Alzheimer's disease is pathologically defined by accumulation of extracellular amyloid-β(Aβ). Approximately 25 mutations in β-amyloid precursor protein(APP) are pathogenic and cause autosomal dominant Alzheimer...Alzheimer's disease is pathologically defined by accumulation of extracellular amyloid-β(Aβ). Approximately 25 mutations in β-amyloid precursor protein(APP) are pathogenic and cause autosomal dominant Alzheimer's disease. To date, the mechanism underlying the effect of APP mutation on Aβ generation is unclear. Therefore, investigating the mechanism of APP mutation on Alzheimer's disease may help understanding of disease pathogenesis. Thus, APP mutations(A673T, A673 V, E682 K, E693 G, and E693Q) were transiently co-transfected into human embryonic kidney cells. Western blot assay was used to detect expression levels of APP, beta-secretase 1, and presenilin 1 in cells. Enzyme-linked immunosorbent assay was performed to determine Aβ_(1–40) and Aβ_(1–42) levels. Liquid chromatography-tandem mass chromatography was used to examine VVIAT, FLF, ITL, VIV, IAT, VIT, TVI, and VVIA peptide levels. Immunofluorescence staining was performed to measure APP and early endosome antigen 1 immunoreactivity. Our results show that the protective A673 T mutation decreases Aβ_(42)/Aβ_(40) rate by downregulating IAT and upregulating VVIA levels. Pathogenic A673 V, E682 K, and E693 Q mutations promote Aβ_(42)/Aβ_(40) rate by increasing levels of CTF99, Aβ_(42), Aβ_(40), and IAT, and decreasing VVIA levels. Pathogenic E693 G mutation shows no significant change in Aβ_(42)/Aβ_(40) ratio because of inhibition of γ-secretase activity. APP mutations can change location from the cell surface to early endosomes. Our findings confirm that certain APP mutations accelerate Aβ generation by affecting the long Aβ cleavage pathway and increasing Aβ_(42/40) rate, thereby resulting in Alzheimer's disease.展开更多
Objective To investigate the impact of sub-chronic Aluminium-maltolate [Al(mal)s] exposure on the catabolism of amyloid precursor protein (APP) in rats. Methods Forty adult male Sprague-Dawley (SD) rats were ran...Objective To investigate the impact of sub-chronic Aluminium-maltolate [Al(mal)s] exposure on the catabolism of amyloid precursor protein (APP) in rats. Methods Forty adult male Sprague-Dawley (SD) rats were randomly divided into five groups: the control group, the maltolate group (7.56 mg/kg BW), and the Al(mal)s groups (0.27, 0.54, and 1.08 mg/kg BW, respectively). Control rats were administered with 0.9% normal saline through intraperitoneal (i.p.) injection. Maltolate and Al(mal)s were administered to the rats also through i.p. injections. Administration was conducted daily for two months. Rat neural behavior was examined using open field tests (OFT). And the protein expressions and their mRNAs transcription related with APP catabolism were studied using enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (RT-PCR). Results The expressions of APP, 13-site APP cleaving enzyme 1 (BACEI) and presenilin-1 (PSi) proteins and their mRNAs transcription increased gradually with the increase of Al(mal)3 doses (P〈0.05). The enzyme activity of BACEI in the 0.54 and 1.08 mg/kg Al(mal)s groups increased significantly (P〈0.05). The expression of 8-amyloid protein (AS) 1-40 gradually decreased while the protein expression of A81-42 increased gradually with the increase of Al(mal)s doses (P〈0.05). Conclusion Result from our study suggested that one of the possible mechanisms that Al(mal)s can cause neurotoxicity is that Al(mal)s can increase the generation of A81-42 by facilitating the expressions of APP, β-, and γ-secretase.展开更多
Curcumin exerts a neuroprotective effect on Alzheimer’s disease;however,it is not known whether microRNAs are involved in this protective effect.This study was conducted using swAPP695-HEK293 cells as an Alzheimer’s...Curcumin exerts a neuroprotective effect on Alzheimer’s disease;however,it is not known whether microRNAs are involved in this protective effect.This study was conducted using swAPP695-HEK293 cells as an Alzheimer’s disease cell model.swAPP695-HEK293 cells were treated with 0,0.5,1,2,5,and 10μM curcumin for 24 hours.The changes in miR-15b-5p,miR-19a-3p,miR-195-5p,miR-101-3p,miR-216b-5p,miR-16-5p and miR-185-5p expression were assessed by real-time quantitative polymerase chain reaction.The mRNA and protein levels of amyloid precursor protein,amyloid-β40 and amyloid-β42 were evaluated by quantitative real-time polymerase chain reaction,western blot assays and enzyme-linked immunosorbent assays.swAPP695-HEK293 cells were transfected with miR-15b-5p mimic,or treated with 1μM curcumin 24 hours before miR-15b-5p inhibitor transfection.The effects of curcumin on amyloid precursor protein,amyloid-β40 and amyloid-β42 levels were evaluated by western blot assays and enzyme-linked immunosorbent assay.Luciferase assays were used to analyze the interaction between miR-15b-5p and the 3′-untranslated region of amyloid precursor protein.The results show that amyloid precursor protein and amyloid-βexpression were enhanced in swAPP695-HEK293 cells compared with HEK293 parental cells.Curcumin suppressed the expression of amyloid precursor protein and amyloid-βand up-regulated the expression of miR-15b-5p in swAPP695-HEK293 cells.In addition,we found a negative association of miR-15b-5p expression with amyloid precursor protein and amyloid-βlevels in the curcumin-treated cells.Luciferase assays revealed that miR-15b-5p impaired the luciferase activity of the plasmid harboring the 3′-untranslated region of amyloid precursor protein.These findings indicate that curcumin down-regulates the expression of amyloid precursor protein and amyloid-βin swAPP695-HEK293 cells,which was partially mediated by miR-15b-5p via targeting of the 3′-untranslated region of amyloid precursor protein.展开更多
The pathological implication of amyloid precursor protein(APP)in Alzheimer’s disease has been widely documented due to its involvement in the generation of amyloid-β peptide.However,the physiological functions of AP...The pathological implication of amyloid precursor protein(APP)in Alzheimer’s disease has been widely documented due to its involvement in the generation of amyloid-β peptide.However,the physiological functions of APP are still poorly understood.APP is considered a multimodal protein due to its role in a wide variety of processes,both in the embryo and in the adult brain.Specifically,APP seems to play a key role in the proliferation,differentiation and maturation of neural stem cells.In addition,APP can be processed through two canonical processing pathways,generating different functionally active fragments:soluble APP-α,soluble APP-β,amyloid-β peptide and the APP intracellular C-terminal domain.These fragments also appear to modulate various functions in neural stem cells,including the processes of proliferation,neurogenesis,gliogenesis or cell death.However,the molecular mechanisms involved in these effects are still unclear.In this review,we summarize the physiological functions of APP and its main proteolytic derivatives in neural stem cells,as well as the possible signaling pathways that could be implicated in these effects.The knowledge of these functions and signaling pathways involved in the onset or during the development of Alzheimer’s disease is essential to advance the understanding of the pathogenesis of Alzheimer’s disease,and in the search for potential therapeutic targets.展开更多
Studies have demonstrated that amyloid precursor protein (APP) expression increases in multiple sclerosis tissues during acutely and chronically active stages. To determine the relationship between axonal injury and...Studies have demonstrated that amyloid precursor protein (APP) expression increases in multiple sclerosis tissues during acutely and chronically active stages. To determine the relationship between axonal injury and regeneration in multiple sclerosis, an animal model of experimental autoimmune encephalomyelitis was induced using different doses of myelin basic protein peptide. APP and growth-associated protein 43 (GAP-43), which is considered a specific marker of neural regeneration, were assessed by western blot analysis. Expression of APP and GAP-43, as well as the correlation between these two proteins, in brain white matter and spinal cord tissues of experimental autoimmune encephalomyelitis rats at different pathological stages was analyzed. Results showed that APP and GAP-43 expression increased during the acute stage and decreased during remission, with a positive correlation between APP and GAP-43 expression in brain white matter and spinal cord tissues. These results suggest that APP and GAP-43 could provide nutritional and protective effects on damaged neurons.展开更多
The deposition of amyloid-beta is a pathological hallmark of Alzheimer's disease, Amyloid-beta is derived from amyloid precursor protein through sequential proteolytic cleavages by β-secretase (beta-site amyloid pr...The deposition of amyloid-beta is a pathological hallmark of Alzheimer's disease, Amyloid-beta is derived from amyloid precursor protein through sequential proteolytic cleavages by β-secretase (beta-site amyloid precursor protein-cleaving enzyme 1) and r-secretase. To further elucidate the roles of beta-site amyloid precursor protein-cleaving enzyme 1 in the development of AIzheimer's disease, a yeast two-hybrid system was used to screen a human embryonic brain cDNA library for proteins directly interacting with the intracellular domain of beta-site amyloid precursor protein-cleaving enzyme 1. A potential beta-site amyloid precursor protein-cleaving enzyme 1- interacting protein identified from the positive clones was divalent cation tolerance protein. Immunoprecipitation studies in the neuroblastoma cell line N2a showed that exogenous divalent cation tolerance protein interacts with endogenous beta-site amyloid precursor protein-cleaving enzyme 1. The overexpression of divalent cation tolerance protein did not affect beta-site amyloid precursor protein-cleaving enzyme 1 protein levels, but led to increased amyloid precursor protein levels in N2a/APP695 cells, with a concomitant reduction in the processing product amyloid precursor protein C-terminal fragment, indicating that divalent cation tolerance protein inhibits the processing of amyloid precursor protein. Our experimental findings suggest that divalent cation tolerance protein negatively regulates the function of beta-site amyloid precursor protein-cleaving enzyme 1. Thus, divalent cation tolerance protein could play a protective role in Alzheimer's disease.展开更多
