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.

High-frequency magnetic stimulation attenuates beta-amyloid protein 1-42 neurotoxicity in organotypic hippocampal slices

Repetitive transcranial magnetic stimulation （rTMS） has been utilized as a therapeutic tool for neurodegenerative disorders including Alzheimer＇s disease. However, the precise mechanisms of its clinical effects remain unknown. β-amyloid （Aβ） exhibits direct neurotoxic effects and is closely related to neuronal degeneration in Alzheimer＇s disease. Therefore, it has been hypothesized that the neuroprotective effects of rTMS are related to the mechanisms of protection against Aβ neurotoxicity. Organotypic hippocampal slices were prepared from 8-day old, Sprague Dawley rats. The tissue slices were exposed to 100 μmol/L Al3142 since day 12 in vitro with and without high-frequency （20 Hz） magnetic stimulation. Magnetic stimulation efficacy was evaluated by measuring neuronal nuclei （NeuN） protein expression and by observing cultures following propidium iodide fluorescence staining and bromodeoxyuridine （BrdU） immunohistochemistry. Lactate dehydrogenase activity was detected in the culture media to evaluate hippocampal neuronal damage. Our results demonstrated that high-frequency magnetic stimulation significantly reversed the reduction of NeuN protein expression because of Aβ1-42 exposure （P 〈 0.05） and significantly reduced the number of damaged cells in the hippocampal slices （P 〈 0.05）. However, lactate dehydrogenase levels and anti-BrdU staining results did not reveal any statistical differences These findings indicate that high-frequency magnetic stimulation might have protective effect on hippocampal neurons from Aβ1-42 neurotoxicity.

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.

S14G-humanin restored cellular homeostasis disturbed by amyloid-beta protein

Humanin is a potential therapeutic agent for Alzheimer’s disease, and its derivative, S14G-humanin, is 1 000-fold stronger in its neuroprotective effect against Alzheimer’s disease-relevant insults. Alt-hough effective, the detailed molecular mechanism through which S14G-humanin exerts its effects remains unclear. Data from this study showed that fibril ar amyloid-beta 40 disturbed cel ular ho-meostasis through the cel membrane, increasing intracel ular calcium, generating reactive oxygen species, and decreasing the mitochondrial membrane potential. S14G-humanin restored these re-sponses. The results suggested that S14G-humanin blocked the effects of amyloid-beta 40 on the neuronal cel membrane, and restored the disturbed cel ular homeostasis, thereby exerting a neuroprotective effect on hippocampal neurons.

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.

Telencephalin protects Paju cells from beta-amyloid protein-induced apoptosis

Previous studies have confirmed that telencephalin （TLN） is a neural glycoprotein that protects axonal disruption induced by the beta-amyloid protein （Aβ42/35） in the neural crest-derived tumor cell line Paju. The present study investigated the effects of TLN on neuronal degeneration induced by Aβ42 in the differentiated Paju cell line. Results demonstrated that after cultivating cells in Aβ42 medium, the survival rate of Paju-TLN cells was significantly higher than that of Paju-neo cells, and that apoptotic rate was noticeably reduced. These results indicate that TLN reduces Paju cell apoptosis induced by Aβ42.

Telencephalin protects PAJU cells from amyloid beta protein-induced apoptosis by activating the ezrin/radixin/moesin protein family/phosphatidylinositol-3-kinase/protein kinase B pathway

