Amyloid-β Uptake By Monocyte And Its Potential Intervention For Alzheimer’s Disease

Background and objectiveAlzheimer’s disease（AD）is the most common neurodegenerative disorder affecting 35million elderly individuals^[1].Aβis deemed as the major pathogenic hallmark of AD.Evidence suggests that there exists imbalance between Aβoverproduction（familial AD）and defective clearance（sporadic AD）in the AD brain.Besides,the balance of Aβis not only regulated by Aβcatabolism in the brain,but also by dynamic exchange of Aβbetween the the brain and peripheral system^[2,3].Previous studies found that approximately 40%-60%of Aβgenerated in the brain is estimated to diffuse into the blood and be cleared in the periphery^[4-6],but it remains undetermined how this brain-derived Aβis cleared in the periphery.Previous evidences demonstrated that deletion or impairment of recruitment of peripheral monocytes will lead to increased plaque burden^[7,8].Monocytes,as the counterparts of microglia in the periphery system,were demonstrated to be more effective at neuroprotection,neuroinflammation regulation and Aβclearance than microglia in AD^[9-11].Based on these findings,monocytes are regarded as an essential role in peripheral Aβclearance in AD.Despite the critical role of monocyte in the pathogenesis of AD,genetic association studies have identified various AD risk loci within the,phagocytic,endosomal and lysosomal pathways in sporadic AD^[12-16].These findings suggest that function of peripheral myeloid cells,may be damaged in AD.However,the role of Aβuptake by monocytes in peripheral Aβclearance and its alterations during ageing and in AD remain unclear.Whether enhancement of the function of monocytes is an effective intervention for AD also needs further investigation.So the present study was aimed to investigate alterations in Aβuptake by peripheral monocytes during ageing in cognitively normal（CN）subjects and in sporadic AD patients and to evaluate the role of peripheral monocytes in the uptake of Aβin the blood.Besides,we intended to investigate the potential intervening effects for AD by enhancement of the Aβcatabolism by monocytes.Materials and methods1.Correlation between Aβ₄₂ uptake by monocytes and Aβlevels in the bloodA total of 25 cognitively normal（CN）subjects were recruited from Daping Hospital between March 2017 and May 2019.Human peripheral blood mononuclear cells（PBMCs）were isolated by density gradient centrifugation using Ficoll-Hypaque.To test the uptake of Aβ,human PBMCs were incubated with FITC-Aβ₄₂（2μg/ml）for overnight.Flow cytometry and imaging flow cytometry were used for screening of monocyte subsets and analysis of Aβ₄₂uptake by each subset.Plasma Aβ₄₂ and Aβ₄₀levels were measured using an ultrasensitive single molecule array（SIMOA）on the Simoa HD-1 Analyser（Quanterix,Lexington,MA）.Covariate correlation analysis was utilized to analyse the association of Aβ₄₂uptake with blood Aβlevels.2.Study on alterations of Aβ₄₂ uptake by monocyte subsets during ageingWe enrolled 104 cognitively normal participants aged 22 to 89 years old from physical examination center of Daping Hospital.Density gradient centrifugation and flow cytometry were used for detection of Aβ₄₂uptake by monocyte subsets.A Spearman correlation analysis was utilized to analyse the association of Aβ₄₂ uptake with age.The trajectory of Aβ₄₂ uptake by monocytes with age was modelled using Fit spline/Lowess（cubic）spline.3.Study on alterations of Aβ₄₂ uptake by monocyte subsets in AD（1）Alterations of Aβ₄₂ uptake by monocyte subsets in ADTo investigate whether the alteration in Aβ₄₂ uptake by monocytes is specific to AD patients,24 AD patients,10 stroke patients and their matched CNs were enrolled.Density gradient centrifugation and flow cytometry were used for analysis of Aβ₄₂uptake by monocyte subsets.Student’s t test was used for comparisons between two groups.