The Functions Of CD147 Involved In The Blood Brain Barrier Injury And Neurodegeneration In Alzheimer’s Disease Mouse Model

The elderly people are extremely prone to Alzheimer’s disease（AD）.The incidence of AD in people over 65 years old is 5%and it increases with age.China is an aging country.According to World Health Organization,the number of AD patients in China will reach 20million by 2050,which will then lead to serious social issues.Beta-amyloid（Aβ）theory is a mainstream hypothesis of AD pathogenesis.The amyloid precursor protein（APP）mutates and is cleaved to form polymeric and neurotoxic Aβ40 and Aβ42.Accumulation of Aβleads to neurodegenerative diseases such as amyloidosis,neuronal death and brain atrophy.The function of blood brain barrier（BBB）is closely related to the pathogenesis and treatment of AD.The brain microvascular endothelial cells（BMEC）with tight junction are the most important component of BBB.BBB restricts the free exchange of substances between peripheral blood and brain tissue,leading to the inability of most drugs to enter the brain interstitium.The incidence of AD and the injury of BBB complement each other,often resulting in ischemia,hypoxia,inflammation and other vascular lesions.Therefore,BBB has become the key point to break through the cure of AD from the perspective of trans-BBB drug delivery and cerebrovascular injury repair.CD147,a transmembrane glycosylated protein encoded by BSG gene,is widely involved in a large number of pathological activities,including tumor proliferation and invasion,SARS-COV-2 infection,inflammation and plasmodium invasion,which is an ideal therapeutic target for a variety of diseases.It is reported that CD147 is highly expressed in BMEC and may have receptor-mediated transcytosis（RMT）function,which has the potential as a"Trojan horse molecule"in the treatment of AD.The brain CD147 expression in the AD models is also found to be at higher levels than in the wild types.In consideration of the pathogenesis of AD,the function of BBB and the role of CD147,we want to explore whether there are correlations among the three,and reveal how CD147plays a role in BBB pathology,what kind of influence it has on the pathogenesis of AD,and what their relationship and mechanism are,so as to provide new ideas and directions for curing AD.This study is divided into the following three parts:Part I:Study on expression and transport function of CD147 in blood brain barrierAt present,an important problem limiting the efficacy of anti-AD drugs is how to efficiently deliver drugs through BBB.Therefore,the main purpose of our study in the first part is to explore whether the expression of CD147 is BBB specific and has the ability of trans-BBB drug delivery.Firstly,immunohistochemistry（IHC）and immunofluorescence（IF）of mouse brain sections were performed and CD147 was found to be stained on endothelial cells and colocalized with CD31,suggesting specific expression of CD147 on mouse BMEC.Secondly,IHC of human brain sections was performed and the expression level of CD147 m RNA in human cerebral cortex was statistically analyzed in 157 cases from The Genotype-Tissue Expression（GTEx）database.The results showed that the expression level of CD147 in cerebral cortex of over age 60 human was higher than that under age 60,suggesting a positive correlation between the level of CD147 and age.In order to study the active transport capacity of CD147 in BBB in vitro,we simulated the blood-brain barrier effect of BBB by stratified co-culture of human microvascular endothelial cells（HCMEC/D3）and human pericytes（HBVP）to establish the BBB model in vitro.We found significantly higher concentration of anti-CD147 antibody influx from HCMEC/D3 side to HBVP side than HBVP side efflux to HCMEC/D3 side,suggesting that CD147 had the function of mediating antibody molecules from the blood to cross the BBB into the brain parenchyma.Conclusion:CD147 is specifically expressed in mouse and human BMEC and has the ability to polarly transport antibody molecules across BBB in vitro.Part II:Study on CD147 involved in the pathology of blood-brain barrier injury in APP/PS1 transgenic miceWe used APP/PS1 mice as the animal model of AD.After IHC staining of brain sections,we found that 12-month-old APP/PS1 mice showed a large amount of Aβstaining,which was widely distributed in endothelial cells and brain interstitium,suggesting that 12-month-old APP/PS1 mice had a typical pathological AD symptom of Aβdeposition.After i.v.injected with angiography agent,the cerebral cortex of the craniotomy mice was observed using two-photon microscopy.It was found that the elderly APP/PS1 mice showed significant accumulation of cerebrovascular amyloid plaques and vascular morphological changes,while the young APP/PS1 mice and the elderly wild-type mice were normal.These results suggested that the pathological changes related to AD in APP/PS1 mice were highly correlated with age.We used type 2/9 AAV-sh CD147 to inject the DG and CA1 regions in the hippocampus of mice to silence the expression of CD147 in the brain of mice.In 8 weeks,the elderly APP/PS1 mice which were CD147 down-regulated had higher BBB integrity,vascular density,blood flow velocity,and lower BBB permeability than the control elderly APP/PS1 mice,suggesting that CD147 was involved in the regulation of BBB morphological lesions in the pathogenesis of AD.However,there was no significant difference in the proportion of BBB amyloid plaque deposition between the two groups,suggesting that CD147 regulation of BBB function did not depend on amyloid protein production or clearance.Conclusion:Down-regulation of brain CD147 level can significantly improve pathological states of BBB in elderly APP/PS1 mice,suggesting that CD147 is involved in the BBB injury with the progression of AD in elderly APP/PS1 mice.Part III:Effect of CD147 down-regulation on cognitive function and neuronal activity in APP/PS1 transgenic miceWe investigated how CD147 levels affect the development of neurodegenerative lesions in AD models from both ethological and molecular imaging perspectives.Firstly,we used ethological tests to assess the cognitive function of young or aged APP/PS1 mice injected by AAV-sh CD147 with wild-type or APP/PS1 mice injected by AAV-Ctrl as controls.In the Y maze experiment,we found that the elderly APP/PS1 mice with down-regulated brain CD147 level had higher motor ability,desire to explore,and lower stationary time than the elderly control APP/PS1 mice,suggesting that the reduction of brain CD147level could improve the motor control ability of elderly AD mice.In the Morris water maze navigation experiment,the aged APP/PS1 mice with down-regulated brain CD147 level were better at taking effective strategies to escape than the control group and the average escape time on Day 5 was shorter.In the spatial search experiment,elderly APP/PS1 mice with reduced brain CD147 level more frequently lingered or swam toward the quadrant of platform location.These results suggested that downregulation of brain CD147 could improve the comprehensive cognitive ability and learning and memory ability of elderly APP/PS1 mice.We also performed PET imaging studies of ¹⁸F-FDG.It was found that in 1of the 3 APP/PS1 mice injected with AAV-sh CD147 8 weeks after injection,the decrease of SUVmax level in the cerebral cortex and hippocampus was significantly lower than that in the other two APP/PS1 mice(the SUVmax of ¹⁸F-FDG represents the neuronal vigorous glucose metabolism level),which was similar to that in the wild mice.These results suggested that the down-regulation of CD147 might help to alleviate neuronal degeneration during AD progression.Conclusion:Down-regulation of brain CD147 level can significantly improve cognitive function and neuronal activity state of of elderly APP/PS1 mice,suggesting that CD147 promotes neurodegeneration of elderly APP/PS1 mice by participating in BBB impairment.Summary:As a transmembrane protein specifically expressed on BMEC,CD147 has the function of transporting antibodies across BBB.CD147 is involved in the BBB injury and promoted the cognitive function and neuronal activity disorders in eldly APP/PS1 mice.Down-regulation of CD147 significantly improves the functions of BBB in aged APP/PS1 mice,thus effectively alleviating AD-related degeneration.Therefore,CD147 is expected to be a new therapeutic target for AD.