The Effects Of Heparan Sulfate And Its Analogues On Aβ Production,Aggregation And Neurotoxicity

Alzheimer’s disease is a kind of central nervous system disease mainly affecting the elderly.With the development of the disease,the cognitive function and mobility of the patient will be significantly reduced,which seriously affects daily life.At present,elucidating the pathogenesis of AD and developing effective treatment agents have become the focus of academic research.At present,the main view is that the imbalance of Aβproduction and the neuronal damage and apoptosis caused by Aβoligomerization are the central link in triggering AD.β-secretase(BACE1)is the rate-limiting enzyme of Aβproduction.Therefore,BACE1 is an ideal drug target to attennate Aβproduction.Many studies have shown that HS is widely involved in the pathogenesis of AD and can directly bind to BACE1.Therefore,illustrating the regulating mechanism between HS and BACE1 might help in understanding the cause of AD and provide new clues for the development of AD therapentics.The main research contents and results of this thesis are as follows:1.The recombinant lentiviral expression plasmid pLVX-IRES-NEO-APP695swe was constructed and transfected into 293T cells together with the lentiviral packaging plasmid.The lentiviral particles were concentrated and then infected the SH-SY5Y neuroblastoma cells.The APP protein expression was confirmed using western blotting method.2.A structural analogue of HS chain was purified from white jade snail(WJS)and named as WJSPS.Results indicated that WJSPS is a homogeneous polysaccharides.Its average molecular weight(Mn)is 244 k Da.Combined ~1H NMR spectrum and ~1H-~1H COSY spectrum and the literature to assign each ~1H signal,we concluded that the polysaccharide was composed of→4)-D-GlcNAc-(1→4)-L-Ido A2S-(1→disaccharide repeating unit.Further study found that WJSPS promoted the aggregation of Aβ42 at low concentration conditions(1 and 10μg/mL),and inhibited the aggregation of Aβ42with high concentration(100 and 500μg/mL).In addition,WJSPS could significantly inhibit the cytotoxicity of SH-SY5Y induced by Aβ42 oligomers.Treatment of SH-SY5Y-APP695swe cells with WJSPS had no effects on the APP and C99 production indicating that WJSPS had no effects on APP processing to Aβ.3.To study the regulatory effects of endogenous HS on BACE1.Different concentrations of xyloside were used to inhibit the synthesis of endogenous HS in SH-SY5Y-APP695swe cells,and the expression level of BACE1 protein was detected by Western blotting technology.It was found that inhibiting the synthesis of endogenous HS chains reduced the expression of BACE1 and the production of C99 protein,but increased the production of C83 fragments.These results indicate that inhibiting the expression of endogenous HS could increase the non-amyloid pathway of APP processing,thereby reducing Aβproduction through the amyloid pathway processing of APP.