Physiological Aβ Clearance Of The Liver And Its Role In Alzheimer’s Disease

Background and objectiveAlzheimer’s disease(AD)is the most common type of dementia,and no disease-modifying treatments are available to halt or slow its progression to date.Amyloid-β(Aβ)deposition in the brain has been widely implicated in AD and regarded as a key pathological event.Impairment of Aβ clearance is considered as the main cause of sporadic AD,which accounts for 99%of all AD cases.Therefore,improving brain Aβ clearance has become a promising therapeutic strategy.Previous studies on Aβ clearance have focused on the central nerve system.Recent studies found that there exists a link between the brain and periphery Aβ,and Aβ clearance in the periphery also contributes substantially to reducing Aβ accumulation in the brain.Indeed,several pathways,including the blood-brain barrier pathway,lymphatic-related pathway have been shown to mediate Aβ effluxes from the brain into the periphery.It has been suggested that 40%-60%of brain-derived Aβ is cleared via peripheral tissue and organ.However,it is unclear where and how these brain-derived Aβ peptides are cleared in the periphery.Hence,understanding the mechanism of how Aβ is cleared in the periphery will provide novel insight into the pathogenesis of AD and present new opportunities for systemic interventions.The liver is the largest metabolism organ and likely be responsible for clearing Aβ from the blood.Genome-wide meta-analysis indicated that AD-associated genes are strongly expressed in immune-related tissues and cell types,which include liver cells.However,the evidence about the hepatic physiological Aβ clearance function is lacking.The current study investigated the physiological function of the liver in clearing Aβ from the blood and brain,explored the role of impaired hepatic Aβ clearance in AD pathogenesis,and the therapeutic potential of enhanced hepatic Aβ clearance for AD intervention.Materials and methodsThis study was approved by the Institutional Review Board of XXX.All participants provided informed consent.The liver sections from human and wild-type(wt)mice and APP/PS1 transgenic(Tg)mice were obtained.A series of 8 μm frozen sections of liver tissue were stained via immunostaining.Female mice and rabbits were used in our study.To demonstrate Aβ uptake by the liver,6-9 months APP/PS1 mice and their age-and weight-matched wt littermates on a C57BL/6 background were selected for establishing parabiosis model following our previous study.In optical near-infrared imaging experiments,3-and 15-month-old APP/PS1 mice received an injection of synthetic Cy5.5-labeled Aβ42 via tail vein,dynamic changes of fluorescence intensity in the hepatic area were detected.3-month-old rabbits were used to explore the hepatic capacity of Aβ clearance.5 milliliters of blood were collected from the hepatic artery,portal vein,and hepatic vein of rabbits.To evaluate hepatocyte Aβ uptake capacity with age,primary hepatocytes of 3-and 15-month-old mice were isolated as previously reported.Hepatocytes are separated from other cells via differential centrifugation and CD45(-)CD146(-)immunomagnetic bead sorting,and then cultured with FITC-labeled Aβ42 followed by Flow Cytometry analysis.To investigate the acute influence of hepatic Aβ clearance on the dynamics of Aβ in the blood and the brain,6-month-old APP/PS1 mice were subjected to hepatic portal vein ligation,interstitial space fluid(ISF)were collected every 2 hours by microdialysis.To investigate the chronic influence of hepatic Aβ clearance on AD pathologies and progression,rAAV2/8-hTBG-shRNA(Lrp1)-eGFP-WPRE-pA virus and rAAV2/8-hTBGshRNA(scramble)-eGFP-WPRE-pA control virus was used to specifically knockdown liver LRP-1 in APP/PS1 mice at 3 months of age to reduce hepatic Aβ clearance;virus AAV2/8-hTBG-Lrp1_mini_DomIV-3HA-pA and control virus A AV2/8-hTB G-eGFPWPRE-pA were used to specifically express functional liver LRP-1 minigene(mLRP-1)in APP/PS1 mice at 6-month-old to enhance hepatic Aβ clearance.Mice were sacrificed and analyzed after 6 months of virus injection.The Morris water maze,Y-maze test,and open-field test were conducted to assess the behavior performance.Aβ burden,tau phosphorylation,neuroinflammation,neurodegeneration in mice were measured by Congo-red staining,immunohistochemistry,immunofluorescence staining,Golgi staining,and enzyme-linked immunosorbent assay(ELISA).The levels of LRP-1 and Aβ clearance-related proteins and enzymes were Western blottings.To investigate the correlations of angiostatin with AD common biomarkers.A cross-sectional study was