Potential Roles Of Naturally Occurring Antibody To BACE1 And PS1 In The Pathogenesis Of Alzheimer’s Disease

Background and objectiveAlzheimer’s disease(AD)is an aging-related neurodegenerative disease characterized by progressive dementia.AD leads to impaired cognitive function and social function in patients,causing a heavy mental and economic burden to the family and the society.At present,there are no effective early diagnosis measures and prevention methods for AD,mainly because the pathogenesis of AD is not fully understood.Amyloid-beta(Aβ)is the core pathogenic substance of AD,and the increased production of Aβ in the brain plays an important role in AD.Aβ peptide is generated through sequential cleavage of amyloid precursor protein(APP)by β-secretases(beta-site amyloid precursor protein cleaving enzyme 1,BACE1)generating a C99 fragment and then y-secretase.BACE1 plays a pivotal role in the production of Aβ.Aging is the most important and well-defined risk factor for AD.During aging,the expression level and enzyme activity of BACE1 increased with age,thereby promoting the production of Aβ and the development of AD.Plasma BACE1 levels can predict the progression from mild cognitive impairment to AD during aging.Aging-related factors such as chronic inflammation,oxidative stress,and energy metabolism disorders all affect the level and activity of BACE1.Therefore,revealing the regulatory mechanism of the elevated BACE1 level and activity during aging is of great scientific significance for elucidating the pathogenesis of AD.As an important part of aging,immunosenescence can lead to abnormal functions of innate and adaptive immunity.On the one hand,this abnormal immune function will cause the immune system to incorrectly process immune information,enhance the recognition and reactivity of self-antigens,increase the level of autoantibodies and lead to autoimmune reactions;on the other hand,cause the ability of immune system to recognize exogenous antigens,necrotic cells and metabolites decreased,so that the body’s function of removing"alien" and excreting "waste" is weakened.In recent years,the role of autoimmunity in the occurrence and development of AD has been gradually revealed.The dysfunction of the immune system during aging is an important factor that promotes the development of AD.Animal experiments have also confirmed that AD mice have immune dysfunction(such as hepatosplenomegaly,systemic inflammation,abnormal humoral immune function,etc.),and this immune dysfunction occurs in AD-related pathological changes and recognition during the aging process.Before the decline of cognitive function,it is suggested that immune dysfunction may be one of the important factors that promote the evolution of aging to AD.Natural autoantibodies against self-antigens are widely present in human blood.Under physiological conditions,these autoantibodies play an important role in immune regulation,reducing inflammation,removing metabolic wastes and abnormal cells,and maintaining homeostasis,and changes in antibody levels may be involved in the disease.There are autoantibodies against various autoantigens in the brain(such as Aβ,tau,nerve cells,neurotransmitters and their receptors)in AD patients.Previous studies have found that there are various autoantibodies on the cortex and hippocampal neuron cell membranes of AD patients,and the level of autoantibodies in the cerebrospinal fluid of AD patients is related to brain atrophy,oxidative stress and energy metabolism in the brain,suggesting that humoral immunity is involved in Aβ metabolism and other AD pathological processes.Recent studies have shown that AD,mild cognitive impairment(MCI)patients and healthy people have different blood autoantibody profiles,which can be used as biomarkers for early diagnosis and clinical staging of AD.The above evidences all indicate that humoral immune dysfunction is involved in the evolution of aging to AD.We previously cooperated with the Australian Imaging,Biomarkers Lifestyle(AIBL)prospective cohort study,found that the blood of AD patients has disordered autoantibody spectrum by the chip screening technology,and the altered autoantibodies target the important proteins of AD pathological circuits such as APP,BACE1 and PS1.The above results suggest that the changes of these autoantibodies during aging may promote the development of AD by regulating important pathological circuits such as Aβ metabolism,deposition and pathophysiological functions.We conducted an initial