After gene mutation, the pcDNA3.1/APP595/596 plasmid was transfected into HEK293 cells to establish a cell model of Alzheimer's disease. The cell model was treated with donepezil or compound Danshen tablets after cul...After gene mutation, the pcDNA3.1/APP595/596 plasmid was transfected into HEK293 cells to establish a cell model of Alzheimer's disease. The cell model was treated with donepezil or compound Danshen tablets after culture for 72 hours. Reverse transcription-PCR showed that the mRNA expression of amyloid protein precursor decreased in all groups following culture for 24 hours, and that there was no significant difference in the amount of decrease between donepezil and compound Danshen tablets. Our results suggest that compound Danshen tablets can reduce expression of the mRNA for amyloid protein precursor in a transgenic cell model of Alzheimer's disease, with similar effects to donepezil.展开更多
Cerebral ischemia was induced using photothrombosis 1 hour after intraperitoneal injection of the p38 mitogen-activated protein kinase (MAPK) inhibitor $B239063 into Swedish mutant amyloid precursor protein (APP/SW...Cerebral ischemia was induced using photothrombosis 1 hour after intraperitoneal injection of the p38 mitogen-activated protein kinase (MAPK) inhibitor $B239063 into Swedish mutant amyloid precursor protein (APP/SWE) transgenic and non-transgenic mice. The number of surviving neurons in the penumbra was quantified using Nissl staining, and the activity of p38 MAPKs was measured by western blotting. The number of surviving neurons in the penumbra was significantly reduced in APP/SWE transgenic mice compared with non-transgenic controls 7 days after cerebral ischemia, but the activity of p38 MAPKs was significantly elevated compared with the non-ischemic hemisphere in the APP/SWE transgenic mice. SB239063 prevented these changes. The APP/SWE mutation exacerbated ischemic brain injury, and this could be alleviated by inhibiting p38 MAPK activity.展开更多
Previous studies have reported that non-human primates and rodents exposed to lead during brain development may become dependent on the deposition of pre-determined β-amyloid protein (Aβ),and exhibit upregulation ...Previous studies have reported that non-human primates and rodents exposed to lead during brain development may become dependent on the deposition of pre-determined β-amyloid protein (Aβ),and exhibit upregulation of β-site amyloid precursor protein expression in old age.However,further evidence is required to elucidate the precise relationship and molecular mechanisms underlying the effects of early lead exposure on excessive Aβ production in adult mammals.The present study investigated the effects of lead exposure on expression of β-amyloid precursor protein cleavage enzyme-1 (BACE-1) in the rat retina and the production of Aβ in early development,using the retina as a window for studying Alzheimer's disease.Adult rats were intraocularly injected with different doses of lead acetate (10μmol/L,100μmol/L,1 mmol/L,10 mmol/L and 100 mmol/L).The results revealed that retinal lead concentration,BACE-1 and its cleavage products β-C-terminal fragment and retina Aβ1-40 were all significantly increased in almost all of the lead exposure groups 48 hours later in a dose-dependent manner.The only exception was the 10μmol/L group.The distribution of BACE-1 in the retina did not exhibit obvious changes,and no distinctive increase in the activation of retinal microglia was apparent.Similarly,retinal synaptophysin expression did not exhibit any clear changes.These data suggest that lead exposure can result in the upregulation of retinal neuron BACE-1 expression in the early period of development and further increase the overproduction of Aβ1-40 in the retina.Our results provided novel insight into the molecular mechanisms underlying environmentally-induced Alzheimer's disease.展开更多
In this study, we employed chromatin immunoprecipitation, a useful method for studying the locations of transcription factors bound to specific DNA regions in specific cells, to investigate amyloid precursor protein i...In this study, we employed chromatin immunoprecipitation, a useful method for studying the locations of transcription factors bound to specific DNA regions in specific cells, to investigate amyloid precursor protein intracellular domain binding sites in chromatin DNA from hippocampal neurons of rats, and to screen out five putative genes associated with the learning and memory functions. The promoter regions of the calcium/calmodulin-dependent protein kinase II alpha and glutamate receptor-2 genes were amplified by PCR from DNA products immunoprecipitated by amyloid precursor protein intracellular domain. An electrophoretic mobility shift assay and western blot analysis suggested that the promoter regions of these two genes associated with learning and memory were bound by amyloid precursor protein intracellular domain (in complex form). Our experimental findings indicate that the amyloid precursor protein intracellular domain is involved in the transcriptional regulation of learning- and memory-associated genes in hippocampal neurons. These data may provide new insights into the molecular mechanism underlying the symptoms of progressive memory loss in Alzheimer's disease.展开更多
BACKGROUND: In vitro cultures of neural stem cells have shown that estrogen can regulate beta-amyloid precursor protein (β-APP) metabolism and reduce amyloid-beta production. OBJECTIVE: To investigate the effects...BACKGROUND: In vitro cultures of neural stem cells have shown that estrogen can regulate beta-amyloid precursor protein (β-APP) metabolism and reduce amyloid-beta production. OBJECTIVE: To investigate the effects of long-term oral administration of compound nylestriol or low-dose 17beta-estradiol on β-APP and mRNA expression in the hippocampus of ovariectomized (OVX) rats. DESIGN, TIME AND SETTING: This randomized and controlled experiment was performed at the Animal Laboratory and Laboratory of Endocrine and Metabolic Disease, Xiangya Second Hospital of Central South University between April 2003 and May 2004. MATERIALS: According to body mass, 50 six-month-old female Sprague-Dawley rats were randomly divided into five groups (n = 10 per group): normal control, sham operation, OVX model, 17beta-estradiol (Sigma, USA), and compound nylestriol tablet (Laboratory of Endocrine and Metabolic Disease, Xiangya Second Hospital of Central South University) groups. METHODS: Rats in OVX plus 17beta-estradiol and OVX plus compound nylestriol tablet groups underwent ovariectomy. On the second day after surgery, rats were intragastrically given 17beta-estradiol (100 μg/kg), once per day or compound nylestriol tablet (0.5 mg/kg) and levonorgestrel (0.15 mg/kg) every 2 days. MAIN OUTCOME MEASURES: β-APP expression in the hippocampus of OVX rats was determined using immunohistochemistry (SABC method) and β-APP mRNA expression was analyzed by in situ hybridization. The results were quantitatively analyzed using cell counting and average optical density. RESULTS: The number and optical density of β-APP-positive neurons in every subregion of the hippocampus of OVX rats was dramatically increased compared with normal and sham operation groups following 35 weeks of administration (P 〈 0.05). Levels of β-APP were decreased following oral administration of compound nylestriol or 17beta-estradiol. In situ hybridization showed that long-term estrogen deficiency and oral administration of compound nylestriol or 17beta-estradiol did not alter the number of β-APP mRNA-positive neurons. CONCLUSION: The results show that long-term estrogen deficiency results in an increase of expression of β-APP though no changes in the expression of β-APP mRNA are detected. Replacement of estrogen with low-dose 17 beta-estradiol or compound nylestriol tablet inhibits the expression of β-APP in the hippocampus to the same extent.展开更多
BACKGROUND: Previous studies have demonstrated that Piper futokadsura stem selectively inhibits expression of amyloid precursor protein (APP) at the mRNA level. In addition, the piperlonguminine (A) and dihydropi...BACKGROUND: Previous studies have demonstrated that Piper futokadsura stem selectively inhibits expression of amyloid precursor protein (APP) at the mRNA level. In addition, the piperlonguminine (A) and dihydropiperlonguminine (B) components (1 : 0.8), which can be separated from Futokadsura stem, selectively inhibit expression of the APP at mRNA and protein levels. OBJECTIVE: Based on previous findings, the present study investigated the effects of β-site amyloid precursor protein cleaving enzyme (BACE1) and APP genes on the production of β-amyloid peptide 42 (Aβ42) in human neuroblastoma cells (SK-N-SH cells) using small interfering RNAs (siRNAs) and A/B components separated from Futokadsura stem, respectively. DESIGN, TIME AND SETTING: A gene interference-based randomized, controlled, in vitro experiment was performed at the Key Laboratory of Cardiovascular Remodeling and Function Research, Ministries of Education and Public Health, and Institute of Pharmacologic Research, School of Pharmaceutical Science & Department of Biochemistry, School of Medicine, Shandong University between July 2006 and December 2007. MATERIALS: SK-N-SH cells were provided by Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences, Shanghai, China; mouse anti-human BACE1 monoclonal antibody was purchased from R&D Systems, USA; mouse anti-human APP monoclonal antibody was purchased from Cell Signaling Technology, USA; and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG was provided by Sigma, USA. METHODS: The human BACE1 cDNA sequence was obtained from NCBI website (www.ncbi.nlm.nih.gov/sites/entrez). Three pairs of siRNAs, specific to human BACE1 gene, were synthesized through the use of Silencer pre-designed siRNA specification, and were transfected into SK-N-SH cells with siPORT NeoFX transfection agent to compare the effects of different concentrations of siRNAs (10-50 nmol/L) on SK-N-SH cells. Futokadsura stem was separated and purified with chemical methods, and the crystal was composed of A/B components, with an A to B ratio of 1:0.8. The A/B (1 : 0.8) components were added to the SK-N-SH cells at different concentrations (13.13, 6.56, and 3.28 mg/mL). MAIN OUTCOME MEASURES: Using RT-PCR and Western blot methods, BACE1 and APP expression at mRNA and protein levels was detected in SK-N-SH cells following treatment with different siRNAs and concentrations of Futokadsura stem-separated A/B components, respectively. Altered Aβ42 secretion by SK-N-SH cells was determined by ELISA. RESULTS: BACE1 mRNA and protein levels were significantly suppressed by 40 and 50 nmol/L siRNAs at 48 hours post-transfection. A/B components (1 : 0.8), which were separated from Futokadsura stem, selectively inhibited mRNA and protein expression of APP in SK-N-SH cells. Aβ42 secretion by SK-N-SH cells was significantly decreased following treatment with siRNAs or A/B components. CONCLUSION: Inhibition of BACE1 and APP genes by various materials and methods efficiently decreased production of Aβ42.展开更多
