None-invasive Biomarkers For Early Diagnosis And Treatment Of Alzhermer's Disease

Alzheimer's disease(AD)is a common neurodegenerative disease which is characterized by progressive decline in cognitive functions.AD has two major pathological changes: senile plaques(SP)and neurofibrillary tangles(NFT).Amyloid-b(Ab),a peptide derived from cleavage of amyloid-b precursor protein(APP),is the major component of SP and NFTs are constituted of aberrantly hyperphosphorylated tau protein.AD develops undiagnosed for many years due to the lack of detectable early biomarkers.Visual dysfunction is prevalent in AD patients and correlates tightly with the severity of cognitive and behavioral defects,and the alterations in the eyes can be none-invasively detected.Clinical and epidemiology data have demonstrated that hyperhomocysteinemia(Hhcy)has been an independent risk factor of AD.Our previous study found that injecting rats of homocysteine(Hcy)via vena caudalis for 14 days can induce the AD-like pathology and memory dysfunction.Simultaneous supplement of folate and vitamin B12 could reduce the plasma Hcy level,attenuate the AD-like pathology and improve the learning and memory function.In our study,Hcy was progressive injected via caudal vein of rats with or without supplement of folate and vitamin B12,and cognitive functions and the changes of related molecular pathways were measured.Part ?Retina Ser262-tau hyperphosphorylation and PP2A demethylation precedes the hippocampus in hyperhomocysteinemia ratsIn this study,we used healthy adult male Sprague-Dawley(SD)rats by injecting Hcy through the tail vein at different times.To search for early biomarkers in the eyes,we characterized here the AD-like pathologies both in retina and the hippocampus along with the progressive administration of homocysteine(Hcy)to induce hyperhomocysteinemia(Hhcy).The concrete methods and results are as follows:1.Hhcy induces memory and synaptic deficits in ratsIn order to investigate the effect of increasing Hcy on memory function in rats,we injected rats with Hcy(400 ?g/kg/day)by vena caudalis for 3,7 and 14 days.The results showed that venous injection of Hcy for 14 days but not for 3 or 7 days induced hyperhomocysteinemia although a gradually increased blood Hcy level was detected.Then,we detected the memory function of rats by new object recognition experiment.We found that rats only had memory dysfunction after 14 days injection of Hcy,while the open field test(OFT)showed that no motor dysfunction and anxiety behavior in the Hcy rats.We further examined the synaptic function during Hcy exposure.Western blotting showed that compared with control,Hcy significantly decreased protein levels of syanpsin-1 and PSD-95 in hippocampal extracts.Long-term potentiation(LTP)results showed that synaptic plasticity decreased only after 14 days injection of Hcy.These data showed that only long-term(14 days)but not short-term(3 days)of Hcy exposure induces synaptic dysfunction.2.The increasing plasma Hcy gradually induces retina impairments in ratsSince the retina is a continuation of the brain,it has a similar neurotransmission system to the brain.A large number of studies have shown that there is a related pathological change in the retina of AD patients.To explore whether the retina will appear AD-like lesions as the same as in the hippocampus,we first detected the rats in each group after Hcy injection at different time points by fundus photography.The results showed that the retinal surface of rats in each group was smooth with no obvious hemorrhage and exudation.The blood vessels were tortuosity and stenosis only appears after 14 days of injection.Hematoxylin-eosin(HE)staining of the retina showed that the retinal histological structure in each group was clear and the retina thickness was not significantly different.However,we observed vacuolar disarrangement and nuclear shrinkage in RGC layer withsignificantly decreased RGC number in rats with 14 days Hcy exposure.Although the cell number in INL also showed a decreasing trend,the difference was statistically not significant in 14 days Hcy rats.Optical coherence tomography(OCT)showed that Hcy exposure did not change the topographic feature of retina,as all the layers were smooth and RNFL thickness was normal in all Hcy-injected groups compared with saline controls.These data suggested that Hcy exposure for 14 days but not 3 and 7 days impairs retinal arteries and induces RGC death without affecting the geomorphic feature of the retina.These results also reminded us that the retinal neurodegeneration was similar to the hippocampus of AD induced by Hcy with the increased level of Hcy in rat's plasma.At the same time,we examined the other ocular tissues to explore whether the relevant AD-like pathological changes also occur at cornea or lens.By enzyme-linked immunosorbent assay(ELISA)we found that levels of Ab42 was significantly increased in retina but not in cornea and lens after 14 days Hcy injection.Western blotting results showed that the phosphorylated tau at Ser396 epitope was significantly increased in retina but not in cornea and lens after 14 days Hcy injection.These data demonstrated that Hcy can increase A b level and tau phosphorylation selectively in retina but not in cornea and lens in 14 days Hcy rats.Therefore,we chose to observe retinal lesions in order to explore the early diagnosis indicators of AD.3.Hcy exposure for 3 days induces retinal Ser262-tau hyperphosphorylation preceding that of the hippocampusTo further investigate whether there was earlier pathological change in the retina than in the hippocampus of Hcy-induced AD model,we first examined the Ab lesions of the two tissues at different time points.By ELISA assay using Ab42-specific antibody,significant elevation of A b 42 in retina was only detected in 14 days Hcy group.Immunohistochemistry and immunofluorescence staining were performed at