（2）Expression of Aβ₄₂ uptake-related receptor TLR2 of monocyte is decreased in AD patientsTo investigate the potential mechanisms underlying the deficient Aβ₄₂ uptake by monocyte subsets in AD,we used immunomagnetic bead separation and flow cytometry to analyse the expressions of the Aβ-uptake mediating receptors in monocytes,including Toll-like receptor 2（TLR2）,triggering receptor expressed on myeloid cells 2（TREM2）,CD36,CD33,and macrophage scavenger receptor 1（MSR1）.In addition,western blot was used to make comparisons of the levels of lysosomal Aβ-degrading enzymes between AD and CN.4.Intervention and mechanism of enhancing Aβcatabolism by monocytes in AD（1）Effects of PSK on catabolism of Aβ₄₂by human monocytes in vitro.Previous study indicates that decreased expression of TLR2,an Aβ-uptake mediating receptor in monocytes,may be involved with deficient Aβuptake in AD.In the present study,we used a novel TLR2 agonist,namely polysaccharide krestin（PSK）,and incubated it with human monocytes.Immunomagnetic bead separation and flow cytometry were utilized for analysis of effect of PSK on Aβuptake and its mediating receptors in monocytes.Confocal microscopy,ELISA and western blot were implied for analysis of effect of PSK on monocyte intracellular Aβprocessing and degradation.（2）Effect of PSK on cognitive function in APP/PS1 mousePrior to initiation of Aβdeposition,APP/PS1 transgenic mice aged 3 month were randomly assigned to be applied with PSK（2mg/mouse,3 times/week for 6 months,n=8 per group）and control applied with PBS（3 times/week for 6 months,n=8 per group）,and age-and sex-matched wild type controls（n=8）were treated with same volume of PBS.Prior to sample collection,all mice were with different treatments were applied with various behavioral tests,including Morris water maze,Y-maze and open field.（3）Effect of PSK on Aβburden in APP/PS1 miceFlow cytometry and imaging flow cytometry were used for analysis of effects of PSK on ratio of monocyte subsets and Aβuptake in APP/PS1 mice.After sample collection,half of the hemisphere per animal was frozen or fixed in 4%paraformaldehyde for biochemical analysis or histological analysis.Histochemical staining and ELISA were used for detection of Aβlevels in brain and blood of APP/PS1 mice.（4）Effect of PSK on AD-related pathologies in the brains of APP/PS1 miceAfter sample collection,half of the hemisphere per animal was frozen or fixed in 4%paraformaldehyde for biochemical analysis or histological analysis.Immunohistochemcal staining,double immunofluorescence staining and ELISA were implied for analysis of neuroinflammation,neurodegeneration and tau phosphorylation in APP/PS1 mice.Results1.Aβ₄₂ uptake by monocytes is negatively correlated with Aβlevels in the blood.We found that all subsets of monocytes have the ability to uptake Aβ₄₂,with the CD14⁺CD16⁺subset having the highest uptake of Aβ₄₂ among the three subsets.After controlling the cofounding factors of age,sex,vascular risk factors and APOEε4 genotype,the partial correlation analysis showed that Aβ₄₂ uptake by total monocytes,CD14⁺CD16^-and CD14^dimCD16⁺subsets were negatively correlated with plasma Aβ₄₂ levels in CN subjects.Moreover,Aβ₄₂ uptake by total monocytes,CD14⁺CD16^-and CD14⁺CD16⁺subsets were negatively correlated with plasma Aβ₄₀ levels2.Alterations of Aβ₄₂ uptake by monocyte subsets during ageingThen,we measured Aβ₄₂ uptake by monocytes in 104 CN subjects aged 22 to 89 years who were not different in sex among the different age groups.Aβ₄₂ uptake by the total monocyte CD14⁺CD16^-and CD14^dimCD16⁺subsets were negatively correlated with age.Aβ₄₂uptake by total monocytes and the CD14⁺CD16^-subset decreased rapidly in the group aged20-40 years,but the reduction rate became relatively slow after 40 years of age.Aβ₄₂ uptake by CD14^dimCD16⁺subset decreased rapidly in the group aged 40-60 years,but the reduction rate became relatively slow after 60 years of age.These results suggest that the decrease in Aβuptake is a life-long process that may be different among the three monocyte subsets and occurs prior to the cerebral deposition of Aβ.3.Alterations of Aβ₄₂ uptake by monocyte subsets in AD（1）Aβ₄₂ uptake by monocytes is decreased in AD but not in stroke patients.To investigate whether the alteration in Aβ₄₂ uptake by monocytes is specific to AD patients,24 AD patients,10 stroke patients and their matched CNs were enrolled.There were no significant differences in sex,age,years of education,APOEε4 genotype,comorbidities including hypertension,hyperlipidaemia,and diabetes mellitus,or medications between the matched groups.We found that Aβ₄₂uptake by total monocytes and the various subsets was lower in AD patients than in CNs.There were no significant differences in Aβ₄₂ uptake by total monocytes and their subsets between stroke patients and CNs.