2O2-treated cells. Knockout of GPR4 was also shown to reduce the cleavage of PARP after MPP<sup>+</sup> treatment. NE52-QQ57, a selective antagonist of GPR4, demonstrated a similar level of the inhibition of GPR4 expression, as was determined through both our immunoblots and RT-PCR.

We further investigated the effect of GPR4 on mitochondrial oxidative stress-induced increases in intracellular ROS generation and MMP. Surprisingly, GPR4-OE was found to significantly increase tricellular ROS generation in SH-SY5Y cells, whereas GPR4-KO generated a lower level of intracellular ROS accumulation, after a high concentration of H2O2 treatment. Similarly, through both a JC-10 assay and fluorescence microscopy, the knockout of GPR4 was found to decrease mitochondrial membrane depolarisation. In JC-10-tagged fluorescence microscopy, knockout of GPR4 was seen to prevent MPP<sup>+</sup> stimulated decrease red fluorescence and increase green fluorescence. The latter was highly increased in the case of GPR4-OE as membrane depolarisation occurs 24 h after MPP<sup>+</sup> treatment.

Besides mitochondrial dysfunction, abnormal protein aggregation and dysregulated Ca2<sup>+</sup> homeostasis are other factors that may be involved in the neurodegeneration observed in individuals with PD [42]. Recent findings suggest that increases in cytosolic Ca2<sup>+</sup> occur at both early and late stages of the apoptotic pathway. In both cases, ER Ca2<sup>+</sup> channels are linked with the release of Ca2<sup>+</sup> to the cytoplasm [33,34]. However, the exact mechanism by which intracellular Ca2<sup>+</sup> modulates mitochondrial oxidative stress-mediated apoptosis remains elusive. Many studies have suggested that MPP<sup>+</sup>- and H2O2-induced apoptosis are associated with an increase in intracellular calcium levels [43]. For example, Sing et al. (2016) demonstrated that the administration of Nimodipine, an L-type calcium channel blocker, protected from MPTP-induced dopaminergic neuronal death in an animal model of PD. More importantly, providing evidence for Nimodipine as a means to improve mitochondrial integrity and function. In the study, Nimodipine attenuated the MPTP-induced loss of tyrosine hydroxylase-positive dopaminergic neurons in the SN. It also improved mitochondrial oxygen consumption and inhibited ROS production, as well as improving mitochondrial integrity and function in striatal mitochondria [43]. These findings provide evidence in support of the notion that calcium signalling is linked with neurotoxin-induced mitochondrial dysfunction and neurodegeneration. GPR4 is a Gs-coupled receptor that signals through adenylate cyclase and also via G proteins G13 and Gq/11. GPR4 is well known for its ability to recognise phospholipase C β (PLCβ) as its canonical target [44]. Gq class α subunits, or Gβγ released by GPCR, activate Ca2<sup>+</sup> signalling through Gβγ-dependent activation of PLCβ. Upon activation, PLC<sup>β</sup> hydrolyses PIP2 to generate IP3. IP3 binds

to the ER-resident IP3 receptors, which act as Ca2<sup>+</sup> release channels to release ER-stored Ca2<sup>+</sup> into the cytoplasm [35]. In this study, overexpression of GPR4 significantly increased the intracellular calcium level in both MPP<sup>+</sup>- and H2O2-treated cells (MPP<sup>+</sup> data not given), whereas knockout generated very little change in the intracellular calcium. These findings were also observed in our study when employing the Fluo-4 AM indicator. To determine how GPR4 can modulate the intracellular calcium level, we investigated GPCR-mediated calcium signalling. We found that GPR4 knockout decreases the breakdown of PIP2, which is a critical step in Ca<sup>2</sup><sup>+</sup> release from the ER to the cytoplasm. Therefore, decreased intracellular Ca2<sup>+</sup> may be responsible for GPR4-mediated neuroprotection against MPP+ or H2O2-induced apoptotic cell death. Our study, for the first time, demonstrated that the knockout of GPR4 protects SH-SY5Y cells from both MPP<sup>+</sup>- and H2O2-stimulated mitochondrial apoptotic cell death, in association with a decrease in intracellular Ca2<sup>+</sup>.

In summary, our study suggests that overexpression of GPR4 potentiates neurotoxin-induced mitochondrial oxidative stress, whereas a knockout or pharmacological inhibition of GPR4 improves the neurotoxin-induced, caspase-dependent mitochondrial apoptosis of dopaminergic neuronal cells. This study has also found that GPR4 can increase intracellular Ca2<sup>+</sup> through the degradation of PIP2. Further investigation is required to determine how GPR4-mediated calcium signalling can mitigate the neuronal cell death seen in neurodegenerative disorders, including PD.