Objective: To investigate the neuroprotective effects of Syzygium aromaticum(S.aromaticum)extract(500 mg/kg) on AlCl_3(300 mg/kg)-induced mouse model of oxidative stress and neurotoxicity.Methods: An ethanolic extract...Objective: To investigate the neuroprotective effects of Syzygium aromaticum(S.aromaticum)extract(500 mg/kg) on AlCl_3(300 mg/kg)-induced mouse model of oxidative stress and neurotoxicity.Methods: An ethanolic extract of S.aromaticum seeds was prepared and the active compounds were identified using nuclear magnetic resonance spectroscopy.BALB/c mice were divided into five groups(negative control, AlCl_3-treated, self-recovery, AlCl_3 + S.aromaticum, S.aromaticum only; n=10) and treated with AlCl_3 and S.aromaticum extract.Expression of oxidative markers [Superoxide dismutase 1(SOD1) and peroxiredoxin 6(Prdx6)] and amyloid precursor protein(APP) in the hippocampus and cortex was evaluated via PCR.Histopathological assessment was performed to investigate the extent of neurodegeneration.Results: It was observed that AlCl_3 exposure increased the expression of APP770 while simultaneously down regulated the expression of APP695.AlCl_3 also induced a significant decrease(P<0.05) and an increase(P<0.05) in the expression level of SOD1 and Prdx6, respectively.A substantial decrease substantial(P<0.05) in the density of Nissl substance was also observed in cortex of the mice treated with AlCl_3.Interestingly, treatment with S.aromaticum extract normalized the alterations in the expression level of SOD1, Prdx6 and APPisoforms and improved the neuronal structural damage.Conclusions: The results showed that S.aromaticum is a promising antioxidant and a neuroprotective agent.展开更多
To analyze whether expressed amyloid precursor protein(APP) existed in hydrophilic(cytoplasmid) or hydrophobic(lipid bilayer) environment in E. coli and to obtain intact APP for study on its function, we investi...To analyze whether expressed amyloid precursor protein(APP) existed in hydrophilic(cytoplasmid) or hydrophobic(lipid bilayer) environment in E. coli and to obtain intact APP for study on its function, we investigated the expression characterization and preparation of the three intact isoforms APP770, APP751, and APP695 in E. coll. The results show that these expressed APPs existed both in hydrophilic cytoplasm region as inclusion bodies and hydrophobic membrane region as membrane-bound state in E. coll. APPs in inclusion bodies were purified on an NTA-Ni^2+ agarose column after dissolving in the urea buffer and APPs in membrane-bound state were obtained by ultracentrifugation. The activity analysis indicates that APP770 and APP751 exhibited strong trypsin-inhibitory activity like the natural ones. These results indicate that E. coli cells can be used as host cells for the expression of human integral membrane protein like APP in either soluble or membrane-bound state unless the interest protein undergone post-translational modification is required.展开更多
BACKGROUND: Previous studies have demonstrated that mutant amyloid precursor protein (APP) or presenilin-1 (PS1) genes increase susceptibility to ischemic brain damage induced by middle cerebral artery occlusion....BACKGROUND: Previous studies have demonstrated that mutant amyloid precursor protein (APP) or presenilin-1 (PS1) genes increase susceptibility to ischemic brain damage induced by middle cerebral artery occlusion. Possible mechanisms include over-production of beta-amyloid peptide (Aβ). OBJECTIVE: Because Aβ is over-produced in the APP/PS1 double-transgenic mouse, the present study focused on mechanisms of increased ischemic damage due to mutant APP and PS1 genes by measuring oxidative stress, mitochondrial function, and calcium homeostasis. DESIGN, TIME AND SETTING: The non-randomized, controlled, in vivo and in vitro experiments were performed at the Medical Research Center, Second Clinical College, Jinan University between May and October 2008. MATERIALS: Male APP transgenic mice carrying the mutant 695swe gene and female PS1 transgenic mice carrying the mutant Leu235Pro gene were donated from the University of Hong Kong. SHSY5Y human neureblastoma cells were purchased from ATCC (Manassas, VA, USA), and Aβ1-42 was obtained from Sigma-Aldrich (St. Louis, MO, USA). METHODS: APP transgenic mice were mated with PS1 transgenic mice to produce APP/PS1 double-transgenic mice and wildtype littermates mice. The photothrombotic stroke model was induced in six APP/PS1 double-transgenic and 6 wildtype littermates mice. SHSY5Y human neuroblastoma cells were cultured in vitro, and were divided into 4 groups: Aβ group, cells were exposed to 5 pmol/L Aβ for 24 hours; oxygen-glucose deprivation (OGD) group, cells were exposed to OGD for 1 hour after treatment with sterile, ultra-pure water for 24 hours; OGD+Aβ group, cells were exposed to OGD and Aβfor 1 hour after treatment with 5 pmol/L Aβ for 24 hours; sham control group: cells were exposed to sterile, ultra-pure water for 25 hours. OGD was achieved by exposing the cells to glucose-free DMEM and placing the cells in an anaerobic chamber flushed with 5% CO2 and 95% N2 (v/v) at 37 ℃ for 1 hour. MAIN OUTCOME MEASURES: TTC staining was used to measure infarct volume 7 days after photothrombotic stroke. Cell viability was evaluated using the MTT kit. Opening of the mitochondrial permeability transition pore, intracellular concentration of superoxide anion, and calcium after OGD were detected with fluorescence intensity of calcein-AM, hydroethidine, and fluo-3/AM. RESULTS: At 7 days after stroke, total infarct volume and cortical infarct volume were significantly greater in the APP/PS1 transgenic mice compared with the wildtype littermates mice (P 〈 0.01). Aβ, OGD, and Aβ + OGD significantly decreased cell viability and increased fluorescence intensity of hydroethidine and fluo-3/AM (P 〈 0.01). Compared with the Aβ or OGD group, Aβ + OGD significantly decreased cell viability (P 〈 0.01) and significantly increased fluorescence intensity of calcein-AM, hydroethidine, and fluo-3/AM (P 〈 0.01 or P 〈 0.05). CONCLUSION: The APP/PS1 double-transgenic mice were more vulnerable to ischemia. The possible mechanisms included enhanced opening of the mitochondrial permeability transition pore, overproduction of superoxide anion due to pore opening, and disturbed calcium homeostasis induced by excess superoxide anion.展开更多
BACKGROUND: The mechanisms of brain injury Following diabetes could be related to amyloid precursor protein (APP) mRNA overexpression. Studies have shown that Gingko biloba leaf extract (EGb) is effective in prom...BACKGROUND: The mechanisms of brain injury Following diabetes could be related to amyloid precursor protein (APP) mRNA overexpression. Studies have shown that Gingko biloba leaf extract (EGb) is effective in promoting functional recovery of the brain after traumatic injury. EGb is also effective in improving central nervous system plasticity and learning and memory functions of the elderly. OBJECTIVE: To study the effects of EGb on learning and memory, as well as hippocampal APP mRNA expression in the brains of diabetic rats, using Morris water maze behavioral testing and reverse transcription polymerase chain reaction (RT-PCR), respectively. DESIGN: Complete random design, controlled experimental study. SETTING: Department of Pharmacology, Pharmaceutical School, Guangxi Medical University. MATERIALS: A total of 70 male Wistar rats (180-220 g), 8 weeks old and specific pathogen free, were used for this study. GbE (containing 24.8% flavone glycosides and 6.2% diterpene lactone) was purchased from Guilin Sitejia Natural Plants Pharmaceutical Factory (Guangxi Province, Lot NO. 200405). Streptozotocin was purchased from Sigma (USA). Protamine zinc insulin injection was purchased from WANBANG Biochemical Pharmaceutical Co., Ltd. (Xuzhou Jiangsu, China). METHODS: The experiment was performed in the Experimental Center of Guangxi Medical University from March to October 2005.(1) Experimental intervention: 70 rats were divided randomly into normal control group, diabetic model group (DM group), diabetic model +10 μ g/kg insulin group (DM + Ins group), diabetic model + 100 mg/kg ginkgo leaf extract group (DM + EGb high-dose group), and diabetic model + 50 mg/kg ginkgo leaf extract group (DM + EGb low-dose group); there were 14 rats in each group. Rats with an intraperitoneal (i.p.) injection of citrate buffer solution (pH 4.4) served as the control group. To establish the diabetes model, rats were treated with i.p. injection of 55 mg/kg streptozotocin. Insulin (10 U/kg) was injected subcutaneously (s.c.) every day for 6 months in the DM group. EGb (100 mg/kg) and EGb (50 mg/kg) was administered intragastrically every day for 6 months in the DM + EGb high-dose group and DM + EGb low-dose group, respectively. The DM group and control group were administered distilled water intragastrically every day for 6 months. Drugs were administered once every morning. (2) Experimental evaluation: Six month after intervention, learning and memory of diabetic rats was tested by Morris water maze. Rats were allowed to train for 4 days, and the escape latency and platform-searching score were measured at days 5 and 8. Changes in hippocampal APP mRNA expression were measured with RT-PCR. MAIN OUTCOME MEASURES: Morris water maze performances and hippocampal APP mRNA expression in rats. RESULTS: A total of 70 Wistar rats were included in the final analysis, without any loss. (1) Learning and memory dysfunction of diabetic rats: After 4 days of Morris water maze training, escape latency was longer in the DM group on days 5 and 8, and the platform-searching score was lower in the DM group compared to the control group. In the DM + EGb group, the escape latency score was shorter and platform-searching score was significantly increased compared to the DM group. (2) APP mRNA expression: in the hippocampus of diabetic rats, a 340 bp mRNA product was amplified, which is comparable to the APP mRNA amplification length of design. The expression of APP mRNA from the hippocampus of diabetic rats with learning and memory dysfunctions was significantly increased. EGb extract significantly inhibited the APP mRNA expression in these rats. CONCLUSION: EGb not only ameliorated the learning and memory dysfunctions in diabetic rats, but also significantly inhibited APP mRNA expression. Results from this study led to the hypothesis that diabetes could be one of the risk factors for AD.展开更多