（2）Expression of Aβ₄₂ uptake-related receptor TLR2 of monocyte is decreased in AD patients.We tested the major Aβ-uptake receptors involved with myeloid cell-mediated physiological uptake of Aβ,including Toll-like receptor 2（TLR2）,triggering receptor expressed on myeloid cells 2（TREM2）,CD36,CD33,and macrophage scavenger receptor 1（MSR1）.The expression of TLR2 was lower in AD patients than CNs.However,no differences were observed in the expressions of other four receptors between AD patients and CNs.To investigate whether internalized Aβ₄₂ was effectively digested by monocytes in AD,we tested the expression of two degrading lysosomal Aβenzymes,including cathepsin D and cathepsin S,and found no significant differences in the protein levels of cathepsin D and cathepsin S,between AD patients and CNs.4.Intervention and mechanism of enhancing Aβcatabolism by monocytes in AD（1）PSK enhances catabolism of Aβ₄₂by human monocytes in vitro.In the present study,we found that uptake of FITC-Aβ₄₂by monocyte was enhanced dependent on PSK concentration（0-200μg/ml,P<0.001）,with 100μg/ml being the best PSK stimulating concentration.Further,we examined whether degradation of internalized Aβ₄₂by monocyte was influenced by PSK and found that the degradation of Aβ₄₂ of monocyte was significantly enhanced by PSK.We tested the major surface receptors involved with myeloid cell-mediated physiological uptake of Aβand found that the only the expression of TLR2 was significantly enhanced by PSK.By using TLR2 antagonist before addition of PSK,we found that stimulating function of PSK was almost counteracted with lower Aβ₄₂uptake than monocytes without TLR2 antagonist.Calculation of the Aβ₄₂immunoactive area within EEA1⁺,Lamp1⁺,and Lamp2⁺revealed that monocyte treated with PSK had greater amounts of Aβ₄₂ in the endosomal-lysosomal pathway,contributing to more intracellular degradation.（2）PSK attenuates cognitive deficits in APP/PS1 mouse.In the Morris water maze,APP/PS1 mice,the mice treated with PSK showed significant decreased time of escape latency in the successive platform learning trials,and greater frequencies of annulus crossing and more time spent in targeted quadrant in the probe trial than PBS treated mice.In novel arm exploration test,PSK treated mice showed more entries into the novel arm than PBS treated mice.In spontaneous alteration test,the mice treated with PSK showed greater total entries than PBS treated mice.We also found a longer distance travelled in the PSK treated group in the open filed test.These results indicate that PSK could prevent cognitive decline in APP/PS1 mice.（3）PSK reduces Aβburden in APP/PS1 mice.To investigate whether PSK affects Aβburden in APP/PS1 mice,Congo red staining and Aβimmunostaining（6E10）for detection of compact amyloid plaques and total amyloid plaques were performed respectively.Compared with APP/PS1 controls,the mice treated with PSK showed significant lower amyloid plaque burden in the brain.By ELISA tests,mice treated with PSK also showed a significant reduction in the levels of Aβin brain homogenates,along with reduced Aβ₄₂level in blood.Next,we investigated the potential mechanisms underlying the reduction of Aβdeposition after PSK treatment.Except for Aβlevel,there were no significant differences in expressions of APP and its metabolites.Moreover,there were no significant difference in enzymes associated with Aβproduction and Aβ-degrading enzymes between the two groups.Interestingly,compared with control,level of Aβtransport receptor RAGE in