Regulator of G protein signaling 6 mediates exercise-induced recovery of hippocampal neurogenesis,learning,and memory in a mouse model of Alzheimer’s disease

Hippocampal neuronal loss causes cognitive dysfunction in Alzheimer’s disease.Adult hippocampal neurogenesis is reduced in patients with Alzheimer’s disease.Exercise stimulates adult hippocampal neurogenesis in rodents and improves memory and slows cognitive decline in patients with Alzheimer’s disease.However,the molecular pathways for exercise-induced adult hippocampal neurogenesis and improved cognition in Alzheimer’s disease are poorly understood.Recently,regulator of G protein signaling 6(RGS6)was identified as the mediator of voluntary running-induced adult hippocampal neurogenesis in mice.Here,we generated novel RGS6fl/fl;APP_(SWE) mice and used retroviral approaches to examine the impact of RGS6 deletion from dentate gyrus neuronal progenitor cells on voluntary running-induced adult hippocampal neurogenesis and cognition in an amyloid-based Alzheimer’s disease mouse model.We found that voluntary running in APP_(SWE) mice restored their hippocampal cognitive impairments to that of control mice.This cognitive rescue was abolished by RGS6 deletion in dentate gyrus neuronal progenitor cells,which also abolished running-mediated increases in adult hippocampal neurogenesis.Adult hippocampal neurogenesis was reduced in sedentary APP_(SWE) mice versus control mice,with basal adult hippocampal neurogenesis reduced by RGS6 deletion in dentate gyrus neural precursor cells.RGS6 was expressed in neurons within the dentate gyrus of patients with Alzheimer’s disease with significant loss of these RGS6-expressing neurons.Thus,RGS6 mediated voluntary running-induced rescue of impaired cognition and adult hippocampal neurogenesis in APP_(SWE) mice,identifying RGS6 in dentate gyrus neural precursor cells as a possible therapeutic target in Alzheimer’s disease.

Observation of amyloid precursor protein cleavage and Aβ generation in living cells by using multiphoton laser scanning microscopy

Objective To investigate the proteolytic mechanism of amyloid precursor protein （APP） and to explore amyloidbeta （Aβ） generation in living neurons. Methods DNA fragments were amplified by PCR or synthesized. The four fragments, CFP, 54bp, YFP and C99 were ligated into pcDNA3.0 vector to construct the recombinant plasmids pcDNA3.0-CFP-54bp- YFP and pcDNA3.0-CFP-54bp-YFP-C99. The SH-SY5Y cells were transiently transfected with pcDNA3.0-CFP-54bp-YFP or pcDNA3.0-CFP-54bp-YFP-C99. The expression of fusion gene was examined under a multiphoton laser scanning microscope. Fluorescence resonance energy transfer （FRET） was used to measure the β cleavage and γ cleavage of APE Aβ generation was confirmed by immunocytochemistry and multiphoton laser scanning microscopy. Cell viability was tested by MTT assay at different time points. Results （1） The double restriction endonuclease digestion and sequencing analysis confirmed the authenticity of the recombinant plasmids pcDNA3.0-CFP-54bp-YFP and pcDNA3.0-CFP-54bp- YFP-C99. （2） Blue and yellow fluorescences were detected in the transfected cells. （3） FRET occurred in pcDNA3.0-CFP- 54bp-YFP-transfected cells but not in pcDNA3.0-CFP-54bp-YFP-C99-transfected cells. （4） Aβ was produced in the pcDNA3.0- CFP-54bp-YFP-C99 transfected cells. （5） Aβ-deposition was widespread in the cell. （6） Cell viability decreased along with the intracellular Aβ deposition. Conclusion C99 is important for the APP β cleavage. Aβ may be generated and deposited in cells at the early stage of Alzheimer＇s disease. Intracellular Aβ accumulation brings deleterious effects on cells.

Compound Danshen tablets downregulate amyloid protein precursor mRNA expression in a transgenic cell model of Alzheimer's disease Effects and a comparison with donepezil

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.

Mutations of beta-amyloid precursor protein alter the consequence of Alzheimer's disease pathogenesis

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.

Effect of glycosides of Cistanche on the expression of mitochondrial precursor protein and keratin type Ⅱ cytoskeletal 6A in a rat model of vascular dementia

Glycosides of Cistanche（GC）is a preparation used extensively for its neuroprotective effect against neurological diseases,but its mechanisms of action remains incompletely understood.Here,we established a bilateral common carotid artery occlusion model of vascular dementia in rats and injected the model rats with a suspension of GC（10 mg/kg/day,intraperitoneally）for 14 consecutive days.Immunohistochemistry showed that GC significantly reduced p-tau and amyloid beta（Aβ）immunoreactivity in the hippocampus of the model rats.Proteomic analysis demonstrated upregulation of mitochondrial precursor protein and downregulation of keratin type II cytoskeletal6A after GC treatment compared with model rats that had received saline.Western blot assay confirmed these findings.Our results suggest that the neuroprotective effect of GC in vascular dementia occurs via the promotion of neuronal cytoskeleton regeneration.

