Role Of Glycogen Synthase Kinase-3β In Alzheimer’s Disease And Type2Diabetes Mellitus

Alzheimer’s disease (AD) is an common age-related neurodegenerative disease of the central nervous system (CNS) which is primarily characterized by a progressive decline in memory and cognitive function. Type2diabetes mellitus (T2DM) is a common endocrine and metabolic disease which is primarily characterized by insulin resistance (IR), hyperglycemia and hyperinsulinemia. T2DM has been identified as an independent risk factor for AD, and AD has been considered as the "type3diabetes mellitus".With the coming acceleration of China’s population aging, the incidence of T2DM and diabetes-related AD will quickly grow. T2DM and AD have become serious public health problems in China.The pathology of AD is characterized by the accumulation of extracellular senile plaques (SP), which primarily consist of β-amyloid, and intracellular neurofibrillary tangles (NFTs), which primarily consist of hyperphosphorylated tau. T2DM and AD have several features in common, including aging-related processes, high cholesterol levels, metabolic disorders, Aβ protein aggregation, tau protein phosphorylation, glycogen synthase kinase-3β (GSK-3β) activation, insulin resistance (IR) and oxidative stress response (OSR). Tau phosphorylation has been considered as an important phosphoprotein in the pathogenesis of AD, which is obviously regulated by tau phosphatases such as protein phosphatase2A (PP2A) and tau kinases such as GSK-3βprotein. GSK-3β protein has previously been suggested to play an important role in the pathogenesis of T2DM and AD, which is activated in the periphery or the brain. But the upstream and downstream factors, and the underlying regulatory mechanisms of GSK-3βin T2DM and AD are still unknown.Magnesium is a key mineral for the human body. Magnesium plays an important role in a host of vital cellular processes, which include glycolysis, oxidative phosphorylation, cellular respiration and protein synthesis. Previous reports have shown that hypomagnesemia is the most common feature in T2DM patients, and magnesium deficiency has been proposed as an independent risk factor for T2DM. The decline of magnesium level is found in the plasma or the brain of AD patients in numerous clinical and laboratory studies. Magnesium depletion, especially in the hippocampus, has been known as an important pathogenic factor in AD.A chronic reduction in dietary magnesium causes memory impairment, and magnesium supplementation in the treatment of dementia patients improves memory performance. Therefore, magnesium plays an important role in the pathogenesis of AD and T2DM, and may serve as a convergent point which links AD and T2DM. Till now, the effect of magnesium and GSK-3β protein on AD which is related to diabetes has not been reported.In our animal study, we investigated the role of magnesium in the GSK-3β regulation and tau hyperphosphorylation, and discussed the underlying mechanisms of cognitive improvement in ICV-STZ rats. In our clinical experiment, we confirmed the influencing factors of T2DM with mild cognitive impairment (MCI) and the significance of platelet GSK-3β in T2DM patients. The main findings are as following:Part ⅠMagnesium improves learning and memory via inhibiting glycogen synthase kinase3β and tau hyperphosphorylation in sporadic AD model ratsClinical studies have shown that hypomagnesemia is the most common feature in AD and T2DM patients, and the administration with nutritional magnesium enhances AD patients’ memory. Animal studies have revealed that magnesium regulates NMDAR signaling pathway, rescues synapse loss, and reverses memory impairments in the aged rats and AD model rats. Cell experiments have confirmed that magnesium influences the AβPP processing, and that in the presence of high extracellular magnesium levels, AβPP processing activates the a-secretase cleavage pathway and reduces amyloid-β production. Magnesium plays an important role in the pathogenesis of AD and diabetes, and may serve as a convergent point that links AD and diabetes. GSK-3β protein is activated in the periphery of T2DM patients or the brain of AD patients, and GSK-30is involved in the pathogenesis of T2DM and AD.But the mechanisms for magnesium and GSK-3β in diabetes-related AD have not been elucidated.[Objective] To clarify whether magnesium improves cognitive impairment in sporadic AD model rats by regulating GSK-3P and tau hyperphosphorylation, and explore the underlying regulatory mechanisms of GSK-3β activity.[Methods] These experiments were performed on male Sprague-Dawley (SD) rats. SAD model was made by an intracerebroventricular (ICV) infusion of streptozotocin (STZ). Magnesium sulfate dissolved in normal saline (NS) was administered to rats via intraperitoneal injection. SD rats were randomly divided into the following six groups (n=12in each group):sham-operated control group (Con), magnesium control group (100mg/kg, Mg), STZ group (STZ), and STZ (STZ+Mg) plus Mg groups (50mg/kg,100mg/kg and200mg/kg, i.p.). The Morris water maze (MWM) was performed to evaluate the learning and memory of rats during the3rd week after the ICV-STZ treatment. The total magnesium in the cerebral cortex and hippocampus were measured using atomic absorption spectroscopy (AAS). Long-term potentiation (LTP) recordings, Golgi staining and Western blotting of synapse-associated proteins were carried out to evaluate synaptic damage. Immunohistochemistry and western blot were applied to detect GSK-3β proteins activity, tau phosphorylation and PI3K/Akt signaling pathway in the hippocampus. Western blot and real time PCR were used to analyse the protein level of INSR and the mRNA levels of INS and INSR in the hippocampus.