Jmjd3 Mediates The Susceptibility To Depression Induced By Early-life Stress By Enhancing The Neuroinflammation Of The Prefrontal Cortex And Hippocampus

1.BackgroundDepressive disorder,as the fourth largest disease in the word,is the most common mental disorder.The main symptoms are low mood,anhedonia and cognitive impairment.At present,depression affects about 4.4%of population all over the world,and brings huge economic burden because of its high incidence and disability rate.So far,the pathogenesis of depression is so complicated that has not been fully elucidated,resulting in limited treatment methods and curative effects.Therefore,it is important to explore the pathogenesis and find new treatment targets in the prevention and treatment of depression.Studies have shown that stress is a critical risk factor for depressive disorder.Early-life stress leads to persistent abnormalities in the structure and function of the brain,which cause persistent changes in individual behaviors and cognition,and increase the susceptibility to depression in adulthood.A large number of studies have confirmed that stress induces the immune activation in the central nervous system and results in the dyregulation of inflammatory cytokines production,and then leads to the neuroinflammation of emotionally related brain regions.This process is one of the important pathogenesis of depression.Microglia,as immune cells involved in the innate immunity in the central nervous system,are the main source of cytokines,and mediate the occurrence of neuroinflammation.Studies have confirmed that stress induces the activation of hypothalamic-pituitary-adrenal(HPA)axis,and a large amount of glucocorticoid(GC)is released into emotional-related brain regions such as the prefrontal cortex and hippocampus.The surface of microglia in these brain regions expresses a large number of glucocorticoid receptors(GR),so the microglia are activated.There are two functional subtypes of activated microglia:the classical activation(M1)and conversion/substitution model(M2).When M1 microglial cells are activated,a large number of pro-inflammatory cytokines related to cytotoxicity are released,such as tumor necrosis factor-a(TNF-α),interleukin-1β(IL-1β)and interleukin-6(IL-6),which cause abnormality of multiple pathogenesis of depression.And recent studies have manifested that microglia have the function of "psychological immune memory".Microglia are primed by early-life stress and remain in pre-activated state for years.The primed microglia have an exaggerated response to later-life stress,thus increasing the susceptibility to mental disorders.It can be seen that neuroinflammation,characterized by microglial activation and pro-inflammatory cytokine over-expression,plays an important role in the susceptibility to depression induced by early-life stress.Epigenetic researches have shown that covalent modifications of histone such as methylation and acetylation determine the transcription of genes.Jumonji domain-containing protein 3(Jmjd3)is a histone demethylase of H3K27me3,which plays an important role in the development and differentiation of macrophages.Recently,it has been found.that Jmjd3 regulate the activation of microglia and participate in many inflammatory processed by enhancing the transcription of pro-inflammatory cytokines GSK-J4,as an inhibitor of Jmjd3,could alleviate the inflammatory processes.This study suggests that Jmjd3 may be a key regulator of microglial activation and pro-inflammatory cytokines expression.Therefore,we speculate that Jmjd3 may play a critical role in the epigenetic mechanism of depression caused by neuroinflammation.Therefore,the current study first focuses on the short-and long-term effects of early-life stress on depression-like behaviors,microglial activation,pro-inflammatory cytokines expression,as well as Jmjd3 and H3K27me3 expression in the prefrontal cortex and hippocampus of rats,in order to reveal the roles of Ml microglial activation and pro-inflammatory cytokines expression in the pathogenesis of depression.Afterwards,the study focuses on the changes of the above-mentioned behavioral and biological indicators when early-life stressed rats encounter the later life challenge,in order to reveal the role of Jmjd3 and neuroinflammation in the susceptibility to depression induced by early-life stress.2.Objectives2.1 To observe