| Background Methamphetamine?METH?is a well-known psychostimulant,the highly abuse of which is a serious worldwide public health issue.Recent studies have shown that METH has a strong neurotoxicity,manifested as a significant adverse change in neural structure and function,leading to pathological changes similar to degenerative diseases of the nervous system,such as Alzheimer's disease?AD?[1,2].In recent years,increasing evidence shows that central insulin resistance is an important cause of AD-like changes,manifested as a disorder of insulin signaling,and thus intervention on central insulin signaling and its important protein substrates has become the third important intervention of AD treatment besides A? and p-tau [3,4].Studies have shown that enhancing insulin signaling pathways by intravenous injection of insulin,intranasal infusion of insulin,and insulin sensitizers can improve cognitive function [5].Based on the changes in aging brain caused by METH abuse[6],we believe that METH may promote the production of AD-like neurodegenerative diseases by inhibiting insulin signaling pathway transduction.Insulin receptor is a key trigger for insulin action and its downstream signaling pathway.After insulin binds to insulin receptors?IRs?,it recruits adaptor proteins such as insulin receptor substrates 1 and 2?IRS1/2?to initiate multi-directional effects through multiple signaling pathways.There are two main signaling pathways related to insulin: First,extracellular signal regulated protein kinase?ERK?is an important rendezvous point of multi-channel[7];second,IRS activates phospho-dylinositol-3 kinase?PI3K?,and then Lead to activation of protein kinase B?PKB,also known as AKT?[8].In the insulin signal transduction pathway,glycogen synthase kinase 3?/??GSK3?/??is located downstream of PI3K/AKT,which in turn initiates or inhibits the expression of AD-like pathological proteins,such as the expression of APP,APP cleavage,p-tau.However,current studies of METH exposure have focused on striatum injury,including neuronal apoptosis,dopaminergic neurotransmitter release,and changes in related receptor expression in this region,while few studies are made on the expression of AD-like marker proteins and their mechanisms.Therefore,this study is based on the important supporting role of insulin signaling in the central system,combined with the central energy metabolism disorder caused by METH exposure,to explore whether METH exposure can change the key signaling pathway of insulin signaling,which provides clinical treatment strategies to the early stage of AD.Object To investigate whether METH exposure can lead disorder of insulin signaling pathway in the neuronal cells.Methods Human neuroblastoma cells?SH-SY5 Y,abbreviated as SY5Y?and rat adrenal medullary pheochromocytoma cells?PC-12?were studied.The SY5 Y cells were treated with different concentrations of METH?0,100,300,1000 ?M?for 12 h,while 300 ?M of METH was co-incubated with SY5 Y for different periods?0,0.5,1,3,6,12,24 h?,the expression of AKT,GSK3?/?,ERK and their phosphorylation level were observed by Western blot?WB?.In PC-12 cells,the expression of GSK3?,p-GSK3? and BACE-1 were observed in the same way,and the immunofluorescence staining of BACE-1 was detected by enzyme linked immunosorbent assay?ELISA?.Results In the SY5 Y cell line,the expression of p-AKT-Ser473,p-GSK3?-Ser9,p-GSK3?-Ser21,and p-ERK1/2 observed at 300 and 1000 ?M were significantly down-regulated after 12 h exposure to different concentrations of METH.After co-incubated with 300 ?M METH for different periods,the expression of p-AKT-Ser473,p-GSK3?-Ser9,p-GSK3?-Ser21,and p-ERK1/2 began to decrease after a short-term increase,which were significantly lower than the control group at 12,24,6,and 12 h respectively.In the PC-12 cell model,the expression of p-GSK3?-Ser21 was decreased in a dose-and time-dependent manner after METH treatment,and the expression of BACE-1 was reversed.After co-cultured with 300 ?M METH for 12 h,immunofluorescence staining of BACE-1 expression revealed that METH treatment significantly enhanced the fluorescent labeling of BACE-1.ResultsConclusion After the exposure of METH in a certain dose for a period of time in SY5 Y cells,both PI3K/AKT and ERK insulin signaling pathways were inhibited,then GSK3?