Melatonin Alleviates Depression-like Symptoms And Cognitive Impairment In Mice By Regulating The Glymphatic System Via PER2-DTNA-AQP4 Pathway

Objective:Major depression disorder(MDD)is a common chronic psychiatric illness,which is resistant to existing medical treatments in some patients.Previous studies indicate that melatonin may alleviate emotional symptoms and sleep disorders caused by MDD,while further studies explaining the mechanisms for its efficacy against depression is missing.Glymphatic system is an astrocyte-dependent neurotoxic waste clearance pathway,constituted of directional cerebrospinal fluid(CSF)in perivascular spaces and polarized aquaporin-4(AQP4)expressed at astrocytic endfeet.Previous studies have indicated the significant role glymphatic system plays in several neurological disorders.Here,the treatment effects of melatonin against depressive symptoms,cognitive dysfunction,and circadian rhythm sleep-wake disorder(CRSWD)in chronic unpredictable mild stress(CUMS)mice model was tested,the regulation effects targeting at glymphatic system and its underlying molecular mechanism was explored.Methods:Depressive behaviors were identified by open field test,sucrose preference test,and tail suspension test,while cognitive abilities by Morris water maze,circadian rhythm sleep-wake state by continuous 24-hour Electroencephalogram/Electromyogram(EEG/EMG)monitor.Interstitial fluid in mice prefrontal cortex was acquired by microdialysis,and the levels of neurotransmitters were measured by liquid chromatograph-mass spectrometer(LC-MS).Glymphatic influx function was measured by multi-scale optical imaging after CSF tracer injected via cisterna magna,while efflux function by enzymelinked immunosorbent assay(ELISA)after stereotaxic injection of exogenous β amyloid into prefrontal cortex.Expressions of Aquaporin 4(AQP4)and circadian proteins were measured by immunostaining and western blot combining with the Donut analysis and the Vertical Line analysis.The relationships between circadian proteins and AQP4 were tested by Pearson regression analysis.The rhythmicity of the proteins was further analyzed by CircaCompare R package.Regulation of Period 2(PER2)expression in primary cultured astrocytes was performed by transfection of small interfering RNA(siRNA)and overexpression plasmid.Results:CUMS mice exhibited depressive symptoms,cognitive impairment,and circadian rhythm sleep-wake disorder,characterized by decreased percentage of non-rapid eye movement(NREM)sleep in the light phase,shortened latency of rapid eye movement(REM)sleep,and shortened sleep bouts duration,which were rescued by melatonin treatment.The expression of melatonin receptor 1(MT1)was increased in the prefrontal cortex of CUMS mice,while melatonin receptor 2(MT2)remained unchanged.Melatonin decreased the expression of MT1,and had no obvious influence on MT2.The levels of 5hydroxytryptamine(5-HT),dopamine,and norepinephrine in the interstitial fluid of CUMS mice prefrontal cortex were obviously decreased,while they remained unchanged after melatonin treatment.The number of astrocytes and the total length of astrocytic processes were decreased after CUMS,while melatonin treatment could not rescue these changes.CUMS led to glymphatic influx and efflux dysfunction and AQP4 depolarization.Melatonin restored the impaired glymphatic influx and efflux function and increased the AQP4 polarization.Furthermore,AQP4 inhibitor TGN020 partly blocked the treatment effects of melatonin on improving glymphatic system function,depressive symptoms,and cognitive disturbance in CUMS mice.The expression of circadian proteins PER2,Brain and Muscle ARNT Like 1(BMAL1),CLOCK,Period 1(PER1),Nuclear Receptor Subfamily 1 Group D Member 1(NR1D1),and AQP4 polarization lost the rhythmicity in the prefrontal cortex of CUMS mice.Melatonin rectified the abnormal expression of PER2,BMAL1,CLOCK,PER1,and AQP4 polarization after CUMS.Among them,there existed a negative correlation between PER2 and AQP4 polarization.Decreasing PER2 expression in primary cultured astrocytes by siRNA transfection led to increased AQP4 polarization and Dystrobrevin alpha(DTNA)expression,while increasing PER2 expression in primary cultured astrocytes by overexpression plasmid transfection led to decreased AQP4 polarization and DTNA expression.Conclusions:Melatonin treatment ameliorated depressive symptom and cognitive impairment in CUMS mice by increasing AQP4 polarization and thus rescuing glymphatic system dysfunction.The regulation of melatonin to AQP4 polarization was mediated by PER2-DTNA-AQP4 pathway.Part Ⅰ:Melatonin ameliorated depressive symptoms,cognitive impairment,and circadian rhythm sleep-wake disorder in CUMS mice modelObjective:To evaluate the depressive symptoms,cognitive impairment,and circadian rhythm sleep-wake disorder in CUMS mice,and to evaluate the therapeutic effects of melatonin on the model