Telencephalin is a neural glycoprotein that reduces apoptosis induced by amyloid beta protein in the human neural tumor cell line PAJU. In this study, we examined the role of the ezrin/radixin/moesin protein family/phosphatidylinositol-3-kinase/protein kinase B pathway in this process. Western blot analysis demonstrated that telencephalin, phosphorylated ezrin/radixin/moesin and phosphatidylinositol-3-kinase/protein kinase B were not expressed in PAJU cells transfected with empty plasmid, while they were expressed in PAJU cells transfected with a telencephalin expression plasmid. After treatment with 1.0 nM amyloid beta protein 42, expression of telencephalin and phosphorylated phosphatidylinositol-3-kinase/protein kinase B in the transfected cells gradually diminished, while levels of phosphorylated ezrin/radixin/moesin increased. In addition, the high levels of telencephalin, phosphorylated ezrin/radixin/moesin and phosphatidylinositol-3-kinase/protein kinase B expression in PAJU cells transfected with a telencephalin expression plasmid could be suppressed by the phosphatidylinositol-3-kinase inhibitor LY294002. These findings indicate that telencephalin activates the ezrin/radixin/moesin family/phosphatidylinositol-3-kinase/protein kinase B pathway and protects PAJU cells from amyloid beta protein-induced apoptosis.

Alterations in amyloid beta-protein and apolipoprotein E in cerebrospinal fluid after subarachnoid hemorrhage

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.

Targeting amyloid precursor protein shuttling and processing-long before amyloid beta formation

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;

Effects of long-term estrogen replacement therapy on beta-amyloid precursor protein and mRNA expression in ovariectomized rat hippocampus

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.

Tau protein,phosphorylated tau protein,and beta-amyloid 42 levels in patients with neurodegenerative diseases complicated by cognitive deficits A non-randomized,concurrent,case-control investigation

BACKGROUND： The differential diagnosis of many neurodegenerative disorders depends primarily on clinical symptoms together with imaging methods. Recently, increased importance has been placed on the use of biomarkers for diagnosing various neurodegenerative disorders. OBJECTIVE： To assess the feasibility of tau-protein, phosphorylated tau-protein, beta-amyloid 42 （Aβ42）, and 14-3-3 protein as biomarkers for diagnosing several neurodegenerative diseases complicated by cognitive deficits. DESIGN, TIME AND SETTING： A non-randomized, concurrent, case-control investigation was performed in three medical centers in the Czech Republic （Department of Neurology at the University Hospital in Hradec Kralove, Department of Neurology at the 2rd Medical Faculty, and the University Hospital Motol） between October 2000 and November 2006. PARTICIPANTS： Eighteen patients with probable AIzheimer＇s disease, 4 patients with Creutzfeldt-Jakob disease, 10 patients with frontotemporal dementia, 9 patients with clinically isolated syndrome suggestive of multiple sclerosis, and 7 patients with multiple sclerosis, as well as 38 race-, nationality-, and age-matched cognitively intact controls, were included in the study. Diagnoses were established based on the following criteria： the criteria for Alzheimer＇s disease proposed by the National Institute of Neurological and Communicative Disorders and Stroke/Alzheimer＇s Disease and Related Disorders Association, WHO criteria for Creutzfeldt-Jakob disease, Neary criteria for frontotemporal dementia, and McDonald＇s criteria for multiple sclerosis. All included patients were confirmed to suffer from various degrees of dementia. METHODS： Enzyme-linked immunosorbent assay was used to measure concentrations of tau-protein, phosphorylated tau-protein, and Aβ42 in cerebrospinal fluid （CSF） samples collected by standard lumbar puncture from each patient. Moreover, 14-3-3 protein was assessed by Western blot in CSF of Creutzfeldt-Jakob disease patients. Cognitive status was assessed using the Mini Mental Scale Examination （MMSE） in all subjects. MAIN OUTCOME MEASURES： Establishment of biomarkers with greatest specificity and sensitivity for the investigated disorders according to Receiver Operating Characteristic curves, which were based on values from patients and controls; correlation between concentrations of given biomarkers and demographic parameters, diagnosis, duration of disease, and level of cognitive deficit. RESULTS： Increased concentrations of total tau protein and phosphorylated tau protein, and decreased levels of Aβ42, in CSF of Alzheimer＇s disease patients reached the required sensitivity/specificity ratio of 80% or greater. A marked elevation in CSF concentrations of total tau protein showed even greater sensitivity than 14-3-3 protein in Creutzfeldt-Jakob disease. There was no association between selected biomarkers and frontotemporal dementia or multiple sclerosis. Phosphorylated tau-protein was the only biomarker that noticeably correlated with MMSE scores for Alzheimer＇s disease.CONCLUSION： Levels of total tau protein, phosphorylated tau protein, and A!342 in the CSF could differentiate patients with Alzheimer＇s disease and Creutzfeldt-Jakob disease from healthy controls and patients with other neurodegenerative disorders. The diversity of absolute values demonstrates the necessity to establish a specific standard for each laboratory.

