Physiological Aβ Clearance Of The Spleen And The Effect Of Its Dysfunction On The Pathogenesis Of Alzheimer’s Disease

BackgroundAlzheimer’s disease(AD)is the most leading cause for dementia,with a high incidence,thereby bringing a grave burden to society and patients.AD contains the main clinical characteristics of progressive cognitive impairment and memory loss.However,due to the obscure mechanisms underlying AD,there is currently no effective treatment avaliable.The prodominent pathological changes of AD are amyloid-β(Aβ)accumulation and neurofibril tangle composed by over phosphorylated tau protein.Most AD patients are sporadic AD,which is caused by insufficient Aβ clearance in brain.Therefore,enhanceing clearance for Aβin brain may hold the promise for curing AD.The Aβ clearance in brain is mainly based on phagocytosis of microglia and degradation of proteases.It is estimated that 40%-60% of brain-derived Aβ is transferred into other tissues outside the brain for clearance,suggesting that the peripheral system plays a vital role in the clearance for brain-derived Aβ.However,the specific action site for Aβ clearance in the peripheral system remains elusive.Our previous study found that radioelement-labeled Aβ was detected in the spleen after being injected into the cerebellar medulla cisterna.Moreover,spleen is a crucial immune organ and comprises numerous mononuclear macrophages.It is shown in previous studies that mononuclear macrophages can effectively phagocytose Aβ.Therefore,we speculate that spleen may have a capacity for Aβ clearance.Our study aimed to investigate whether spleen can physiologically scavenge Aβ and the impact of physiological Aβ clearing function of spleen on blood and brain Aβ levels.MethodsStudy 1: Investigation on the physiological scavenging capacity of Aβ of spleen(1)Mononuclear macrophages isolated from by immunomagnetic beads were co-cultured with FITC-labeled Aβ.Flow cytometry was ultilized to dectect Aβ and CD115double-positive cells.(2)Comparison of Aβ levels between splenic artery and splenic vein were conducted by enzyme Linked immunosorbent assay(ELISA).(3)FITC labeled Aβ was injected into the tail vein of mice,and spleen was collected 1 hour after injection.After frozen sections were prepared a stained with F4/80 antibody,FITC and F4/80 double positive cells were captured by confocal laser microscopy.Study 2: Effects of physiological Aβ clearance dysfunction in the spleen on blood immune cells in AD mice4-month-old APP/PS1 mouse models of splenectomy were established to simulate the physiological Aβ clearance dysfunction of spleen.Five months after splenectomy,flow cytometry was performed to analyze the frequency of immune cells in peripheral blood.Study 3: Effects of physiological Aβ clearance dysfunction in the spleen on Aβ levels,AD-related pathology,and cognitive function in AD miceThe experimental groups were as follows :(1)wild-type mice(WT)sham group;(2)WT mice splenectomy group;(3)AD mice sham group;(4)AD mice splenectomy group.Five months after splenectomy,Aβ42 and Aβ40 levels in both blood and brain were detected by ELISA.Y maze test,morris water maze test,etc.were used to analyze the cognitive function.Aβ burden,neuroinflammation,neurodegeneration and Tau phosphorylation in brain of AD mice were detected by immunohistochemistry,immunofluorescence staining and western blot.ResultsStudy 1: Investigation on the physiological scavenging capacity of Aβ of spleenSplenic mononuclear macrophages can directly engulf fluorescent-labeled Aβ in vivo and in vitro.Level of Aβ in splenic artery were significantly higher than that in splenic vein(P<0.05).Study 2: Effects of physiological Aβ clearance dysfunction in the spleen on blood immune cells in AD miceCompared with WT sham mice,the proportion of blood T cells in AD sham mice was significantly decreased(P<0.05),while splenectomy had no striking effect on that in AD mice(P>0.05).In addition,the proportion of blood mononuclear macrophages was significantly intensified in the AD sham group in comparision with WT sham group(P<0.01),wherease splenectomy significantly attenuated the proportion of blood mononuclear macrophages and B cells in the AD mice(P<0.01).Study 3: Effects of physiological Aβ clearance dysfunction in the spleen on Aβ levels,AD-related pathology,and cognitive function in AD miceSplenectomy had no significant impact on cognitive function of WT mice;Compared with the AD sham group,AD splenectomy mice had significantly worsened cognitive impairment,increased Aβ levels in blood and brain,enchanced neuroinflammation,neuronal degeneration and apoptosis as well as more phosphorylated Tau protein in brain.ConclusionIn conclusion,our study shows that the spleen can exert physiological Aβ scavenging effect through mononuclear macrophages.The decreased clearing ability for Aβ in spleen may promote the development of AD.