Amyloid precursor-like protein 2 C-terminal fragments upregulate S100A9 gene and protein expression in BV2 cells

The murine microglial cell line BV2 has neuroprotective effects, but is toxic to neurons by secret-ing inlfammatory cytokines, and is an important target in the treatment of nerve inlfammation and neurodegenerative diseases. In the present study, we observed the effects of transfecting three amyloid precursor-like protein 2 （APLP2） C-terminal fragments （CTFs; C57, C50 and C31） in the pEGFP-N1 vector on S100A9 expression in BV2 cells. Reverse transcription-PCR, western blot assay and immunocytochemistry revealed that S100A9 protein and mRNA expression was greater in BV2 cells after CTF transfection than after mock transfection with an empty vector. Furthermore, transfection of full-length APLP2-751 resulted in low levels of S100A9 protein ex-pression. Our results show that APLP2-CTFs upregulate S100A9 protein and mRNA expression in BV2 cells, and identify a novel pathway involved in neuronal injury and apoptosis, and repair and protection 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.

Effect of Panax notoginseng saponins on the expression of beta-amyloid protein in the cortex of the parietal lobe and hippocampus, and spatial learning and memory in a mouse model of senile dementia

BACKGROUND： The pharmacological actions of Panax notoginseng saponins （PNS） lie in removing free radicals, anti-inflammation and anti-oxygenation. It can also improve memory and behavior in rat models of Alzheimer＇s disease. OBJECTIVE： Using the Morris water maze, immunohistochemistry, real-time PCR and RT-PCR, this study aimed to measure improvement in spatial learning, memory, expression of amyloid precursor protein （App） and β -amyloid （A β ）, to investigate the mechanism of action of PNS in the treatment of AD in the senescence accelerated mouse-prone 8 （SAMP8） and compare the effects with huperzine A. DESIGN, TIME AND SETTING： A completely randomized grouping design, controlled animal experiment was performed in the Center for Research ＆ Development of New Drugs, Guangxi Traditional Chinese Medical University from July 2005 to April 2007. MATERIALS： Sixty male SAMP8 mice, aged 3 months, purchased from Tianjin Chinese Traditional Medical University of China, were divided into four groups： PNS high-dosage group, PNS low-dosage group, huperzine A group and control group. PNS was provided by Weihe Pharmaceutical Co., Ltd. （batch No.： Z53021485, Yuxi, Yunan Province, China）. Huperzine A was provided by Zhenyuan Pharmaceutical Co., Ltd. （batch No.： 20040801, Zhejiang, China）. METHODS： The high-dosage group and low-dosage group were treated with 93.50 and 23.38 mg/kg PNS respectively per day and the huperzine A group was treated with 0.038 6 mg/kg huperzine A per day, all by intragastric administration, for 8 consecutive weeks. The same volume of double distilled water was given to the control group. MAIN OUTCOME MEASURES： After drug administration, learning and memory abilities were assessed by place navigation and spatial probe tests. The recording indices consisted of escape latency （time-to-platform）, and the percentage of swimming time spent in each quadrant. The number of A β 1-40, A β 1-42 and App immunopositive neurons in the brains of SAMP8 mice was analyzed by immunohistochemistry. The mRNA content ofApp, tau, acetylcholinesterase, and synaptophysin （Syp） was tested by real time PCR and RT-PCR. RESULTS： The PCR results show that PNS can downregulate the expression of the App gene and upregulate the expression of the Syp gene in the parietal cortex and hippocampus of SAMP8 mice. The therapeutic effects of the PNS high-dosage group were greater than those of the PNS low-dosage group and the huperzine A group （P 〈 0.05）. The results of the Morris water maze and immunohistochemistry indicated that PNS can improve the capacity for spatial learning and memory in SAMP8 mice, and reduce the content of A β 1-40, A β 1-42 and expression of App in the brains of SAMP8 mice. The therapeutic effects of the PNS high-dosage group were greater than that of the PNS low-dosage group and the huperzine A group （P 〈 0.05）. CONCLUSION： These results support the hypothesis that PNS plays a therapeutic and protective role on the pathological lesions and learning dysfunction of Alzheimer＇s disease. The therapeutic effects of PNS for Alzheimer＇s disease are possibly achieved through downregulating the expression of the App gene and upregulating the expression of the Syp gene. The therapeutic effects of PNS are dose-dependent and are greater than the effect of huperzine A.

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.

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.