Impact of Sub-chronic Aluminium-maltolate Exposure on Catabolism of Amyloid Precursor Protein in Rats

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.

Amyloid precursor protein and growth-associated protein 43 expression in brain white matter and spinal cord tissues in a rat model of experimental autoimmune encephalomyelitis

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.

Beta-amyloid precursor protein cleavage enzyme-1 expression in adult rat retinal neurons in the early period after lead exposure

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.

miR-15b-5p targeting amyloid precursor protein is involved in the anti-amyloid eflect of curcumin in swAPP695-HEK293 cells

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.

Physiological effects of amyloid precursor protein and its derivatives on neural stem cell biology and signaling pathways involved

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.

Divalent cation tolerance protein binds to β-secretase and inhibits the processing of amyloid precursor protein

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.

Inhibiting p38 mitogen-activated protein kinase attenuates cerebral ischemic injury in Swedish mutant amyloid precursor protein transgenic mice

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.

Expression and Immunological Analysis of Capsid Protein Precursor of Swine Vesicular Disease Virus HK/70

VP1, a capsid protein of swine vesicular disease virus, was cloned from the SVDV HK/70 strain and inserted into retroviral vector pBABE puro, and expressed in PK15 cells by an retroviral expression system. The ability of the VP1 protein to induce an immune response was then evaluated in guinea pigs. Western blot and ELISA results indicated that the VP1 protein can be recognized by SVDV positive serum, Furthermore, anti-SVDV specific antibodies and lymphocyte proliferation were elicited and increased by VP1 protein after vaccination. These results encourage further work towards the development of a vaccine against SVDV infection.

Inhibition of beta-site amyloid precursor protein-cleaving enzyme and beta-amyloid precursor protein genes in SK-N-SH cells

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.

Two memory associated genes regulated by amyloid precursor protein intracellular domain Novel insights into the pathogenesis of learning and memory impairment in Alzheimer's disease

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.

Expression of the Capsid Precursor Protein gene of Foot-and-mouth Disease Virus and Green Fluorescent Protein Gene in BHK-21 Cells Mediated by Retroviral Vector

We have constructed a retroviral vector mediated mammalian cell expression system of the capsid precursor protein of foot-and-mouth disease virus(FMDV).The recombinant retroviral vector pBABEpuro-P1-2A-EGFP was constructed by sequentially inserting capsid precursor protein gene(P1) of FMDV and enhanced green fluorescent protein gene(EGFP) into pBABEpuro.The recombinant retroviral vector and the pVSV-G plasmid were co-transfected into packaging cells(GP2-293) by liposomemediated transduction to produce the pseudovirus.The pseudovirus was used to infect BHK-21 cells and resistant cells were screened with puromycin.Green fluorescent proteins were observed by fluorescence microscopy and expression of the capsid precursor protein gene of FMDV was detected by indirect immunofluorescence.The recombinant retroviral vector pBABEpuro-P1-2A-EGFP was constructed successfully.The capsid precursor protein of FMDV and green fluorescent protein were expressed in BHK-21 cells.The mammalian cell expression system for the capsid precursor protein of FMDV has been constructed successfully,which lays the foundation of development of a FMDV subunit vaccine.

Syzygium aromaticum ethanol extract reduces AlCl_3-induced neurotoxicity in mice brain through regulation of amyloid precursor protein and oxidative stress gene expression

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.

Expression Characterization and Preparation of Human Amyloid Precursor Protein in Escherichia coli

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.

Schisandrin B protects PC12 cells by decreasing the expression of amyloid precursor protein and vacuolar protein sorting 35

PC12 cell injury was induced using 20 μM amyloid β-protein 25-35 to establish a model of Alzheimer＇s disease. The cells were then treated with 5, 10, and 25 μM Schisandrin B. Methylthiazolyldiphenyl-tetrazolium bromide assays and Hoechst 33342 staining results showed that with increasing Schisandrin B concentration, the survival rate of PC12 cells injured by amyloid β-protein 25-35 gradually increased and the rate of apoptosis gradually decreased. Reverse transcription-PCR, immunocytochemical staining and western blot results showed that with increasing Schisandrin B concentration, the mRNA and protein expression of vacuolar protein sorting 35 and amyloid precursor protein were gradually decreased. Vacuolar protein sorting 35 and amyloid precursor protein showed a consistent trend for change. These findings suggest that 5, 10, and 25 μM Schisandrin B antagonizes the cellular injury induced by amyloid β-protein 25-35 in a dose-dependent manner. This may be caused by decreasing the expression of vacuolar protein sorting 35 and amyloid precursor protein.

Mutant amyloid precursor protein and presenilin-1 genes effect on ischemia vulnerability via calcium homeostasis disturbance

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.