exploration of the clinical relevance and pathophysiological functions of autoantibodies against important AD pathologic proteins such as BACE1 and PS1.Materials and methods1.Detection of plasma autoantibodies in AD patients.The Chinese cohort and the Australian AIBL cohort were included in this study.HuProtTM protein microarray was used to detect the levels of autoantibodies against about 20000 human autoantigens in the blood of 5 AD patients and 5 healthy controls from the Chinese cohort.Then,a customized chip(customized protein chip anchored autoantigens with the significant differences screened in stage 1)was used to further detect the levels of autoantibodies in the AIBL cohort(including 30 Clinical AD,70 Preclinical AD and 100 CN).2.The level and pathological mechanisms of plasma NAbs-BACE1 in AD2.1 The level and clinical relevance of plasma NAbs-BACE1 in AD(1)Detection of plasma NAbs-BACE1:The synthesized BACE1 protein was subjected to Western Blotting(WB),Intravenous Immunoglobulin(IVIg)and plasma of 3 random samples were used as primary antibodies,while monoclonal BACE1 antibody was used as positive control.(2)Quantitative detection of plasma NAbs-BACE1 and its clinical correlation with AD:The levels of blood AD biomarkers(Aβ42,Aβ40,Tau)in Chinese cohort were detected by the Single-molecule array(SIMOA)technology.Enzyme-linked immunosorbent assay(ELISA)was used to detect plasma levels of NAbs-BACE1 in Chinese cohort and AIBL cohort.The correlations between the levels of NAbs-BACE1 and MMSE score,PiB-PET standard uptake value ratio(SUVR)and the biomarkers were analyzed.2.2 The pathological mechanisms of NAbs-BACE1 in AD(1)Binding of NAbs-BACE1 with endogenous BACE1:NAbs-BACE1 was obtained from IVIg by the separation chromatography,and its specificity was verified.(2)The impact of NAbs-BACE1 on the cognitive function and the pathology of AD mice:8-month-old APP/PS1 mice were randomly divided into the administration group(n=8)and the control group(n=8).For the administration group,7μg of isolated NAbs-BACE1 was injected into the right lateral ventricle of mice by the brain stereotaxic injection technique.The control group was injected with the same amount of anti-rabbit IgG by the same method.This was done once a week for 8 weeks.Age-and sex-matched wild-type WT mice(n=8)were used as control.Morris water maze,Y maze and open field test were performed on the mice.Blood and brain tissue were then sampled from the mice.RNA sequencing was performed on the brain tissues.While Aβ deposition,Aβ metabolism,Tau protein pathology,neuroinflammation,neuron apoptosis and synaptic degeneration were assessed by Immunohistochemistry(IHC)and WB.(3)BACE1 expression in SY5Y-APP cells treated with different concentrations of NAbs-BACE1 was analyzed by the Immunofluorescence(IF).The level of Aβ and its metabolites were evaluated by the WB.Mitochondrial staining was used to detect the number of mitochondria and cell activity.And the CCK-8 cell survival assay was used to detect the cell proliferation.3.Detection of plasma NAbs-PS1 levels in AD patients.The study included the Chinese cohort.Participants’ demographics(including gender,age,years of education,etc.),medical history,fasting blood samples were collected,and cognitive function was assessed by MMSE.The levels of AD biomarkers(Aβ42,Aβ40,Tau)in the blood of subjects were detected by SIMOA technology.The plasma NAbs-PS1 levels of the subjects were detected by the ELISA,and then the correlations between the levels of NAbs-PS1 and subjects’ MMSE score,SUVR,and the biomarkers were analyzed.4.The relevance of the Severe acute respiratory syndrome coronavirus 2(SARSCoV-2)infection and cognitive function.This cross-sectional study recruited 1539 COVID-19 inpatients aged over 60 years who were discharged from three COVID-19-designated hospitals in Wuhan,China,from February 10 to April 10,2020.In total,466 uninfected spouses of COVID-19 patients were selected as controls.The current cognitive status was assessed using a Chinese version of the Telephone Interview of Cognitive Status-40(TICS-40)and the longitudinal cognitive decline was assessed using an Informant Questionnaire on Cognitive Decline in the Elderly(IQCODE).Cognitive assessments were performed 6 months after patient discharge.Results1.Changes in autoantibody profiles in ADAbout 150 different autoantibodies were screened out by HuProtTM protein chip technology.By evaluating the goodness of fit model,it was found that the sensitivity and specificity