mice.Methods:In CUMS mice,depressive behaviors were identified by open field test,sucrose preference test,and tail suspension test.Cognitive ability was identified by Morris water maze test.Circadian rhythm sleep-wake state was identified by continuous 24 hours EEG/EMG monitor.Results:Open field test,sucrose preference test,and tail suspension test indicated that CUMS mice exhibited obvious depressive behaviors,characterized by decreased total distance in the open field,decreased preference to sucrose,and shortened immobility latency during tail suspension,while melatonin rescued the above symptoms.Morris water maze indicated cognitive impairment in CUMS mice,characterized by prolonged platform latency and decreased percentage in platform zone,while melatonin improved the cognitive ability.Continuous 24 hours EEG/EMG monitor indicated the circadian rhythm sleep-wake disturbance in CUMS mice,characterized by decreased percentage of NREM sleep in the light phase,shortened latency of REM sleep,and shortened sleep bouts duration,while melatonin ameliorated the disorders.Conclusions:CUMS mice manifested depressive symptoms,cognitive impairment and circadian rhythm sleep-wake disorder,which were similar in clinical major depression disorder patients.Melatonin ameliorated the above symptoms.Part Ⅱ:Melatonin improved depressive symptoms and cognitive impairment in CUMS mice by regulating the Glymphatic functionObjective:To explore the possible mechanism in which melatonin improved depressive symptoms and cognitive impairment in CUMS mice.Methods:Expression of MT1 and MT2 in mice prefrontal cortex was measured by Western Blot.Interstitial fluid in mice prefrontal cortex was acquired by microdialysis,and the levels of neurotransmitters were measured by LC-MS.Glymphatic influx function was measured by multi-scale optical imaging after CSF tracer injected via cisterna magna,while efflux function by ELISA after stereotaxic injection of exogenous β amyloid into prefrontal cortex.AQP4 polarization were detected by immunostaining of AQP4,vascular endothelial marker CD31,and astrocytic marker GFAP combining with the Donut analysis and the Vertical Line analysis.The glymphatic function was blocked by polarized AQP4 inhibitor TGN020.Results:The expression of MT1 was increased in the prefrontal cortex of CUMS mice,while MT2 remained unchanged.Melatonin decreased the expression of MT1,and had no obvious influence on MT2.The levels of 5-HT,dopamine,and norepinephrine in the interstitial fluid in CUMS mice prefrontal cortex were obviously decreased,while they remained unchanged after melatonin treatment.The number of astrocytes and the total length of astrocytic processes were decreased after CUMS,while melatonin treatment could not rescue these changes.The influx and efflux function of glymphatic system were decreased in CUMS mice,and melatonin rescued the impaired influx and efflux glymphatic system.AQP4 depolarization was occurred in CUMS mice,and melatonin improved the AQP4 polarization.After inhibiting polarized AQP4 by TGN020,the therapeutic effects of melatonin on glymphatic system,depressive symptoms,and cognitive disturbance in CUMS mice were partly blocked.Conclusions:Melatonin improved CUMS depressive symptoms and cognitive impairment by restoring glymphatic dysfunction,which was mediated by increased AQP4 polarization.Part Ⅲ:Melatonin regulated AQP4 polarization via PER2-DTNA-AQP4 pathwayObjective:To explore the possible molecular mechanism how melatonin regulated AQP4 polarization and thus regulated glymphatic system function.Methods:The expression rhythms of circadian proteins including CLOCK,BMAL1,PER1,PER2,CRY1,CRY2,NR1D1,AQP4 total amount and polarization in prefrontal cortex were detected by western blots and immunofluorescence staining at certain zeitgeber times(ZT).The rhythmicity of the proteins was further analyzed by CircaCompare R package.The correlations between circadian proteins and AQP4 were calculated by Pearson correlation analysis.The expression of circadian protein PER2 was regulated by transfection of siRNA or overexpression plasmid in primary cultured astrocytes.Results:The expression of circadian proteins PER2,BMAL1,CLOCK,PER1,NR1D1 and AQP4 polarization lost the rhythmicity in the prefrontal cortex of CUMS mice.Melatonin rectified the abnormal expression of PER2,BMAL1,CLOCK,PER1,and AQP4 polarization after CUMS.Among them,PER2 was negatively relevant to AQP4 polarization.Decreasing PER2 expression in primary cultured astrocytes by siRNA transfection led to increased AQP4 polarization and DTNA expression,while increasing PER2 expression in primary cultured astrocytes by overexpression plasmid transfection led to decreased AQP4 polarization and DTNA expression.Conclusions:Melatonin rectified the rhythmicity of certain circadian proteins and AQP4 polarization in CUMS mice.Circadian protein PER2 regulated AQP4 polarization via DTNA.