Inhibitory Effects Of H102 On The Agregation And Fibril Formation Of Amyloid-β-Protein (Aβ) In Vitro And In Vivo

BackgroundAlzheimer's disease(AD) is a major progressive neurodegenerative disorder occurred in center nervous system.The main clinical features of this disease are congnitive decline and mental deterioration.Its hisitopathological hall marks consist of senile plaques(SP),neurofibrillary tangles(NFT) and neuronal loss.Amyloid-βprotein (Aβ),a 4.2KD polypeptide,is the primary component of SP.It is postulated to be a major determinant of AD.The prevalence of AD is strongly associated with age.It is approximately 10%in persons over 65 years old,and increases to 50%in those over the age of 85.Following the growth of the life span of human beings,the incidence of AD are progressively increased.Because the pathogenesis of AD has not been well understood,it is very difficult to develop an effective pharmacologic therapy of AD. The present therapeutic medicines of AD such as inhibitors of acetyl cholinesterase (AchE),neurotrophic factors(NTF) and activators of neuronal metablism are not ideal drugs because of their suspicious effect and inevitable toxicity.It is necessary to find new drugs that have low toxicity and high therapeutic effect.It has been shown that the aggregation of Aβcauses the neurotoxic change of the peptide.Therefore, inhibiton of this process seems to be an effective therapeutic strategy for AD.Based on the stereochemistry structure and aggregation of Aβ1-42,our group designed severalβ-sheet breakers.The aim of our present study is to find out the best one and test its neuroprotective effects via experiments both in vitro and in vivo.Method1.Drug screeningWe compared the pharmaceutical effects of sixβ-sheet breakers on inhibitive aggregation and fibril formation ofβ-amyloid peptide.A solution of Aβ1-42 peptide 11.07μmol/L 20μl was incubated for 24h at 37℃.Same solutions of Aβ1-42 22.15μmol/L 10μl were respectively added into solutions of sixβ-sheet breakers or VE(88.61μmol/L 10μl) then co-incubated for 24h.For thioflavine-T(Th-T) fluorescent assay,a 980μl sample were added to 3.0μmol/L of Th-T in a final volume of 1.0ml 50mM phosphate buffer pH(6.0).The fluorescence was monitored at excitation 453nm and emission 478～486nm,using a F4500 spectroflurometer.2.Dose response courseThe fluorescence of the solution of Aβ1-42 peptides with H102 and VE at different concentration 20μmol/L,40μmol/L,88.61μmol/L,200μmol/L was monitored as above.3.Time response courseA solution of Aβ1-42 peptide(22.15μmol/L) were respectively added up solution of H102 and VE(88.61μmol/L) and co-incubated for 12h,1d,3d,5d,7d.The fluorescence was monitored as above.4.Drugs inhibit aggregation and fibril formation of Aβ1-42 by TEMA solution of Aβ1-42 peptide was incubated for 5d at 37℃in phosphate-buffered saline(PBS pH7.4).Solution of Aβ1-42(22.15μmol/L 10μl) were respectively added up solution of sixβ-sheet breakers or VE(88.61μmol/L 10μl) then co-incubated as above.After co-incubation,each sample was placed on Formvar-carbon coated 300-mesh copper grids for 15 min and then was negatively stained with 2% uranylacetate for 2 min.The grids were examined on a transmission electron microscope.5.Selecting the drugs and Checking the effect of H102 on SH-SY5YThe best candidate was selected via measuring MTT metabolic rate.Human neuroblastoma cells SH-SY5Y were cultured and separated into 3 groups:normal control group,damaged group(Aβ1-42 5μmol/L),and protection group(Aβ1-42 5μmol/L+H102 20μmol/L).Cell count,MTT metabolic rate,LDH leakage rate were used as indication.6.H102's influence to APP and AβThe protection efficiency of H102 on neurons in vivo is observed via the model of APP transgenic mouse.The transgenic mice were randomly divided into model group, H102 peptide injection group,normal control adopted the same age and background mice C57BL/6J mice.The animals were injected with H102 peptide and 0.9%NaCl solution for 10dObserving:Stained Congo red after formalin fixationAPP,Aβchanges by immuno-histochemistry after formalin fixation.Result1.Drug screening preliminary analysisWith de-background intensity of fluorescence from aged group as a result of 100 percent,inhibitory rate of each group for aggregation and fibril formation of Aβwere accounted.Finally we found that H102 poses strongest inhibitory effect on Aβaggregation and fibril formation.2.Dose response courseH102 and VE poses obvious inhibitory effect on aggregation and fibril formation of Aβ,and these inhibitory effects are dose-dependent.However,inhibitory rate of H102 group at each concentration level is higher than that of VE,so H102 group has the strongest inhibitory effect.3.Time response courseTime response curve in H102 group shows lowest fluorescence intensity.The semi-effective time(T1/2) in Aβ1-42 group was at day 3,but was delayed to day 4 in both H102 group and VE group.Aggregation and fibril formation of Aβ1-42 by TEMAmorphous and fibrillar Aβwere observed by TEM.There are rich Aβfibrils in the solution of Aβincubated for 5d.The fibrils with a diameter of 8～10nm are crystal-like aggregated,branch and web-like fibrils,similar to amyloid-like fibrils in SP of AD brains;There are more Aβfibrils in the solution of Aβ1-42 co-incubated with K7 or H100;However,only a few fibrils were observed in the solution of Aβ1-42 co-incubated with H101 or H103;Some slightness fibrils occurred in the solution of Aβ1-42 with L5.Some less slightness fibrils observed in H102 added up. 4.Selecting the drugs and Checking the influence of H102 via SH-SY5Y4.1 H102 is the best breaker peptide among six candidates.4.2 H102 promoted neuronal growth,increased cell survival rate,enhanced axonal growth and dendritic density,decreased cell death rate4.3 Compared with normal control group,Aβ1-42 damaged group,showed reduced cell count and MTT metabolic rate,increased LDH leakage rate,while the addition of H102 peptide alleviated the above damages..5.H102's influence to APP and Aβ5.1 APP immunohistochemistry;density of positive cell in the CA1 region of hippocampus of control group were less than model group.H102 peptide reduced the area,and density of positive cells.5.2 Aβimmunohistochemistr:We stained the brain sections with specific antibody to Aβ1-42 by immunohistochemistry.Both area and density of positive cells in control group were less than model group.H102 decreased the area and density of positive cells in the CA1 region of hippocampus.5.3 Congo red staining:there were lots of amyloid plagues in the brains of model mice but not in the brains of normal control.H102 significantly decreased the amyloid plagues.ConclusionsOur experiments screened theβsheet breaker,as shown in both vivo and vitro that among 6 tested candidates,H102 is the most effective one in inhibiting the aggregation of Aβand protecting SH-SY5Y and decreasing the level of APP,Aβin APP transgenic mice.