Effect Of Extracellular Vesicle Delivered-circDYM On Major Depressive Disorders And The Underlying Mechanisms

Aims:Major depressive disorder（MDD）is a complex non-organic mental disease with high prevalence,recurrence,and disability,which seriously affects the patients’psychosocial function and quality of life.Current antidepressants have disadvantages such as individual variations in drug response,side effects and blood-brain barrier（BBB）limitation.Therefore,it is of great significance to develop new antidepressants and deliver them safely and effectively to the site of action.Previous laboratory studies have found that circ DYM is involved in the process of depression.Present study is based on a novel drug delivery system,extracellular vesicles（EVs）that can target delivery of circ DYM into the brain,to explore an effective treatment strategy with antidepressant and clinical translational value.Methods:Engineered rabies glycoprotein（RVG）-circ DYM-EVs based on modified RVG-Lamp2b-EVs targeting brain were generated to deliver circ DYM.Transmission electron microscope（TEM）,nanoparticle tracking analysis（NTA）,western blot,and absolute quantitative polymerase chain reaction（q PCR）were used to characterize RVG-circ DYM-EVs.The uptake of RVG-circ DYM-EVs in primary microglia was assessed in vitro using a live cell workstation.In vivo imaging system（IVIS）and absolute q PCR were used to analyze the distribution and uptake of RVG-circ DYM-EVs in mice.The safety of RVG-circ DYM-EVs was evaluated by CCK8 assay and flow cytometry（FCM）.Normal mice were injected with different doses of RVG-circ DYM-EVs to evaluate the expression of circ DYM level in the brain of mice.RVG-circ DYM-EVs were intravenously injected into chronic unpredictable stimulation（CUS）mice and depressive-like behaviors was assessed.Enzyme-linked immunosorbent assay（ELISA）was applied to assess pro-inflammatory cytokines（IL-6,IL-1β,MCP-1 and TNF-α）levels,and western blot was used to examine the expression of inducible nitric oxide synthase（i NOS）and glial fibrillary acidic protein（GFAP）in the hippocampus of mice.The number and morphology of microglia and astrocytes were determined by FCM and diaminobenzidine（DAB）combined with sholl analysis.Western blot and immunofluorescence assay were used to evaluate the effects of RVG-circ DYM-EVs on the BBB.The infiltration of peripheral immune cells into the central nervous system was analyzed by FCM.Transcriptomics combined with bioinformatics,immunofluorescence assay,RNA binding protein immunoprecipitation（RIP）,RNA pull-down assay,cytoplasmic and nuclear separation,fluorescence in-situ hybridization（FISH）and chromatin immunoprecipitation（Ch IP）assay were used to identify the molecular mechanism（s）of circ DYM inhibiting neuroinflammation.Results:The engineered EVs were physically homogenous,with a size distribution peaking at 115.2 nm in diameter.Brain targeted RVG-Lamp2b fusion protein was expressed on the surface of EV,and circ DYM was overexpressed in the RVG-EVs.RVG-circ DYM-EVs efficiently delivered circ DYM into primary microglia in vitro.RVG-circ DYM-EVs safely and effectively delivered circ DYM into the brain of mice in vivo.The expression level of circ DYM in the brain of normal mice increased with the dose of RVG-circ DYM-EVs,and RVG-circ DYM-EVs significantly alleviated depressive-like behaviors in CUS mice.Cellular mechanism studies showed that RVG-circ DYM-EVs inhibited microglia activation,reduced astrocyte loss,reduced BBB damage,as well as reduced infiltration of peripheral immune cells(CD4⁺T cells,CD8⁺T cells,B220⁺B cells and Ly6G^-Ly6C^low cells)in CUS mice.Elucidating molecular mechanism that circ DYM bound to transcription factor TATA-box binding protein associated factor 1（TAF1）,affected subcellular distribution of TAF1,and then inhibited transcription of TAF1 downstream pro-inflammatory target genes（Trpm6,Cyp39a1）,thereby alleviating depressive-like behaviors in mice.Conclusions:Based on the RVG-circ DYM-EV delivery system,this study reveals that RVG-circ DYM-EVs can effectively inhibit depressive-like behaviors in mice by inhibiting neuroinflammation,and elucidates the molecular and cellular mechanisms of how circ DYM can be therapeutically exploited to alleviate depressive-like behaviors.This study will provide a basis for development of effective treatment for depression.