The Relationship Between CCDC152 And Neuronal Plasticity And Related Molecules

Neuroplasticity resulted in the changes in neuronal structure and/or function.Therefore,a variety of neurological and psychiatric diseases are related to neuroplasticity.The hippocampus is closely related to the advanced cognitive function such as plasticity of nerve structure,learning,memory and emotion.The regulation of neuro plasticity involves a variety of signal pathways,such as G protein receptor(GPCR)signaling,Wnt signaling,Hippo signaling,RhoA/ROCK signaling,and so on.CCDC152(Coiled-coil Domain Containing Protein 152)is a member of CCDC family and contains 4 coiled-coil domains,but its function is unknown.Transcriptomic sequencing revealed that CCCDC152 may particupate in the regulation of multiple signaling pathways.In this study,we studied the role of CCDC 152 in the neuroplasticity regulation and the related pathways as well.The main research methods and conclusions are as follows:Research methods:1.Transfected with pGL2-CCDC152 luciferase-reporter vector,which bears CCDC 152 promoter.2.Western Blot was used to detect the expression of CCDC 152 in response to GPCR agonist stimulation,such as DOI(2,5-dimethoxy-4-iodo-ampHetamines),LPA(lysopHospHatidic acid)and other GPCR receptor agonists.3.CCDC 152 was overexpressed or knockout in the HT22 cells,cAMP-ELISA kit was then used to detect cAMP level under DOI stimulation.4.CCDC 152 was overexpressed or knockout in HT22 cells,flow cytometry was used to detection calcium ion content under the treatment of DOI.5.Overexpression of CCDC152 in the HT22 cells,the expression of Egr-1,SYP,Neurabin-?,HSP70,HSPA6 and Calpain 1 were detected by Western Blot.6.To study the expression of CCDC 152 in signal system of GPCR,Wnt,Hippo and Integrin,Western blot was used to detect the downstream Egr-1,Calpain 1,TEF-1 and Vinclin molecules.7.To study the role CCDC 152 in Rho/ROCK signaling,LPA or ROCK inhibitor Y-27632 was used to treat HT22 cells and HT22-CCDC152KO cells,Western Blot was used to detect the expression of key downstream proteins such as p-Cofilin and p-LIMK in RhoA/ROCK system.8.To sudy the effect of CCDC152 on cytoskeleton,LPA or ROCK inhibitor Y-27632 was used to treat HT22 cells and HT22-CCDC152KO cells,changes of cytoskeleton F-actin were studied by PHalloidin staining.Experimental results:1.The promoter activity of CCDC152 is regulated by DOI,exhibited an activation with a low concentration of DOI whereas an inhibition with a high concentration.Therefore,10uM was chosen as the experimental concentration.Therefor,cells treated for 5-30 min showed high promoter activity whereas low activity 2-48 h,which is related to the desensitization of serotonin receptors.2.Gai inhibitor PTX has an effect on DOI-regulated CCDC152 expression,but does not affect the overall trend of its expression.KCI,PMA,CTX or PTX,which durgs a variety of intracellular signaling pathways,will affect DOI-induced CCDC152 expression.In contrast,overexpression or knockout CCDC152 regulated DOI induced the oscillation of cAMP and Ca2+.3.CCDC152 expression showed inhibitory effect on the expression of neuronal apoptosis-related factors Calpain 1,Cas 9,neuronal protective factors HSPA6,HSP70 and neuronal plasticity and memory regulatory factors SYP,Egr-1,EGR 3,but showed activation effect on the expression of Neurabin-II.4.The expression of CCDC152 in HT22 cells is regulated by GPCR agonist LPA:a short term treatment with LPA(up to 1 hour),CCDC152 expression showed an augmentation with a fluctuation;a long term LPA treatment(up to 48 hours),the expression of CCDC152 was significantly decreased,which was related to the desensitization of the receptor.5.The effect of CCDC152 on LPA activated RhoA/ROCK targeted proteins in HT22 cells:the expression CCDC152 affected the PHospHorylation of p-Cofilin;in both HT22 cells and HT22-CCDC152KO knockout cells,LPA could activate the phosphorylation of ERK1/2,and ROCK inhibitor Y-27632 showed no effect on LPA.6.Effects of CCDC152 on the LPA induced F-actin dynamics:In the HT22 cells,LPA induced Rho/ROCK activation and consequently enhanced phphrylation of Cofilin and MLC2,which trigged the stress fiber formation and filament stabilization,cells presented as neurite retraction.In contrast,HT22-CCDC152KO cells a shrinkened cell body but elonged axons.Experimental conclusion:1.CCDC152 expression induced by DOI in HT22 hippocampal cells is related to the activation of Gaq and other signaling pathways.2.CCDC152 plays a regulatory role in neurotic plasticity,memory and neurological protection,which involved in Wnt,Hippo,and RhoA/ROCK signalings.3.CCDC152 played no role in the phsphrylation of ERK1/2 by LPA.4.CCDC152 regulates neuroplasticity through inhibiting Rho/ROCK activity.