homogenates was lower in PSK-treated mice with,suggesting that PSK reduces the receptor-mediated influx of Aβfrom periphery into brain through the BBB.Next,we used flow cytometry analysis to investigate the effects of PSK on Aβ₄₂uptake of monocyte in APP/PS1 mice in vivo.The mice monocytes were identified and gated as Ly6C^high and Ly6C^low/neg subsets.Although Aβ₄₂ uptake of Ly6C^low/negsubset was greater than Ly6C^highsubset,there was no significant difference in proportion of monocyte subsets between groups.Both Aβ₄₂ uptake and expression of TLR2 of mice monocytes were greater in PSK treated mice.But we didn’t find significant difference in Aβ₄₂uptake of microglia and TLR2expression in AD mouse brain.（4）PSK rescues AD-related pathologies in the brains of APP/PS1 mice.Compared with PBS control,microgliosis（detected by CD68 antibody）and astrocytosis（detected by GFAP antibody）in the PSK treated group were significantly decreased in the cortex.Additionally,the levels of proinflammatory cytokines in brain homogenates in the treatment group were also lower than PBS controls.APP/PS1 mice treated with PSK showed markedly increased staining area fraction of Neu N（neurons）,MAP2（dendrites）and decreased neuronal apoptosis（detected by caspase-3）in the hippocampus.Moreover the area fractions of Tau-phospho-Ser231–positive neurons in the neocortex of PSK-treated APP/PS1mice were significantly lower than those of the controls.Western blots further showed that Tau-phosphorylation at the site of serine 396 was significantly diminished in the brain of PSK treated mice than controls,with no statistically difference in expression of total Tau（Tau5）between the two groups.DiscussionIn the current study,we found that the Aβ₄₂ uptake ability is correlated with blood Aβlevels in cognitively normal subjects,that is,the greater the Aβuptake ability is,the lower the blood Aβlevels.This finding suggests that monocytes may play a critical role in clearing Aβfrom the blood.However,Aβ₄₂ uptake by monocytes decreased as with age.It is worth noting that the decrease in Aβuptake by classic monocyte subset and non-classic subset began at the age of20 years and 40 years respectively,suggesting that the decrease in Aβuptake ability is a life-long process that may be different among monocyte subsets and occur prior to brain Aβdeposition.Despite the impact of ageing,the Aβuptake ability of monocytes is further decreased in AD patients,implying that compromised Aβuptake by monocytes is involved in AD pathogenesis.Unlike ageing that Aβuptake by monocyte was mainly decreased in the classic and non-classic subsets,the Aβuptake ability was decreased in all subsets in AD patients,including the most effective intermediate subset as reflected by our findings.Besides,the intermediate subset is the main producer of IL-10 upon stimulation of Toll-like receptors.So dysfunctional CD14⁺CD16⁺subset not only affect Aβclearance in AD,but also may lead to reduction of IL-10,which is an anti-inflammatory cytokine and protective factor for neurogenesis and cognitive preservation,thus exacerbating the pathogenesis of AD.These results imply that the mechanisms underlying the alteration in Aβuptake ability by monocytes in AD patients are different from those associated with ageing.Additionally,the decrease in Aβuptake by monocytes seems specific to AD,as it was not changed in stroke patients compared with cognitively normal controls in our study.The mechanisms underlying the decreased Aβuptake ability by monocytes during ageing and AD remain to be investigated.We found that the decrease of Aβuptake in AD may be partially due to deficits in Aβrecognition by monocytes,as reflected by the reduced expression of TRL2 in monocytes.TRL2 belongs to a type I transmembrane pattern recognition receptor and acts as a natural innate immune receptor for Aβuptake through the formation of a receptor complex with CD14.Deletion or inhibition of the CD14-TLR receptor complex will impair fibrillary Aβ₄₂ uptake in human monocytes and delay cognitive decline in a mouse model of AD.Previous studies