Summary: Over-expression of APP and Swedish mutation could cause some familial early onset AD. In this study, a primary screening was conducted of effective small interference RNAs (siRNAs) targeted wild type APP ...Summary: Over-expression of APP and Swedish mutation could cause some familial early onset AD. In this study, a primary screening was conducted of effective small interference RNAs (siRNAs) targeted wild type APP (APPwt) and Swedish mutant APP (APPswe). One siRNA targeting APPwt and the other siRNA targeting APPswe were designed, All these siRNAs were endogenously expressed by siRNAs expressing plasmids, COS-7 cells were transiently co-transfected with APP-GFP recombinant plasmids and siRNA expression vector, The silencing effect of each siRNA was quantitatively assessed by the level of expression of green fluorescent protein (GFP). It was found that the siRNAs silenced APPwt and APPswe to different degrees, siRNA directed against APPswe was more effective in suppressing the expression of fusion gene of APPswe than that of APPwt. The silencing effect of siRNA directed against APPswe indicating allele-specific silencing property of the siRNAs. Therefore, siRNAs directed against APP play an important role both in the therapeutic study of Alzheimer disease and functional exploration ofAPP gene.展开更多
BACKGROUND: The findings about the alterations in cerebrospinal fluid beta-amyloid protein (Aβ ) and apolipoprotein E (APOE) after subarachnoid hemorrhage indicate that they have significant correlation with pro...BACKGROUND: The findings about the alterations in cerebrospinal fluid beta-amyloid protein (Aβ ) and apolipoprotein E (APOE) after subarachnoid hemorrhage indicate that they have significant correlation with prognosis of patients. OBJECTⅣE: To observe the alterations in cerebrospinal fluid Aβ and ApoE after subarachnoid hemorrhage (SAH). DESIGN: Contrast observation. SETTING: Department of Neurosurgery, the First Hospital of Lanzhou University. PARTICIPANTS: A total of 25 SAH patients including 16 males and 9 females aged from 13 to 72 years were selected form Department of Neurosurgery, the First Affiliated Hospital of Lanzhou University from October 2003 to February 2004. The Hunt-Hess grade ranged from Ⅰ to Ⅳ, and patients admitted hospital in 24 hours after invasion, affirmed by the brain CT scan and lumbar vertebra puncture, no other severe complications and important organs' functional defect and severe infection, no hematological system disease. METHODS- All admitted patients were collected CSF by lumbar vertebra puncture in 24 hours. The cerebrospinal fluid (CSF) of control group came from the admitted 15 patients of our hospital that have no nervous system disease. Aβ content was detected by enzyme linked immunosorbent assay (ELISA), the kit was provided by the Central Laboratory of the First Hospital of Lanzhou University; ApoE concentration was detected by monoclone enzyme linked immunosorbent assay (ELISA), the kit was provided by the Immunotechnique Research Institute of the Fourth Military Medical University. S100B concentration was detected by enzyme linked immunosorbent assay double antibody sandwich method, the kit was provided by the Physiological Research Room of the Fourth Military Medical University. The data were indicated on Mean±SD and were analyzed by SPSS 10.0 statistical package. All data were handled through test of significance variance analysis, and groups were compared through independent sampler t test. The concentration was handled through Pearson correlation analysis between Aβ and ApoE. The relationship between Aβ, ApoE concentration with pathogenetic condition and prognosis of the patients was handled through Spearman ranking correlation analysis. MAIN OUTCOME MEASURES:① The concentration of ApoE, Aβ and S100B after SAH in contrast to the control group in CSF by different Hunt-Hess and Glasgow Outcome Scale (GOS) grades; ② The level of correlation between ApoE and Aβ ; ③Correlation between ApoE and Aβ in pathogenetic condition and prognosis of the patients. RESULTS: All 25 SAH patients and 15 controls were involved in the final analysis. ① The concentration of ApoE, Aβ and S100B in CSF: The concentration of ApoE decreased after SAH in contrast to the control group [(0.46±0.007), (0.85±0.11) μg/L, P 〈 0.01], the concentration of ApoE decreased after SAH in contrast to the control group [(5.36± 1.19), (8.41± 1.60) μg/L, P 〈 0.01], and the concentration of S100B increased after SAH in contrast to the control group [(18.60±7.31), (6.56±1.02) pg/L, P 〈 0.01]. ② The concentration of ApoE, Aβ and S100B in CSF after SAH on different Hunt-Hess and GOS grades: The concentration of Aβ in Hunt-Hess Ⅰ -Ⅲ grade was higher than Hunt-Hess Ⅳ, Ⅴ grade [(6.63 ± 1.25), (3.35± 1.02) μg/L, P 〈 0.01], and the concentration of ApoE in Hunt-Hess Ⅰ- Ⅲ grade was higher than Hunt-Hess Ⅳ, Ⅴ grade [(0.56±0.07), (0.38±0.04) μg/L, P 〈 0.05], the concentration of S100B in Hunt-Hess Ⅰ - Ⅲ grade was lower than Hunt-Hess Ⅳ - Ⅴ grade [(16.32±5.58), (22.85±8.10) pg/L, P 〈 0.01]; the concentration of Aβ in GOS Ⅰ - Ⅲ grade was lower than GOS Ⅳ, Ⅴ grade [(3.76± 1.04), (5.89±1.20) μg/L, P 〈 0.01], and the concentration of ApoE in GOS Ⅰ - Ⅲ grade was lower than GOS Ⅳ, Ⅴ grade [(0.32±0.02), (0.58±0.07) μg/L, P 〈 0.011, and the concentration of S100B in GOS Ⅰ - Ⅲ grade was higher than GOS Ⅳ, Ⅴ grade [(25.36±9.70), (14.33±6.69) pg/L, P 〈 0.01].③ The results of Pearson correlation analysis and Spearman ranking correlation analysis: There was significantly positive correlation between CSF Aβ concentration and clinical outcome (r=0.65, P 〈 0.01), and the decrease in CSF Aβ concentration correlated significant with that of ApoE (r =0.85, P 〈 0.01). CONCLUSION: There is a significant decrease in both Aβ and ApoE in the CSF after SAH, and there is significant correlation between CSF Aβ and ApoE concentration with clinical outcome, the interactions between these proteins may have important effects on SAH, ApoE and Aβ as surrogate markers for the outcome of patients with SAH.展开更多
Targeting early steps in amyloid-beta production:Alzheimer’s disease(AD)has a long history as the"amyloid deposit"disorder.Many disorders are now known to be caused by proteinβ-sheet misfolding and aggregation...Targeting early steps in amyloid-beta production:Alzheimer’s disease(AD)has a long history as the"amyloid deposit"disorder.Many disorders are now known to be caused by proteinβ-sheet misfolding and aggregation(e.g.,Parkinson’s disease:α-synuclein;Huntington’s disease:Huntingtin;展开更多
Pharmacological studies reveal APP and Aβ have interactions with glutamate and calcium, cytokines, copper/zinc chelators, secretases and presenilins, nicotinic receptors, acetycholinesterase, neurotrophins, non-stero...Pharmacological studies reveal APP and Aβ have interactions with glutamate and calcium, cytokines, copper/zinc chelators, secretases and presenilins, nicotinic receptors, acetycholinesterase, neurotrophins, non-steroidal anti-inflame-matory drugs, monoclonal antibodies to Aβ, protease inhibitors, oestrogen, homocysteine, immediate early genes such as c-fos or c-jun and cholesterol. These functional and pharmacological observations highlight the need for greater understanding of APP and Aβ in brain function and have implications for clinical trials.展开更多
基金funded by the National Natural Science Foundation of China,No.81671268(to HQ)partially supported by a grant from the Ministry of Science and Technology of China,No.2013YQ03059514(to HQ)a grant from Key Laboratory for Neurodegenerative Disease of Ministry of Education of China,No.2015SJBX05(to HQ),2015SJZS01(to HQ)
文摘Alzheimer's disease is pathologically defined by accumulation of extracellular amyloid-β(Aβ). Approximately 25 mutations in β-amyloid precursor protein(APP) are pathogenic and cause autosomal dominant Alzheimer's disease. To date, the mechanism underlying the effect of APP mutation on Aβ generation is unclear. Therefore, investigating the mechanism of APP mutation on Alzheimer's disease may help understanding of disease pathogenesis. Thus, APP mutations(A673T, A673 V, E682 K, E693 G, and E693Q) were transiently co-transfected into human embryonic kidney cells. Western blot assay was used to detect expression levels of APP, beta-secretase 1, and presenilin 1 in cells. Enzyme-linked immunosorbent assay was performed to determine Aβ_(1–40) and Aβ_(1–42) levels. Liquid chromatography-tandem mass chromatography was used to examine VVIAT, FLF, ITL, VIV, IAT, VIT, TVI, and VVIA peptide levels. Immunofluorescence staining was performed to measure APP and early endosome antigen 1 immunoreactivity. Our results show that the protective A673 T mutation decreases Aβ_(42)/Aβ_(40) rate by downregulating IAT and upregulating VVIA levels. Pathogenic A673 V, E682 K, and E693 Q mutations promote Aβ_(42)/Aβ_(40) rate by increasing levels of CTF99, Aβ_(42), Aβ_(40), and IAT, and decreasing VVIA levels. Pathogenic E693 G mutation shows no significant change in Aβ_(42)/Aβ_(40) ratio because of inhibition of γ-secretase activity. APP mutations can change location from the cell surface to early endosomes. Our findings confirm that certain APP mutations accelerate Aβ generation by affecting the long Aβ cleavage pathway and increasing Aβ_(42/40) rate, thereby resulting in Alzheimer's disease.