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.

Effects of natural-cerebrolysin-containing serum on neurotoxicity and synaptogenesis in amyloid-beta 1-40-induced Alzheimer's disease in vitro models

BACKGROUND： Neuronal loss, synapse mutilation, and increasing malnourished axons are pathologically related to Alzheimer＇s disease. Microtubule-associated protein 2 （MAP2） is of importance for neuronal, axonal, and dendritic generation, extension, and stabilization, as well as for the regulation of synaptic plasticity. OBJECTIVE： To investigate the antagonistic effects of natural-cerebrolysin-containing serum on beta amyloid protein 1-40 （Aβ1-40）-induced neurotoxicity from the standpoints of cell proliferation, synaptogenesis, and cytoskeleton formation （MAP2 expression）. DESIGN, TIME AND SETTING： A paralleled, controlled, neural cell, and molecular biology experiment was performed at the Institute of Integrated Chinese and Western Medicine, Shenzhen Hospital, Southern Medical University between February 2006 and April 2008. MATERIALS： PC12 cells, derived from the rat central nervous system, were purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences, China. A β1-40 was provided by Sigma, USA. Natural-cerebrolysin was provided by Shenzhen Institute of Integrated Chinese and Western Medicine, China. The natural-cerebrolysin was predominantly composed of Renshen （Radix Ginseng）, Tianma （Rhizoma Gastrodiae）, and Yixingye （Ginkgo Leaf） in a proportion of 1：2：2. Following conventional water extraction technology, an extract （1：20） was prepared. Each gram of extract equaled 20 grams of crude drug. In a total of 12 adult male New Zealand rabbits, six underwent intragastric administration of natural-cerebrolysin extract for 1 month to prepare natural-cerebrolysin-containing serum, and the remaining six rabbits received intragastric administration of physiological saline to prepare normal blank serum. METHODS： An AIzheimer＇s disease in vitro model was induced in PC12 cells using Aβ1-40. The cells were incubated with varying doses of natural-cerebrolysin-containing serum （2.5%, 5%, and 10%）. Normal blank serum-treated PC12 cells served as a blank control group. MAIN OUTCOME MEASURES： Through the use of inverted phase contrast microscope, cell morphology and neurite growth were observed, neurite length was measured, and the percentage of neurite-positive cells was calculated. Cell proliferation rate was determined by MTT assay, and MAP 2 expression was detected by fluorescent immunocytochemistry. RESULTS： Following Aβ1-40 treatments, some PC12 cells were apoptotic/dying, and only a few short neurites were observed. Following interventions with natural-cerebrolysin-containing serum, the PC12 cells proliferated, there was an increased number of neurites, and neurite length was enhanced. After middle- and high-dose natural-cerebrolysin treatments, the percentage of neurite-positive cells, as well as the average length of neurites, was significantly greater than the normal blank serum-treated PC12 cells （P 〈 0.05 or P 〈 0.01）. Compared with the blank control group, MAP2 expression in the Aβ1-40-treated PC12 cells was significantly inhibited, and the cell proliferation rate was significantly decreased （P 〈 0.01）. Following incubations with natural-cerebrolysin-containing serum, MAP2 expression and cell proliferation rate in the PC12 cells were significantly increased in a dose-dependent manner, compared with treatments with blank control serum （P 〈 0.05 or P 〈 0.01 ）. CONCLUSION： Natural-cerebrolysin exhibited antagonistic effects on neurotoxicity in Aβ1-40 induced Alzheimer＇s disease in vitro models. These effects were likely related to cell proliferation and the upregulation of intracellular MAP2 expression.

