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At least 973 records · Page 54Linked to original sources
PubMed1997

Altered expression of amyloid protein precursor mRNA in the rat hippocampus following trimethyltin intoxication: an in situ hybridization study.

Previous studies have reported increased levels of amyloid protein precursor (APP) and APP mRNA in the hippocampus and basal forebrain of patients with Alzheimer's disease. Similar changes have been found in the brains of aged rodents and transgenic mice. It now appears that alterations in the expression of individual isoforms of APP mRNA may have a role to play in amyloid-pathogenesis. Here we examined the effect of acute administration of the limbic system neurotoxin trimethyltin (TMT) (8 mg/kg i.p.) on APP-751 and APP-695 mRNA expression in the rat hippocampus (CA1, CA2, CA3 and CA4) using in situ hybridization techniques. We found that following TMT treatment the expression of APP-751 mRNA was increased in CA1 pyramidal cells while that of APP-695 mRNA remained unchanged. TMT also increased the numbers of APP-751 and APP-695 mRNA positively hybridized cells in the CA1 pyramidal layer. These findings suggest that an alteration in APP mRNA expression is involved in the response of the rodent brain to TMT intoxication.

Amyloid beta-Protein Precursor↗
PubMed1999

Nicergoline stimulates protein kinase C mediated alpha-secretase processing of the amyloid precursor protein in cultured human neuroblastoma SH-SY5Y cells.

We investigated the ability of the antidementia agents, nicergoline, aniracetam and hydergine to stimulate PKC mediated alpha-secretase amyloid precursor protein (APP) processing in cultured human neuroblastoma SH-SY5Y cells. Western immunoblotting of cell conditioned media using the Mabs 22C11 and 6E10 revealed the presence of 2 bands with molecular mass of 90 and 120 kDa, corresponding to possible alternatively glycosylated forms of secreted APP (APPs). Short-term (30 min and 2 h) treatment of cells with nicergoline gave an increased intensity of both bands, compared to non-treated cells. Maximal nicergoline effects, of the order of 150-200% over basal APPs release, were seen at concentrations between 1 and 10 microM. Under the same condition, 1 microM PdBu, used as a positive control, gave 500-1000% increases of basal APPs release. In contrast, aniracetam and hydergine, did not show any effect on APPs secretion. 2 h treatment with nicergoline had no effect on cellular full-length APP levels, as determined by immunoblotting of cell extracts with 22C11 and CT15 antibodies. Immunoblotting with PKC isoform specific antibodies of soluble and membrane fractions prepared from 2 h treated cells, showed that nicergoline (50 microM) and PdBu (1 microM) both induced translocation of PKC alpha, gamma and epsilon, but not PKC beta. The involvement of PKC in mediating nicergoline stimulated APPs release was also studied using specific inhibitors. 1 microM calphostin C, a broad range PKC inhibitor, significantly reduced both PdBu (1 microM) and nicergoline (10 microM) induced APPs release. In contrast, Go6976 (1 microM), a selective PKC alpha and beta1 inhibitor, as well as the cAMP-dependent protein kinase inhibitor, H89 (1 microM) were without effect. These results indicate that nicergoline can modulate alpha-secretase APP processing by a PKC dependent mechanism that is likely to involve the gamma and epsilon isoforms of this enzyme.

Amyloid Precursor Protein Secretases↗
PubMed2003

Roles of amyloid precursor protein and its fragments in regulating neural activity, plasticity and memory.

Amyloid-beta precursor protein (APP) is a membrane-spanning protein with a large extracellular domain and a much smaller intracellular domain. It is the source of the amyloid-beta (Abeta) peptide found in neuritic plaques of Alzheimer's disease (AD) patients. Because Abeta shows neurotoxic properties, and because familial forms of AD promote Abeta accumulation, a massive international research effort has been aimed at understanding the mechanisms of Abeta generation, catabolism and toxicity. APP, however, is an extremely complex molecule that may be a functionally important molecule in its full-length configuration, as well as being the source of numerous fragments with varying effects on neural function. For example, one fragment derived from the non-amyloidogenic processing pathway, secreted APPalpha (sAPPalpha), is neuroprotective, neurotrophic and regulates cell excitability and synaptic plasticity, while Abeta appears to exert opposing effects. Less is known about the neural functions of other fragments, but there is a growing interest in understanding the basic biology of APP as it has become recognized that alterations in the functional activity of the APP fragments during disease states will have complex effects on cell function. Indeed, it has been proposed that reductions in the level or activity of certain APP fragments, in addition to accumulation of Abeta, may play a critical role in the cognitive dysfunction associated with AD, particularly early in the course of the disease. To test and modify this hypothesis, it is important to understand the roles that full-length APP and its fragments normally play in neuronal structure and function. Here we review evidence addressing these fundamental questions, paying particular attention to the contributions that APP fragments play in synaptic transmission and neural plasticity, as these may be key to understanding their effects on learning and memory. It is clear from this literature that APP fragments, including Abeta, can exert a powerful regulation of key neural functions including cell excitability, synaptic transmission and long-term potentiation, both acutely and over the long-term. Furthermore, there is a small but growing literature confirming that these fragments correspondingly regulate behavioral learning and memory. These data indicate that a full account of cognitive dysfunction in AD will need to incorporate the actions of the full complement of APP fragments. To this end, there is an urgent need for a dedicated research effort aimed at understanding the behavioral consequences of altered levels and activity of the different APP fragments as a result of experience and disease.

