Deletion Of Leptin Promotes Neuropathological Changes And Its Mechanism In Alzheimer's Disease Mice

Alzheimer's disease is a neurodegenerative disease that frequently occurs in the elderly.The main pathological features of AD include the formation of senile plaques(SP)induced by A? aggregation between neurons,neurofibrillary tangles caused by tau phosphorylation as well as the loss of synapse and cholinergic neurons in specific brain regions.Diabetes mellitus(DM)is a metabolic disease characterized by hyperglycemia,and it can be classified into type 1 diabetes mellitus(T1DM)and type 2 diabetes mellitus(T2DM)according to the cause of the disease.A large number of clinical epidemiological studies have revealed that both T1DM and T2DM can lead to cognitive and memory impairment.The morbidity of AD is also significantly higher in people with a history of DM.In addition,the results of autopsy showed that both A? deposition and tau hyperphosphorylation were detected in the pancreas and brains of DM patients.Therefore,some scholars refer to as AD "type 3 diabetes",in order to emphasize the internal relationship between these two diseases.However,due to the lack of reliable animal models,there is a contradictory understanding of the effects with insulin resistance/hyperglycemia on AD pathology.Therefore,whether there is a mutual relationship between DM and AD remains to be elucidated.In this study,we have bred APP/PS1-ob/ob model mouse,an animal model with both T2DM and AD by hybridization between leptin knockout(ob/ob+/-)mice and APP/PS1 transgenic mice.Subsequently,behavioural,morphological,and molecular biology methods were used to explore whether or not T2DM could aggravate the pathological process of AD.The implementation of this project has an important theoretical value for guiding the DM population to prevent the occurrence and development of AD.Previous studies have shown that leptin knockout mice can be used as a reliable animal models of T2DM,which are characterized by chronic hyperglycemia induced by insulin resistance.In order to confirm the T2DM characteristics of the model mouse,glucose and insulin metabolism in 3-month-old mice were examined.The results showed that leptin depletion mice exhibited marked hyperglycemia symptoms.After depletion of leptin gene,APP/PS1 mice showed marked symptoms of insulin resistance in insulin tolerance test(ITT)and glucose tolerance test(GTT).The results of insulin detection indicated that leptin knockout mice had increased levels of insulin in the serum and brain.Taken together these results suggest that APP/PS1-ob/ob mice have obvious T2DM symptoms and can be used as an important animal model to explore the relationship between T2DM and AD.To investigate the effects of T2DM on memory and cognitive impairments in APP/PS1 mice,we evaluated the behavioral performance of 6-month-old mice by Morris water maze(MWM)and nest construction tests.The results indicated that the escape latency and escape distance of APP/PS1 mice in the water maze increased significantly after leptin knockout.Although APP/PS1 mice did not exhibit significant cognitive impairment,mice in the APP/PS1-ob/ob group performed worse than the APP/PS1 group mice in nest construction test.Our behavioral experiments have suggested that diabetes induced by leptin depletion could aggravate memory and cognitive impairments in APP/PS1 mice.Given the essential role of APP processing and A? deposition in abnormal cognitive function,we initially sought to elucidate the effects of a chronic diabetic state on A? generation and deposition in APP/PS1-ob/ob mice.Immunohistochemistry results showed a significant difference in the SP number between APP/PS1 and APP/PS1-ob/ob mice,and the size of the A?-immunoreactive SP in both the cortex and hippocampus of the APP/PS1-ob/ob mice was also markedly increased.In addition,Western blot showed that T2DM could induce an increased expression of APP and a decreased expression of?-secretase and its shear products,sAPP? and CTF?.Therefore,we can determine that the cerebral chronic diabetic condition promotes the metabolism of APP during amyloidosis,which subsequently leads to the generation and deposition of A?.Tau hyperphosphorylation and synaptic dysfunction are thought to be early manifestation of AD.To further investigate the effects of chronic diabetes mellitus on neurofibrillary tangles and synaptic damage in the brain of AD mice,multiple tau phosphorylation sites and synapses levels were detected by immunohistochemistry and Western blot in this study.Our results showed that the levels of tau phosphorylation at the location of Ser396,Ser202 and Thr231 were significantly increased in the brain of APP/PS 1 mice after leptin gene knockout.Consistent with the immunohistochemical results,the expression levels of NMDAR and PSD95 in the brains of APP/PS1-ob/ob mice were significantly decreased,compared with APP/PS1 group.Studies of the mechanisms on tau phosphorylation and synaptic damage have showed significantly increased phosphorylation of CDK5 and mTOR accompanied with a down regulated CREB in the brain of APP/PS1-ob/ob mice when compared with APP/PS1 mice.This process is regulated by two calcium related protein kinases,CaMK? and calpain1.Immediately,we detected the calcium content in each group of mice brain by atomic absorption spectrometry,and it was found that diabetic symptoms contributed to the increase in calcium content in the brains of APP/PS1 mice.These results suggest that chronic insulin resistance may induce mitochondrial dysfunction,and lead to a disequilibrium of calcium homeostasis.The activation of calpain1/CDK5 and CAMKII/mTOR pathways promote the phosphorylation of tau and synaptic damage.To further investigate the effects of insulin resistance on the inflammatory response in the brain of AD mice,we examined ROS level and inflammation related pathways in mice brain.Our study found that the ROS production in the brains of mice with diabetes was significantly higher than in the WT and APP/PS 1 mice,AGEs/RAGE pathway and its downstream factors ERK/NF?B were further activated in APP/PS 1 mice with leptin gene knockout.In addition,Western blot and Real-Time PCR showed that chronic diabetic conditions significantly increased the expression of inflammatory factors such as TNFa and IL-1? in the brain of AD mice.The results above were further confirmed by fluorescence co-localization of A?/GFAP and A?/Iba1.In summary,the diabetes induced by leptin gene depletion can inhibit the non-amyloid pathway in the brain of APP/PS 1 mice.This process may lead to A?production and accumulation.In addition,T2DM can lead to the aggravation of calcium overload in the brain of APP/PS1 mice and further activate calcium related protein kinase,such as calpain1 and CAMKII,thereby promoting tau phosphorylation and synaptic damage.Finally,chronic insulin resistance exacerbates neuroinflammation and apoptosis in AD mice brain by activating AGEs/RAGE and its downstream ERK/NF?B pathway.