The Protective Effect Of Lithium On Aβ_1-42-Induced Hippocampal Neuronal Injury By Suppressing Astrocytes Glutamate Release

ObjectiveSynaptic loss is the cardinal feature linking neuropathology to cognitive decline in Alzheimer’s disease（AD）,however,the mechanism of synaptic damage remains incompletely understood,inhibition of Glutamate-induced neurotoxicity has potential therapeutic benefit for the treatment or prevention of early AD,but there is still a lack of effective treatments and drug targets.Our previous study had shown that pretreatment with lithium chronic 2 weeks could both significant suppressed Ad A and 4-CMC-evoked the increase of intracellular Ca²⁺concentration.In this study,we observe the inhibitory effect and mechanism of lithium chloride on glutamate release from astrocytes induced by Aβ_1-42,and also its protective effect on hippocampal neuronal.MethodsPrimary cultures of astrocytes were prepared from the neopallia of the cerebral hemispheres of newborn CD-1 mice,after astrocytes mature,the cells were treated with lithium chloride(0.25,0.5,1 mmol×L^-1)for 2 weeks,then,minipuls pumb was used at a rate of 0.5ml/min to stimulate astrocyte,the released glutamate induced by Aβ_1-42(250 nmol×L^-1)was measured by HPLC method,Cultured astrocytes grown on coverslips coated with polylysine were incubated with fura-2 for 30 min,then,Olympus IX71 live cell imaging fluorescence microscope was used to record fluorescence intensity of fura-2to determine intracellular Ca²⁺concentration,The Ca²⁺channel protein expressions of NCX1,Cav1.2 and TRPC1 were measured by western blot.The primary cultured astrocytes were exposed to Aβ_1-42(250 nmol×L^-1)for 48 h after with and without pretreatment with various concentrations of lithium chloride(0.25,0.5,1 mmol×L^-1)for 2 weeks,as a control,Aβ_1-42was incubated in astrocyte culture medium,in the absence of cells,for 48 h.Then,astrocytic conditioned medium（ACM）was added respectively to cultured hippocampal neurons for 24 h,the supernatants of neurons were collected and NO generation was measured by Griess method,the total dendrite branch length（TDBL）,number of primary-order dendrite（PDN）,maximum branch order（MBO）of hippocampal neurons were observed by inverted phase-contrast microscopy.ResultsAfter administration of 250 nmol×L^-1Aβ_1-42,Ca²⁺fluorescenceintensity and glutamate release of astrocytes were significantly increased,Aβevoked the increase of intracellular Ca²⁺concentration both from L-type VDCC and internal stores.Lithium chronic 2 weeks could significant suppressed Ab-evoked the increase of intracellular Ca²⁺concentration and capacitative calcium influx,Lithium chronic 2 weeks also suppressed the consequent Ca²⁺-dependent release of glutamate from astrocyte（P<0.05）.Compared with control group astrocytes,the protein levels of TRPC1 in lithium treatment group cells were significantly decreased（P<0.05）,but there were no obvious effects on the expression of NCX1,cav1.2 proteins.Cultured hippocampal neurons treated with ACM promote NO generation,and also accompanied the decreases in neuronal TDBL,PDN,MBO,Astrocytes pretreatment with Lithium decrease NO generation and also revers the ingury changes of neurons.ConclutionThe possible inhibitory mechanism of lithium on Aβ-induced Ca²⁺-dependent astrocyte glutamate release was by decrease the protein expression of TRPC1,which may be related to the neuroprotective effect.