基金supported by the National Natural Science Foundation of China (30972512)the Graduate Innovation Fund of Academic Degree Committee Office of the Shanxi Provincial Government (20093014)+1 种基金Doctor Start-up Fund from Shanxi Medical University (B03201209)the College Students Innovation Fund of Shanxi Medical University (2010-25)
文摘Objective To investigate the impact of sub-chronic Aluminium-maltolate [Al(mal)s] exposure on the catabolism of amyloid precursor protein (APP) in rats. Methods Forty adult male Sprague-Dawley (SD) rats were randomly divided into five groups: the control group, the maltolate group (7.56 mg/kg BW), and the Al(mal)s groups (0.27, 0.54, and 1.08 mg/kg BW, respectively). Control rats were administered with 0.9% normal saline through intraperitoneal (i.p.) injection. Maltolate and Al(mal)s were administered to the rats also through i.p. injections. Administration was conducted daily for two months. Rat neural behavior was examined using open field tests (OFT). And the protein expressions and their mRNAs transcription related with APP catabolism were studied using enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (RT-PCR). Results The expressions of APP, 13-site APP cleaving enzyme 1 (BACEI) and presenilin-1 (PSi) proteins and their mRNAs transcription increased gradually with the increase of Al(mal)3 doses (P〈0.05). The enzyme activity of BACEI in the 0.54 and 1.08 mg/kg Al(mal)s groups increased significantly (P〈0.05). The expression of 8-amyloid protein (AS) 1-40 gradually decreased while the protein expression of A81-42 increased gradually with the increase of Al(mal)s doses (P〈0.05). Conclusion Result from our study suggested that one of the possible mechanisms that Al(mal)s can cause neurotoxicity is that Al(mal)s can increase the generation of A81-42 by facilitating the expressions of APP, β-, and γ-secretase.
基金supported by the Science and Technology Planning Project of Guangdong Province of China,No.2016A020226022(to HYL)the Medical and Health Technology Project of Guangzhou of China,No.20161A011068(to HYL)the Guangzhou Science Technology and Innovation Commission of China,No.201704020043(to QCG)
文摘Curcumin exerts a neuroprotective effect on Alzheimer’s disease;however,it is not known whether microRNAs are involved in this protective effect.This study was conducted using swAPP695-HEK293 cells as an Alzheimer’s disease cell model.swAPP695-HEK293 cells were treated with 0,0.5,1,2,5,and 10μM curcumin for 24 hours.The changes in miR-15b-5p,miR-19a-3p,miR-195-5p,miR-101-3p,miR-216b-5p,miR-16-5p and miR-185-5p expression were assessed by real-time quantitative polymerase chain reaction.The mRNA and protein levels of amyloid precursor protein,amyloid-β40 and amyloid-β42 were evaluated by quantitative real-time polymerase chain reaction,western blot assays and enzyme-linked immunosorbent assays.swAPP695-HEK293 cells were transfected with miR-15b-5p mimic,or treated with 1μM curcumin 24 hours before miR-15b-5p inhibitor transfection.The effects of curcumin on amyloid precursor protein,amyloid-β40 and amyloid-β42 levels were evaluated by western blot assays and enzyme-linked immunosorbent assay.Luciferase assays were used to analyze the interaction between miR-15b-5p and the 3′-untranslated region of amyloid precursor protein.The results show that amyloid precursor protein and amyloid-βexpression were enhanced in swAPP695-HEK293 cells compared with HEK293 parental cells.Curcumin suppressed the expression of amyloid precursor protein and amyloid-βand up-regulated the expression of miR-15b-5p in swAPP695-HEK293 cells.In addition,we found a negative association of miR-15b-5p expression with amyloid precursor protein and amyloid-βlevels in the curcumin-treated cells.Luciferase assays revealed that miR-15b-5p impaired the luciferase activity of the plasmid harboring the 3′-untranslated region of amyloid precursor protein.These findings indicate that curcumin down-regulates the expression of amyloid precursor protein and amyloid-βin swAPP695-HEK293 cells,which was partially mediated by miR-15b-5p via targeting of the 3′-untranslated region of amyloid precursor protein.
基金supported by grants from the Ministerio de Ciencia e Innovación-Instituto de Salud Carlos Ⅲ(PI-10/00291 and MPY1412/09)Ministerio de Economía y Competitividad(SAF2015-71140-R)+2 种基金Comunidad de Madrid(Neurostem-Comunidad de Madrid consortium S2010/BMD-2336)supported by grants from Plan de Empleo Juvenil-Ministerio de Economía y Competitividad
文摘The pathological implication of amyloid precursor protein(APP)in Alzheimer’s disease has been widely documented due to its involvement in the generation of amyloid-β peptide.However,the physiological functions of APP are still poorly understood.APP is considered a multimodal protein due to its role in a wide variety of processes,both in the embryo and in the adult brain.Specifically,APP seems to play a key role in the proliferation,differentiation and maturation of neural stem cells.In addition,APP can be processed through two canonical processing pathways,generating different functionally active fragments:soluble APP-α,soluble APP-β,amyloid-β peptide and the APP intracellular C-terminal domain.These fragments also appear to modulate various functions in neural stem cells,including the processes of proliferation,neurogenesis,gliogenesis or cell death.However,the molecular mechanisms involved in these effects are still unclear.In this review,we summarize the physiological functions of APP and its main proteolytic derivatives in neural stem cells,as well as the possible signaling pathways that could be implicated in these effects.The knowledge of these functions and signaling pathways involved in the onset or during the development of Alzheimer’s disease is essential to advance the understanding of the pathogenesis of Alzheimer’s disease,and in the search for potential therapeutic targets.
基金the National Natural Science Foundation of China,No. 30873230Beijing Natural Science Foundation,No. 7092014+1 种基金Scientific Research Common Program of Beijing Municipal Education Commission,No. KM2007100025015Fund-ing Project for Academic Human Resources Devel-opment in Institutions of Higher Learning Under the Jurisdiction of Beijing Mu-nicipality,No. PHR201008401
文摘Studies have demonstrated that amyloid precursor protein (APP) expression increases in multiple sclerosis tissues during acutely and chronically active stages. To determine the relationship between axonal injury and regeneration in multiple sclerosis, an animal model of experimental autoimmune encephalomyelitis was induced using different doses of myelin basic protein peptide. APP and growth-associated protein 43 (GAP-43), which is considered a specific marker of neural regeneration, were assessed by western blot analysis. Expression of APP and GAP-43, as well as the correlation between these two proteins, in brain white matter and spinal cord tissues of experimental autoimmune encephalomyelitis rats at different pathological stages was analyzed. Results showed that APP and GAP-43 expression increased during the acute stage and decreased during remission, with a positive correlation between APP and GAP-43 expression in brain white matter and spinal cord tissues. These results suggest that APP and GAP-43 could provide nutritional and protective effects on damaged neurons.