Key gene and protein changes in the beta-amyloid pathway following Longyanshen polysaccharides treatment in a mouse model of Alzheimer's disease

BACKGROUND： During onset and development of Alzheimer＇s disease, β-amyloid （Aβ） precursor protein （APP）, β-site amyloid precursor protein cleaving enzyme （BACE）, and β-amyloid are key genes and proteins in the Aβ pathway, and over-expression of these genes can lead to Aβ deposit/on in the brain. OBJECTIVE： To observe the influence of Longyanshen polysaccharides on expression of BACE, APP, and Aβ in the senescence-accelerated mouse prone/8 （SAMP8） brain, and to compare these effects with huperzine A treatment. DESIGN, TIME AND SETTING： A randomized, controlled, neurobiochemical experiment was performed at the Department of Pharmacology and Scientific Experimental Center of Guangxi Medical University from September 2005 to January 2008. MATERIALS： Longyanshen polysaccharfdes powder was extracted from the dried slices of the medicinal plant Longyanshen. The active component, Longyanshen polysaccharides, was provided by the Department of Pharmacology, Guangxi Medical University; huperzine A was purchased from Yuzhong Drug Manufactory, China. METHODS： Healthy SAMP8 mice were used to establish a model of Alzheimer＇s disease. A total of 50 SAMP8 mice were randomly assigned to 5 groups （n = 10）： SAMP8, huperzine A, low-, middle-, and high-dose polysaccharides. In addition, 10 senescence-accelerated mouse resistant 1 （SAMR1） mice were selected as normal controls. SAMP8 and SAMR1 mice were administered 30 mL/kg normal saline; the huperzine A group was administered 0.02 mg/kg huperzine A; the low-, middle-, and high-dose polysaccharides groups were respectively administered 45, 90, and 180 mg/kg Longyanshen polysaccharides. Each group was treated by intragastric administration, once per day, for 50 consecutive days. MAIN OUTCOME MEASURES： One hour after the final administration, immunohistochemical analysis was used to determine Aβ expression in the cortex and hippocampus of SAMP8 mice. Reverse-transcription polymerase chain reaction was used to determine mRNA levels of BACE and APP in SAMP8 brain tissue. RESULTS： Compared with the SAMR1 group, Aβ expression in the cerebral cortex and hippocampus, as well as expression of BACE, APP mRNA in the brain was significantly increased in the SAMP8 group （P 〈 0.05-0.01）. Compared with the SAMP8 group, Aβ expression, as well as BACE and APP mRNA expression, were significantly decreased in the cerebral cortex and hippocampus of huperzine A and low-, middle-, and high-dose polysaccharides groups （P 〈 0.05-0.01）. In particular, the effect of high-dose polysaccharides was the most significant （P 〈 0.05-0.01 ）. CONCLUSION： Longyanshen polysaccharides reduced or inhibited over-expression of BACE, APP, and Aβ in SAMP8 mice in a dose-dependent manner, and the effect was not worse than huperzine A.

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.

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.

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.

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.

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.

Effects of natural cerebrolysin on protective proteins and pro-apoptotic molecules in mesenchymal stem cells following beta-amyloid peptide1-40-induced endoplasmic reticulum stress

