Early Prediction Of Alzheimer’s Disease And Its Study On Mice Model

【Background】Alzheimer’s disease(AD)is the most common neurodegenerative disease.The effective measures to reduce the prevalence of AD are early detection and early intervention.However,there is no method for early diagnosis and treatment of AD.Therefore,it is of great significance to find peripheral markers related to cognitive decline from high-risk population of AD.Type2 diabetes mellitus(T2DM)and mild cognitive impairment(MCI)are two independent risk factors for AD.Epidemiological data showed that T2 DM was closely related to cognitive impairment and late-onset AD.In recent years,the incidence of T2 DM has increased sharply and it is tended to affect younger populations.MCI is a state between normal aging and AD,which can provide a window period for early intervention of AD.Our previous studies have found that cognitive impairment in T2 DM patients was associated with impaired olfactory,Apo E ε4 genotype,increased plasma Aβ1-42/Aβ1-40 ratio(r Aβ42/40),and increased platelet GSK-3β activity(Total/p S9-GSK-3b,r GSK-3b).【Objective】By follow-up our previously established T2 DM cohorts,we investigated whether the decreased olfactory function,Apo E ε4 genotype,elevated plasma Aβ42/Aβ40 level,and the elevated platelet GSK-3β activity could be used to predict the transition from T2 DM to MCI or AD.【Methods】This is an average of 19-or 30-months follow-up study.A total of 159 T2 DM volunteers were divided into two groups: with normal cognitive function(n MCI)group(n=95)and MCI group(n=64)at baseline level.The patients’ general information,past history,medication status,MMSE score,olfactory function,Apo E genotype,plasma Aβ level and platelet GSK-3βactivity were collected.Logistic regression was used for multivariate analysis and ROC curve was used to conduct the prediction model.【Results】1.Results of a follow-up study of 95 T2 DM patients without MCI at baseline1)The patients who progressed to MCI during follow-up had worse olfactory function,higher GSK-3β activity,and higher Aβ42/Aβ40 ratio than those who did not progress to MCI: Among 95 T2 DM patients with normal baseline cognitive function,35 of them developed MCI and the rest 60 maintained normal cognitive function during 30 months followup period.Compared with the group with normal cognitive function,the baseline characteristics of patients who convert to MCI had worse olfactory function(7.46 ± 1.66 vs6.72 ± 1.25,P=0.015),higher platelet GSK-3β activity(1.08 ± 1.11 vs 0.62 ± 0.38,P=0.023),and higher plasma Aβ42/Aβ40 ratio(0.72 ± 0.80 vs 0.39 ± 0.31,P=0.023).There were no significant differences in age,sex,hypertension,hyperlipidemia,coronary heart disease,family history of diabetes,years of diabetes,insulin therapy,Hb A1 c,Apo E genotyping,follow-up time,and MMSE score at baseline.It is suggested that olfactory dysfunction,activated GSK-3β and increased Aβ42/Aβ40 may predict cognitive decline in patients with T2 DM.2)Olfactory dysfunction,GSK-3β activation and increased Aβ42/Aβ40 ratio can predict MCI in T2 DM patients: The olfactory score,r GSK-3β and r Aβ42/40 were analyzed by ROC curve and binary regression for predicting MCI.The results showed that the cut off of olfactory score was 6.25,with sensitivity,specificity,accuracy and AUC of 0.771,0.450,56.8%and 0.634(95%CI: 0.517-0.751),respectively.The cut off value,sensitivity,specificity,accuracy and AUC of platelet GSK-3β activity were respectively 1.058,0.486,0.883,73.7%,and 0.651(95%CI: 0.528-0.773).The cut off value,sensitivity,specificity,accuracy and AUC of plasma Aβ42/Aβ40 ratio were 0.543,0.600,0.850,75.8%,and 0.664(95%CI: 0.539-0.789),respectively.These results confirm that the changes of olfactory score,platelet r GSK-3β and plasma Aβ42/Aβ40 can predict MCI in T2 DM patients.3)The increased platelet GSK-3β activity and plasma Aβ42/Aβ40 ratio were independent risk factors for cognitive decline in patients with T2DM: after adjustment for other confounding factors,the OR value of platelet GSK-3β activity was 4.426(95%CI: 1.247-15.708,P = 0.021)and the OR value of plasma Aβ42/Aβ40 was 6.188(95%CI: 