基金supported by the National Natural Science Foundation of China, No. 81171192XMU Basic Training Program of Undergraduate, No. CXB2011019Visiting Scholar Fellowship of Key Laboratory of Ministry of Education for Cell Biology and Tumor Cell Engineering of Xiamen University, No. 201101
文摘The deposition of amyloid-beta is a pathological hallmark of Alzheimer's disease, Amyloid-beta is derived from amyloid precursor protein through sequential proteolytic cleavages by β-secretase (beta-site amyloid precursor protein-cleaving enzyme 1) and r-secretase. To further elucidate the roles of beta-site amyloid precursor protein-cleaving enzyme 1 in the development of AIzheimer's disease, a yeast two-hybrid system was used to screen a human embryonic brain cDNA library for proteins directly interacting with the intracellular domain of beta-site amyloid precursor protein-cleaving enzyme 1. A potential beta-site amyloid precursor protein-cleaving enzyme 1- interacting protein identified from the positive clones was divalent cation tolerance protein. Immunoprecipitation studies in the neuroblastoma cell line N2a showed that exogenous divalent cation tolerance protein interacts with endogenous beta-site amyloid precursor protein-cleaving enzyme 1. The overexpression of divalent cation tolerance protein did not affect beta-site amyloid precursor protein-cleaving enzyme 1 protein levels, but led to increased amyloid precursor protein levels in N2a/APP695 cells, with a concomitant reduction in the processing product amyloid precursor protein C-terminal fragment, indicating that divalent cation tolerance protein inhibits the processing of amyloid precursor protein. Our experimental findings suggest that divalent cation tolerance protein negatively regulates the function of beta-site amyloid precursor protein-cleaving enzyme 1. Thus, divalent cation tolerance protein could play a protective role in Alzheimer's disease.
基金supported by the Bureau of Traditional Chinese Medicine of Guangdong Province, No. 2010463the National Science and Technology"12~(th) Five-years"Major Special-purpose Foundation,No.2011ZX09201-201-01
文摘After gene mutation, the pcDNA3.1/APP595/596 plasmid was transfected into HEK293 cells to establish a cell model of Alzheimer's disease. The cell model was treated with donepezil or compound Danshen tablets after culture for 72 hours. Reverse transcription-PCR showed that the mRNA expression of amyloid protein precursor decreased in all groups following culture for 24 hours, and that there was no significant difference in the amount of decrease between donepezil and compound Danshen tablets. Our results suggest that compound Danshen tablets can reduce expression of the mRNA for amyloid protein precursor in a transgenic cell model of Alzheimer's disease, with similar effects to donepezil.
基金supported by the National Natural Science Foundation of China, No. 81171191Shenzhen Bureau of Science Technology and Information, No. 201002013+1 种基金Guangdong Province Medical Science Fund, No. A2008601 and Jinan University Scientific Research Foundation for Creation and Cultivation, No. 21609708
文摘Cerebral ischemia was induced using photothrombosis 1 hour after intraperitoneal injection of the p38 mitogen-activated protein kinase (MAPK) inhibitor $B239063 into Swedish mutant amyloid precursor protein (APP/SWE) transgenic and non-transgenic mice. The number of surviving neurons in the penumbra was quantified using Nissl staining, and the activity of p38 MAPKs was measured by western blotting. The number of surviving neurons in the penumbra was significantly reduced in APP/SWE transgenic mice compared with non-transgenic controls 7 days after cerebral ischemia, but the activity of p38 MAPKs was significantly elevated compared with the non-ischemic hemisphere in the APP/SWE transgenic mice. SB239063 prevented these changes. The APP/SWE mutation exacerbated ischemic brain injury, and this could be alleviated by inhibiting p38 MAPK activity.
基金the National Natural Science Foundation of China,No.30900773the National University Basic Research Foundation of China,No.2010QZZD022
文摘Previous studies have reported that non-human primates and rodents exposed to lead during brain development may become dependent on the deposition of pre-determined β-amyloid protein (Aβ),and exhibit upregulation of β-site amyloid precursor protein expression in old age.However,further evidence is required to elucidate the precise relationship and molecular mechanisms underlying the effects of early lead exposure on excessive Aβ production in adult mammals.The present study investigated the effects of lead exposure on expression of β-amyloid precursor protein cleavage enzyme-1 (BACE-1) in the rat retina and the production of Aβ in early development,using the retina as a window for studying Alzheimer's disease.Adult rats were intraocularly injected with different doses of lead acetate (10μmol/L,100μmol/L,1 mmol/L,10 mmol/L and 100 mmol/L).The results revealed that retinal lead concentration,BACE-1 and its cleavage products β-C-terminal fragment and retina Aβ1-40 were all significantly increased in almost all of the lead exposure groups 48 hours later in a dose-dependent manner.The only exception was the 10μmol/L group.The distribution of BACE-1 in the retina did not exhibit obvious changes,and no distinctive increase in the activation of retinal microglia was apparent.Similarly,retinal synaptophysin expression did not exhibit any clear changes.These data suggest that lead exposure can result in the upregulation of retinal neuron BACE-1 expression in the early period of development and further increase the overproduction of Aβ1-40 in the retina.Our results provided novel insight into the molecular mechanisms underlying environmentally-induced Alzheimer's disease.
基金supported by the Natural Science Foundation of Guangdong Province,China,No.8151051501000004
文摘In this study, we employed chromatin immunoprecipitation, a useful method for studying the locations of transcription factors bound to specific DNA regions in specific cells, to investigate amyloid precursor protein intracellular domain binding sites in chromatin DNA from hippocampal neurons of rats, and to screen out five putative genes associated with the learning and memory functions. The promoter regions of the calcium/calmodulin-dependent protein kinase II alpha and glutamate receptor-2 genes were amplified by PCR from DNA products immunoprecipitated by amyloid precursor protein intracellular domain. An electrophoretic mobility shift assay and western blot analysis suggested that the promoter regions of these two genes associated with learning and memory were bound by amyloid precursor protein intracellular domain (in complex form). Our experimental findings indicate that the amyloid precursor protein intracellular domain is involved in the transcriptional regulation of learning- and memory-associated genes in hippocampal neurons. These data may provide new insights into the molecular mechanism underlying the symptoms of progressive memory loss in Alzheimer's disease.
基金the National Key Medical Technologies Research and Development Program of China during the Tenth Five-Year Plan Period, No. 2001BA702B03
文摘BACKGROUND: In vitro cultures of neural stem cells have shown that estrogen can regulate beta-amyloid precursor protein (β-APP) metabolism and reduce amyloid-beta production. OBJECTIVE: To investigate the effects of long-term oral administration of compound nylestriol or low-dose 17beta-estradiol on β-APP and mRNA expression in the hippocampus of ovariectomized (OVX) rats. DESIGN, TIME AND SETTING: This randomized and controlled experiment was performed at the Animal Laboratory and Laboratory of Endocrine and Metabolic Disease, Xiangya Second Hospital of Central South University between April 2003 and May 2004. MATERIALS: According to body mass, 50 six-month-old female Sprague-Dawley rats were randomly divided into five groups (n = 10 per group): normal control, sham operation, OVX model, 17beta-estradiol (Sigma, USA), and compound nylestriol tablet (Laboratory of Endocrine and Metabolic Disease, Xiangya Second Hospital of Central South University) groups. METHODS: Rats in OVX plus 17beta-estradiol and OVX plus compound nylestriol tablet groups underwent ovariectomy. On the second day after surgery, rats were intragastrically given 17beta-estradiol (100 μg/kg), once per day or compound nylestriol tablet (0.5 mg/kg) and levonorgestrel (0.15 mg/kg) every 2 days. MAIN OUTCOME MEASURES: β-APP expression in the hippocampus of OVX rats was determined using immunohistochemistry (SABC method) and β-APP mRNA expression was analyzed by in situ hybridization. The results were quantitatively analyzed using cell counting and average optical density. RESULTS: The number and optical density of β-APP-positive neurons in every subregion of the hippocampus of OVX rats was dramatically increased compared with normal and sham operation groups following 35 weeks of administration (P 〈 0.05). Levels of β-APP were decreased following oral administration of compound nylestriol or 17beta-estradiol. In situ hybridization showed that long-term estrogen deficiency and oral administration of compound nylestriol or 17beta-estradiol did not alter the number of β-APP mRNA-positive neurons. CONCLUSION: The results show that long-term estrogen deficiency results in an increase of expression of β-APP though no changes in the expression of β-APP mRNA are detected. Replacement of estrogen with low-dose 17 beta-estradiol or compound nylestriol tablet inhibits the expression of β-APP in the hippocampus to the same extent.
