Expression of mutant amyloid precursor proteins induces apoptosis in PC12 cells

The cause of neuronal loss in Alzheimer disease is unknown. We investigated the effects on survival of PC12 cells expressing A692G, E693Q, and V717F mutant amyloid precursor proteins (APP). Differentiated cells expressing mutant APPs exhibited somal shrinkage, followed by cell detachment from the plates. Increased levels of oligonucleosome-sized DNA ladders and TUNEL-positive nuclei were observed, and electron microscopy revealed extensive plasma membrane blebbing, margination of condensed chromatin, and well-preserved organelles in these transfectants. The levels of TUNEL-positive cells, analyzed by a flow-cytometric method, were increased by four- to sevenfold in mutant APP transfectants, but less than twofold in wild-type APP transfectants relative to untransfected cells. Our results provide evidence that expression of mutant APPs in differentiated PC12 cells induces cell death via an apoptotic pathway. J. Neurosci. Res. 47:253–263, 1997. © 1997 Wiley-Liss, Inc.     

The effects of perturbed energy metabolism on the processing of amyloid precursor protein in PC12 cells

Summary. The mismetabolism of amyloid precursor protein (APP), favouring the production of Aβ, is considered to be central to the pathogenesis of Alzheimer's disease (AD). However it remains to be established whether the causative factor is the reported toxicity of Aβ or reduced production of secretory derivatives of APP which may have trophic or neuroprotective properties. One possible contributory factor to an imbalance in APP metabolism is the impaired cellular energy availability described in AD. The aim of this study was to investigate processing of APP-like proteins following inhibition of oxidative energy metabolism in PC12 cells. Under these conditions, intracellular and secreted APP-like proteins were significantly reduced. Treatment of energy perturbed cells with the lysosomotropic agent chloroquine restored intracellular concentrations of APP-like proteins to the control range, while the secretion was completely restored by activation of protein kinase C. These findings raise the possibility that energy related metabolic stress may lead to altered metabolism of APP-like proteins favouring a potentially amyloidogenic pathway. Furthermore, the observation that activation of PKC is able to overcome this potentially pathogenic process has important implications for treatment of AD with the current generation of cholinomimetic drugs, suggesting that such drugs may slow disease progression as well as improve cognitive dysfunction. Received December 12, 1997; accepted March 24, 1998     

Evidence for intracellular cleavage of the Alzheimer's amyloid precursor in PC12 cells.

The Alzheimer's amyloid precursor (APP) is cleaved by an unidentified enzyme (APP secretase) to produce soluble APP. Fractionation of PC12 cell homogenates in a detergent-free buffer showed the presence of the Kunitz protease inhibitor (KPI)-containing soluble APP (nexin II) in the particulate fraction. Digitonin or sodium carbonate treatment of this fraction solubilized nexin II suggesting that it is contained in the lumen of vesicles. Nexin II production was not affected by lysosomotropic agents, suggesting that APP secretase is not a lysosomal enzyme. Labelling of cell surface proteins by iodination failed to detect full-length APP on the surface of PC12 cells, suggesting that most of this protein is located intracellularly. Furthermore, pulse-chase experiments showed that nexin II is detected in cell extracts before it appears in the culture medium. Cellular nexin II was detected at zero time of chase after only 5 min of pulse labelling with 35S-sulfate, indicated that APP secretase cleavage takes place immediately after APP is sulfated. Temperature block, pulse-chase, and 35S-sulfate-labelling experiments suggested that APP is cleaved by APP secretase intracellularly in the trans-Golgi network (TGN) or in a post-Golgi compartment.     

Altered processing of a mutant amyloid precursor protein in neuronal and endothelial cells

Altered proteolysis of the amyloid precursor protein (APP) may play an important role in Alzheimer disease (AD). To better understand the role of mutant APP in the pathogenesis of the disease, we stably overexpressed the mutant APP717F approximately twofold vs. the endogenous wild-type gene in several cell types. The processing of APP was examined by Western blot analysis and immunoprecipitation. We observed distinctive patterns of APP metabolites among various cell lines. Neuronal and endothelial cells expressing mutant APP717F generated higher levels of large, potentially amyloidogenic carboxyl terminal fragments, which were enhanced upon treatment of the cells with leupeptin. These results suggest that mutations in the APP gene shift the protein processing towards the amyloidogenic pathway in neuronal and endothelial cells possibly involving the endosomal-lysosomal system. © 1995 Wiley-Liss, Inc.     