have explored the intracellular processing and degradation of Aβwithin monocyte.After receptor-mediated Aβendocytosis,early endosomes are formed and further combined with lysosomes for degradation.Thereinto,cathepsin D and cathepsin S,which are the two main lysosomal aspartic and cysteine proteases,were demonstrated to mediate Aβdegradation.However,we did not find any differences in the expression of these two Aβ-degrading enzymes between AD patients and cognitively normal controls.These results indicate that dysfunctional Aβrecognition could be crucial for the disability in Aβuptake by monocytes in AD,and activation of TLR2 may promote the Aβuptake of monocyte,thus alleviating Aβ-related pathogenesis.Previous study found that polysaccharide krestin（PSK）,an extract of Trametes versicolor,can both stimulate innate immune pathway and activate TLR2,thus facilitating the Aβuptake of monocyte.Our current study found that PSK,an approved prescription for treatment of multiple cancers,not only can stimulate TLR2-mediated uptake and intracellular metabolism of Aβby monocytes,but also can attenuate cognitive impairment in AD mouse model.In this research,we found that both the uptake and degradation of Aβby monocyte was enhanced by PSK.Among the major surface receptors involved with myeloid cell-mediated physiological uptake of Aβ,TLR2 expression was specifically increased with PSK,and this stimulating function of PSK was almost counteracted with TLR2 antagonist,suggesting that enhanced Aβuptake of monocyte by PSK may be mediated by activation of TLR2.Evaluation of immunoreactive area of colocalized Aβwith EEA1,LAMP1 and LAMP2 revealed that PSK treated monocyte had greater amounts of Aβin the endosomal-lysosomal pathway,indicating greater intracellular processing and degradation of Aβwith PSK.It is noteworthy that Aβlevel in brain and blood were significantly decreased by injection of PSK in APP/PS1 mice prior to onset of Aβdeposition.We didn’t find any difference in expression of APP and its metabolites,enzymes associated with Aβproduction and Aβ-degrading enzymes in brain of AD mice between the PSK treatment group and the control,which suggests that PSK may not take part in reduction of Aβproduction or enhancement of Aβdegradation in brain.The decreased level of Aβinflux transport receptors across the BBB（RAGE）were lower in PSK-treated mice than control indicates that PSK reduces the influx of Aβfrom periphery into brain through the BBB,making more room for PSK activated monocyte to play its role in peripheral Aβclearance.These results indicate that PSK may play its role mainly by enhancing Aβrecognition and processing of monocytes in the periphery,thus leading more brain soluble Aβoutflowed and cleared in the periphery.As expected,we found that PSK also plays role in neuroprotection and anti-inflammation reflected by increased neurons and down-regulation of neuronal apoptosis,dendritic spine shrinkage,microgliosis and astrocytosis in response to Aβ.Another hallmark of AD is neurofibrillary tangles（NFT）,which is caused by hyperphosphorylation of Tau protein and consistently correlated with cognitive function of patients.In the current study,we found that PSK can supress the hyperphosphorylation of Tau at various sites in response to Aβ.These findings indicate that PSK can suppress the toxicity of Tau hyperphosphorylation triggered by Aβ.ConclusionOur findings are of significance to the understanding of the pathogenesis of sporadic AD.Aβin the brain can be transported to the peripheral blood,and the clearance of Aβin the periphery has been suggested to substantially contribute to the clearance of Aβfrom the brain.Therefore,the decrease in Aβuptake by monocytes could play a significant role in the development of sporadic AD.Besides,we suggest the potential application of PSK for intervention of AD via Aβclearance by peripheral monocytes.Because PSK is currently used for treatment of cancer and proven safe in humans,the promising data from our current study warrant future clinical trials to test its therapeutic efficacy for AD.