Developmental expression of GPR3 in rodent cerebellar granule neurons is associated with cell survival and protects neurons from various apoptotic stimuli.
G-protein coupled receptor 3 (GPR3), GPR6, and GPR12 belong to a family of constitutively active Gs-coupled receptors that activate 3'-5'-cyclic adenosine monophosphate (cAMP) and are highly expressed in the brain. Among these receptors, the endogenous expression of GPR3 in cerebellar granule neurons (CGNs) is increased following development. GPR3 is important for neurite outgrowth and neural maturation; however, the physiological functions of GPR3 remain to be fully elucidated. Here, we investigated the survival and antiapoptotic functions of GPR3 under normal and apoptosis-inducing culture conditions. Under normal culture conditions, CGNs from GPR3-knockout mice demonstrated lower survival than did CGNs from wild-type or GPR3-heterozygous mice. Cerebellar sections from GPR3-/- mice at P7, P14, and P21 revealed more caspase-3-positive neurons in the internal granular layer than in cerebellar sections from wild-type mice. Conversely, in a potassium-deprivation model of apoptosis, increased expression of these three receptors promoted neuronal survival. The antiapoptotic effect of GPR3 was also observed under hypoxic (1% O2/5% CO2) and reactive oxygen species (ROS)-induced apoptotic conditions. We further investigated the signaling pathways involved in the GPR3-mediated antiapoptotic effect. The addition of the PKA inhibitor KT5720, the MAP kinase inhibitor U0126, and the PI3 kinase inhibitor LY294002 abrogated the GPR3-mediated antiapoptotic effect in a potassium-deprivation model of apoptosis, whereas the PKC inhibitor Gö6976 did not affect the antiapoptotic function of GPR3. Furthermore, downregulation of endogenous GPR3 expression in CGNs resulted in a marked reduction in the basal levels of ERK and Akt phosphorylation under normal culture conditions. Finally, we used a transient middle cerebral artery occlusion (tMCAO) model in wild-type and GPR3-knockout mice to determine whether GPR3 expression modulates neuronal survival after brain ischemia. After tMCAO, GPR3-knockout mice exhibited a significantly larger infarct area than did wild-type mice. Collectively, these in vitro and in vivo results suggest that the developmental expression of constitutively active Gs-coupled GPR3 activates the ERK and Akt signaling pathways at the basal level, thereby protecting neurons from apoptosis that is induced by various stimuli.
Department of Molecular and Pharmacological Neuroscience, Institute of Biomedical & Health Sciences, Hiroshima University, Hiroshima 734-8551, Japan; Department of Clinical Neuroscience and Therapeutics, Institute of Biomedical & Health Sciences, Hiroshima University, Hiroshima 734-8551, Japan. Electronic address: tanakamd@hiroshima-u.ac.jp.
Shigeru
S
Tanaka
Department of Molecular and Pharmacological Neuroscience, Institute of Biomedical & Health Sciences, Hiroshima University, Hiroshima 734-8551, Japan.
Tatsuhiro
T
Miyagi
Department of Clinical Neuroscience and Therapeutics, Institute of Biomedical & Health Sciences, Hiroshima University, Hiroshima 734-8551, Japan.
Eisuke
E
Dohi
Department of Molecular and Pharmacological Neuroscience, Institute of Biomedical & Health Sciences, Hiroshima University, Hiroshima 734-8551, Japan.
Takahiro
T
Seki
Department of Molecular and Pharmacological Neuroscience, Institute of Biomedical & Health Sciences, Hiroshima University, Hiroshima 734-8551, Japan.
Izumi
I
Hide
Natural Science Center for Basic Research and Development, Hiroshima University, Hiroshima 734-8551, Japan.
Yusuke
Y
Sotomaru
Saeki Hospital, Toyama 939-8015, Japan.
Yoshinaga
Y
Saeki
Harvey Cushing Neuro-Oncology Laboratories, Department of Neurosurgery, Institute for the Neurosciences at the Brigham, Brigham and Women's/Faulkner Hospital and Center for Neuro-Oncology, Dana-Farber Cancer Institute, Boston, MA 02115, USA.
E
E
Antonio Chiocca
Department of Clinical Neuroscience and Therapeutics, Institute of Biomedical & Health Sciences, Hiroshima University, Hiroshima 734-8551, Japan.
Masayasu
M
Matsumoto
Department of Molecular and Pharmacological Neuroscience, Institute of Biomedical & Health Sciences, Hiroshima University, Hiroshima 734-8551, Japan.
Norio
N
Sakai
D000367:Age Factors; D000818:Animals; D017209:Apoptosis / Q000235:genetics*; D002316:Cardiotonic Agents / Q000494:pharmacology; D002470:Cell Survival / Q000235:genetics; D002531:Cerebellum / Q000166:cytology*; D005576:Colforsin / Q000494:pharmacology; D017077:Culture Media, Conditioned / Q000494:pharmacology; D004195:Disease Models, Animal; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects / Q000235:genetics*; D007511:Ischemia / Q000378:metabolism / Q000473:pathology; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D043562:Receptors, G-Protein-Coupled / Q000172:deficiency* / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics
D002316:Cardiotonic Agents; D017077:Culture Media, Conditioned; D004791:Enzyme Inhibitors; C494613:GPR3 protein, mouse; D043562:Receptors, G-Protein-Coupled; D005576:Colforsin
Apoptosis; Brain ischemia; Cerebellar granular neurons; GPR3; Neuronal protection; cAMP