Tmem74 In BLA Neurons Regulates Anxiety-like Behavior

Background:Anxiety disorders are the most prevalent psychiatric disorders,which bring great pressure and economic burden to individuals and society.The etiology of anxiety disorder is diverse and its pathogenesis is complex.Meanwhile,the biological mechanisms underlying anxiety have not yet been fully elucidated.The main clinical treatments for anxiety disorders include psychotherapy,behavioral therapy and drug therapy.Drug therapy runs through the whole process.At present,there are several drugs mainly used for anxiety disorders:benzodiazepines(BDZs),selective serotonin reuptake inhibitors(SSRIs),serotonin-norepinephrine reuptake inhibitors(SNRIs)and monoamine oxidase inhibitors.However,these drugs have side effects such as dependence,slow onset,poor compliance and withdrawal reaction,which greatly limit their use.While continuing to explore the molecular mechanisms of anxiety disorders,researchers are still trying to find and develop new drug targets for it.A large number of studies showed that the anxiety disorder was caused by abnormal neuronal activity in the amygdala,which disturbing the balance of excitation and inhibition.Therefore,searching for new molecules that regulate the function of excitatory neurons is helpful to find fast and effective anxiolytic targets.TMEMs(Transmembrane protein)are a kind of transmembrane proteins.In recent years,many TMEMs have been reported to play an important role in neuropsychiatric disorders such as panic disorder and anxiety disorder.TMEM74,first reported in 2008,was considered to be an autophagy-inducing molecule associated with cancer signaling pathways.Current knowledge of the pathophysiological importance of TMEM74 is predominantly based on cell culture studies.However,research on TMEM74 in vivo is lacking.Notably,RNA expression of TMEM74 is expressed at especially high levels in human brain tissue(The Human Protein Atlas),presuming that it plays an important role in nervous system disease such as anxiety.Therefore,studying the key role of TMEM74 in the brain,demonstrating whether it has a regulatory effect on anxiety disorders,and how the regulatory mechanism works.may provide a new drug target for the diagnosis and treatment of anxiety disordersObjective:To explore the signaling pathways of TMEM74 which participates in the regulation of the pathological process of anxiety disorders.Here,we investigated the regulation of TMEM74 in the samples from clinical anxiety patients and anxiety model animal.Then we studied the changes of behavioral phenotype and neuronal function mediated by the knockout of Tmem74 in mice.Moreover,investigating the molecular mechanisms of Tmem74 in regulating neuronal activity mediating anxiety disorder Finally,we regulated the expression of Tmem74 after anxiety to explore whether the re-expression could effectively reverse anxiety phenotype and repair neuronal dysfunction.This study will provide experimental basis for exploring the molecular mechanisms of anxiety disorder.Meanwhile,it will provide new drug targets for the prevention and treatment of anxiety disordersMethods and Results:Firstly,we collected the serum samples of clinical anxiety patients and brain tissue samples of anxiety model mice.The changes of candidate TMEMs protein in the pathological process of anxiety were investigated by immunoblotting and immunofluorescence staining.It was showed that the expression of TMEM74 in the anxiety patients and anxiety mice was significantly decreased.To further validate the function of Tmem74 in anxiety disorders,we injected adeno-associated viral(AAV)vector carrying Tmem74 into BLA region.It was f’ound that overexpression of Tmem74 could effectively reverse the anxious phenotype of anxiety mice.Secondly.Tmem74 knock out mice were constructed to investigate the changes of behavioral phenotype and neuronal function induced by Tmem74 deletion by several behavioral tasks and electrophysiological patch clamp recordings.CRISPR-Cas9 technique was used to investigate whether specific knockout of Tmem 74 in BLA pyramidal neurons could also induce anxiety-like behaviors.The results showed that both global knock out Tmem74 and conditional knockout increased the excitability of pyramidal neurons to induce anxiety behavior,suggesting that Tmem74 plays an important role in the pathological process of anxiety.Thirdly,we found that Tmem 74-/-mice showed impaired In function and decreased the membrane expression of HCN1 by electrophysiological patch clamp recording and immunoblotting.Moreover,pharmacological regulation confirmed that loss of Tmem74 resulted in increased neuronal excitability mediated by HCN channel proteins.In addition,Tmem74-eGFP,Tmem74-ΔTM1-ΔTM2-eGFP,Tmem74-ΔTM1-eGFP and Tmem74-ΔTM2-eGFP were generated and transfected into HEK293 cells or N2a cells,to investigate the regulation between Tmem74 and HCN1 by immunoprecipitation,and to verify the role of different transmembrane domains in the biological function of Tmem74.The results showed that the membrane localization of Tmem74 and its co-localization with HCN1 were mediated by TM1 transmembrane domain,while Tmem74 regulated the expression of HCN 1 membrane protein and ion channel function through direct binding.Finally,to investigate whether the abnormality of behavior phenotype and electrophysiological function of Tmem74-/-mice was reversible by overexpression of Tmem74.The results showed that the specific overexpression of Tmem74 in BLA pyramidal neurons could effectively reverse the increased neuronal excitability and the impaired function of HCN channel,rescuing the anxiety phenotype mediated by the knockout of Tmem 74.Conclusion:For the first time,we reported that the expression of TMEM74 in clinical anxiety patients and anxiety mice was decreased,and overexpression of Tmem74 reversed the anxious phenotype of CIS mice.Moreover,Tmem74 participated in the regulation of anxiety disorder,but had no significant effect on motor coordination,muscle function and cognitive function.Anxiety-like behaviors were ameliorated by increasing the excitability of pyramidal neurons in BLA region.Furthermore,the dysfunction of HCN channels had a major e ffect on neuronal excitability in Tmem 74-/-mice.Tmem74 interacted with the HCN1 channel at domain TM1 to mediate the Ih current.Finally,the regulation of Tmem74 on anxiety was continuous and reversible Re-expression of Tmem74 repaired the damaged neuron function and reversed the anxiety phenotype of Tmem74-/-mice.