Neurodegenerative changes associated with beta-amyloid deposition in the brains of mice carrying mutant amyloid precursor protein and mutant presenilin-1 transgenes

Mutations of amyloid precursor protein (APP) and presenilin-1 (PS1) lead to an increase in beta-amyloid (Abeta) production. Despite the fact that a number of transgenic mice develop cerebral Abeta plaques, few have been subjected to ultrastructural investigation and the sequence of events leading to Abeta plaque formation is unclear. We therefore investigated the doubly transgenic (mutant APP(K670N,M671L)-mutant PS1(M146L)) mouse, which develops Abeta deposits much earlier than singly transgenic littermates. Widespread Abeta plaques with or without a distinct core were found in gray matter. Abeta plaques were also present in white matter. Astrocytosis was greater around gray matter plaques than around white matter plaques. In some plaques, Abeta cores were associated with cell profiles containing prominent endoplasmic reticulum and a homogeneous cytoplasm that appeared to be neuronal. The morphology and location of other profiles indicated them to be microglia or oligodendrocytes. Some Abeta fibrils appeared to lie within these profiles, but they may have been simply surrounded by the cell profile since the profile membrane was not always visible. Dark atrophic neurons, whose morphology suggested that they were apoptotic, were present around gray matter plaques. Cerebrovascular Abeta deposition was also observed in the brains of APP/PS1 transgenic mice. Thus, the amyloid deposition and neuropathology observed in APP/PS1 mouse brain are similar to those in Alzheimer's disease and they appear to develop earlier and become more severe than in the other transgenic models currently available.     

黄连解毒汤对APP/PS1双转基因阿尔茨海默病模型小鼠脑组织的保护

Huanglian Jiedu decoction(HLJDD) has been shown to improve cerebral blood flow,and reduce lipid peroxidation damage to the brain and its energy metabolism.The present study was designed to observe the cerebroprotective effect of HLJDD on an Alzheimer’s disease rodent model,prese-nilin-1/amyloid protein precursor double transgenic mice.HLJDD reduced serum interleukin-6 and interleukin-1β levels,decreased β-amyloid precursor protein gene and senile plaque expression,resisted oxidation,and reduced free radical-induced injury,thereby improving the learning and memory of these mice.Moreover,HLJDD at 433 mg/kg per day exhibited better effects compared with that at 865 or 216 mg/kg per day,and donepezil hydrochloride at 30 mg/kg per day.Thus,these results suggest that HLJDD may have protective effects against Alzheimer’s disease.     

Hyperphosphorylated tau and paired helical filament-like structures in the brains of mice carrying mutant amyloid precursor protein and mutant presenilin-1 transgenes

Senile plaques composed mainly of beta-amyloid (Abeta) and neurofibrillary tangles principally composed of hyperphosphorylated tau are the major pathological features of Alzheimer's disease (AD). Despite the fact that increased expression of amyloid precursor protein (APP) and presenilin-1 (PS1) transgenes in mice lead to increased Abeta deposition in plaquelike structures in the brain, little is known about the nature and distribution of tau in these mice. Therefore the relationship between Abeta and hyperphosphorylated tau was investigated in mice carrying mutant APP and mutant PS1 transgenes using both light (LM) and electron microscopy (EM) with immunocytochemistry. LM immunocytochemistry revealed cerebral Abeta deposits to be present from 8 weeks of age, whereas hyperphosphorylated tau was not detected until 24 weeks of age, when it appeared as punctate deposits in close association with the Abeta deposits in the cortex and hippocampus. However, dystrophic neurites were not as heavily immunolabeled as they are in AD brain. EM revealed that aggregations of straight filaments (10-12 nm wide) were present in some cellular processes at the periphery of Abeta plaques in 8-month-old APP/PS1 mice. In one such mouse, single filaments and paired filaments showing a helical configuration (50-55 nm half-period, 25 nm max. width) were present in a dark, atrophic hippocampal neuron. Immunogold labeling of APP/PS1 mouse brain revealed hyperphosphorylated tau epitopes in some dystrophic neurites from 24 weeks of age that were similar to those present in AD. These results suggest that hyperphosphorylated tau appears in APP/PS1 mouse brain after the onset of Abeta deposition and although it is associated with Abeta deposits, its distribution is not identical to that in AD.     

