Glutamate-stimulated secretion of amyloid precursor protein from cortical rat brain slices

The aim of this study was to determine whether L-glutamate, a major excitatory transmitter in the cerebral cortex, modulates the proteolytic cleavage of the amyloid precursor protein (APP) in the brain through specific receptor activation. Native rat brain cerebral cortical slices were stimulated either with L-glutamate or various glutamate receptor agonists, and the soluble APP derivatives released into the incubation medium were assayed by Western blot analysis. Immunoprecipitation with specific antibodies revealed that in the medium only secretory forms of APP lacking intact C-terminus were present, whereas in the brain slices both C- and N-terminal intact APP products were detectable. L-glutamate induced the release of secretory APP from cortical slices in a concentration-dependent but biphasic manner, with the highest release at 50 μM L-glutamate and smaller effects at higher glutamate concentrations. To determine whether the effect of L-glutamate is mediated through distinct glutamate receptor subtypes, brain slices were incubated in the presence of various specific glutamate receptor agonists. Activation of the alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) receptor with 50 nM (RS)-bromohomoibotenic acid resulted in a reduced release of secretory APP by 17%±3 (P<0.01, one tailed Student's t-test) compared to the incubation without any drug. Stimulation of the metabotropic glutamate receptor with 50 nM (2S,3S,4S)--(carboxycyclopropyl)-glycine (L-CCG-I) led to an enhanced release of secretory APP by 39%±3 (P<0.001), whereas activation of the N-methyl-D-aspartate (NMDA) receptor with 50 nM (1R,3R)-1-aminocyclopentane-1,3-dicarboxylic acid ((1R,3R)-ACPD) did not significantly change the secretion of APP compared to the incubation without any drug. The data suggest that: (i) cortical glutamatergic neurotransmission is involved in APP metabolism; and (ii) the stimulation of APP cleavage in cerebral cortical brain slices is mainly mediated by the metabotropic but not the NMDA glutamate receptor subtype, whereas the AMPA receptor subtype seems to inhibit the secretory path of APP processing.     

Ultrastructural localization of amyloid β/A4 protein precursor in the normal rat brain

The ultrastructural localization of amyloidβ/ A4 protein precursor (APP) was studied immunohistochemically in normal rat brains using antibodies against different portions of APP. In cerebral cortical neurons and Purkinje cells, APP reaction products were located in the cytoplasm and on cell surface membranes. Some Golgi apparatuses and rough endoplasmic reticulum also showed APP immunoreactivity on their membranes and some vesicles near the trans face of the Golgi apparatuses were stained. In the neuropil of the cerebral cortex and the cerebellar molecular layer, many cell processes, which surrounded synapses and were considered to be astrocytic, were APP-positive. Foot processes around capillaries and subpial astrocytic processes were also immuno-positive. At the ultrastructural level, APP-positive astrocytic processes were identified.     

Amyloid Precursor Protein Metabolism in Primary Cell Cultures of Neurons, Astrocytes, and Microglia

Abstract: Amyloid precursor protein (APP) gives rise by proteolytic processing to the amyloid β peptide (Aβ) found abundantly in cerebral senile plaques of individuals with Alzheimer's disease. APP is highly expressed in the brain. To assess the source of cerebral Aβ, the metabolism of APP was investigated in the major cell types of the newborn rat cerebral cortex by pulse/chase labeling and immunoprecipitation of the APP and APP metabolic fragments. We describe a novel C-terminally truncated APP isoform that appears to be made only in neurons. The synthesis, degradation, and metabolism of APP were quantified by phosphorimaging in neurons, astrocytes, and microglia. The results show that although little APP is metabolized through the amyloidogenic pathways in each of the three cultures, neurons appear to generate more Aβ than astrocytes or microglia.     

Correlation between beta-amyloid peptide production and human APP-induced neuronal death

The production of amyloid peptide (Abeta) from its precursor (APP) plays a key role in Alzheimer's disease (AD). However, the link between Abeta production and neuronal death remains elusive. We studied the biological effects associated with human APP expression and metabolism in rat cortical neurons. Human APP expressed in neurons is processed to produce Abeta and soluble APP. Moreover, human APP expression triggers neuronal death. Pepstatin A, an inhibitor of aspartyl proteases that reduces Abeta production, protects neurons from APP-induced neurotoxicity. This suggests that Abeta production is likely to be the critical event in the neurodegenerative process of AD.     

