The Screening Of Small Molecule Compound W90761 And The Study Of Mechanism Of This Compound Facilitates Apolipoprotein E Lipidation

Alzheimer disease(AD)is a progressive neurodegenerative disease associated with cognitive decline and is the most common form of dementia in the elderly.Apolipoprotein E(Apo E)ε4 allele is the strongest risk factor of AD.Mounting evidence demonstrates that apo E4 contributes to AD pathogenesis by modulating the metabolism and aggregation of amyloid-β peptide.Apo E is a major cholesterol carrier that supports lipid transport,it binds to several cell-surface receptors to deliver lipids and also to amyloid-β(Aβ)peptide,they can form apo E/Ab complex,which is thought to initiate toxic events that lead to synaptic dysfunction and neurodegeneration in AD.Apo E has three different isoforms,apo E2,apo E3 and apo E4.Apo E isoforms differentially regulate Aβaggregation and clearance in the brain,and have distinct functions in regulating brain lipid transport,glucose metabolism,neuronal signalling,and mitochondrial function.Lipidated apo E increases apo E stability and its lipidation is modulated by ABCA1.Transcription of ABCA1 is regulated by Liver X Receptor(LXR),Retinoic X Receptor(RXR)and Peroxisome Proliferator-activated Receptor(PPAR).At present,medicines in clinic used to treat AD patient just alleviate the symptoms and increase cognitive function.In fact,treatment with liver X receptor(LXR)agonists or retinoidd X receptor(RXR)agonists consistently improves memory and reduces Aβ levels by increasing apo E level and lipidation in amyloid model mice.However,despite their efficacy and tolerability in animals,the controversy on drug toxicity still precludes their translation into human clinical trials.To identify novel compounds that can regulate apo E production,we performed a drug screening using a Known Bioactives Library,which contains 500 compounds with defined biological activity and a culture system of immortalized astrocytes from human apo E3 targeted replacement(TR)mice.ELISA,MTT were used to measure apo E level and cell viability in this stage.After two rounds of screening,we choose small molecule compound W90761 as our research object.Experimental methods and machines such as ELISA,Western blot,Real time PCR,FPLC and FACS were uesd to study the related mechanism of this compound,and finally got these data:1.Through the primary screening of all 500 compounds,21 compounds were found to increase secreted apo E level,while 56 compounds decreased the apo E level in the medium.In the 21 compounds,we found that small molecule compound W90761 and compound W90737 significantly increased apo E secretion more than 2 times in a concentration-dependent manner.After thinking about the cell toxicity of these two compounds.Finally,we choose compound W90761 as our research object.Then we measured the apo E m RNA levels by q RT-PCR.The data shows that compound W90761 didn’t affect APOE transcription.This compound also increases apo E secretion in primary culture of astrocytes from apo E-TR mice.2.In the following mechanism study,we found small molecule compound W90761 increased apo E lipidation by stimulating ABCA1 m RNA and protein expression.The cholesterol level also increased with the increasing apo E level.It means that this compound facilitate apo E lipidation in the brain.We also found LDLR and LRP1 expression at both protein and m RNA levels were not changed by tested compound,indicating that cellular apo E clearance pathways are unlikely involved in this mechanism.Compound W90761 and bexarotene facilitate apo E level and lipidation through activation of the LXR/RXR pathways,which may require RAR.3.After treatment with small molecule compound W90761,we assess the ability of cellular Ab uptake.Compound W90761 was shown to reduce the uptake of Ab in astrocytes.But further investigations are necessary to determine whether the suppression of cellular Ab uptake in astrocytes facilities or prevents Ab clearance.In summary,we found small molecule compound W90761 modulate apo E lipidation through LXR/RXR/RAR pathways and increase apo E level in immortalized astrocytes.It may be an effective medicine for AD treatment compare to others.