GPR4 Knockout Improves the Neurotoxin-Induced, Caspase-Dependent Mitochondrial Apoptosis of the Dopaminergic Neuronal Cell
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 t...
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MDPI AG
2020-10-01
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| Series: | International Journal of Molecular Sciences |
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| author | Md Ezazul Haque Mahbuba Akther Shofiul Azam Dong-Kug Choi In-Su Kim |
| author_facet | Md Ezazul Haque Mahbuba Akther Shofiul Azam Dong-Kug Choi In-Su Kim |
| author_sort | Md Ezazul Haque |
| collection | DOAJ |
| description | 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<sup>+</sup>) and hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>)-treated apoptotic cell death of stably GPR4-overexpressing and stably GPR4-knockout human neuroblastoma SH-SY5Y cells. In GPR4-OE cells, MPP<sup>+</sup> and H<sub>2</sub>O<sub>2</sub> 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<sup>+</sup> 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, H<sub>2</sub>O<sub>2</sub> treatment significantly increased the intracellular calcium ions (Ca<sup>2+</sup>) 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<sup>+</sup>- or H<sub>2</sub>O<sub>2</sub>-induced apoptotic cell death. Moreover, the knockout of GPR4 decreased the proteolytic degradation of phosphatidylinositol biphosphate (PIP<sub>2</sub>) and subsequent release of the endoplasmic reticulum (ER)-stored Ca<sup>2+</sup> 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 PIP<sub>2</sub> degradation-mediated calcium signalling. Therefore, GPR4 presents a potential therapeutic target for neurodegenerative disorders such as Parkinson’s disease. |
| first_indexed | 2024-03-10T15:41:56Z |
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| spelling | doaj.art-f57775c1bcdd4c8eba19602779b6e4f22023-11-20T16:47:02ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672020-10-012120751710.3390/ijms21207517GPR4 Knockout Improves the Neurotoxin-Induced, Caspase-Dependent Mitochondrial Apoptosis of the Dopaminergic Neuronal CellMd Ezazul Haque0Mahbuba Akther1Shofiul Azam2Dong-Kug Choi3In-Su Kim4Department of Applied Life Science, Graduate School, Konkuk University, Chungju 27478, KoreaDepartment of Applied Life Science, Graduate School, Konkuk University, Chungju 27478, KoreaDepartment of Applied Life Science, Graduate School, Konkuk University, Chungju 27478, KoreaDepartment of Applied Life Science, Graduate School, Konkuk University, Chungju 27478, KoreaDepartment of Biotechnology, Research Institute of Inflammatory Disease (RID), College of Biomedical and Health Science, Konkuk University, Chungju 27478, KoreaIn 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<sup>+</sup>) and hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>)-treated apoptotic cell death of stably GPR4-overexpressing and stably GPR4-knockout human neuroblastoma SH-SY5Y cells. In GPR4-OE cells, MPP<sup>+</sup> and H<sub>2</sub>O<sub>2</sub> 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<sup>+</sup> 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, H<sub>2</sub>O<sub>2</sub> treatment significantly increased the intracellular calcium ions (Ca<sup>2+</sup>) 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<sup>+</sup>- or H<sub>2</sub>O<sub>2</sub>-induced apoptotic cell death. Moreover, the knockout of GPR4 decreased the proteolytic degradation of phosphatidylinositol biphosphate (PIP<sub>2</sub>) and subsequent release of the endoplasmic reticulum (ER)-stored Ca<sup>2+</sup> 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 PIP<sub>2</sub> degradation-mediated calcium signalling. Therefore, GPR4 presents a potential therapeutic target for neurodegenerative disorders such as Parkinson’s disease.https://www.mdpi.com/1422-0067/21/20/7517apoptosisneurodegenerationGPR4 receptorMPP<sup>+</sup>Parkinson’s diseaseCRISPR/cas9 |
| spellingShingle | Md Ezazul Haque Mahbuba Akther Shofiul Azam Dong-Kug Choi In-Su Kim GPR4 Knockout Improves the Neurotoxin-Induced, Caspase-Dependent Mitochondrial Apoptosis of the Dopaminergic Neuronal Cell International Journal of Molecular Sciences apoptosis neurodegeneration GPR4 receptor MPP<sup>+</sup> Parkinson’s disease CRISPR/cas9 |
| title | GPR4 Knockout Improves the Neurotoxin-Induced, Caspase-Dependent Mitochondrial Apoptosis
of the Dopaminergic Neuronal Cell |
| title_full | GPR4 Knockout Improves the Neurotoxin-Induced, Caspase-Dependent Mitochondrial Apoptosis
of the Dopaminergic Neuronal Cell |
| title_fullStr | GPR4 Knockout Improves the Neurotoxin-Induced, Caspase-Dependent Mitochondrial Apoptosis
of the Dopaminergic Neuronal Cell |
| title_full_unstemmed | GPR4 Knockout Improves the Neurotoxin-Induced, Caspase-Dependent Mitochondrial Apoptosis
of the Dopaminergic Neuronal Cell |
| title_short | GPR4 Knockout Improves the Neurotoxin-Induced, Caspase-Dependent Mitochondrial Apoptosis
of the Dopaminergic Neuronal Cell |
| title_sort | gpr4 knockout improves the neurotoxin induced caspase dependent mitochondrial apoptosis of the dopaminergic neuronal cell |
| topic | apoptosis neurodegeneration GPR4 receptor MPP<sup>+</sup> Parkinson’s disease CRISPR/cas9 |
| url | https://www.mdpi.com/1422-0067/21/20/7517 |
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