of autoantibodies in the diagnosis of AD can reach 88.9%and 95.5%,and the sensitivity and specificity of autoantibodies in the early diagnosis of AD can reach 100%and 86.7%.Pathway enrichment analysis showed that differential autoantibodies target important circuits such as cognition formation and synaptic transmission,including autoantibodies against APP,BACE 1,and PS 1.2.The level and the possible pathological mechanism of plasma NAbs-BACE1 in AD2.1 Increased plasma levels of NAbs-BACE1 in AD patients.The presence and relative level of NAbs-BACE1 in human plasma were confirmed by WB and ELISA.Plasma NAbs-BACE1 levels in AD patients were significantly higher than those in non-AD dementia and cognitively normal groups in the Chinese cohort.In the AIBL cohort,the level of NAbs-BACE1 in Clinical AD patients,then healthy controls.The plasma level of NAbs-BACE1 in all subjects was positively correlated with the amyloid load PiBPET SUVR(in the Chinese cohort)/the PET Amyloid Becket Score(in the AIBL study cohort)in the brain and negatively correlated with the MMSE score.Higher NAbs-BACE1 levels at baseline were positively correlated with the rate of the Aβ deposition and the rate of the cognitive decline.2.2 NAbs-BACE1 can promote the pathological progression of AD(1)WB confirmed that NAbs-BACE1 purified from IVIg can bind specifically to synthetic BACE1 protein,as well as BACE1 protein in human brain and mouse brain.(2)Compared with control APP/PS1 mice and WT mice,NAbs-BACE1 treated mice performed poorly in the Morris water maze and Y maze experiments.Compared with the control group,the expressions of AD-related pathological proteins such as BACE1,PS1,and GSK3β were up-regulated after NAbs-BACE1 treatment.Compared with the control group,the levels of the Aβ burden and the AD-related pathology(including Tau protein pathology,neuroinflammation,neuronal apoptosis and synaptic degeneration,etc.)in the brain of the NAbs-BACE1 treated mice were higher.(3)NAbs-BACE1 can promote the metabolism of Aβ and the neuronal apoptosis by upregulating BACE1 expression in vitro:Compared with the control group,the amyloid cleavage pathway products Aβ and CTF-β in the cell lysate of the NAbs-BACE1 treated group were significantly increased,and NAbs-BACE1 could promote the expression of BACE1 in a dose-dependent manner.In addition,the level of cell survival in the NAbsBACE1 treated group was decreased than the control group.3.Decreased plasma levels of NAbs-PS1 in AD patients.The plasma level of NAbs-PS1 in Amyloid-PET+patients was lower than that in the Amyloid-PET-group,and the plasma level of NAbs-PS1 in AD patients was lower than that in non-AD dementia patients and healthy controls.In all subjects,The plasma level of NAbsPS1 was negatively correlated with the amyloid load PiB-PET SUVR in the brain and positively correlated with the MMSE score.4.Cognitive decline in COVID-19 patients.Compared with controls,COVID-19 patients had lower TICS-40 scores and higher IQCODE scores.Severe COVID-19 patients had lower TICS-40 scores and higher IQCODE scores than non-severe COVID-19 patients and controls.Severe COVID-19 patients had a higher proportion of cases with current cognitive impairment and longitudinal cognitive decline than non-severe COVID-19 patients and controls.COVID-19 severity,delirium and COPD were risk factors of current cognitive impairment.Low education level,severe COVID-19,delirium,hypertension and COPD were risk factors of longitudinal cognitive decline.ConclusionsIn this study,we first screened the differential autoantibody profiles between AD and healthy controls by HuProtTM protein chip technology,including autoantibodies against BACE1,PS1 and other important molecules in AD pathological process.The clinical correlation of BACE1 and PS1 autoantibodies in AD was further discussed.It was found that BACE1 autoantibodies increased during the development of AD,and further experimental studies showed that BACE1 autoantibodies promoted the production of Aβ by up-regulating the expression of BACE1,thus aggravated the pathological process of AD.Population-based studies also confirmed that the higher level of BACE1 autoantibody at baseline,the faster Aβdeposition in the brain and cognitive decline in patients.These results suggest that the elevated level of BACE1 autoantibody is an important pathological event in the development of AD.In addition,we also found that PS1 autoantibodies were elevated in AD patients and negatively correlated with AD severity.