/? were activated.In PC-12 cells,GSK3? was activated,while the activity of BACE-1 was also enhanced.BackgroundAlzheimer's disease?AD?,commonly known as senile dementia,is a neurodegenerative disease characterized by progressive cognitive and memory loss.With the prolongation of human life,Alzheimer's disease has become the third biggest killer of human health after cardiovascular and tumor.It has also become a younger trend in recent years and has become a hot topic in the field of public health.According to a statistics in 2012,there are 35 million Alzheimer's patients worldwide,while the number of China has exceeded 1/6 of the global number of cases,and many countries have or are about to enter the aging society.By 2030,the global number of AD patients can reach 67 million,the incidence of AD in the population over 65 years old is 10%,and the number over 85 years old is as high as 50%.It is a progressive disease that mainly affects the hippocampus and cortical areas of the brain.There are two hypotheses in its pathogenesis,namely the formation of extracellular amyloid plaques and intracellular tangles?NFTs?.A? is a peptide substance of different sizes formed by sequential hydrolysis and cleavage of amyloid precursor protein?APP?[9],and carboxy terminal fragments?CTFs?are key hydrolysates of APP [10].The up-regulation of the above pathological proteins currently has various mechanisms,such as oxidative stress,mitochondrial damage,endoplasmic reticulum stress,and inflammatory response.Recently,some biochemical,molecular and cellular abnormalities have emerged in the formation and development of AD.The brain's abnormalities in glucose and energy metabolism are abnormal in AD,even in the early stages of cognitive impairment [11].As a key component of the central nervous system,the brain accounts for only about 2% of the body's weight,but consumes 20% of the total oxygen consumption of the human body and 25% of the total energy.In the process,insulin?Insulin,IN?and its related pathways play a decisive role in regulating glucose homeostasis and energy metabolism.It has been previously thought that the regulation of glucose homeostasis in the brain is only achieved by the transport of insulin-related receptors in peripheral insulin,as the brain is not sensitive to insulin.In recent years,more and more evidence has shown that the brain itself can also produce and secrete insulin and insulin-like growth factors,but the role of insulin signaling in this "insulin-insensitive" tissue remains poorly understood.With the development and integration of interdisciplinary studies such as imaging,multimodality,bioinformatics,and molecular probes,people have a long-standing understanding of the role of glucose homeostasis and insulin signaling in the brain.Using Fluorodeoxyglucose as a ligand to construct molecular probes,imaging with PET technology,and integration of multi-modal techniques,it was found that AD and even mild cognitive impairment?MCI?patients showed signs of decreased glucose metabolism in the cerebral palsy and hippocampus in early stages[12],suggesting energy metabolic "regulatory systems"-insulin signaling may play an important role in the progression of AD.This part of the study is based on the potential role of METH in central insulin signaling.It is hypothesized that insulin signaling pathway disorder may be associated with METH-induced changes in downstream neurodegenerative pathological proteins.This study can provide a pathological basis for neurological damage characterized by neurodegenerative changes caused by METH abuse,thus providing a preliminary basis for the mechanism.ObjectTo investigate the effect of METH on the expression of early pathological proteins in AD.MethodsIn this part of the experiment,SY5 Y and PC-12 with general neuron characteristics were used as cell models.SY5 Y cells were treated with different concentrations of METH?0,100,300,1000 ?M?for 12 h,while 300 ?M of METH was co-incubated with SY5 Y for different times?0,0.5,1,3,6,12,24 h?,western blot was used to detect changes in the expression of APP and tau phosphorylation on Thr205 site.The expression of APP,CTFs and A?1-42 was observed in the same manner in PC-12 cells.ResultsIn the SY5 Y cell line,the expression of APP and p-tau-Thr205 was significantly up-regulated after METH treatment