Lentiviral Aβ_1-42 Affects The Expression Of CV3CL1 And Regulates The Activation Of Microglia

Alzheimer’s disease（AD）is a common neurodegenerative disease in the elderly people.The etiology of AD is still unclear,and it is lack of effective diagnosis and treatment measures at present.Aβ（amyloidβ）,the main toxic fragments being Aβ₄₂,is mainly deposited in neurons at the early stage of AD,and gradually accumulated extracellularly with the disease progress.Microglial activation induced by Aβis one of the most important mechanisms of AD.CX3C chemokine ligand 1（CX3CL1）contacts with its sole receptor CX3CR1 is an important signal between neurons and microglia,regulating microglia activation and the release of inflammatory cytokines.Our previous studies found that microinjection of Aβ_1-42-42 Lentivirus in brain induced increased Aβ₄₂ deposition in neurons,while decreased Aβ₄₂ positive microglial in APOE4-TR mice,and Aβ₄₂2 mainly located intraneurons in young mice,both intraneuronal and extracellular Aβ₄₂2 was observed in senile mice.These results indicated there is a dynamic process that Aβ₄₂ deposition from intracellular to extracellular in the progression of AD and the balance of Aβ₄₂2 between neurons and microglial changing.The current study involved the effects of Aβ₄₂deposition on the expression of CX3CL1/CX3CR1 and the activation of microglia in the pathological procession that Aβ₄₂ from intraneuronal deposition to extracellular accumulation in AD.Firstly,we detected the distribution of Aβ₄₂ at the two weeks after microinjection of Aβ_1-42-42 Lentivirus into cortex of young and old C57 mice.Secondly,we observed the distribution of Aβ₄₂ in the primary neuron and microglia co-culture system on day 1,2,4,7,10,12 after treated with Aβ_1-42 Lentivirus.Subsequently,Microglia number,morphology,CD86（M1）and CD206（M2）,inflammatory factors TNF-α、IL-1βand anti-inflammatory IL-10 were also measured to test the effect of Aβ₄₂2 on microglia activitation.Besides,the mRNA and protein expression detection of CX3CL1 and CX3CR1,the effects of CX3CR1 neutralizing antibody on Aβ₄₂ induced microglia activation were detected to analyse Aβ₄₂ induced signal transmission between neuron and microglia.Two weeks after microinjection of Aβ_1-42-42 Lentivirus in the cortex of C57 mice,Aβ₄₂2 mainly located in intraneurons of young mice,and extracellular Aβ₄₂2 was observed in senile mice in addition to intraneuronal deposition of Aβ₄₂.Consistently,in the primary neuron and microglia co-culture system,Aβ₄₂ deposition occurred in neurons at the next day,significantly increased on the fourth day,and extracellular Aβ₄₂deposition increase gradually with the extension of time.Aβ₄₂ deposition appeared in microglia at the fllowing day and then persisted.Moreover,lentiviral Aβ_1-42 induced tau phosphoralation was also observed in vitro.In the primary neuron and microglia co-culture system,microglia were normally in resting state（small cell body,synaptic slender）.Microglia shifted to an activated state（bigger cell body,shorter and enlargement synapses）after the treatment of lentiviral Aβ_1-42 while mostly remained in resting state in lentiviral lacz control group.Compared with lentiviral lacz control,the expression of CD206（M2）did not changed obvious at day 1-4 and the expression of CD86（M1）increased at day 2-4 after treated with Aβ_1-42 Lentivirus,and both of them decreased significantly from the 7th day.Moreover,lentiviral Aβ_1-42treatment significantly increased IL-1βexpression at day 4-7,increased TNF-αexpression at day 4 and decreased its expression at day 12,increased IL-10 expression at day 4-7 and decreased its expression at the12th day.RT-PCR and western blot showed CX3CL1 and its receptor CX3CR1 expression did not changed obviously from 1to 4 days after the treatment of Aβ₄₂ Lentivirus and began to decline at day 7.Furthermore,the administration of CX3CR1 neutralizing antibody enhanced Aβ₄₂induced microglia activation and the expression of inflammatory factors at the early stage and subsequently decreased the M2 microglia activation.In conclusion,the present study set up an in vitro model that could imitate the AD pathological process of Aβ₄₂ accumulation from intraneurons to the extracellular,which can be used sepecifically to study the Aβ₄₂ deposition and subsequent microglia activation for AD pathogenesis,prevention and treatment.And based on this model,we found intraneuronal deposition of Aβ₄₂2 stimulate the activation of both M1 and M2 microglia,promote extracellular Aβ₄₂ accumulation,and CX3CL1/CX3CR1 is involved in the process and regulate the activation of microglia.