A monoclonal antibody to amyloid precursor protein induces neuronal apoptosis

Although there is considerable evidence suggesting that altered metabolism of beta-amyloid precursor protein (APP) and accumulation of its beta-amyloid fragment are key features of Alzheimer's disease (AD), the normal physiological function of APP remains elusive. We investigated the potential role of APP in neurons using the monoclonal antibody 22C11, which binds to the extracellular domain of the human, rat, or mouse APP. Exposure of cortical neurons to 22C11 induced morphological changes including neurite degeneration, nuclear condensation, and internucleosomal DNA cleavage that were consistent with neurons dying by apoptosis. Supporting a role for 22C11-mediated apoptosis occurring by binding to APP were data demonstrating that preincubation of 22C11 with either purified APP or a synthetic peptide (APP(66-81)) that contains the epitope for 22C11 significantly attenuated neuronal damage induced by 22C11. The specificity of 22C11 was further supported by data showing no apparent effects of either mouse IgG or the monoclonal antibody P2-1, which is specific for the aminoterminal end of human but not rat APP. In addition, biochemical features indicative of apoptosis were the formation of 120- and 150-kDa breakdown products of fodrin following treatment of cortical neurons with 22C11. Both the morphological and the biochemical changes induced by 22C11 were prevented following pretreatment of neurons with the general caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(O-methyl)-fluoromethyl ketone. Prior incubation of cortical neurons with GSH ethyl ester (GEE), a cell-permeable form of GSH, resulted in complete protection from the 22C11 insult, thus implicating an oxidative pathway in 22C11-mediated neuronal degeneration. This was further supported by the observation that prior treatment of neurons with buthionine sulfoximine, an inhibitor of gamma-glutamylcysteinyl synthetase, potentiated the toxic effects of 22C11. Finally, with use of compartmented cultures of hippocampal neurons, it was also demonstrated that selective application of 22C11 caused local neuritic degeneration that was prevented by the addition of GEE to the neuritic compartment. Thus, the binding of a monoclonal antibody to APP initially triggers neurite degeneration that is followed by caspase-dependent apoptosis in neuronal cultures and illustrates a novel property of this protein in neurons that may contribute to the profound neuronal cell death associated with AD.     

Large quantities of Abeta peptide are constitutively released during amyloid precursor protein metabolism in vivo and in vitro

The metabolism of the amyloid precursor protein (APP) has been extensively investigated because its processing generates the amyloid-β-peptide (Aβ), which is a likely cause of Alzheimer disease. Much prior research has focused on APP processing using transgenic constructs and heterologous cell lines. Work to date in native neuronal cultures suggests that Aβ is produced in very large amounts. We sought to investigate APP metabolism and Aβ production simultaneously under more physiological conditions in vivo and in vitro using cultured rat cortical neurons and live pigs. We found in cultured neurons that both APP and Aβ are secreted rapidly and at extremely high rates into the extracellular space (2-4 molecules/neuron/s for Aβ). Little APP is degraded outside of the pathway that leads to extracellular release. Two metabolic pools of APP are identified, one that is metabolized extremely rapidly (t1/2;) = 2.2 h), and another, surface pool, composed of both synaptic and extrasynaptic elements, that turns over very slowly. Aβ release and accumulation in the extracellular medium can be accounted for stoichiometrically by the extracellular release of β-cleaved forms of the APP ectodomain. Two α-cleavages of APP occur for every β-cleavage. Consistent with the results seen in cultured neurons, an extremely high rate of Aβ production and secretion from the brain was seen in juvenile pigs. In summary, our experiments show an enormous and rapid production and extracellular release of Aβ and the soluble APP ectodomain. A small, slowly metabolized, surface pool of full-length APP is also identified.     