Alzheimer Disease↗
PubMed1999

Protein kinase C and amyloid precursor protein processing in skin fibroblasts from sporadic and familial Alzheimer's disease cases.

Non-amyloidogenic alpha-secretase processing of amyloid precursor protein (APP) is stimulated by protein kinase C (PKC). Levels and activity of PKC are decreased in sporadic Alzheimer's disease skin fibroblasts. We investigated whether alterations in PKC and PKC-mediated APP processing occur also in fibroblasts established from individuals with familial Alzheimer's disease APP KM670/671NL, PS1 M146V and H163Y mutations. These pathogenic mutations are known to alter APP metabolism to increase Abeta. PKC activities, but not levels, were decreased by 50% in soluble fractions from sporadic Alzheimer's disease cases. In contrast, familial Alzheimer's disease fibroblasts showed no significant changes in PKC enzyme activity. Fibroblasts bearing the APP KM670/671NL mutation showed no significant differences in either PKC levels or PKC-mediated soluble APP (APPs) secretion, compared to controls. Fibroblasts bearing PS1 M146V and H163Y mutations showed a 30% increase in soluble PKC levels and a 40% decrease in PKC-mediated APPs secretion. These results indicate that PKC deficits are unlikely to contribute to increased Abeta seen with APP and PS1 mutations, and also that PS1 mutations decrease alpha-secretase derived APPs production independently of altered PKC activity.

Adult↗
PubMed2000

Relationship between permeability status of the blood-brain barrier and in vitro permeability coefficient of a drug.

OBJECTIVE: The aim was to test the hypothesis that the assessment of basal and drug-induced changes in permeability of the blood-brain barrier (BBB) during in vitro drug transport assays is essential for an accurate estimation of the permeability coefficient of a drug. METHODS: An in vitro BBB model was used, comprising of brain capillary endothelial cells (BCEC) and astrocytes co-cultured on semi-permeable filter inserts. Experiments were performed under control and challenged experimental circumstances, induced to simulate drug effects. The apparent BBB permeability coefficient for two markers for paracellular drug transport, sodium fluorescein (P(app,FLU), M(w) 376 Da) and FITC-labeled dextran (P(app,FD4), M(w) 4 kDa), was determined. Transendothelial electrical resistance (TEER) was used to quantify basal and (simulated) drug-induced changes in permeability of the in vitro BBB. The relationship between P(app) and TEER was determined. Drug effects were simulated by exposure to physiologically active endogenous and exogenous substances (i.e., histamine, deferroxamine mesylate, adrenaline, noradrenaline, bradykinin, vinblastine, sodium nitroprusside and lipopolysaccharide). RESULTS: P(app,FLU) and P(app,FD4) in control experiments varied from 1.6 up to 17.6 (10(-6)cm/s) and 0.3 up to 7. 3 (10(-6)cm/s), respectively; while for individual filters P(app, FLU) was 4 times higher than P(app,FD4) (R(2)=0.97). As long as TEER remained above 131.Omega cm(2) for FLU or 122.Omega cm(2) for FD4 during the transport assay, P(app) remained independent from the basal permeability of the in vitro BBB. Below these TEER values, P(app) increased exponentially. This nonlinear relationship between basal BBB permeability and P(app) was described by a one-phase exponential decay model. From this model the BBB permeability status independent permeability coefficients for FLU and FD4 (P(FLU) and P(FD4)) were estimated to be 2.2+/-0.1 and 0.48+/-0.03 (10(-6)cm/s), respectively. In the experimentally challenged experiments, a reliable indication for P(FLU) and P(FD4) could be estimated only after the (simulated) drug-induced change in BBB permeability was taken into account. CONCLUSIONS: The assessment of basal BBB permeability status during drug transport assays was essential for an accurate estimation of the in vitro permeability coefficient of a drug. To accurately extrapolate the in vitro permeability coefficient of a drug to the in vivo situation, it is essential that drug-induced changes in the in vitro BBB permeability during the drug transport assay are determined.

Animals↗
PubMed2000

Amyloid precursor protein in unique cholesterol-rich microdomains different from caveolae-like domains.