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.     

Progressive, age-related behavioral impairments in transgenic mice carrying both mutant amyloid precursor protein and presenilin-1 transgenes

This study provides a comprehensive behavioral characterization during aging of transgenic mice bearing both presenilin-1 (PS1) and amyloid precursor protein (APP(670,671)) mutations. Doubly transgenic mice and non-transgenic controls were evaluated at ages wherein beta-amyloid (Abeta) neuropathology in APP+PS1 mice is low (5-7 months) or very extensive (15-17 months). Progressive cognitive impairment was observed in transgenic mice for both water maze acquisition and radial arm water maze working memory. However, transgenicity did not affect Y-maze alternations, circular platform performance, standard water maze retention, or visible platform recognition at either age, nor did transgenicity affect anxiety levels in elevated plus-maze testing. In sensorimotor tasks, transgenic mice showed a progressive increase in open field activity, a progressive impairment in string agility, and an early-onset impairment in balance beam. None of these sensorimotor changes appeared to be contributory to any cognitive impairments observed, however. Non-transgenic mice showed no progressive behavioral change in any measure evaluated. Given the age-related cognitive impairments presently observed in APP+PS1 transgenic mice and their progressive Abeta deposition/neuroinflammation, Abeta neuropathology could be involved in these progressive cognitive impairments. As such, the APP+PS1 transgenic mouse offers unique opportunities to develop therapeutics to treat or prevent Alzheimer's Disease through modulation of Abeta deposition/neuroinflammation.     

β分泌酶抑制剂对冈田酸诱导PC12细胞淀粉样蛋白前体蛋白代谢的影响

目的 探索β分泌酶抑制剂对冈田酸(OA)诱导的PC12细胞淀粉样蛋白前体蛋白(APP)代谢的影响. 方法 10 nmol/LOA作用4 h、8 h、16 h、24h及48 h诱导PC12细胞tau磷酸化,加β分泌酶抑制剂干预,MTT法测定细胞抑制率,免疫细胞化学法及Western blot检测全长APP和β-C末端片段(βCTF). 结果 10 nmol/L OA对PC12细胞的抑制作用呈时间依赖性,β分泌酶抑制剂可明显减轻该作用.OA诱导PC12细胞内全长APP和13CTF含量增加,B分泌酶抑制剂进一步增加细胞内全长APP含量,并减少βCTF含量. 结论 OA诱导PC12细胞中APP主要经β分泌酶途径代谢,生成具有神经毒性的βCTF.β分泌酶抑制剂通过维持细胞存活和减少13CTF从而减轻OA诱导的神经毒性,但使细胞内APP进一步增多.     

Cognitive correlates of Abeta deposition in male and female mice bearing amyloid precursor protein and presenilin-1 mutant transgenes

Several transgenic mouse models of Alzheimer's disease (AD) have been developed that exhibit beta-amyloid (Abeta) neuropathology and behavioural deficits. However, not all studies have investigated the relationship between the development of cognitive impairment and neuropathology. Therefore, temporal changes in cognition were investigated in male and female double-mutant APPswexPS1.M146V (TASTPM) transgenic mice using an object recognition test and correlated with the development of cerebral Abeta neuropathology. Both male and female TASTPM mice exhibited similar significant cognitive impairment at 6, 8 and 10 months of age in the object recognition test, compared to wild-type littermates. There was no such cognitive impairment at 3 or 4 months of age. Quantitative immunohistochemistry using a battery of Abeta antibodies demonstrated that cerebral Abeta deposition was first apparent in 3-month-old mice, and it increased with age. The early appearance of cerebral Abeta deposits in the double-transgenic TASTPM mice supports the evidence that mutations in the PS1 gene accelerate Abeta deposition. The cerebral Abeta load was greater in female than in male TASTPM mice at all ages investigated. In the electron microscope, mature Abeta plaques comprising a fibrillar core surrounded by degenerating neurites and reactive glia were first observed in the cortex of TASTPM mice at 6 months of age, the same age at which cognitive impairment became apparent. These results suggest that the cognitive impairment in TASTPM mice is related to the disruption of neural connectivity and not simply Abeta deposition, which first occurs 3 months earlier.     

