Regulation Of A? Degrading Enzymes In The Pathogenesis Of Alzheimer's Disease And Pilot Drug Research

Alzheimer's disease(AD)is a progressive neurodegenerative disease which is closely associated with aging.AD is mainly characterized by neurofibrillary tangles and senile plaques.The neurofibrillary tangles consist of hyperphosphorylated tau,and the senile plaques are composed of ?-amyloid(A?)peptide.Imbalance of A? production and removal may lead to A? accumulation,and then result in the formation of senile plaque in brain.Currently,the role of A? clearance has been increasingly studied in the progression of AD.As a key pathway of A? clearance,A? degrading enzymes have been regarded as important in the filed,especially neprilysin(NEP)and endothelin converting enzyme(ECE)that are described with a range of abilities to degrade A?,however,their expression,regulation and interaction in the progression and development of AD remains unclear.Following works were presented in the thesis:1)Previously,we found NEP mRNA,protein level,and activity decreased,while ECE expression increased in AD brains when compared with no cognitive impairment(NCI).This results suggested A? degrading enzymes NEP played important roles in the development of AD,and ECE might compensatory secured A? metabolism when the function of NEP was damaged.Due to the sporadic AD in clinic usually found in advanced stages,which makes hard to observe the whole process of AD.Thus,we used APP/PS1 double transgenic mice to study the alteration of NEP and ECE protein and activity in the progression and development of AD,exploring the relationship between Ap degrading enzymes and A? deposit,and to discuss the possible target of drug,which might provide a clue for Alzheimer's disease treatment.Firstly,we focused the study on the alternation of NEP and ECE protein and enzyme activity with age using APP/PS1 mice,and evaluated the role of them in A? degradation.We further explored the relationship between NEP or ECE regulation and severity of AD.The results showed a significant increase in the levels of A?40 and A?42 levels with age in the cerebral cortex and hippocampus of APP/PS1 mice(3,6,9,12 M),and the results of A?plaques were similar to Ap levels,while the data of Morris Water Maze showed APP/PS1 mice displayed a significant cognitive deficit at 9 month.Furthermore,NEP protein displayed a marked decrease with age in the cerebral cortex and hippocampus of APP/PS1 mice,ECE protein,however,was up-regulated with age.Double immunofluorescence staining demonstrated that ECE and NEP highly colocalized in cytoplasmic and membrane.However,NEP and ECE activity showed a significantly decrease with age in the cerebral cortex and hippocampus of APP/PS1 mice older than 6 months of age.Correlation analysis showed the negative correlations between enzyme(NEP or ECE)activity and A? levels in the cerebral cortex and hippocampus of APP/PS1 mice,which was closely correlated with A? accumulation and influenced AD development,which is similar to AD patients.2)It was predicted the subcellular distribution of membrane-bound NEP might be different and influence A? metablism in the progression of AD,which might lead to decreasing the degradation efficiency of NEP.Thus,we investigated the subcellular distribution of NEP protein and activity in brains of human individuals in different stages of AD,APP/PS1 mice as well as human neuroblastoma SH-SY5Y cells.In subcellular extracts,NEP protein decreased in cytoplasmic fractions in brains of mild cognitive impairment(MCI)and AD,but increased in membrane fractions,with a significant increase in the membrane/cytoplasmic ratio of NEP protein in AD brains.By contrast,NEP activity was decreased in AD.Similar results were observed in AD-mimic transgenic mice.Studies of SH-SY5Y neuroblastoma showed an up-regulation of NEP protein in the cytoplasmic compartment induced by 4-hydroxy-2-nonenal(HNE)or A?,however,NEP activity decreased in cytoplasmic fractions.Activity of NEP in membrane fractions increased at 48 hours and then significantly decreased at 72 hours after treatment with HNE and A?.The membrane/cytoplasmic ratio of NEP protein increased at 24 hours and then decreased in both HNE and A? treated cells.These results demonstrated both HNE and A? up-regulated NEP expression,but NEP enzyme activity did not show the same increase,possibly indicating that the major NEP activity was associated with membrane fractions.HNE,a by-product of lipid peroxidation can oxidative modification of some molecular including NEP,which might be one of the major causes for NEP deactivation.Therefore,LTQ Velos Pro-Orbitrap Elite mass spectrometer was performed to identify the sites of HNE-modified NEP,we identified that NEP was covalently adducted at Lys 93 and Lys 472 by HNE via Michael addition.These results demonstrated NEP and ECE played crucial roles against the accumulation of overproduced A?.Up-regulated ECE owing to the damage of NEP might compensatory ameliorate dysfunction of A? metabolism in the progression of AD.NEP in membrane fractions played a major role in A? degradation.In addition,the identification of HNE modified the specific sites of NEP provided some experiment bases for search compounds against the modified sites.3)Pilot drugs exploration:? In order to search possible effective drugs targeting A? degrading enzymes,we initially explored the effect of Chidamide,a HD AC inhibitor,on the NEP expression and activity.Chidamide up-regulated NEP protein,increased NEP activity in membrane fractions,and decreased A? levels.Thus Chidamide might be a potential candidate for AD in future.This preliminary findings provided a valuable clue for further research.? Peroxisome proliferator-activated receptors(PPARs)are ligand-activated nuclear transcription factors that regulate peripheral lipid and glucose metabolism.PPARs family comprises PPARa,PPAR?/? and PPAR?.PPAR? is the most extensively studied among three isoforms.Recently,accumulating evidence demonstrated PPAR? agonists thiazolidinedione drugs(TZDs),like rosiglitazone and pioglitazone,attenuated neurodegeneration,repressed the expression of proinflammatory cytokines and improved cognition in patients and mouse models with mild-to-moderate AD.Thus,PPAR? agonists might be a therapeutic target in AD.Based on a lead compound TZDs,we designed and synthesized a series of 2-thioxo-4-thiazolidinone derivatives and evaluated them on PPAR? binding activities.Through the biological assays,compounds 18 and 38 were highlighted with Ki values of 12.15 nM and 14.46 nM,respectively.Then structure-activity relationship(SAR)was analyzed to screen privileged structural modifications.Moreover,molecular fitting of these compounds in the PPARy ligand binding domain was performed to elucidate the SAR and explore potential receptor-ligand interactions.These results demonstrated that the 2-thioxo-4-thiazolidinones might be considered as new promising compound with excellent binding activities to PPAR?.This work provided more evidences for the regulation and the drug intervention of A?degrading enzymes NEP and ECE in the development of AD,and provided a new direction for AD treatment with compounds targeting HDAC inhibitor and PPAR?.