the short-and long-term effects of adolescent chronic unpredictable stress on depressive-like behaviors,cognitive function and microglial activation,pro-inflammatory cytokines and Jmjd3/H3K27me3 expression in the prefrontal cortex and hippocampus of rats.2.2 To observe the short-and long-term effects of microglial inhibitor minocycline administration on depression-like behaviors,cognitive function and Jmjd3/H3K27me3 expression in the prefrontal cortex and hippocampus of stressed rats.2.3 To probe the roles of microglial activation and dysregulation of pro-inflammatory cytokines and Jmjd3 expression in susceptibility to depression induced by adolescence chronic unpredictable stress.2.4 To investigate the short-and long-term effects of maternal separation on depressive-like behaviors,cognitive function,microglial activation and the expression of pro-inflammatory cytokines and NF-κB,as well as Jmjd3 and H3K27me3 in the prefrontal cortex and hippocampus of rats.2.5 To investigate the effects of later-life challenge on depressive-like behaviors,cognitive function and neuroinflammation,as well as Jmjd3 and H3K27me3 expression in the prefrontal cortex and hippocampus of adult maternally separated rats.2.6 To investigate whether Jmjd3 inhibitor GSK-J4 administration can improve the behavioral changes and prefrontal and hippocampal neuroinflammation induced by early-life stress and later-life challenges.2.7 To probe the roles of neuroinflammation,Jmjd3 and H3K27me3 in the susceptibility to depressive disorder induced by early-life stress.3.Methods3.1 Dynamic changes of microglial activation and expression of cytokines and Jmjd3/H3K27me3 in the prefrontal cortex and hippocampus of rats induced by adolescent stress3.1.1 Experimental animals and groupingSixty 21-day-old male Wistar rats were randomly divided into three group of twenty rats namely the control group(C),chronic unpredictable mild stress(CUMS)group(S)and CUMS+Minocycline group(S+M).After 7 days of acclimatization,the animal model of depression was established using CUMS,and minocycline(40 mg/kg)was injected intraperitoneally for intervention.After modeling and drug administration,10 rats from each group were selected randomly for behavioral tests and later molecular biological tests.And the remaining rats were raised into adulthood and subjected to the same behavioral tests and molecular biological tests.3.1.2 Experimental methods(1)We developed the animal model of depression using CUMS for 21 consecutive days.There are seven stressors,including heat stress(45 ℃,5 min),radio noise(8 h),light/dark cycle reversal,food deprivation(24 h),water deprivation(24 h),pinching tail(1 min),and foot shock(30 mV,10 s duration for total of 10 min).(2)The sucrose preference test was used to evaluate the level of anhedonia.The open filed test was conducted to detect the exploratory behaviors and autonomous activities of rats in a novel environment.Application of elevated plus maze and Morris water maze were detected the level of anxiety and learning and memory in all rats.(3)The expression of pro-inflammatory cytokines in the prefrontal cortex and hippocampus was detected by enzyme-linked immunosorbent assay(ELISA).(4)Immunofluorescence and immunohistochemical were used to detect changes of the number and morphology of microglial cells and microglial activation phenotype.(5)Expressions of Jmjd3 and H3K27me3 in the prefrontal cortex and hippocampus were detected by western blotting.3.2.Jmjd3 is involved in the susceptibility to depression induced by maternal separation via enhancing the neuroinflammation in the prefrontal cortex and hippocampus of male rats3.2.1 Experimental animals and groupingEighty male rat pups were randomly divided into 2 groups(40 pups in each group)on postnatal day(PND)2:control group and MS group.After 20-day maternal separation(MS),10 rats from each group were selected randomly for behavioral tests and molecular biological tests.The remaining rats were raised into adulthood and each group was divided into 3 subgroups randomly,a total of 6 groups(10 rats in each group):control(control)group,MS(MS)group,lipopolysaccharide(LPS)(Con+lps)group,MS+LPS(MS+lps)group,LPS+GSK-J4(CL+GSK-J4)group and MS+LPS+GSK-J4(ML+GSK-J4)group.Seven-day LPS(600 μg/kg)administration and GSK-J4(3.5 mg/kg)intervention were used in LPS groups and LPS+GSK-J4 groups.After modeling and drug administration,all rats were subjected to behavioral tests and sacrificed for molecular biological tests.3.2.2 Experimental methods(1)We developed the animal model of depression using maternal separation.Pups in MS group were separated from their mothers for 4 h per day(from 8:00 AM to 12:00 AM)from PND2 to PND20.