[Results] A simultaneous treatment of magnesium sulfate significantly enhanced the brain magnesium levels and efficiently attenuated the ICV-STZ-induced cognitive impairments. Additionally, simultaneous supplementation of magnesium sulfate(100mg/kg) reversed the LTP impairment, restored the number of dendritic branches and mushroom spines percentage, and rescued the expression of synaptic protein. Magnesium administration attenuated tau protein hyperphosphorylation at the Thr205, Thr231,Ser396, Ser214and Ser404sites. Magnesium arrested ICV-STZ-induced GSK-3P activation via an increase in inhibitory phosphorylation at the Ser9site. Magnesium supplementation promoted the protein expression of INSR, the mRNA expressions of INS and INSR in ICV-STZ-induced rats, which was prone to stimulate the PI3K/Akt signaling pathway.[Conclusion) We found that simultaneous administration of magnesium sulfate significantly increased the brain magnesium levels, rescued the LTP impairment, improved synaptic efficacy, enhanced insulin sensitivity, decreased tau protein hyperphosphorylation through PI3K/Akt/GSK-3β signaling pathway, and attenuated learning and memory decline in ICV-STZ rats. Altogether, our results provide novel insights suggesting that magnesium treatment at the early stage may decrease the risk for cognitive impairment in AD patients. Part ⅡInfluencing factors for mild cognitive impairment and the significance of platelet GSK-3p in T2DM patientsAlzheimer’s disease (AD) is the most common neurodegenerative disorder of the central nervous system (CNS). At present, AD is still lack of effective treatment measures. The progression of AD from the early stages to symptomatic stages often occurs over a long period of time. Therefore, early detection and intervention of AD may help to develop and target effective treatment strategies. It is difficult to make an early diagnosis of sporadic AD due to its insidious onset, which accounts for over99%of the AD cases. Epidemiologic studies have shown that T2DM is an important risk factor which can trigger or aggravate the development of AD. The rate of conversion of the mild cognitive impairment (MCI) to AD in T2DM patients was three times higher than that in healthy people. At present, early diagnosis or independent predictors for the conversion of MCI into AD have not been elucidated. The change of olfactory function usually occurs in the early stage of AD. GSK-3β plays an important role in the pathogenesis of T2DM and AD. ApoE ε4is a major prevalent genetic risk factor for AD. Therefore, our study investigated the correlations for cognitive dysfunction, olfactory function, platelet GSK-3β activity and ApoE genepolymorphisms in T2DM patients.[Objective] To explored the influencing factors of T2DM with MCI and the significance of platelet GSK-30in T2DM patients.[Methods] A sample of206T2DM patients were divided into two groups:137subjects in T2DM without MCI group (T2DM-nMCI) and69subjects in T2DM with MCI group (T2DM-MCI). All subjects had finished the neuropsychological test (MMSE, CDR) and olfactory threshold determination (Connecticut Chemosensory Clinical Research Center, CCCRC method). Biochemical indicators including fasting blood glucose, postprandial blood glucose, serum insulin, hemoglobin Alc (HbAlc) and serum magnesium were measured the next morning. An immunologic test (ELISA) was applied to determine the serum levels of GSK-3β and AGEs protein. GSK-3β activity in platelet was detected by Western blot and enzyme activity kit. The expressions of platelet GSK-3β (total-GSK-3p and Ser-9phosphorylated GSK-3β) were measured by Western blot and Dot blot. GSK-3β activity was indirectly assessed by means of the proportion between phospho-GSK-3β to total GSK-3β (GSK-3β ratio), the former representing the inactive form of the enzyme. Genomic DNA was extracted from peripheral blood leukocytes with DNA extraction kit according to the manufacturer’s protocol. Amplification refractory mutation system (ARMS) was applied to detect ApoE gene polymorphism.[Results] Our results showed that the MMSE score and olfactory threshold determination in T2DM with MCI group (T2DM-MCI)were lower than those in T2DM without MCI group (T2DM-nMCI). The HbAlc concentration in T2DM-MCI group was higher than that in T2DM-nMCI group. Compared to T2DM-nMCI group, T2DM-MCI group had higher percentages of ε4allele and lower e3allele. The expressions of AGEs and GSK-3β in serum of T2DM-MCI group were higher than those in T2DM-nMCI group. The proportion between phospho-GSK-3β to total GSK-3β (GSK-3β ratio) was significantly reduced in T2DM-MCI group as compared to that in T2DM-nMCI group, which indicated the higher GSK-3β activity. ApoE ε4gene, olfactory threshold and Ser9-GSK-3β/GSK-3β ratio were closely associated with MCI in T2DM patients. The OR values (95%CI) were2.72(1.19-6.18),0.51(0.38-0.69) and0.40(0.18-0.91) respectively. The ROC (Receiver operator characteristic curve) analysis showed that combined index had higher specificity and sensitivity than single index.[Conclusion] ApoE ε4gene, olfactory threshold and Ser9-GSK-3β/GSK-3β were independent influence factors for MCI in T2DM. Determination of platelet GSK-3β activity can be used as a diagnostic index for MCI in T2DM patients. The combination of indexes can improve the sensitivity and specificity of MCI diagnosis in T2DM.