The Study On The Mechanism Of DGCR2,a Risk Factor For Schizophrenia,Regulating Dendritic Spine Development And The Role Of Perineuronal Nets In Anxiety

Anxiety is a transient negative emotional state that drives an adaptive response to potential threats.However,excessive and persistent anxiety contributes to the development of many mental diseases,such as anxiety disorders.At present,the pathological mechanisms underlying anxiety disorders are still unclear.The 22q11.2 deletion syndrome(22q11DS)is a neurogenetic condition caused by a microdeletion in chromosome 22,with an incidence of 1 in 2,000–4,000 live births.22q11 DS is a high risk for developing mental diseases like schizophrenia and anxiety disorders.DGCR2(Di George syndrome critical region gene 2)is a deleted gene among the critical region of 22q11 DS and is also associated with schizophrenia.DGCR2 expression is reduced in schizophrenic brains.However,little is known about the functions of DGCR2 in the brain.We used DGCR2-deficient(mt)and their control wild-type mice for behavioral tests.In the open-field test(OFT),duration in the center area was reduced in mt mice.In the elevated plus maze(EPM),entries and duration in the opened arms were decreased in mt mice.These results suggest that DGCR2 deficiency induces anxiety-like behaviors.Next,we examined DGCR2 expression in the mouse brain.β-galactosidase staining with DGCR2-Lac Z reporter mice showed a global expression of DGCR2 in the brain.Immunoblotting with brain lysates indicated an increased expression of DGCR2 during neuronal development.And immunostaining with brain slices suggested expressions of DGCR2 in both excitatory and inhibitory neurons.Moreover,DGCR2 was distributed in the soma and dendrites,and colocalized with postsynaptic density-95(PSD-95)puncta in cultured hippocampal neurons.Biochemical studies demonstrated that DGCR2 could interact with PSD-95 and was enriched in the PSDs.We furtherly used RNA interference to knock down DGCR2 in hippocampal neurons by calcium phosphate precipitation transfection or in utero electroporation.The spine density was reduced in DGCR2 knockdown neurons.And Golgi staining also found a decreased spine density in hippocampal CA1 pyramidal neurons of mt mice.Accordingly,the frequency of miniature excitatory postsynaptic currents(m EPSCs),as well as long-term potential(LTP)was reduced.These results suggest DGCR2 regulates spine development,glutamatergic transmission,and synaptic plasticity.DGCR2 is a putative adhesion receptor protein with a single transmembrane domain.By coimmunoprecipitation(co-IP)and cell aggregation assay,we found that the extracellular domain(ECD)of DGCR2 can transcellularly interact with the presynaptic cell adhesion molecule NRXN1.The binding of DGCR2 with NRXN1 increased NRXN1-NLGN1 interaction,thus promoting dendritic spine development.Taken together,our results suggest DGCR2 regulates dendritic spine development through its interaction with NRXN1,and its deficiency induces anxiety-like behaviors.Inhibitory circuit deficiency is a hallmark of many mental disorders like anxiety and depression.Perineuronal nets(PNNs)are specialized extracellular matrix structures that surround interneurons and regulate synaptic transmission and plasticity.It has been reported that PNNs expressions in the orbitofrontal cortex(OFC)are inversely correlated with basal anxiety levels of mice,suggesting PNNs may contribute to anxiety-like behaviors.To explore the role of PNNs in stress-induced anxiety-like behaviors,we subjected wild-type mice to chronic restraint stress(CRS).Duration in the center area of OFT,as well as entries and duration in the opened arms of EPM were reduced in mice after CRS,suggesting CRS induces anxiety-like behaviors.Wisteria floribunda agglutinin(WFA)staining showed decreased PNNs expression in the medial prefrontal cortex(m PFC)of CRS mice.The m RNA and protein levels of aggrecan(ACAN),a core component of PNNs,were also reduced.In parallel,frequencies of m EPSCs and miniature inhibitory postsynaptic currents(m IPSCs)were decreased in the m PFC pyramidal neurons of CRS mice.And interestingly,enzymatic digestion of PNNs in m PFC by injecting chondroitinase ABC(ch ABC)leads to anxiety-like behaviors.These results suggest that PNNs regulate anxiety-like behaviors in mice.Fluoxetine(FXT)is a clinically prescribed antidepressant/anxiolytic drug.We intraperitoneally(i.p.)injected FXT into CRS mice and found that FXT treatment ameliorated anxiety-like behaviors,restored m PFC PNNs and ACAN expressions,and rescued abnormal synaptic transmission in CRS mice.These results suggest that PNNs may be a new therapeutic target for anxiety disorders.Our study investigated the roles of DGCR2 in excitatory synapse development and PNNs in anxiety-like behaviors induced by CRS at the molecular level.These results provide new insight into the pathogenesis of anxiety disorders.