BACKGROUND： Studies have demonstrated that β-amyloid peptide （Aβ）, a characteristic pathological product of Alzheimer＇s disease （AD）, results in neuronal endoplasmic reticulum stress （ERS）. However, the mechanisms of traditional Chinese medicine against ERS in AD are poorly understood. OBJECTIVE： To measure expression levels of protective proteins （GRP78 and GRP94） of ER molecular partners and pro-apoptotic Caspase-12 ER membrane expression following application of traditional Chinese medicine natural cerebrolysin （NC） to treat Aβ1-40-induced ERS. DESIGN, TIME AND SETTING： A parallel-controlled study was performed at the Institute of Integrated Western and Traditional Chinese Medicine, Shenzhen Hospital of Southern Medical University between September 2006 and November 2008. MATERIALS： Sprague Dawley male rats, 6-8 weeks old, were used to harvest tibial and femoral bone marrow. Isolation and purification of mesenchymal stem cells （MSCs） were established from the whole bone marrow by removing non-adherent cells in primary and passage cultures. Aβ1-40 was provided by Sigma, USA. NC was provided by Shenzhen Institute of Integrated Chinese and Western Medicine, China. NC was predominantly composed of Renshen （Radix Ginseng）, Tianma （Rhizoma Gastrodiae）, and Yinxingye （Ginkgo Leaf） in a proportion of 1 ： 2： 2. Following conventional water extraction technology, an extract （1 ： 20） was prepared. Six adult, male, New Zealand rabbits underwent intragastric administration of NC extract （0.976 g/kg per day） for 1 month to prepare NC-positive serum, and the remaining 6 rabbits received intragastric administration of physiological saline to prepare normal blank serum. METHODS： A total of 500 nmol/L Aβ1-40 was used to establish ERS models of primary cultured MSCs. AD cell models were incubated with different doses of NC-positive serum （2.5%, 5%, and 10%）. MSCs treated with normal blank serum served as normal blank controls. MAIN OUTCOME MEASURES： Reverse transcription-polymerase chain reaction and fluorescent immunocytochemistry were respectively used to measure mRNA and protein expression levels of GRP78, GRP94, and Caspase-12 in MSCs. RESULTS： Following Aβ1-40 exposure, mRNA and protein expression levels of GRP78 and GRP94, as well as Caspase-12, significantly increased （P 〈 0.05）, suggesting successful establishment of ERS models. Following NC-positive serum application, mRNA and protein expression levels of GRP78 and GRP94 in MSCs significantly increased （P 〈 0.05 or P 〈 0.01）. However, mRNA and protein expression levels of Caspase-12 significantly decreased （P 〈 0.05, or P 〈 0.01） compared with the ERS model group. These effects were dose-dependent. CONCLUSION： NC downregulated Caspase-12 expression and upregulated GRP78 and GRP94 expression in MSCs in a dose-dependent manner under the state of Aβ1-40-induced ERS.

The prognostic value of serum C-reactive protein-bound serum amyloid A in early-stage lung cancer

Background:Elevated levels of serum C-reactive protein(CRP) have been reported to have prognostic significance in lung cancer patients.This study aimed to further identify CRP-bound components as prognostic markers for lung cancer and validate their prognostic value.Methods:CRP-bound components obtained from the serum samples from lung cancer patients or healthy controls were analyzed by differential proteomics analysis.CRP-bound serum amyloid A(CRP-SAA) was evaluated by coimmunoprecipitation(IP).Serum samples from two independent cohorts with lung cancer(retrospective cohort,242patients;prospective cohort,222 patients) and healthy controls(159 subjects) were used to evaluate the prognostic value of CRP-SAA by enzyme-linked immunosorbent assay.Results:CRP-SAA was identified specifically in serum samples from lung cancer patients by proteomic analysis.CRP binding to SAA was confirmed by co-IP in serum samples from lung cancer patients and cell culture media.The level of CRP-SAA was significantly higher in patients than in healthy controls(0.37 ± 0.58 vs.0.03 ± 0.04,P < 0.001).Elevated CRP-SAA levels were significantly associated with severe clinical features of lung cancer.The elevation of CRPSAA was associated with lower survival rates for both the retrospective(hazard ration[HR]= 2.181,95%confidence interval[CI]= 1.641-2.897,P < 0.001) and the prospective cohorts(HR = 2.744,95%CI = 1.810-4.161,P < 0.001).Multivariate Cox analysis showed that CRP-SAA was an independent prognostic marker for lung cancer.Remarkably,in stages l-ll patients,only CRP-SAA,not total SAA or CRP,showed significant association with overall survival in two cohorts.Moreover,univariate and multivariate Cox analyses also showed that only CRP-SAA could be used as an independent prognostic marker for early-stage lung cancer patients.Conclusion:CRP-SAA could be a better prognostic marker for lung cancer than total SAA or CRP,especially in earlystage patients.

Relationship between β-amyloid protein 1-42, thyroid hormone levels and the risk of cognitive impairment after ischemic stroke