Reduced expression of amyloid precursor protein, presenilin-1 and rab3a in cortical brain regions in Alzheimer's disease

To study the role of amyloid precursor protein (APP) in the pathogenesis of Alzheimer's disease (AD), the level of APP was analysed by quantitative immunoblotting in 6 AD patients and 6 age-matched controls in 9 brain regions. These were associative cortices (orbital frontal cortex, inferior temporal cortex, inferior parietal cortex), primary cortex (occipital cortex), limbic structures (anterior cingulate gyrus, hippocampus), subcortical structures (putamen, thalamus) and cerebellum. To assess a potential relationship between APP and presenilin-1 (PS-1) and/or synaptic proteins, the levels of PS-1 and rab3a, a specific synaptic vesicle protein, were also determined in the same tissue samples. The level of APP was almost the same in the association cortical regions, primary cortex, and limbic structures and in the subcortical structures, while the lowest level was found in the cerebellum. There were more marked differences in the level of PS-1 and rab3a between different brain regions. The highest levels of PS-1 and rab3a were found in the association cortical areas, while intermediate levels were found in primary cortex, limbic structures and subcortical structures. As for APP, the lowest level was found in cerebellum. We found significantly reduced levels of all three proteins in the association cortices and in hippocampus in AD. Our data show that the protein levels are reduced in specific areas, restricted to neuronal populations that are known to degenerate in AD. Due to the similarity of the expression of APP, PS-1 and rab3a, it is tempting to speculate whether there is a functional relationship between these proteins.     

Proteolytic processing of the Alzheimer's disease amyloid precursor protein in brain and platelets

Proteolytic processing of the amyloid precursor protein by beta -and gamma-secretases results in the production of Alzheimer's disease (AD) Abeta amyloid peptides. Modulation of secretase activity is being investigated as a potential therapeutic approach. Recent studies with human brain have revealed that the beta-secretase protein, BACE, is increased in cortex of AD patients. Analysis of betaCTF (or C99), the amyloid precursor protein (APP) product of BACE cleavage that is the direct precursor to Abeta, shows it is also elevated in AD, underlying the importance of beta-secretase cleavage in AD pathogenesis. The C-terminal product of gamma-secretase cleavage of APP, epsilonCTF (or AICD), is enriched in human brain cortical nuclear fractions, a subcellular distribution appropriate for a putative involvement of APP cytosolic domain in signal transduction. Analysis of AD cortex samples, particularly that of a carrier of a familial APP mutation, suggests that processing of APP transmembrane domain generates an alternative CTF product. All these particularities observed in the AD brain demonstrate that APP processing is altered in AD. The transgenic mouse model Tg2576 seems to be a promising laboratory tool to test potential modulators of Abeta formation. Indeed, C-terminal products of alpha-, beta-, and gamma-secretase cleavage are readily detectable in the brain of these transgenic mice. Finally, the finding of the same secretase products in platelets and neurons make platelets a potentially useful and easily accessible clinical tool to monitor effects of novel therapies based on inhibition of beta- or gamma-secretase.     

Aberrant accentuation of neurofibrillary degeneration in the hippocampus of Alzheimer's disease with amyloid precursor protein 717 and presenilin-1 gene mutations

This study reports correlation of the hippocampal neurofibrillary tangles (NFT) density with beta-amyloid (Abeta) precursor protein (APP) 717 mutation, presenilin (PS)-1 mutation and apolipoprotein E (Apo-E) e4 alleles (E4), being graded as 3 forms (no-E4, one-E4 and two-E4) in autopsied brains from patients with familial and non-familial Alzheimer's disease (AD). We studied the density of NFT-free neurons, intracellular NFT (I-NFT), extracellular NFT (E-NFT) and total NFT (I-NFT plus E-NFT) in the six hippocampal subdivisions: cornu ammonis (CA) 1-CA4, subiculum and entorhinal cortex. The APP mutation cases showed significantly higher total NFT density in the CA1-CA2 region, and the PS-1 mutation cases also showed higher density of total NFT in the CA1-CA3 than non-familial cases. Moreover, high densities of the E-NFT contributed to these high total NFT densities. Non-familial AD cases showed a stereotypical NFT distribution with entorhinal accentuation in the hippocampus irrespective of E4 frequency. Thus, APP and PS-1 mutations predominantly affect the CA regions with profound neurodegeneration, which contributes early and severe clinical features of familial AD.     

