The Mechanisms Of The Depression-like Phenotypes Induced By The Deficiency Of Cend1

Objective:Depression is a high prevalence,recurrent and often life-threatening mental illness accompanied with symptoms such as depressed mood,fatigue and helplessness.However,the etiology of depression is to be confirmed.Recently,hippocampal neurogenesis hypothesis quickly became popular because of the hippocampus atrophy found in patients with depressive disorder.Cell cycle exit and neuronal differentiation protein 1(Cendl)is a neuronal specific protein that participated in the regulation of cell cycle exit and neuronal precursors differentiation.Hence,the purpose of this study is to explore the behavioral and hippocampal neurogenesis changes in Cendl knockout(KO)mice and to further explore possible related molecular mechanisms.Methods:In this study,PCR and sequencing analysis were used to detect the knocking out of Cend1 gene in KO mice.Western Blot analysis was used to detect the deficiency of Cendl protein in KO mice.Behavioral tests such as Elevated plus maze test,Open field test,Sucrose preference test,Tail suspension test and Forced swimming test were used to examine the anxiety-like and depression-like phenotypes in mice.Three-week chronic restraint stress was used to induce the model of depression.Western Blot analysis was used to detect the expression of Cend1 in different brain regions.Immunofluorescence was used to detect the hippocampal neurogenesis in adult mice.RNA sequencing and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment were used to forecast Cendl-related signaling pathway.Real-time quantitative PCR(qPCR)was used to examine the mRNA expression of specific genes.Immunofluorescence was used to detect the intracellular location of proteins.Cytoplasmic and nuclear proteins in hippocampus were separated by nuclear plasma separation kit.And Western blot analysis was used to detect the nuclear protein levels.Results:The result of PCR amplification and sequencing analysis showed that Cend1 gene sequence was successfully knocked out.And the expression of Cend1 protein were reconfirmed by Western Blot analysis in Cendl KO mice.In behavioral tests,the Cend1 KO mice displayed a depressive-like phenotype rather than an anxiety-like phenotype.After 3 weeks of chronic restraint stress treatment,the hypothalamus,hippocampus,prefrontal cortex and brain stem of control and stressed mice were separated.Western Blot analysis showed that compared to control group,the expression of Cendl was significantly decreased in the hippocampus of stressed group;however,there was no significant changes in other brain regions.Immunofluorescent staining in the hippocampus of WT and Cendl KO mice showed that compared to WT mice,the number of neural stem cells remained unchanged whereas the number of immature neurons and the new-born mature neurons significantly decreased in the SGZ of Cend1 KO mice.RNA sequencing and KEGG enrichment revealed that Cendl deficiency influenced the Notch signaling pathway.And the result of qPCR showed that the mRNA levels of the downstream target genes Hes family bHLH transcription factor 5(Hes5)and Hairy/enhancer-of-split related with YRPW motif-like(Heyl)in the Notch pathway were significantly increased.After 3 weeks of chronic restraint stress treatment,ICR mice showed a depressive-like phenotype.The result of nuclear and cytoplasmic protein extraction and Western Blot showed that the nuclear Cendl protein levels were decreased in stressed group compared to control group.In PC 12 cells,immunofluorescent staining showed that overexpression of Abelson helper integration site-1(Ahi1)up-regulated the expression of Cend1 in nucleus but knocking out the nuclear localization sequence of Ahi1 gene impaired this effect.The result of nuclear and cytoplasmic protein extraction and Western Blot showed that the expression of Cend1 protein and its nuclear localization were reduced in Ahi1 KO mice;however,in Cend1 KO mice,the Ahi1 protein level and intracellular localization did not change significantly.Conclusions:The deficiency of Cendl could lead to depressive-like phenotypes and impaired adult hippocampal neurogenesis.And its nuclear translocation,which is regulated by Ahi1,might play an important role in stress-induced depression.This study provided a new mechanism for the hippocampal neurogenesis hypothesis.