BACKGROUND Post-stroke cognitive impairment(PSCI)is not only a common consequence of stroke but also an important factor for adverse prognosis of patients.Biochemical indicators such as blood lipids and blood pressure are affected by many factors,and the ability of evaluating the progress of patients with PSCI is insufficient.Therefore,it is necessary to find sensitive markers for predicting the progress of patients and avoiding PSCI.Recent studies have shown thatβ-amyloid protein 1-42(Aβ1-42)and thyroid hormone levels are closely related to PSCI,which may be the influencing factors of PSCI,but there are few related studies.AIM To investigate the relationship between serum levels of Aβand thyroid hormones in acute stage and PSCI and its predicted value.METHODS A total of 195 patients with acute cerebral infarction confirmed from June 2016 to January 2018 were enrolled in this study.Baseline data and serological indicators were recorded to assess cognitive function of patients.All patients were followed up for 1 year.Their cognitive functions were evaluated within 1 wk,3 mo,6 mo and 1 yr after stroke.At the end of follow-up,the patients were divided into PSCI and non-PSCI according to Montreal cognitive assessment score,and the relationship between biochemical indexes and the progression of PSCI was explored.RESULTS Compared with patients with non-PSCI,the levels of Aβ1-42,triiodothyronine(T3)and free thyroxin were lower in the patients with PSCI.Repeated measures analysis of variance showed that the overall content of Aβ1-42 and T3 in PSCI was also lower than that of the non-PSCI patients.Further analysis revealed that Aβ1-42(r=0.348),T3(r=0.273)and free thyroxin(r=0.214)were positively correlated with disease progression(P<0.05),suggesting that these indicators have the potential to predict disease progression and outcome.Cox regression analysis showed that Aβ1-42 and T3 were important factors of PSCI.Then stratified analysis showed that the lower the Aβ1-42 and T3,the higher risk of PSCI in patients who were aged over 70,female and illiterate.CONCLUSION Aβ1-42 and T3 have the ability to predict the progression of PSCI,which is expected to be applied clinically to reduce the incidence of PSCI and improve the quality of life of patients.

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.

Amyloid-beta-dependent phosphorylation of collapsin response mediator protein-2 dissociates kinesin in Alzheimer＇s disease

Alzheimer’s disease（AD）is a neurodegenerative disorder characterized by accumulation of amyloid plaques and neurofibrillary tangles.Prior to the development of these characteristic pathological hallmarks of AD,anterograde axonal transport is impaired.However,the key proteins that initiate these intracellular impairments remain elusive.The collapsin response mediator protein-2（CRMP-2）plays an integral role in kinesin-1-dependent axonal transport and there is evidence that phosphorylation of CRMP-2releases kinesin-1.Here,we tested the hypothesis that amyloid-beta（Aβ）-dependent phosphorylation of CRMP-2 disrupts its association with the kinesin-1（an anterograde axonal motor transport protein）in AD.We found that brain sections and lysates from AD patients demonstrated elevated phosphorylation of CRMP-2 at the T555 site.Additionally,in the transgenic Tg2576 mouse model of familial AD（FAD）that exhibits Aβaccumulation in the brain with age,we found substantial co-localization of p T555CRMP-2and dystrophic neurites.In SH-SY5Y differentiated neuronal cultures,Aβ-dependent phosphorylation of CRMP-2 at the T555 site was also elevated and this reduced the CRMP-2 association with kinesin-1.The overexpression of an unphosphorylatable form of CRMP-2 in neurons promoted the re-establishment of CRMP-2-kinesin association and axon elongation.These data suggest that Aβ-dependent phosphorylation of CRMP-2 at the T555 site may directly impair anterograde axonal transport protein function,leading to neuronal defects.

Effects of Ginsenoside Rg1 on nuclear factor-kappa B activity in beta amyloid protein-treated neural cells