基金the National Natural Science Foundation of China,No. NSFC-3027164
文摘BACKGROUND: Previous studies have demonstrated that Piper futokadsura stem selectively inhibits expression of amyloid precursor protein (APP) at the mRNA level. In addition, the piperlonguminine (A) and dihydropiperlonguminine (B) components (1 : 0.8), which can be separated from Futokadsura stem, selectively inhibit expression of the APP at mRNA and protein levels. OBJECTIVE: Based on previous findings, the present study investigated the effects of β-site amyloid precursor protein cleaving enzyme (BACE1) and APP genes on the production of β-amyloid peptide 42 (Aβ42) in human neuroblastoma cells (SK-N-SH cells) using small interfering RNAs (siRNAs) and A/B components separated from Futokadsura stem, respectively. DESIGN, TIME AND SETTING: A gene interference-based randomized, controlled, in vitro experiment was performed at the Key Laboratory of Cardiovascular Remodeling and Function Research, Ministries of Education and Public Health, and Institute of Pharmacologic Research, School of Pharmaceutical Science & Department of Biochemistry, School of Medicine, Shandong University between July 2006 and December 2007. MATERIALS: SK-N-SH cells were provided by Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences, Shanghai, China; mouse anti-human BACE1 monoclonal antibody was purchased from R&D Systems, USA; mouse anti-human APP monoclonal antibody was purchased from Cell Signaling Technology, USA; and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG was provided by Sigma, USA. METHODS: The human BACE1 cDNA sequence was obtained from NCBI website (www.ncbi.nlm.nih.gov/sites/entrez). Three pairs of siRNAs, specific to human BACE1 gene, were synthesized through the use of Silencer pre-designed siRNA specification, and were transfected into SK-N-SH cells with siPORT NeoFX transfection agent to compare the effects of different concentrations of siRNAs (10-50 nmol/L) on SK-N-SH cells. Futokadsura stem was separated and purified with chemical methods, and the crystal was composed of A/B components, with an A to B ratio of 1:0.8. The A/B (1 : 0.8) components were added to the SK-N-SH cells at different concentrations (13.13, 6.56, and 3.28 mg/mL). MAIN OUTCOME MEASURES: Using RT-PCR and Western blot methods, BACE1 and APP expression at mRNA and protein levels was detected in SK-N-SH cells following treatment with different siRNAs and concentrations of Futokadsura stem-separated A/B components, respectively. Altered Aβ42 secretion by SK-N-SH cells was determined by ELISA. RESULTS: BACE1 mRNA and protein levels were significantly suppressed by 40 and 50 nmol/L siRNAs at 48 hours post-transfection. A/B components (1 : 0.8), which were separated from Futokadsura stem, selectively inhibited mRNA and protein expression of APP in SK-N-SH cells. Aβ42 secretion by SK-N-SH cells was significantly decreased following treatment with siRNAs or A/B components. CONCLUSION: Inhibition of BACE1 and APP genes by various materials and methods efficiently decreased production of Aβ42.
基金supported by research grant by National University of Sciences and Technology (NUST), Islamabad, Pakistan
文摘Objective: To investigate the neuroprotective effects of Syzygium aromaticum(S.aromaticum)extract(500 mg/kg) on AlCl_3(300 mg/kg)-induced mouse model of oxidative stress and neurotoxicity.Methods: An ethanolic extract of S.aromaticum seeds was prepared and the active compounds were identified using nuclear magnetic resonance spectroscopy.BALB/c mice were divided into five groups(negative control, AlCl_3-treated, self-recovery, AlCl_3 + S.aromaticum, S.aromaticum only; n=10) and treated with AlCl_3 and S.aromaticum extract.Expression of oxidative markers [Superoxide dismutase 1(SOD1) and peroxiredoxin 6(Prdx6)] and amyloid precursor protein(APP) in the hippocampus and cortex was evaluated via PCR.Histopathological assessment was performed to investigate the extent of neurodegeneration.Results: It was observed that AlCl_3 exposure increased the expression of APP770 while simultaneously down regulated the expression of APP695.AlCl_3 also induced a significant decrease(P<0.05) and an increase(P<0.05) in the expression level of SOD1 and Prdx6, respectively.A substantial decrease substantial(P<0.05) in the density of Nissl substance was also observed in cortex of the mice treated with AlCl_3.Interestingly, treatment with S.aromaticum extract normalized the alterations in the expression level of SOD1, Prdx6 and APPisoforms and improved the neuronal structural damage.Conclusions: The results showed that S.aromaticum is a promising antioxidant and a neuroprotective agent.
基金Supported by the Fund from Science & Technology Department of Jilin Province, China(Nos.20060725, 20070926-02).
文摘To analyze whether expressed amyloid precursor protein(APP) existed in hydrophilic(cytoplasmid) or hydrophobic(lipid bilayer) environment in E. coli and to obtain intact APP for study on its function, we investigated the expression characterization and preparation of the three intact isoforms APP770, APP751, and APP695 in E. coll. The results show that these expressed APPs existed both in hydrophilic cytoplasm region as inclusion bodies and hydrophobic membrane region as membrane-bound state in E. coll. APPs in inclusion bodies were purified on an NTA-Ni^2+ agarose column after dissolving in the urea buffer and APPs in membrane-bound state were obtained by ultracentrifugation. The activity analysis indicates that APP770 and APP751 exhibited strong trypsin-inhibitory activity like the natural ones. These results indicate that E. coli cells can be used as host cells for the expression of human integral membrane protein like APP in either soluble or membrane-bound state unless the interest protein undergone post-translational modification is required.
基金Supported by: Shenzhen Science Technology Project from Shenzhen Bureau of Science Technology and Information, No. 200702029Medicial Science Technology Research Fund of Guangdong Province, No. A2008601 & A2007570
文摘BACKGROUND: Previous studies have demonstrated that mutant amyloid precursor protein (APP) or presenilin-1 (PS1) genes increase susceptibility to ischemic brain damage induced by middle cerebral artery occlusion. Possible mechanisms include over-production of beta-amyloid peptide (Aβ). OBJECTIVE: Because Aβ is over-produced in the APP/PS1 double-transgenic mouse, the present study focused on mechanisms of increased ischemic damage due to mutant APP and PS1 genes by measuring oxidative stress, mitochondrial function, and calcium homeostasis. DESIGN, TIME AND SETTING: The non-randomized, controlled, in vivo and in vitro experiments were performed at the Medical Research Center, Second Clinical College, Jinan University between May and October 2008. MATERIALS: Male APP transgenic mice carrying the mutant 695swe gene and female PS1 transgenic mice carrying the mutant Leu235Pro gene were donated from the University of Hong Kong. SHSY5Y human neureblastoma cells were purchased from ATCC (Manassas, VA, USA), and Aβ1-42 was obtained from Sigma-Aldrich (St. Louis, MO, USA). METHODS: APP transgenic mice were mated with PS1 transgenic mice to produce APP/PS1 double-transgenic mice and wildtype littermates mice. The photothrombotic stroke model was induced in six APP/PS1 double-transgenic and 6 wildtype littermates mice. SHSY5Y human neuroblastoma cells were cultured in vitro, and were divided into 4 groups: Aβ group, cells were exposed to 5 pmol/L Aβ for 24 hours; oxygen-glucose deprivation (OGD) group, cells were exposed to OGD for 1 hour after treatment with sterile, ultra-pure water for 24 hours; OGD+Aβ group, cells were exposed to OGD and Aβfor 1 hour after treatment with 5 pmol/L Aβ for 24 hours; sham control group: cells were exposed to sterile, ultra-pure water for 25 hours. OGD was achieved by exposing the cells to glucose-free DMEM and placing the cells in an anaerobic chamber flushed with 5% CO2 and 95% N2 (v/v) at 37 ℃ for 1 hour. MAIN OUTCOME MEASURES: TTC staining was used to measure infarct volume 7 days after photothrombotic stroke. Cell viability was evaluated using the MTT kit. Opening of the mitochondrial permeability transition pore, intracellular concentration of superoxide anion, and calcium after OGD were detected with fluorescence intensity of calcein-AM, hydroethidine, and fluo-3/AM. RESULTS: At 7 days after stroke, total infarct volume and cortical infarct volume were significantly greater in the APP/PS1 transgenic mice compared with the wildtype littermates mice (P 〈 0.01). Aβ, OGD, and Aβ + OGD significantly decreased cell viability and increased fluorescence intensity of hydroethidine and fluo-3/AM (P 〈 0.01). Compared with the Aβ or OGD group, Aβ + OGD significantly decreased cell viability (P 〈 0.01) and significantly increased fluorescence intensity of calcein-AM, hydroethidine, and fluo-3/AM (P 〈 0.01 or P 〈 0.05). CONCLUSION: The APP/PS1 double-transgenic mice were more vulnerable to ischemia. The possible mechanisms included enhanced opening of the mitochondrial permeability transition pore, overproduction of superoxide anion due to pore opening, and disturbed calcium homeostasis induced by excess superoxide anion.