Yizhijiannao Granule and a combination of its effective monomers,icariin and Panax notoginseng saponins,inhibit early PC12 cell apoptosis induced by beta-amyloid(25-35)

One of our previous studies showed that Yizhijiannao Granule,a compound Chinese medicine, effectively improved the clinical symptoms of Alzheimer’s disease.In the present study,we established a model of Alzheimer’s disease using beta-amyloid（25-35）in PC12 cells,and treated the cells with Yizhijiannao Granule and its four monomers,i.e.,icariin,catechin,Panax notoginseng saponins,and eleutheroside E.Flow cytometry showed that Yizhijiannao Granule-containing serum, icariin,Panax notoginseng saponins,and icariin＋Panax notoginseng saponins were protective against beta-amyloid（25-35）-induced injury in PC12 cells.Icariin in combination with Panax notoginseng saponins significantly inhibited early apoptosis of PC12 cells with beta-amyloid （25-35）-induced injury compared to icariin or Panax notoginseng saponins alone.The effects of icariin＋Panax notoginseng saponins were similar to the effects of Yizhijiannao Granule.The findings indicate that two of the effective monomers of Yizhijiannao Granule,icariin and Panax notoginseng saponins,can synergistically inhibit early apoptosis of PC12 cells induced by beta-amyloid（25-35）.

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.

Scutellaria baicalensis stem-leaf total flavonoid reduces neuronal apoptosis induced by amyloid beta-peptide (25-35)

Scutellaria baicalensis stem-leaf total flavonoid might attenuate learning/memory impairment and neuronal loss in rats induced by amyloid beta-peptide. This study aimed to explore the effects of Scutellaria baicalensis stem-leaf total flavonoid on amyloid beta-peptide-induced neuronal apoptosis and the expression of apoptosis-related proteins in the rat hippocampus. Male Wistar rats were given intragastric administration of Scutellaria baicalensis stem-leaf total flavonoid, 50 or 100 mg/kg, once per day. On day 8 after administration, 10 pg amyloid beta-peptide （25-35） was injected into the bilateral hippocampus of rats to induce neuronal apoptosis. On day 20, hippocampal tissue was harvested and probed with the terminal deoxyribonucleotidyl transferase-mediated biotin-16-dUTP nick-end labeling assay. Scutellaria baicalensis stem-leaf total flavonoid at 50 and 100 mg/kg reduced neuronal apoptosis induced by amyloid beta-peptide （25-35） in the rat hippocampus. Immunohistochemistry and western blot assay revealed that expression of the pro-apoptotic protein Bax, cytochrome c and caspase-3 was significantly diminished by 50 and 100 mg/kg Scutellaria baicalensis stem-leaf total flavonoid, while expression of the anti-apoptotic protein Bcl-2 was increased. Moreover, 100 mg/kg Scutellana baicalensis stem-leaf total flavonoid had a more dramatic effect than the lower dosage. These experimental findings indicate that Scutellaria baicalensis stem-leaf total flavonoid dose-dependently attenuates neuronal apoptosis induced by amyloid beta-peptide in the hippocampus, and it might mediate this by regulating the expression of Bax, cytochrome c, caspase-3 and Bcl-2.

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.

LPS Regulates Apolipoprotein E and A<i>β</i>Interactionswith Effects on Acute Phase Proteins and Amyloidosis