The survival of rat cerebral cortical neurons in the presence of trophic APP peptides

One function of Alzheimer amyloid protein precursor (APP) is the regulation of growth and differentiation in several types of cells, including fibroblasts, PC12 cells, and neurons. This activity is represented by a small stretch of amino acids in the center of the molecule around RERMS. The APP 17-mer peptide containing the RERMS domain supported survival and neurite extension of rat cortical neurons in a dose-dependent and sequence-specific manner. The APP fragment synthesized in Escherichia coli supported the survival and neurite extension of rat cortical neurons, whereas the mutant APP fragment lacking the 30 amino acids around the RERMS domain had drastically reduced activity to support the survival and neurite extension. The current study established APP as a neuron survival factor and determined that the sequence around RERMS is important for this function. © 1994 John Wiley & Sons, Inc.     

Evidence for binding of the ectodomain of amyloid precursor protein 695 and activated high molecular weight kininogen

To identify ligands that bind to the N-terminal portion of human amyloid precursor protein (APP), we sought binding partners for a fragment of the ectodomain of human APP695 (sAPP(695)T). The probe bound to fragments of high molecular weight kininogen (HK) in rat cortical membrane preparations in vitro. Laser confocal microscopy indicated that APP and HK colocalize near cerebral blood vessels, in the neuropil, and in many neurons of rat brain. sAPP(695)T bound to human activated kininogen (HKa) (K(d)=0.3+/-0.1 nM), but not to inactivated or low molecular weight kininogen. Binding was specific for the light chain sequence of HKa. Biotinylated human HKa also bound to sAPP(695) (K(d)=0.3+/-0.5 nM). sAPP(695) and HKa form tight complexes in solution that can be coimmunoprecipitated. These results support the hypothesis that forms of APP and kininogen can interact in brain tissue. Considering the implications of APP in neurite outgrowth, the APP-HKa interaction could modulate neurogenesis.     

Brain oscillations,medium spiny neurons,and dopamine

1. The striatum is part of a multisynaptic loop involved in translating higher order cognitive activity into action. The main striatal computational unit is the medium spiny neuron, which integrates inputs arriving from widely distributed cortical neurons and provides the sole striatal output.2. The membrane potential of medium spiny neurons' displays shifts between a very negative resting state (down state) and depolarizing plateaus (up states) which are driven by the excitatory cortical inputs.3. Because striatal spiny neurons fire action potentials only during the up state, these plateau depolarizations are perceived as enabling events that allow information processing through cerebral cortex – basal ganglia circuits. In vivo intracellular recording techniques allow to investigate simultaneously the subthreshold behavior of the medium spiny neuron membrane potential (which is a reading of distributed patterns of cortical activity) and medium spiny neuron firing (which is an index of striatal output).4. Recent studies combining intracellular recordings of striatal neurons with field potential recordings of the cerebral cortex illustrate how the analysis of the input–output transformations performed by medium spiny neurons may help to unveil some aspects of information processing in cerebral cortex – basal ganglia circuits, and to understand the origin of the clinical manifestations of Parkinson's disease and other neurologic and neuropsychiatric disorders that result from alterations in dopamine-dependent information processing in the cerebral cortex – basal ganglia circuits.     

Immunohistochemical investigation of amyloid beta-protein (Abeta) in the brain of aged cats

To clarify the immunohistochemical features of amyloid deposits and cerebral amyloid angiopathy (CAA), the distribution of the amyloid beta-protein subtypes Abeta40, Abeta42, Abeta43 and Abeta precursor protein (APP) were examined in the brains of fourteen aged cats (7.5-21 year-old). Two types of plaques were detected. The first type was characterized by Ass positive antigenic material and detected in the cortical layers of the frontal and parietal lobes of all examined cats. The second type was characterized by diffuse positive immune staining representing diffuse plaques, which were detected only in the very aged cats (17-21 years old) and distributed throughout the cortical layers of the parietal lobes. Vascular amyloid and the amyloid deposits were strongly positive-stained with the antibody Abeta42. APP was exhibited in neurons and axons while the staining was stronger in the very aged cats (17-21 years old). Our findings suggest that the feline forms a spontaneous model for understanding the early changes of normal brain aging and the early stage of amyloid beta-protein deposition.     