基金the Natural Science Foundation of Guangxi Science and Technology Bureau, No.0542073
文摘BACKGROUND: The mechanisms of brain injury Following diabetes could be related to amyloid precursor protein (APP) mRNA overexpression. Studies have shown that Gingko biloba leaf extract (EGb) is effective in promoting functional recovery of the brain after traumatic injury. EGb is also effective in improving central nervous system plasticity and learning and memory functions of the elderly. OBJECTIVE: To study the effects of EGb on learning and memory, as well as hippocampal APP mRNA expression in the brains of diabetic rats, using Morris water maze behavioral testing and reverse transcription polymerase chain reaction (RT-PCR), respectively. DESIGN: Complete random design, controlled experimental study. SETTING: Department of Pharmacology, Pharmaceutical School, Guangxi Medical University. MATERIALS: A total of 70 male Wistar rats (180-220 g), 8 weeks old and specific pathogen free, were used for this study. GbE (containing 24.8% flavone glycosides and 6.2% diterpene lactone) was purchased from Guilin Sitejia Natural Plants Pharmaceutical Factory (Guangxi Province, Lot NO. 200405). Streptozotocin was purchased from Sigma (USA). Protamine zinc insulin injection was purchased from WANBANG Biochemical Pharmaceutical Co., Ltd. (Xuzhou Jiangsu, China). METHODS: The experiment was performed in the Experimental Center of Guangxi Medical University from March to October 2005.(1) Experimental intervention: 70 rats were divided randomly into normal control group, diabetic model group (DM group), diabetic model +10 μ g/kg insulin group (DM + Ins group), diabetic model + 100 mg/kg ginkgo leaf extract group (DM + EGb high-dose group), and diabetic model + 50 mg/kg ginkgo leaf extract group (DM + EGb low-dose group); there were 14 rats in each group. Rats with an intraperitoneal (i.p.) injection of citrate buffer solution (pH 4.4) served as the control group. To establish the diabetes model, rats were treated with i.p. injection of 55 mg/kg streptozotocin. Insulin (10 U/kg) was injected subcutaneously (s.c.) every day for 6 months in the DM group. EGb (100 mg/kg) and EGb (50 mg/kg) was administered intragastrically every day for 6 months in the DM + EGb high-dose group and DM + EGb low-dose group, respectively. The DM group and control group were administered distilled water intragastrically every day for 6 months. Drugs were administered once every morning. (2) Experimental evaluation: Six month after intervention, learning and memory of diabetic rats was tested by Morris water maze. Rats were allowed to train for 4 days, and the escape latency and platform-searching score were measured at days 5 and 8. Changes in hippocampal APP mRNA expression were measured with RT-PCR. MAIN OUTCOME MEASURES: Morris water maze performances and hippocampal APP mRNA expression in rats. RESULTS: A total of 70 Wistar rats were included in the final analysis, without any loss. (1) Learning and memory dysfunction of diabetic rats: After 4 days of Morris water maze training, escape latency was longer in the DM group on days 5 and 8, and the platform-searching score was lower in the DM group compared to the control group. In the DM + EGb group, the escape latency score was shorter and platform-searching score was significantly increased compared to the DM group. (2) APP mRNA expression: in the hippocampus of diabetic rats, a 340 bp mRNA product was amplified, which is comparable to the APP mRNA amplification length of design. The expression of APP mRNA from the hippocampus of diabetic rats with learning and memory dysfunctions was significantly increased. EGb extract significantly inhibited the APP mRNA expression in these rats. CONCLUSION: EGb not only ameliorated the learning and memory dysfunctions in diabetic rats, but also significantly inhibited APP mRNA expression. Results from this study led to the hypothesis that diabetes could be one of the risk factors for AD.
文摘Summary: Over-expression of APP and Swedish mutation could cause some familial early onset AD. In this study, a primary screening was conducted of effective small interference RNAs (siRNAs) targeted wild type APP (APPwt) and Swedish mutant APP (APPswe). One siRNA targeting APPwt and the other siRNA targeting APPswe were designed, All these siRNAs were endogenously expressed by siRNAs expressing plasmids, COS-7 cells were transiently co-transfected with APP-GFP recombinant plasmids and siRNA expression vector, The silencing effect of each siRNA was quantitatively assessed by the level of expression of green fluorescent protein (GFP). It was found that the siRNAs silenced APPwt and APPswe to different degrees, siRNA directed against APPswe was more effective in suppressing the expression of fusion gene of APPswe than that of APPwt. The silencing effect of siRNA directed against APPswe indicating allele-specific silencing property of the siRNAs. Therefore, siRNAs directed against APP play an important role both in the therapeutic study of Alzheimer disease and functional exploration ofAPP gene.
文摘BACKGROUND: The findings about the alterations in cerebrospinal fluid beta-amyloid protein (Aβ ) and apolipoprotein E (APOE) after subarachnoid hemorrhage indicate that they have significant correlation with prognosis of patients. OBJECTⅣE: To observe the alterations in cerebrospinal fluid Aβ and ApoE after subarachnoid hemorrhage (SAH). DESIGN: Contrast observation. SETTING: Department of Neurosurgery, the First Hospital of Lanzhou University. PARTICIPANTS: A total of 25 SAH patients including 16 males and 9 females aged from 13 to 72 years were selected form Department of Neurosurgery, the First Affiliated Hospital of Lanzhou University from October 2003 to February 2004. The Hunt-Hess grade ranged from Ⅰ to Ⅳ, and patients admitted hospital in 24 hours after invasion, affirmed by the brain CT scan and lumbar vertebra puncture, no other severe complications and important organs' functional defect and severe infection, no hematological system disease. METHODS- All admitted patients were collected CSF by lumbar vertebra puncture in 24 hours. The cerebrospinal fluid (CSF) of control group came from the admitted 15 patients of our hospital that have no nervous system disease. Aβ content was detected by enzyme linked immunosorbent assay (ELISA), the kit was provided by the Central Laboratory of the First Hospital of Lanzhou University; ApoE concentration was detected by monoclone enzyme linked immunosorbent assay (ELISA), the kit was provided by the Immunotechnique Research Institute of the Fourth Military Medical University. S100B concentration was detected by enzyme linked immunosorbent assay double antibody sandwich method, the kit was provided by the Physiological Research Room of the Fourth Military Medical University. The data were indicated on Mean±SD and were analyzed by SPSS 10.0 statistical package. All data were handled through test of significance variance analysis, and groups were compared through independent sampler t test. The concentration was handled through Pearson correlation analysis between Aβ and ApoE. The relationship between Aβ, ApoE concentration with pathogenetic condition and prognosis of the patients was handled through Spearman ranking correlation analysis. MAIN OUTCOME MEASURES:① The concentration of ApoE, Aβ and S100B after SAH in contrast to the control group in CSF by different Hunt-Hess and Glasgow Outcome Scale (GOS) grades; ② The level of correlation between ApoE and Aβ ; ③Correlation between ApoE and Aβ in pathogenetic condition and prognosis of the patients. RESULTS: All 25 SAH patients and 15 controls were involved in the final analysis. ① The concentration of ApoE, Aβ and S100B in CSF: The concentration of ApoE decreased after SAH in contrast to the control group [(0.46±0.007), (0.85±0.11) μg/L, P 〈 0.01], the concentration of ApoE decreased after SAH in contrast to the control group [(5.36± 1.19), (8.41± 1.60) μg/L, P 〈 0.01], and the concentration of S100B increased after SAH in contrast to the control group [(18.60±7.31), (6.56±1.02) pg/L, P 〈 0.01]. ② The concentration of ApoE, Aβ and S100B in CSF after SAH on different Hunt-Hess and GOS grades: The concentration of Aβ in Hunt-Hess Ⅰ -Ⅲ grade was higher than Hunt-Hess Ⅳ, Ⅴ grade [(6.63 ± 1.25), (3.35± 1.02) μg/L, P 〈 0.01], and the concentration of ApoE in Hunt-Hess Ⅰ- Ⅲ grade was higher than Hunt-Hess Ⅳ, Ⅴ grade [(0.56±0.07), (0.38±0.04) μg/L, P 〈 0.05], the concentration of S100B in Hunt-Hess Ⅰ - Ⅲ grade was lower than Hunt-Hess Ⅳ - Ⅴ grade [(16.32±5.58), (22.85±8.10) pg/L, P 〈 0.01]; the concentration of Aβ in GOS Ⅰ - Ⅲ grade was lower than GOS Ⅳ, Ⅴ grade [(3.76± 1.04), (5.89±1.20) μg/L, P 〈 0.01], and the concentration of ApoE in GOS Ⅰ - Ⅲ grade was lower than GOS Ⅳ, Ⅴ grade [(0.32±0.02), (0.58±0.07) μg/L, P 〈 0.011, and the concentration of S100B in GOS Ⅰ - Ⅲ grade was higher than GOS Ⅳ, Ⅴ grade [(25.36±9.70), (14.33±6.69) pg/L, P 〈 0.01].③ The results of Pearson correlation analysis and Spearman ranking correlation analysis: There was significantly positive correlation between CSF Aβ concentration and clinical outcome (r=0.65, P 〈 0.01), and the decrease in CSF Aβ concentration correlated significant with that of ApoE (r =0.85, P 〈 0.01). CONCLUSION: There is a significant decrease in both Aβ and ApoE in the CSF after SAH, and there is significant correlation between CSF Aβ and ApoE concentration with clinical outcome, the interactions between these proteins may have important effects on SAH, ApoE and Aβ as surrogate markers for the outcome of patients with SAH.
文摘Targeting early steps in amyloid-beta production:Alzheimer’s disease(AD)has a long history as the"amyloid deposit"disorder.Many disorders are now known to be caused by proteinβ-sheet misfolding and aggregation(e.g.,Parkinson’s disease:α-synuclein;Huntington’s disease:Huntingtin;
文摘Pharmacological studies reveal APP and Aβ have interactions with glutamate and calcium, cytokines, copper/zinc chelators, secretases and presenilins, nicotinic receptors, acetycholinesterase, neurotrophins, non-steroidal anti-inflame-matory drugs, monoclonal antibodies to Aβ, protease inhibitors, oestrogen, homocysteine, immediate early genes such as c-fos or c-jun and cholesterol. These functional and pharmacological observations highlight the need for greater understanding of APP and Aβ in brain function and have implications for clinical trials.