(2)Seven-day systemic LPS(600μg/kg)administration in adulthood was utilized as later-life challenge,and GSK-J4(3.5 mg/kg)injection was used as an intervention.(3)The sucrose preference test was used to evaluate the level of anhedonnia.The open filed test was used to detect the exploratory behaviors and autonomous activities of rats.Elevated plus maze was used to detected the level of anxiety.Y maze and Morris water maze were detected learning and memory in all rats.(4)The expression of pro-inflammatory cytokines in the prefrontal cortex and hippocampus was detected by ELISA.(5)Immunofluorescence was used to detect changes of the number and morphology of microglial cells and microglial activation phenotype.(6)Expressions of NF-κB,Jmjd3 and H3K27me3 in the prefrontal cortex and hippocampus were detected by western blotting.4.Results4.1 Dynamic changes of microglial activation and expression of cytokines and Jmjd3/H3K27me3 in the prefrontal cortex and hippocampus of rats induced by adolescent stress4.1.1 Chronic stress in early adolescence induced depressive-and anxiety-like behaviors and memory impairment in both adolescent and adult rats.4.1.2 Chronic stress in early adolescence induced increased number of microglia and increased expression of iNOS,IL-1β and IL-6 in the prefrontal cortex and hippocampus of both adolescent and adult rats.4.1.3 Chronic stress in early adolescence resulted in increased Jmjd3 expression and decreased H3K27me3 expression in the prefrontal cortex and hippocampus of both adolescent and adult rats.4.1.4 The microglial inhibitor minocycline significantly improved depressive-like behaviors,anxiety-like behaviors,and memory function in rats,and reduced the number of microglia,especially M1 microglia,and normalized the expression of pro-inflammatory cytokines,Jmjd3 and H3K27me3.4.2 Jmjd3 is involved in the susceptibility to depression induced by maternal separation via enhancing the neuroinflammation in the prefrontal cortex and hippocampus of male rats4.2.1 MS resulted in depressive-like behaviors,anxiety-like behaviors and memory impairment in 27-day-old rats.Meanwhile,MS resulted in microglial activation and pro-inflammatory cytokines over-expression,as well as increased expression of NF-κB and Jmjd3 and decreased H3K27me3 expression in the prefrontal cortex and hippocampus.4.2.2 MS-induced behavioral and biological alterations could persist into adulthood.And seven-day LPS administration in adult rats induced the similar changes of behaviors and biomarkers.4.2.3 LPS administration in MS experienced rats resulted in severer depressive-like behaviors and neuroinflammatory status,higher levels of NF-κB and Jmjd3 expression,and lower levels of H3K27me3 expression.4.2.4 Treatment with GSK-J4(Jmjd3 inhibitor)could relieve the depressive-like behaviors and neuroinflammation induced by MS and LPS administration.5.Conclusions5.1 Chronic unpredictable stress in adolescence induced depressive-like behaviors,anxiety-like behaviors,and impaired learning and memory in adolescent rats,which persist into adulthood.5.2 Microglial activation and dysregulation of pro-inflammatory cytokines expression in the prefrontal cortex and hippocampus play important role in susceptibility to depression induced by early adolescent stress.5.3 Jmjd3 might be involved in the susceptibility to depression by regulating the microglial activation and pro-inflammatory cytokines expression.5.4 MS induced depressive-like behaviors,anxiety-like behaviors,and impairment of learning and memory function in infant rats,which could also lead to increased susceptibility to depressive disorder when rats faced later-life challenges.5.5 Prefrontal and hippocampal neuroinflammation in rats,characterized by microglial activation and pro-inflammatory cytokines over-expression,play an important role in susceptibility to depression induced by early-life stress.5.6 Jmjd3 is involved in the susceptibility to depression induced by early-life stress via enhancing the neuroinflammation in the prefrontal cortex and hippocampus of rats.