Chemorepulsion of neuronal migration by Slit2 in the developing mammalian forebrain.

Newborn cerebral cortical neurons migrate along radial glia to the cortical plate. Experiments using a collagen gel assay revealed that the choroid plexus repelled cerebral cortical neurons and olfactory interneuron precursors, which were mimicked by Neuro-2A cells. Fractionation of Neuro-2A-conditioned medium identified a protein of 190 kDa, equivalent to full-length Slit proteins. Indeed, it cross-reacted with an antibody against Slit2, suggesting that it is either Slit2 or another Slit protein. Further, Slit2, expressed in COS cells, repelled cerebral cortical neurons and olfactory interneuron precursors. Thus, Slit2, which is expressed by the choroid plexus and the septum, acts as a chemorepulsive factor for neuronal migration. These results suggest chemorepulsion as a guidance mechanism for neuronal migration in the developing forebrain.     

肝细胞生长因子对氧糖剥夺/再灌注神经元的保护作用

本文旨在探讨肝细胞生长因子（hepatocyte growth factor,HGF）对神经元氧糖剥夺/再灌注损伤的影响。取原代培养12d的Sprague-Dawley大鼠大脑皮层神经元,无糖、无氧（95%N2＋5%CO2）孵育2h后,换含25mmol/L葡萄糖的培养液、常氧培养0-24h,以MTT比色法检测细胞活力、乳酸脱氢酶（lactate dehydrogenase,LDH）漏出率作为细胞损伤指标,建立体外氧糖剥夺/再灌注损伤细胞模型;用流式细胞仪和Hoechst33258染色分析细胞凋亡率;用RT-PCR和Western blot分别检测大鼠脑皮层神经元HGF受体c-Met mRNA和蛋白的表达。于氧糖剥夺2h/再灌注24h处理前2h,加入不同终浓度（5-120ng/mL）的HGF,观察HGF对皮层神经元的影响。结果显示,c-Met表达于皮层神经元,氧糖剥夺2h/再灌注24h后,c-Met mRNA和蛋白表达均显著上调,神经元细胞活力明显降低,LDH漏出率和细胞凋亡率显著增高。HGF预处理明显促进氧糖剥夺/再灌注损伤神经元的存活,降低LDH漏出率,最大效应剂量为80ng/mL。流式细胞术和Hoechst33258染色结果均显示,HGF（80ng/mL）显著降低氧糖剥夺/再灌注神经元的细胞凋亡率。此外,c-Met抑制剂SU11274（5μmol/L）完全阻断HGF的神经保护作用。结果表明,HGF对皮层神经元氧糖剥夺/再灌注损伤具有直接的保护作用,呈一定的剂量依赖关系,并能有效对抗神经元凋亡。     

Huntingtin associated protein 1 regulates trafficking of the amyloid precursor protein and modulates amyloid beta levels in neurons

J. Neurochem. (2012) 122, 1010-1022. ABSTRACT: Amyloid precursor protein (APP) is involved in the pathogenesis of Alzheimer's disease. It is axonally transported, endocytosed and sorted to different cellular compartments where amyloid beta (Aβ) is produced. However, the mechanism of APP trafficking remains unclear. We present evidence that huntingtin associated protein 1 (HAP1) may reduce Aβ production by regulating APP trafficking to the non-amyloidogenic pathway. HAP1 and APP are highly colocalized in a number of brain regions, with similar distribution patterns in both mouse and human brains. They are associated with each other, the interacting site is the 371-599 of HAP1. APP is more retained in cis-Golgi, trans-Golgi complex, early endosome and ER-Golgi intermediate compartment in HAP1-/- neurons. HAP1 deletion significantly alters APP endocytosis and reduces the re-insertion of APP into the cytoplasmic membrane. Amyloid precursor protein-YFP(APP-YFP) vesicles in HAP1-/- neurons reveal a decreased trafficking rate and an increased number of motionless vesicles. Knock-down of HAP1 protein in cultured cortical neurons of Alzheimer's disease mouse model increases Aβ levels. Our data suggest that HAP1 regulates APP subcellular trafficking to the non-amyloidogenic pathway and may negatively regulate Aβ production in neurons.     