at different concentrations for 12 h.After co-incubated with 300 ?M METH for different periods,APP increased significantly from 3 h,while the peak was reached at 6 h.Meanwhile,p-tau-Thr205 was up-regulated from 6 h and peaked at 12 h.In the PC-12 cell model,the expression of APP and A?1-42 was increased significantly after exposure to different concentrations of METH for 12 h,and the CTFs was only significantly up-regulated at 1000 ?M?P < 0.001?.At different times of 300 ?M METH treatment,we found that APP and CTFs increased significantly from 6 h,peak at 12 and 24 h respectively,while A?1-42 increased significantly from 3 h.ConclusionIn SY5 Y cells,METH promoted hyperphosphorylation of the Thr205 site in tau and increased APP production.In PC-12 cells,METH led an increase in the level of APP,A?1-42,and CTFs.BackgroundInsulin?IN?/insulin-like growth factor?IGF?plays an important role in maintaining energy metabolism in the brain,neuronal survival,learning and memory.The insulin receptor?IR?and the insulin-like growth factor receptor?IGFR?are highly similar in structure and function.When IN/IGF-1 binds to both receptors,it activates the downstream insulin receptor substrate?IRS?,delivering insulin signals.Monte et al found that IN/IGF and its related receptors IR,IGF-1R,and IGF-2R were widely expressed in hippocampus,but compared to normal controls,IGF-1R in hippocampus of AD patients were 8-10 times lower,while IN expression decreased by 4 times and IGF-2 decreased by 3 times [13].Arnold's group found that IN/IR-IRS-1-PI3 K and IGF-IR-IRS-2-PI3 K insulin signals in the hippocampus of AD patients by immunoprecipitation,the binding ability of IN/IGF-1 and IR/IGF-1R decreased,in addition,IRS-1 serine phosphorylation levels increased and tyrosine phosphorylation levels decreased [14].Glycogen synthase kinase-3?GSK-3?is involved in the pathological process of insulin resistance and cognitive abnormalities,promotes the production of A? and p-tau,and interferes with insulin signaling.Peroxisome proliferator-activated receptors?PPARs?are ligand-activated transcription factors known to play important role in the regulation of glucose absorption,homeostasis of lipid metabolism and further involves in repressing the expression of genes related to inflammation.In recent,both clinical and preclinical studies show that use of Peroxisome proliferator-activated receptor gamma?PPAR??agonist improves both learning and memory along with other AD related pathology[15].Studies found that PPAR? agonists in T2 D and AD rats can diminish the aggregation of A?40 and A?42,then improve learning and cognitive dysfunction[16].However,it has not been reported whether rosiglitazone can improve insulin signaling pathway against METH-induced AD-like changes.ObjectBased on METH-induced insulin resistance and AD-like pathology,the insulin sensitizer Ros was used to observe the expression of insulin signaling pathway-related proteins and the changes of downstream neuropathic proteins.In this way,we may determine whether the reduction of the pathological proteins in AD was associated with the improvement of insulin signaling,which can further elucidate the regulation of insulin signaling during the expression of AD-like neuropathic proteins.MethodsIn this experiment,SY5 Y cells were observed.The cells were divided into six groups: blank control group,Ros low dose group?10 ?M?,Ros high dose group?50 ?M?,METH group?300 ?M?,METH + Ros low dose group,as well as METH + Ros high dose group.The expression levels of insulin signaling cascades proteins,APP and p-tau were detected by western blot after 12 hours of incubation.ResultsCompared with the METH group,the resistance of insulin signaling pathway and the expression of pathological proteins in METH + Ros low and high dose groups were significantly improved.In METH + Ros co-treatment groups,the expression of p-IR-Tyr1355,p-IRS1-Tyr896,p-AKT-Ser473,p-GSK3?/?-Ser21/9,p-ERK were up-regulated,while the expression of APP and p-tau-Thr205 were significantly decreased.ConclusionIn SY5 Y cells,the insulin signaling pathway may be the target of Ros intervention in the AD process,suggesting that Ros has potential value in the prevention and treatment of central AD-like pathology induced by METH. |
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