conducted including 35 cognitively normal controls(CN)and 59 amyloid-positive AD dementia patients.Characteristics and medical history were collected.Fasting blood was sampled.The levels of angiostatin in plasma,and Aβ and tau in CSF,were examined by enzyme-linked immunosorbent assay(ELISA).A single-molecule array(SIMOA)was used to detect plasma Aβ levels.The data are expressed as the mean ± SEM.All analyses were completed using SPSS 20.0 software.Comparisons between two groups were made by two-tailed Student’s t-test,paired t-test,or Mann-Whitney U test as appropriate.Comparisons among multiple groups were made by one-way ANOVA followed by the least significant difference(LSD)test.Normality and equal variance tests were performed for all assays.A p-value<0.05 was considered statistically significant.Analyses in this study were done with blind evaluation.All figures were plotted using GraphPad Prism software.Results1.The physiological function of hepatic Aβ clearance.(1)Aβ deposits in the liver:Aβ staining was detected in the liver of both human and AD mice,especially in hepatocytes.Then,using the parabiosis model of AD and WT mice,considerable human Aβ40 and Aβ42 peptides were detected in the liver homogenates of paraWT mice.Furthermore,accumulation of Cy5.5-labeled Aβ increased in the liver after intravenous injection and the fluorescence intensity of Cy5.5-Aβ in the liver gradually decreased after reaching the peak at 4-7h.(2)The liver physiologically clears Aβ:Aβ40 and Aβ42 levels in liver outflow blood were respectively 13.9%and 8.9%lower than those in the inflow blood.2.The function of hepatic Aβ clearance declined with age.(1)The impact of age on hepatic Aβ clearance:The dynamic changes of intravenously injected Cy5.5-Aβ42 in the liver in young(3-month-old)and aged(15-month-old)mice were compared.The max fluorescence intensity of the hepatic area was weaker and the peak arrival time was longer in aged mice than those in young mice.Besides,the increased concentration ratio of Aβ in liver outflow and inflow blood(Qout/in)were found in aged rabbits compared with young ones.(2)The mechanism of compromised hepatic Aβ clearance capability in aging:The total fluorescence intensity and the percentage of FITC-positive hepatocytes were lower in aged mice than those in young mice in Flow Cytometry analysis.Reduced LRP-1 levels in liver homogenates of aged mice compared to young ones.3.The effect of hepatic Aβ clearance on the levels of Aβ in blood and the brain.The blood and brain Aβ levels increased rapidly after the liver portal vein ligation to block the hepatic blood flow in part.4.The effect of chronic hepatic Aβ clearance dysfunction on cognitive impairment and AD-related pathological damage in the brain.(1)Chronic hepatic Aβ clearance dysfunction aggravates cognitive impairments:AD-AAVshLRP1 mice performed worse in cognitive-behavioral tests than AD-AAVvehicie mice.(2)Chronic hepatic Aβ clearance dysfunction aggravates AD-type pathologies:Blood Aβ40 and Aβ42 levels of the AD-AAVshLRP1 mice were higher than those of controls.AD-AAVshLRP1 mice showed a higher Aβ burden in the brain than controls.The liver-specific LRP-1 knockdown aggravates AD pathologies.5.The effect of enhancing hepatic Aβ clearance on cognitive-behavioral deficits and AD-related pathologies.(1)Enhancing hepatic Aβ clearance alleviates cognitive impairments:AD-AAVmLRP1 mice performed better in cognitive-behavioral tests than AD-AAVeGFP mice.(2)Enhancing hepatic Aβ clearance alleviates AD-type pathologies:Brain Aβ burdens were decreased in the AD-AAVmLRP1 mice.Besides,AD-AAVmLRP1 mice had lower levels of AD pathologies.6.Associations of plasma angiostatin and Aβ and tau levels in Alzheimer’s disease.Plasma angiostatin levels were decreased in AD dementia patients compared to CN.Plasma angiostatin levels were negatively correlated with plasma Aβ42 and Aβ40 levels in AD and positively correlated with CSF total tau(t-tau)and t-tau/Aβ42 levels in APOEε4+AD dementia patients.In addition,plasma angiostatin levels had the potential to distinguish AD from CN.ConclusionThe liver can physiologically clear circulating Aβ,and this capability decreases with aging.Comprised hepatic Aβ clearance increases blood and brain Aβ levels,aggravates AD-type pathologies and cognitive-behavioral deficits.Enhanced hepatic Aβ clearance reduces blood and brain Aβ levels,alleviates AD-type pathologies and cognitive-behavioral impairments in AD mice.Our findings indicate that impaired hepatic Aβ clearance affects AD pathogenesis,and enhancing hepatic Aβ clearance may provide a novel approach for AD intervention.