To determine the localization of the amyloid precursor protein (APP) on the cellular membrane, we performed membrane fractionation of cultured cells including that of Madin-Darby canine kidney (MDCK) and P19 cells transfected with human APP cDNA, non-transfected SH-SY5Y cells, and rat cerebral cortices. In MDCK cells, APP was exclusively present in abundance in the supernatant following solubilization of the plasma membranes using Triton X-100, and in high-density fractions of sucrose density gradient fractionation (SDGF) following Triton X-100 solubilization of whole cellular membranes. Caveolin-1 was not cofractionated with APP. In experiments using P19 cells and rat cerebral cortices, we detected two isoforms of APP. The APP with the apparently lower molecular weight (immature type) coexisted in abundance with integrin in the high-density fractions, whereas the APP with the apparently higher molecular weight (mature type) was recovered predominantly in the low-density fractions with cholesterol and GM1 gangliosides, the concentrations of which were higher than those in the bulk plasma membranes, but lower than those in caveolae-like domains (CLDs), following SDGF of Triton X-100-solubilized cellular membranes. The results of this study suggest the following; first, APP is not present in abundance in caveolae or CLDs, but is in unique cholesterol-rich microdomains; second, the targeting of APP to these unique microdomains may be linked to the maturation of APP in some cells.

Amyloid beta-Protein Precursor↗
PubMed1998

Laser light scattering evidence for a common wormlike growth structure of mixed micelles in bile salt- and straight-chain detergent-phosphatidylcholine aqueous systems: relevance to the micellar structure of bile.

We employed quasielastic and static light scattering to measure apparent values of the mean hydrodynamic radii (Rh)app, molecular weights (Mapp), and radii of gyration (Rg)app in solutions containing mixed micelles composed of bile salts (cholate and taurochenodeoxycholate, both cholanoyl derivatives) and the glycoacyl chain detergent, octyl glucoside, with egg yolk phosphatidylcholine (EYPC) as functions of total lipid concentration (0.1-10 g/dL), EYPC/detergent molar ratio (0-1.2), and ionic strength (0.15-0.4 M NaCl) at 20 degreesC and 1 atm. As the mixed micellar phase boundaries were approached by dilution, (Rh)app, Mapp, and (Rg)app values increased markedly by up to 20-fold. For each micellar system, the scaling ratios (Rh)app/Mapp1/2 and (Rg)app/(Rh)app remained essentially constant at 0.018 nm/(g/mol)1/2 and 1.5 (dimensionless), respectively, despite large variations in total lipid concentration, detergent molecular species, and ionic strength. Refined data analysis is inconsistent with a flat "mixed-disc" model for bile salt-EYPC micelles [Mazer, N. A., Benedek, G. B., and Carey, M. C. (1980) Biochemistry 19, 601] and octyl glucoside-EYPC micelles principally because the numerical value of (Rh)app/Mapp1/2 corresponds to a hypothetical disk thickness of approximately 1 nm, which is 4-fold smaller than the bimolecular width of EYPC molecules, and for a disk, (Rg)app/(Rh)app ratios should be close to 1 at low total lipid concentrations. Assuming disc-shaped micelles, we show that intermicellar excluded volume interactions would have only a minor effect on Mapp and cannot account for the unrealistic disk thickness. Instead, locally cylindrical, semiflexible wormlike micelles of diameter d = 4 nm and persistence length xip = 17 nm in solution are compatible with the observed (Rh)app/Mapp1/2 and (Rg)app/(Rh)app values when intermicellar excluded-volume interactions are considered. With EYPC/taurochenodeoxycholate = 0.6 and EYPC/cholate = 1.0 in 0.15 M NaCl, independent micelles grow upon dilution and use of the second virial coefficient [Egelhaaf, S. U., and Schurtenberger, P. (1994) J. Phys. Chem. 98, 8560] is adequate for estimating micellar weights. The systems EYPC/cholate = 1.0 in 0.4 M NaCl, EYPC/cholate = 1.2 in 0.15 M NaCl, and EYPC/octyl glucoside = 0.13 in 0.15 M NaCl all form highly overlapping, semidilute polymer solutions, which mimic the observed scaling ratios. In such semidilute systems, use of the second virial coefficient alone to account for intermicellar interactions is inadequate for estimating micellar weights. The results of the present study, in combination with locations of known phase boundaries of the ternary bile salt-EYPC-water phase diagram at high dilution, suggest that elongation, as well as entanglement of wormlike mixed micelles may occur at concentrations approaching the micellar phase limit.

Bile↗
PubMed2001

The C-terminal fragment of the Alzheimer's disease amyloid protein precursor is degraded by a proteasome-dependent mechanism distinct from gamma-secretase.