Survival and plasticity of basal forebrain cholinergic systems in mice transgenic for presenilin-1 and amyloid precursor protein mutant genes

The basalo-cortical cholinergic system was characterized in mice expressing mutant human genes for presenilin-1 (PS1), amyloid precursor protein (APP), and combined PS/APP. Dual immunocytochemistry for ChAT and A beta revealed swollen cholinergic processes within cortical plaques in both APP and PS/APP brains by 12 months, suggesting aberrant sprouting or redistribution of cholinergic processes in response to amyloid deposition. At 8 months, cortical and subcortical ChAT activity was normal (PS/APP) or elevated (PS, APP frontal cortex), while cholinergic cell counts (nBM/SI) and receptor binding were unchanged. ChAT mRNA was up-regulated in the nBM/SI of all three transgenic lines at 8 months. The data indicate that the basal forebrain cholinergic system does not degenerate in mice expressing AD-related transgenes, even in mice with extreme amyloid load. The     

荧光标记的突变型淀粉样前体蛋白裂解过程的细胞研究

目的 研究淀粉样前体蛋白(amyloid precursor protein,APP)酶解过程,构建含有Swedish和APP717两种突变的荧光真核表达系统.方法 以pcDNA3.0-APP为模板,通过聚合酶链式反应(PCR)得到含有APP717突变的APP最后300个碱基片段(C99);以pcDNA3.0-CFP-CaM-YFP酶切产物为模板,通过PCR分别得到编码蓝色荧光蛋白(CFP)和黄色荧光蛋白(YFP)碱基序列;生物合成含有Swedish突变的APP中间54个碱基片段(54 bp).利用基因工程技术将CFP、54 bp、YFP、C99片段克隆至载体质粒pcDNA3.0中,通过酶切、PCR、测序鉴定最终得到重组质粒pcDNA3.0-CFP-54bp-YFP-C99和pcDNA3.0-CFP-54 bp-YFP,并将其转染至人神经母细胞瘤(SH-SY5Y)细胞中,利用多光子共聚焦显微镜观察荧光表达,检测荧光共振能量转移(FRET)以及免疫细胞化学染色观察B淀粉样蛋白(Aβ).结果 (1)基因序列分析证明重组质粒构建成功.(2)利用多光子共聚焦显微镜观察转染细胞,显示融合基因能够准确表达蓝色和黄色荧光.(3)表达CFP-54bp-YFP的细胞有FRET现象,而表达CFP-54bp-YFP-C99的细胞中观察不到FRET现象.(4)利用多光子共聚焦显微镜发现CFP-54bp-YFP-C99转染的细胞中有YFP标记的A13产生并沉积在胞质胞膜以及细胞间隙中.(5)免疫细胞化学检测证实CFP-54bp-YFP-C99经过裂解可以产生Aβ,Aβ在细胞膜、细胞质、细胞间隙聚集沉积.结论 (1)融合基因的表达产物能够完成APP的有序裂解产生Aβ.(2)Aβ有可能产生于APP由胞质至胞膜的运输过程中.(3)研究显示C99对于APP被裂解有重要意义,可能起到信号肽样的引导定位作用.(4)在细胞外形成沉积之前,Aβ已经在细胞内聚集导致细胞形态改变.     

突变型早老素-1致维甲酸诱导PC12细胞ERp29表达增强及其意义

目的 探讨突变型早老素-1(PS-1)对维甲酸(RA)诱导PC12细胞基因表达的影响,寻找与阿尔茨海默病发病机制相关的基因.方法 分别建立表达突变型和野生型PS-1的经RA诱导的基因工程化PC12细胞,应用银染mRNA差异显示技术研究突变型PS-1对RA诱导的PC12细胞基因差异表达的影响.结果 成功建立了表达突变型和野生型PS-1的经RA诱导的基因工程化PC12细胞后,应用银染mRNA差异显示技术发现29型内质网蛋白(ERp29)在表达突变型PS-1的基因工程化PC12细胞中高表达,并与细胞凋亡密切相关.结论 突变型PS-1致细胞内ERp29的表达增强可能是内质网应激的结果,ERp29表达增强与阿尔茨海默病的发生与发展可能存在着密切关系.