Rasd₂-mediated Antidepressant Like Effect Of Energy Restriction And Its Mechanism In Female Mice

Depression is a common mental system disease,which is mainly characterized by depressed mood,lack of interest,loss of appetite,loss of pleasure,energy decline and other symptoms,accompanied by a strong sense of self blame,guilt,worthlessness,even suicide and other extreme self mutilation.The pathogenesis of depression has not been fully elucidated.There are multiple hypotheses for the pathogenesis of depression,which may be related to monoamine neurotransmitters,changes in the hypothalamus-pituitary-adrenal axis,inflammation,neuroplasticity and neurogenesis,structural and functional changes in the brain,and genetic,environmental,and epigenetic changes.Dopamine is a monoamine neurotransmitter closely related to depression.Clinic studies have found that patients with depression have changes in the level of dopamine and its metabolites.Meanwhile,Rasd₂?also known as Rhes?is a thyroid hormone-regulated target gene that is highly specifically expressed in striatum multipolar neurons?MSNs?.It has an important regulatory effect on dopamine neurons.However,it is not clear whether rasd₂ has an ameliorative effect on depression.The sustained rise in the incidence rate of depression has brought enormous financial burden to the society.To study the pathogenesis of depression is an important topic in the field of neuroscience.This project explores the role of transcription factor Rasd₂-mediated energy restriction in improving depression-like behavior through transcriptomics,behavioral,and molecular biology-related experiments.A large number of studies have shown that energy restriction can significantly improve depression like behavior.However,its detailed mechanism of action is not fully clear.In this study,we first used high-throughput sequencing to analyze the transcriptome of mouse prefrontal cortex and hippocampus.The results showed that energy restriction significantly changed the gene expression level in prefrontal cortex and hippocampus.Further analysis of go enrichment showed that energy restriction had a significant regulatory effect on 12 genes including rasd₂ and D2 receptor.The enrichment analysis of KEGG pathway showed that the energy limitation may be closely related to PI3K-Akt and the signal pathway of the interaction of neuroactive ligand receptor.Based on the results of transcriptome screening,we proposed the hypothesis that"rasd₂ mediates the effect of energy restriction on depressive behavior through dopamine D2 receptor".Therefore,we used D2 receptor antagonists to explore the mechanism of rasd₂ in energy limited antidepressant.The experiment was divided into four groups:control group,fasting group,dopamine D2 receptor antagonist,dopamine D2 receptor antagonist and fasting group.The open field experiment excluded the interference of the spontaneous movement of the perimenopausal depression model mice.In the open field experiment,there was no significant difference between the groups.In the forced swimming experiment,compared with the control group,the immobility time in the fasting group was significantly reduced and the swimming time was significantly increased,while the dopamine D2 receptor antagonist group was significantly increased in immobility time and swimming time was significantly reduced;compared with the fasting group,dopamine D2 receptor antagonist and fasting group significantly increased immobility time and significantly reduced swimming time.Therefore,the results of behavioral experiments suggest that:energy restriction can produce antidepressant effect on mice,while dopamine D2receptor antagonists reverse the antidepressant like effect of energy restriction,and also suggest that dopamine plays an important role in the regulation of depression like behavior.We then used Western blotting to study the mechanism of antidepressant like effect of energy restriction,and the results showed that fasting promotes the expression of Rasd₂ and D2 receptor in both the prefrontal and hippocampus of mice,while the effect was reversed by dopamine D2 receptor antagonist.It is suggested that rasd₂ and D2 receptor are closely related to the antidepressant like effect of energy restriction.In addition,energy restriction can protect the nervous system by regulating the CREB?cAMP response element binding protein?/BDNF?brain-derived neurotrophic factor?signal pathway and the release of endorphins.CREB is a member of a large group of transcription factors related to function and structure,involved in the regulation of neuronal plasticity and neurotransmitter release.BDNF is a brain-derived neurotrophic factor,and its expression level is closely related to neurogenesis and depression like behavior.Clinical data showed that the increase of BDNF expression was related to the decrease of depressive symptoms.Our results showed that energy restriction significantly increased the levels of CREB and BDNF in prefrontal cortex and hippocampus,while dopamine D2 receptor antagonist could partially or completely antagonized the effect of energy restriction on CREB/BDNF signaling pathway,suggesting that the antidepressant effect of energy restriction might be closely related to the activation of CREB/BDNF signaling pathway mediated by dopamine D2 receptor.In addition,our study showed that energy restriction also significantly changed the expression level of Akt protein.It is suggested that Akt related signaling pathway is also involved in the antidepressant like effect of energy restriction.Based on the close relationship between the expression levels of rasd₂ and D2receptor,we continue to study the effect of the expression changes of rasd₂ on the depression like behavior of perimenopausal depression mice.We first tested the expression of rasd₂ in perimenopausal depression mice.It was found that the expression level of rasd₂ decreased significantly after ovariectomy,suggesting that the down-regulation of rasd₂ may be related to the pathogenesis of depression.Therefore,we used adenovirus specific overexpression of rasd₂ in the hippocampus of depressed mice.Behavioral studies showed that the overexpression of rasd₂ could significantly shorten the immobility time and increase the swimming time of depressed mice in forced swimming experiment,but not affect the spontaneous movement of mice in open field experiment,which indicated that the overexpression of rasd₂ had a certain improvement effect on depression like behavior.In Western blot,we found that rasd₂overexpression regulated the expression of CREB in hippocampus.Moreover,the overexpression of rasd₂ can significantly increase the expression level of phosphorylated CREB protein in the prefrontal cortex and hippocampus of mice,suggesting that rasd₂ may play an antidepressant-like effect by regulating CREB phosphorylation.In addition,the overexpression of rasd₂ will lead to a significant decrease in Akt expression in prefrontal cortex and hippocampus,but the relationship between Akt expression and CREB phosphorylation still needs to be confirmed in subsequent experiments.In summary,in this study we uses transcriptomics,behavioral,and molecular biology research methods to find that the transcription factor Rasd₂-mediated energy restriction plays a role in antidepressant-like behavior in ovariectomized mice.Energy restriction has the advantages of simple operation and almost no side effects,so it has a bright future as an assistant treatment of antidepressant.This project provides a new theoretical basis for the treatment of energy restriction antidepressant,especially for female depression.