Molecular Mechanisms Of CCDC152 Involving In The Regulation Of Plasticity Of Hippocampal Cells

According to some phenomena in clinical practice, biologists and physicians have long recognized the nervous system plasticity. Neuroplasticity refers to adaptive changes of neuronal structure and function that is produced in nervous system in response to various internal and external stimuli signaling molecules.Neuroplasticity includes plasticity of synaptic, plasticity of neurotransmitter chemical properties, and plasticity of neuronal electrical activity. Neuroplasticity ensures the accuracy of the complex behavior of animals and the ability to adapt to environmental changes. Neuroplasticity is involved in pathogenesis of depression and other psychotic diseases. Early growth response factor-1 (Egr-1) is one of proteins that play important roles in the regulation of synaptic plasticity of neurons.Egr-1 is one of gonadotropin-releasing hormone receptor(GnRHR) coupled effector molecules. GnRHR belongs to the G protein-coupled receptor(GPCR) family and activates PLC-IP3-Ca2+ second messenger systems by coupling and activating Gq/11. It is mainly expressed in the pituitary glands, mammary gland and the ovaries and other tissues. It is capable of regulating the secretion of luteining hormone(LH) and follicle stimulating hormone(FSH)and plays a role in the reproductive process. In addition, GnRHR is also expressed in Hippocampal neurons and can regulate the growth and development of nerve cell. GPCRs are important signal molecule receptors within the organisms. The desensitization and termination of GPCR is regulated mainly by intracellular β-arrestin and regulator of G protein signaling protein(RGS).CCDC152 belongs to Coiled-coil Domain Containing Protein family, which plays an important role in many aspects, for example, themetabolic regulation of cell, muscle stretching and contraction, membrane channel. However, CCDC152 function is unclear. Our previous transcriptomic sequencing results showed that CCDC152 is involved in the regulation of GnRHR downstream effector molecules.This study investigated the expression of CCDC152 in GnRHR signaling pathway, the role of CCDC 152 in the passivation and stop of GnRHR and in the plasticity.Research aims and methods:1. GnRH affects the expression of CCDC 152:After HT22 cells were treated with GnRH, the change of promoter activity of CCDC 152 was measured using Luciferase method and the expression of CCDC152was measured using the Western Blot method.2. The signaling pathway of GnRH affecting CCDC152 expression:After HT22 cells were treated with Gai inhibitor and GnRH,the expression of CCDC152was measured using the Western Blot method;After HT22 cells were treated with GnRH, the expression of CCDC152 and Gaqwere measured using the Western Blot method; After HT22 cells were treated with CaCl2 the change of promoter activity of CCDC152 was measured using Luciferase method;After HT22 cells were treated with CaCl2, the expression of CCDC152 was measured using the Western Blot method.3. The mechanism of CCDC152 in GnRHR-G protein-coupled signal:After HT22 cells were transiently transfected CCDC152 overexpression vector, Control vector N3-pEGFP and CCDC152-CRISPR/Cas9 KO and were treated with GnRH, the expression of RGS7 and Gαq were tested by Western Blot and the change of concentration of the second messenger cAMP and Ca2+ were detected using ELISA method and flow cytometry method.4. GnRH affects the expression of Egr-1:After HT22 cells were treated with GnRH, the expression of Egr-1 was measured using the Western Blot method; After HT22 cells were transiently transfected CCDC152 overexpression vector, Control vector N3-pEGFPandCCDC152-CRISPR/Cas9 KO and were treated with GnRH, the expression of Egr-1 was measured using the Western Blot method.Results:1. Luciferase reporter gene assay fruits revealed that transcriptional activity of CCDC152 promoter is significantly increased by low concentration of GnRH and is inhibited by high concentration of GnRH. Western Blot showed GnRH can increase the expression of CCDC152.2. Western Blot showed GnRH can increase the expression of CCDC152 and Gaq and can aslo increase the expression of CCDC152 with Gai inhibitor. In addition, Luciferase reporter gene assay fruits revealed that transcriptional activity of CCDC152 promoter is affected by Ca2+. Western Blot showed Ca2+ can aslo increase the expression of CCDC152.3. Western Blot showedCCDC152 can increasethe expression of RGS7 and reduce the expression of Gaq after HT22 cells were treated with GnRH. ELISA and flow cytometry results revealed thatCCDC152 can affect the concentration change of cAMP and Ca2+ after HT22 cells were treated with GnRH.4. Western Blot showed GnRH can increase the expression of Egr-1 and CCDC152 can decrease the expression ofEgr-lafter HT22 cells were treated with GnRH.Conclusions:GnRHR-Gaq coupling can increase the expression of CCDC152 by inducing PLC-IP3-Ca2+ second messenger system. In turn, CCDC152 expression can activate RGS7 and inhibit Gaq, and thus plays a negative feedback regulation of GnRHR-Gaq coupling. In addition, CCDC152 can regulate the expression of Egr-1 in hippocampal cells by a negative feedback regulation.This study may indicate that CCDC152 is involved in the regulation of hippocampal neuroplasticity.