1.255-30.499,P = 0.025).These results suggest that the increased r GSK-3β and r Aβ42/40 are independent risk factors for transition to MCI in T2DM-n MCI population.4)Predictive models for progression to MCI in patients with T2DM: Based on age,the olfactory score,Apo E ε4,platelet GSK-3β activity and plasma Aβ42/Aβ40,we established MCI prediction model in T2 DM cohort as follow: logit(MCI)=-4.129-0.007 × Age-0.197×(Apo E ε4=1)+ 0.352 × Olfactory score + 1.150 × r GSK-3β(Total/S9)+ 1.321 × Aβ42/40.The sensitivity,specificity,accuracy and AUC of the model were respectively 0.629,0.833,75.8% and 0.751(95% CI: 0.640 0.862).2.Results of a follow-up study of 64 T2 DM patients with MCI at baselineAfter a mean follow-up of 19 months,the olfactory function was further impaired(7.63 ±1.72 vs 8.09 ± 1.37)and the plasma Aβ1-40 level was further decreased(249.61 ± 194.50 vs202.40 ± 138.25)in T2DM-MCI patients.However,the MMSE score,platelet GSK-3β activity,and plasma Aβ1-42 levels were not significantly changed during the follow-up period.【Conclusion】Elevated platelet GSK-3β activity and plasma Aβ42/Aβ40 ratio were independent risk factors for cognitive decline in T2 DM patients.Olfactory dysfunction,elevated platelet r GSK-3β and elevated plasma Aβ42/Aβ40 ratio can effectively predict the transition to MCI or AD in patients with T2 DM.【Background】Alzheimer’s disease(AD)is a neurodegenerative disease characterized by progressive memory loss and cognitive decline,synaptic degeneration of neurons and massive loss of brain neurons,which eventually leads to complete loss of personality and death.At present,the cause of the disease is not fully understood.Epidemiological studies have shown that high plasma homocysteine(Hcy)levels are associated with the development of AD,and high homocysteinemia(HHcy)is a strong and independent risk factor for AD.Hcy is a precursor of methionine and an intermediate of sulfur amino acids in methylation and antisulfidation pathways.There are three major dietary cofactors in Hcy metabolism: vitamin B6,B12,and folic acid.One of the mechanisms by which homocysteine exites its toxic effect is by interfering with the methylation of some functional proteins.Thus,it has been speculated that elevated plasma homocysteine leads to demethylation of PP2 A,resulting in hyperphosphorylation of Tau.Aging is an independent risk factor for AD,and brain aging in animals induced by Dgalactose injection is similar in many ways to human brain aging.D-galactose is an aldohexose that is found as a reducing sugar in foods such as cheese,butter,honey and cherries,as well as in the human body.D-galactose can be converted to glucose at normal concentrations,but at higher concentrations,it reacts with free amines of amino acids in proteins and peptides to form advanced glycation end products,causing oxidative damage to the body.In our previous study,we found that homocysteine injected into tail vein for 14 days can induce HHcy in rats,accompanied by learning and memory impairment.The operation of caudal venous injection in mice is very difficult.Therefore,it is of great practical value to investigate whether intraperitoneal injection of homocysteine or simultaneous injection of D-galactose can also replicate the mouse model of AD-like pathological and behavioral abnormalities.【Objective】To explore whether intraperitoneal injection of homocysteine or D-galactose in mice can induce AD-like brain pathological changes and cognitive dysfunction.【Methods】The effect of intraperitoneal injection of different concentrations of Hcy on mice: 12-week-old C57 male mice were randomly divided into four groups and weighed before injection: Control group,intraperitoneal injection of normal saline 150μl;low concentration Hcy group,according to body weight 360μg In the medium concentration Hcy group,150 μl of Hcy solution was injected intraperitoneally at a dose of 720μg/kg according to body weight;in the high concentration Hcy group,150μl of Hcy solution was injected intraperitoneally at a dose of 1440μg/kg according to body weight.Behavioural experiments such as open field experiment,cross elevated