大鼠大脑皮质星形胶质细胞条件培养液促进小脑皮质神经元的生长

本研究从大鼠大脑皮质分离、纯化星形胶质细胞,再经培养后收集星形胶质细胞的无血清条件培养液。用盖玻片培养法与快速自动比色微量分析法研究了星形胶质细胞条件培养液对小脑皮质神经元生存以及神经元活力的影响。发现星形胶质细胞条件培养液能够明显提高小脑皮质神经元的体外存活率,增强神经元的活力。表明星形胶质细胞具有神经营养性作用。     

Geniposide Increases Unfolded Protein Response-Mediating HRD1 Expression to Accelerate APP Degradation in Primary Cortical Neurons

Altered proteostasis induced by amyloid peptide aggregation and hyperphosphorylation of tau protein, is a prominent feature of Alzheimer’s disease, which highlights the occurrence of endoplasmic reticulum stress and triggers the activation of the unfolded protein response (UPR), a signaling pathway that enforces adaptive programs to sustain proteostasis. In this study, we investigated the role of geniposide in the activation of UPR induced by high glucose in primary cortical neurons. We found that high glucose induced a significant activation of UPR, and geniposide enhanced the effect of high glucose on the phosphorylation of IRE1α, the most conserved UPR signaling branch. We observed that geniposide induced the expression of HRD1, an ubiquitin-ligase E3 in a time dependent manner, and amplified the expression of HRD1 induced by high glucose in primary cortical neurons. Suppression of IRE1α activity with STF-083010, an inhibitor of IRE1 phosphorylation, prevented the roles of geniposide on the expression of HRD1 and APP degradation in high glucose-treated cortical neurons. In addition, the results from RNA interfere on HRD1 revealed that HRD1 was involved in geniposide regulating APP degradation in cortical neurons. These data suggest that geniposide might be benefit to re-establish proteostasis by enhancing the UPR to decrease the load of APP in neurons challenged by high glucose.     

Basal Forebrain Cholinergic Dysfunction in Alzheimer’s Disease – Interrelationship with β-amyloid, Inflammation and Neurotrophin Signaling

Alzheimer’s disease, the most common neurodegenerative disorder of senile dementia, is characterized by two major morpho-pathological hallmarks. Deposition of extracellular neuritic, β-amyloid peptide-containing plaques (senile plaques) in cerebral cortical regions of Alzheimer patients is accompanied by the presence of intracellular neurofibrillary tangles in cerebral pyramidal neurons. Basal forebrain cholinergic dysfunction is also a consistent feature of Alzheimer’s disease, which has been suggested to cause, at least partly, the cognitive deficits observed in patients with Alzheimer’s disease. Impaired cortical cholinergic neurotransmission may also contribute to β-amyloid plaque pathology in Alzheimer’s disease by affecting expression and processing of the β-amyloid precursor protein (APP). Vice versa, low level of soluble β-amyloid has been observed to inhibit cholinergic synaptic function. Deposition of β-amyloid plaques in Alzheimer’s disease is also accompanied by a significant plaque-associated glial up-regulation of interleukin-1, which has been attributed to affect expression and metabolism of APP and to interfere with cholinergic transmission. Understanding the molecular mechanisms underlying the interrelationship between cortical cholinergic dysfunction, β-amyloid formation and deposition, as well as local inflammatory upregulation, would allow to derive potential treatment strategies to pharmacologically intervene in the disease-causing signaling cascade.     