Interactions between apolipoprotein E (apo E) and amyloid beta (Aβ) are associated with the peripheral clearance of Aβ and are important to the development of neurodegenerative diseases. Interests in acute phase proteins (APP) as biomarkers for the early progression of Alzheimer’s disease indicate that the peripheral Aβ metabolism is perturbed and the role of nutritional diets are important to reduce APPs to maintain peripheral Aβ clearance with relevance to hepatic cholesterol homeostasis and brain amyloidosis. The role of nutriproteomic diets that reverse the effects of high fat diets are associated with the reduction in APPs, cholesterol homeostasis and improved clearance of Aβ. Nutritional diets that reduce the increase in plasma endotoxins (gut microbiotica) such as lipopolysaccarides (LPS) reduce the effects of LPS on cell membranes and increase the cellular uptake of Aβ by interactions with apo E. LPS alter hepatic lipid metabolism with an increase hepatic cytokines and APPs in plasma. Interactions between apo E and Aβ are altered by LPS with increased binding of LPS to apo E with effects on electrostatic alterations in Aβ oligomers. The role of LPS in neurodegenerative diseases includes the effects of LPS on alpha-synuclein metabolism with relevance to Parkinson’s disease and Alzheimer’s

Gengnianchun recipe inhibits apoptosis of pheochromocytoma cells from beta-amyloid 25-35 insult, better than monotherapies and their compounds

This study aims to determine and compare the protective effects of Gengnianchun recipe drug serum and compounds of its representative drug monotherapies against sympathetic nerve pheochromocytoma cell line PC12 cells damaged by beta-amyloid 25-35 at the cellular apoptosis and related signal pathway levels. PC12 cells cultured with medicated rat serum showed enhanced cell viability and reduced cellular apoptosis rates compared with those of monotherapies and their compounds. Furthermore, Gengnianchun recipe up-regulated expressions of anti-apoptotic protein Bcl-2, estrogen receptor-beta and phosphorylated extracellular-signal-regulated kinase 1/2; and down-regulated expressions of pro-apoptotic proteins Bax and caspase-3. Gengnianchun recipe was superior to representative drug monotherapies, such as paeoniflorin, berberine, timosaponin A-III, icariine and their compounds in protecting PC12 cells. Mitogen-activated protein kinase blocker and estrogen receptor antagonist were found to reverse the above effects of Gengnianchun recipe. The experimental findings indicate that, Gengnianchun recipe protects PC12 cells from beta-amyloid 25-35 insult; its inhibitory effect on apoptosis may be achieved through the mitogen-activated protein kinase and estrogen receptor pathways.

Hyperoside protects the blood-brain barrier from neurotoxicity of amyloid beta 1–42

Mounting evidence indicates that amyloid β protein（Aβ） exerts neurotoxicity by disrupting the blood-brain barrier（BBB） in Alzheimer＇s disease. Hyperoside has neuroprotective effects both in vitro and in vivo against Aβ. Our previous study found that hyperoside suppressed Aβ1-42-induced leakage of the BBB, however, the mechanism remains unclear. In this study, bEnd.3 cells were pretreated with 50, 200, or 500 μM hyperoside for 2 hours, and then exposed to Aβ1-42 for 24 hours. Cell viability was determined using 3-（4,5-dimethyl-2-thiazolyl）-2,5-diphenyl-2-H-tetrazolium bromide assay. Flow cytometry and terminal deoxynucleotidyl transferase-mediated d UTP nick-end labeling assay were used to analyze cell apoptosis. Western blot assay was carried out to analyze expression levels of Bax, Bcl-2, cytochrome c, caspase-3, caspse-8, caspase-9, caspase-12, occludin, claudin-5, zonula occludens-1, matrix metalloproteinase-2（MMP-2）, and MMP-9. Exposure to Aβ1-42 alone remarkably induced bEnd.3 cell apoptosis; increased ratios of cleaved caspase-9/caspase-9, Bax/Bcl-2, cleav ed caspase-8/caspase-8, and cleaved caspase-12/caspase-12; increased expression of cytochrome c and activity of caspase-3; diminished levels of zonula occludens-1, claudin-5, and occludin; and increased levels of MMP-2 and MMP-9. However, hyperoside pretreatment reversed these changes in a dose-dependent manner. Our findings confirm that hyperoside alleviates fibrillar Aβ1-42-induced BBB disruption, thus offering a feasible therapeutic application in Alzheimer＇s disease.