experiment,water maze experiment,and conditioned fear experiment were performed immediately after 14 days of continuous administration to detect anxiety and depression emotions,learning and memory function of mice.Then,the mouse brain tissue was taken to detect the Tau at different phosphorylation sites in the mouse brain by Western blotting experiment and the Aβ content in the mouse brain by Dot blotting experiment.The effect of the combination of homocysteine and D-galactose on mice: 8 weeks old C57 male mice were randomly divided and weighed before injection: Control group,intraperitoneal injection of normal saline 300μl;Hcy group,according to body weight 150μl of Hcy solution at 720μg/kg and 150μl of normal saline were injected intraperitoneally for 14 days;Hcy+Dgalactose group,150μl of Hcy solution at a dose of 720μg/kg and 150μl of D-galactose at a dose of 120mg/kg according to body weight Continuous intraperitoneal injection for 14 days;D-galactose group,according to body weight with a dose of 120mg/kg D-galactose 150μl and normal saline 150μl continuous intraperitoneal injection for 14 days.On the 1/14/21/28 days after the continuous administration of 14 days,the mice were subjected to open-field experiments,new thing recognition and other behavioral experiments to detect the mice’s anxiety,depression and memory function.Mice were less irritated,so Morris water maze was used to test the learning and memory function of mice.Subsequently,3 mice in each group were collected using eyeball enucleation to collect mouse blood and centrifuged.After centrifugation,the plasma was collected and stored in a refrigerator at minus 80°C.Then,the brain tissues of these mice were taken to detect different phosphorylation in the mouse brain by Western blotting.The Tau protein at the site and the Aβ content in the mouse brain were detected by Dot blotting.In addition,3 mice in each group were perfused and the brains were embedded in paraffin.【Results】1.The low(360μg/kg),medium(720μg/kg)and high(1440μg/kg)concentration of homocysteine were administered by intraperitoneal injection for 14 consecutive days.In the Morris water maze experiment,the escape latency of mice in the high-concentration group was prolonged on the 3-5 days during the learning phase of the Morris water maze experiment,while the number of times of crossing the platform was significantly reduced in the lowconcentration group and the high-concentration group during the detection phase.The mouse hippocampus was taken for Western blotting,and it was found that the phosphorylated Tau content of Thr231 in the hippocampus of the high-concentration group increased.At the same time,the Aβ content in the hippocampus of mice in the low concentration group also increased significantly.2.On the first day after the continuous intraperitoneal injection of Hcy and D-galactose for 14 days,the level of Aβ in the hippocampus of mice increased,and the level of phosphorylated Tau at Ser262 site in the hippocampus of mice in the Hcy group increased.On the 14 th day after the end of the administration,the open field experiment showed that the mice in the Hcy+D-galactose group had a shorter residence time in the central area.On the 28 th day after the end of the administration,the content of Tau phosphorylated at Thr231 in the hippocampus of the Hcy+D-galactose group increased.The Morris water maze experiment found that the Hcy+D-galactose group mice had longer escape latency on the 4th and 5th day of the learning phase.【Conclusion】Intraperitoneal injection of high concentration of Hcy(1440μg/kg)can cause damage to the learning and memory function of mice.At the same time,the level of phosphorylated Tau protein at Thr231 can be seen in the hippocampus of mice.The mice injected with Hcy and Dgalactose showed anxiety and depression-like emotions and impaired learning and memory function,accompanied by increased levels of Aβ and Tau protein phosphorylated at Thr231 in the hippocampus.Intraperitoneal injection of high concentration Hcy or combined application with D-galactose may partially mimic AD-like pathological and behavioral changes.