Effect of estradiol on neuronal Swedish-mutated beta-amyloid precursor protein metabolism: reversal by astrocytic cells

Alzheimer's disease is the most frequent neurodegenerative disorder in the aged population and is characterized by the deposition of the 40/42-residue amyloid beta protein (Abeta), a proteolytic fragment of the beta-amyloid precursor protein (APP). Recently, it has been shown that physiological doses of estradiol reduce the generation of endogenous Abeta in primary cortical neurons. Here we investigate the influence of estrogen in amyloidogenesis and sAPPalpha secretion in the CNS. By means of primary cortical neurons overexpressing humanized APP(695) bearing the Swedish mutation (hAPP(695sw)), we analyzed APP maturation in the absence or in the presence of estrogen. We show that estrogen at a 2 microM concentration increases the release of the neuroprotective sAPPalpha fragment but does not reduce the release of Abeta in primary neurons overexpressing the Swedish-mutated form of APP. Furthermore, neurons cocultured with astrocytic cells or grown with astrocytes conditioned media do not exhibit the estrogen-induced increase in sAPPalpha secretion. Altogether, our data indicate that astrocytes interfere with estrogen in the regulation of sAPPalpha secretion, probably via secreted factor(s).     

Statins Reduce Amyloid β-Peptide Production by Modulating Amyloid Precursor Protein Maturation and Phosphorylation Through a Cholesterol-Independent Mechanism in Cultured Neurons

Statins, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, have been reported to attenuate amyloid-β peptide (Aβ) production in various cellular models. However, the mechanisms by which statins affect neuronal Aβ production have not yet been clarified. Here, we investigated this issue in rat primary cortical neurons using two statins, pitavastatin (PV) and atorvastatin (AV). Treatment of neurons with 0.2–2.5 μM PV or AV for 4 days induced a concentration- and time-dependent reduction in the secretion of both Aβ40 and Aβ42. Moreover, Western blot analyses of cell lysates showed that treatment with PV or AV significantly reduced expression levels of the mature form of amyloid precursor protein (APP) and Thr668-phosphorylated APP (P-APP), but not immature form of APP; the decreases in P-APP levels were more notable than those of mature APP levels. The statin treatment did not alter expression of BACE1 (β-site APP-cleaving enzyme 1) or γ-secretase complex proteins (presenilin 1, nicastrin, APH-1, and PEN-2). In neurons overexpressing APP via recombinant adenoviruses, PV or AV similarly reduced Aβ secretion and the levels of mature APP and P-APP. Statins also markedly reduced cellular cholesterol content in neurons in a concentration-dependent manner. Co-treatment with mevalonate reversed the statin-induced decreases in Aβ secretion and mature APP and P-APP levels, whereas co-treatment with cholesterol did not, despite recovery of cellular cholesterol levels. Finally, cell-surface biotinylation experiments revealed that both statins significantly reduced the levels of cell-surface P-APP without changing those of cell surface mature APP. These results suggest that statins reduce Aβ production by selectively modulating APP maturation and phosphorylation through a mechanism independent of cholesterol reduction in cultured neurons.     

Downregulation of amyloid precursor protein inhibits neurite outgrowth in vitro

The amyloid precursor protein (APP) is a transmembrane protein expressed in several cell types. In the nervous system, APP is expressed by glial and neuronal cells, and several lines of evidence suggest that it plays a role in normal and pathological phenomena. To address the question of the actual function of APP in normal developing neurons, we undertook a study aimed at blocking APP expression using antisense oligonucleotides. Oligonucleotide internalization was achieved by linking them to a vector peptide that translocates through biological membranes. This original technique, which is very efficient and gives direct access to the cell cytosol and nucleus, allowed us to work with extracellular oligonucleotide concentrations between 40 and 200 nM. Internalization of antisense oligonucleotides overlapping the origin of translation resulted in a marked but transient decrease in APP neosynthesis that was not observed with the vector peptide alone, or with sense oligonucleotides. Although transient, the decrease in APP neosynthesis was sufficient to provoke a distinct decrease in axon and dendrite outgrowth by embryonic cortical neurons developing in vitro. The latter decrease was not accompanied by changes in the spreading of the cell bodies. A single exposure to coupled antisense oligonucleotides at the onset of the culture was sufficient to produce significant morphological effects 6, 18, and 24 h later, but by 42 h, there were no remaining significant morphologic changes. This report thus demonstrates that amyloid precursor protein plays an important function in the morphological differentiation of cortical neurons in primary culture.