different time points after injection.Compared with the controls,A? staining was started to show in INLand GCL layers of the retina at 7 days Hcy group,and significant A? accumulation was only detected in 14 days Hcy group.We also observed that elevation of APP,PS1 and BACE-1 in retina was only detected in 14 days Hcy group by western blotting.Interestingly,although the increase of A? in total hippocampal extracts was only detected by ELISA at 14 days group,a remarkably increased A? expression in hippocampal CA3 was detected in 3 days Hcy group.These data suggest that A? and its producing system are activated in both retina and hippocampus during Hcy exposure,and the retinal A?pathology lags behind hippocampus.Next,we examined the tau pathological changes of the two tissues at different time points.Western blotting was used to detect the phosphorylation of tau at the Ser262,Ser356,Ser396,Ser214,Ser422 and Thr212 sites(PS262,PS356,PS396,PS214,PS422 and PT212)in the retina and hippocampus of rats at different time points.The results showed that retina PS262 began to show a marked increase after 3 days of injection,while hippocampal PS262 increased only after 14 days of injection.There was no significant difference of the other sites between the retina and the hippocampus.By immunohistochemistry and immunofluorescence staining,we observed accumulation of pS262-tau in INL after 3 days Hcy injection,and the accumulation was extended to the inner plexiform layer(IPL)and GCL of the retina at 14 days Hcy group.These data together demonstrate that increase of pS262-tau in retina precedes that of the hippocampus,suggesting that pS262-tau may serve as a feasible biomarker for early diagnosis of AD.4.Hcy exposure for 3 days decreases PP2 A methylation and increases PP2 A demethylation in retina preceding that of the hippocampusActivation of protein kinases and/or inhibition of protein phosphatases are the direct cause of tau hyperphosphorylation,among various kinases and phosphatases,GSK-3? and PP2 A are the most implicated.Therefore,we measured the levels of GSK-3? and PP2 A.We observed that Hcy did not change the total GSK-3? or the Ser9-phosphorylatedGSK-3? in retina and hippocampus.Hcy exposure for 3 days significantly decreased methylated PP2Ac(M-PP2 Ac,the active form)with an increased demethylation(DM-PP2 Ac,inactive form)and unchanged total PP2 Ac level in retina.In hippocampus,the significant reduction of M-PP2 Ac and increase of DM-PP2 Ac were only detected in 14 days Hcy group.Additionally,the phosphorylation of PP2 Ac at Y307(pY307-PP2 Ac,the inactive form)increased in both retina and hippocampus of the 14 days Hcy group.By immunohistochemical and immunofluorescent staining,we also observed that Hcy exposure for 3 days significantly increased DM-PP2 Ac in GCL and INL,while Hcy increased hippocampal DM-PP2 Ac only at 14 days group.These data suggest that the reduced M-PP2 Ac and increased DM-PP2 Ac in retina may also serve as biomarkers for early diagnosis of hyperhomocysteinemia-induced AD.ConclusionWe found in the present study that Hhcy induces AD-like hallmark pathologies in retina.Among them,the increased pS262-tau and DM-PP2 Ac and decreased M-PP2 Ac can serve as feasibly visualized biomarkers for early diagnosis of hyperhomocystinemia-induced AD.Part ? The treatment effectiveness of homocysteine-induced Alzheimer disease can be assessed through retinaIn this study,we injected the rats with Hcy by caudal vein for 14 days to reproduce the HHcy model.At the same time,folic acid and vitamin B12(FB)were supplement to explore whether the retina could evaluate the effectiveness of AD treatment.The concrete methods and results are as follows:1.Folate and vitamin B12 reverse Hhcy-induced retinal vascular pathological changes in rat retinaTo investigate the feasibility of AD treatment through the retina,we used adult SD rats to receive Hcy(400 ?g/kg/day)injection with or without folic acid and vitamin B12 supplementation.The results showed that FB can reduce plasma Hcy levels.Retinal fundus photograph results showed that FB can reverse the retinal vascular tortuous narrow pathological phenomenon induced by Hcy.2.Folate and vitamin B12 reduces Hhcy-induced Ab accumulation in rat retinalThe level of Ab42 in the retina of rats in each group was detected by ELISA.It was found that FB could reduce the increasing level of A b 42 induced by Hcy.Immunohistochemistry and immunofluorescence staining with 6E10 antibody were performed after injection.We found that FB could also reduce the A? accumulation.We also observed that FB could downregulate of APP,PS1 and BACE-1 in retina by western blotting.These results showed that FB supplementation reduce the increase of A? induced by Hcy.3.Folate and vitamin B12 decreased Hhcy-induced tau hyperphosphorylation in rat retinaNext,we examined the phosphorylation of tau in the retina.Tau-1,PS262 and PS356 of retina in three groups were detected by Western blotting.The results showed that FB could reverse the pathological changes induced by Hcy.Immunohistochemistry and immunofluorescence staining with PS262 showed the results consistent with the above results.4.Folate and vitamin B12 ameliorates Hhcy-induced protein kinase and protein phosphatase unbalanced in rat retinaWe further examined the changes of protein kinase and lipase in the retina.We detected the GSK-3?,GSK-3?-S9,PP2 Ac,M-PP2 Ac and DM-PP2 Ac in the retinas of three groups by Western blotting.It showed that FB could reverse the abnormal changes in protein kinases and lipases induced by Hcy.Immunohistochemistry and immunofluorescence staining of DM-PP2 Ac in retina showed the same results as mentioned before.ConclusionWe found that folate and vitamin B12 treatment could reverse Hhcy-induced AD-like tau and A ? pathologies in retina in rats.These data indicate that retina can serve as a window for evaluating the effectiveness of the drug for AD.