BACKGROUND： Modern pharmacological studies have shown that Ginsenoside Rgl is one of the active components of ginseng that promote intelligence in the nervous system. Ginsenoside Rgl can improve memory and learning in mouse models of β-amyloid protein （Aβ）-induced dementia. OBJECTIVE： To investigate whether effects of Ginsenoside Rgl against Aβ are associated with activity of nuclear factor-kappa B （NF-κB）. DESIGN, TIME AND SETTING： The randomized performed at the DME Center, Institute of Clinica controlled, cell biological experiment was Pharmacology, Guangzhou University of Chinese Medicine, China from July 2005 to May 2006. MATERIALS： Beta-amyloid fragment 25-35 （Aβ25-35） was supplied by the Neural Biochemical Laboratory, Xuanwu Hospital, Capital Medical University, China. Ginsenoside Rgl was obtained from National Institute for the Control of Pharmaceutical and Biological Products, China. Rabbit anti-rat NF-κB p65 antibody was purchased from Santa Cruz Biotechnology, USA. METHODS： Hippocampal neurons and cortical astrocytes of neonatal Sprague Dawley rats were harvested and treated with various concentrations （0, 5, 10, 20, and 40 μmol/L） of Aβ for 6, 12, and 24 hours to establish cellular models of Alzheimer＇s disease. Cellular models were pretreated with various concentrations of Ginsenoside Rgl （1,2, 4, 8, and 16 μmol/L）. According to cell morphology and activity, the following conditions were selected： 40 μmol/L Aβ for 24 hours, as well as 2, 4, and 8 μmol/L Ginsenoside Rg1. NF-κB activity was observed using immunofluorescence and cytochemical staining. MAIN OUTCOME MEASURES： Morphology and viability of hippocampal neurons and cortical astrocytes, and activities of NF-κB were measured. RESULTS： Hippocampal neuron activity was significantly greater in the normal and 2 and 4 μmol/L Ginsenoside Rgl groups compared with the model group （P 〈 0.05）. Astrocyte activity was significantly greater in the normal, 1,2, 4, 8, and 16 μmol/L Ginsenoside Rgl groups compared with the model group （P 〈 0.05）. NF-κB activity of hippocampal neurons was significantly greater in the normal, 2, 4, and 8 μmol/L Ginsenoside Rgl groups compared with the model group （P 〈 0.01）. NF-κB activity of astrocytes was significantly less in the normal, 2, 4, and 8 μmol/L Ginsenoside Rgl groups compared with the model group （P 〈 0.01 or P 〈 0.05）. No significant difference in NF-κB activity was determined between the 2 μmol/L Ginsenoside Rgl and normal groups （P 〉 0.05）. CONCLUSION： Ginsenoside Rgl protected neural cells by upregulating NF-κB activity in neurons and downregulating NF-κB activity in astrocytes. Ginsenoside Rgl （2 μmol/L） maintained cell activity and NF-κB activity at normal levels.

THE PROTECTIVE EFFECTS OF THE TOTAL SAPONIN OF DIPSACUS ASPEROIDES ON THE APOPTOSIS OF HIPPOCAMPAL NEURONS INDUCED BY β-AMYLOID PROTEIN

Objective To investigate the effects of the total saponin of Dipsacus asperoides (tSDA) and ginsenoside Rb1 (GRb1) on the apoptosis of primary cultured hippocampal neurons induced by β-amyloid protein (Aβ). Methods Primary cultured hippocampal neurons, the cultures were pretreated with tSDA and GRb1 on 10d for 24 hours respectively. Then the cultures were treated with 35 μmol·L -1 Aβ25-35 for 24 hours, observed the changing of survival rate of neurons and the apoptosis of neurons with biochemical analysis combining immunofluorescent cytochemical double-staining technique. Results Hippocampal neurons were treated with 35 μmol·L -1 Aβ for 24 hours, and survival rate of neurons downed to 52.6%. When neurons were pretreated by tSDA and GRb1, survival rate of neurons increased 11% to 15%. The findings of immunofluorescent cytochemical double-staining indicated that apoptotic neurons were obviously more than that of the blank group, reaching 43.9%.When neurons were pretreated by tSDA and GRb1, apoptotic neurons were downed to 16.6%, 10.8% respectively. Conclusion tSDA had the same effects as GRb1, protecting the neurons, antagonizing neurotoxicity of Aβ, increasing survival rate of neurons, and reducing apoptotic neurons induced by Aβ.

Overexpression of estrogen receptor beta alleviates the toxic effects of beta-amyloid protein on PC12 cells via non-hormonal ligands

After binding to the estrogen receptor, estrogen can alleviate the toxic effects of beta-amyloid protein, and thereby exert a therapeutic effect on Alzheimer＇s disease patients. Estrogen can increase the incidence of breast carcinoma and endometrial cancer in post-menopausal women, so it is not suitable for clinical treatment of Alzheimer＇s disease. There is recent evidence that the estrogen receptor can exert its neuroprotective effects without estrogen dependence. Real-time quantitative PCR and flow cytometry results showed that, compared with non-transfected PC12 cells, adenovirus-mediated estrogen receptor β gene-transfected PC12 cells exhibited lower expression of tumor necrosis factor a and interleukin 1β under stimulation with beta-amyloid protein and stronger protection from apoptosis. The Akt-specific inhibitor Abi-2 decreased the anti-inflammatory and anti-apoptotic effects of estrogen receptor β gene-transfection. These findings suggest that overexpression of estrogen receptor β can alleviate the toxic effect of beta-amyloid protein on PC12 cells, without estrogen dependence. The Akt pathway is one of the potential means for the anti-inflammatory and anti-apoptotic effects of the estrogen receptor.