The beta-amyloid protein (Abeta) is derived by proteolytic processing of the amyloid protein precursor (APP). Cleavage of APP by beta-secretase generates a C-terminal fragment (APP-CTFbeta), which is subsequently cleaved by gamma-secretase to produce Abeta. The aim of this study was to examine the cleavage of APP-CTFbeta by gamma-secretase in primary cortical neurons from transgenic mice engineered to express the human APP-CTFbeta sequence. Neurons were prepared from transgenic mouse cortex and proteins labelled by incubation with [35S]methionine and [35S]cysteine. Labelled APP-CTFbeta and Abeta were then immunoprecipitated with a monoclonal antibody (WO2) specific for the transgene sequences. Approximately 30% of the human APP-CTFbeta (hAPP-CTFbeta) was converted to human Abeta (hAbeta), which was rapidly secreted. The remaining 70% of the hAPP-CTFbeta was degraded by an alternative pathway. The cleavage of hAPP-CTFbeta to produce hAbeta was inhibited by specific gamma-secretase inhibitors. However, treatment with proteasome inhibitors caused an increase in both hAPP-CTFbeta and hAbeta levels, suggesting that the alternative pathway was proteasome-dependent. A preparation of recombinant 20S proteasome was found to cleave a recombinant cytoplasmic domain fragment of APP (APPcyt) directly. The study suggests that in primary cortical neurons, APP-CTFbeta is degraded by two distinct pathways, one involving gamma-secretase, which produces Abeta, and a second major pathway involving direct cleavage of APP-CTFbeta within the cytoplasmic domain by the proteasome. These results raise the possibility that defective proteasome function could lead to an increase in Abeta production in the AD brain.

Alzheimer Disease↗
PubMed1998

Profiles of amyloid precursor and presenilin 2-like proteins are correlated during development of the mouse hypothalamus.

The amyloid precursor protein (APP) and APP-like (APLP) material, as visualized with the Mab22C11 antibody, have previously been shown to be associated with radial glia in hypothalamus, which are known to promote neurite outgrowth. By Northern blot analysis, APP 695 mRNA levels increased steadily over hypothalamic development, APP 770 mRNA was transiently expressed at 12 days postnatally, and APLP mRNA was only weakly expressed in the hypothalamus. The developmental pattern of APP moeities in mouse hypothalamus and in fetal hypothalamic neurons in culture was compared with a presenilin 2 (PS2) related protein using an antibody developed against the N-terminal part of PS2. By Western blot analysis, APP and PS2-like immunoreactivity were visualized as a 100-130 and 52 kDa bands, respectively. An APP biphasic increase was observed during hypothalamic development in vivo. APP immunoreactivity was equally detected in neuronal and glial cultures, while PS2-like material was more concentrated in neurons. A correlation between APP/APP-like and PS2-like levels was observed during development in vivo. While APP was mostly associated with membrane fractions, a significant portion of PS2-like material was also recovered from cytosolic fractions in vitro. In contrast to native PS2 in COS-transfected cells, the PS2-like material did not aggregate after heating for 90 s at 90 degrees C. These results indicate a close association between APP and PS2-like material during hypothalamic development in vivo, and suggest that neuronal and glial cultures may provide appropriate models to test their interactions.

Amyloid beta-Protein Precursor↗
PubMed1998

The deletion of the C-terminal tail and addition of an endoplasmic reticulum targeting signal to Alzheimer's amyloid precursor protein change its localization, secretion, and intracellular proteolysis.

The metabolic pathway of Alzheimer's amyloid precursor protein (APP) involves restricted intracellular proteolysis by secretases, which leads to the secretion of the N-terminal soluble APP (sAPP) and the generation of a cell-associated C-terminal fragment. The precise cellular sites at which these processes occur remain unknown. In this report, we describe the route of APP sorting and the processing site using novel systems with and without sorting signals on the APP molecule. One system involves the replacement of the C-terminal ten amino acids of APP with Adenoviral E19 protein containing an endoplasmic reticulum (ER) retrieval signal (APPE19); the other involves deleting the last ten amino acids corresponding to the replaced site (APPdeltaC10). APPE19 localized mainly within the cis/medial Golgi compartment and exclusively suppresses the secretion of APP. In contrast, deletion of the C-terminal tail promotes sAPP secretion by a constitutive secretion pathway. Metabolic labeling followed by immunoprecipitation with anti-APP antibody revealed that APPE19 is rapidly degraded within 30 min and that the subsequent intracellular turnover rate is decreased with 40% of the protein retained within the cells even after a chase period a 3 h. In contrast, APPdeltaC10 is rapidly eliminated from the intracellular compartments and secreted into the culture medium. The surface internalization and recycling processes of this protein are relatively impaired compared with wild-type APP. The ratios of the levels of production to secretion of sAPP alpha, the N-terminal, soluble APP fragment released by alpha-secretase, are proportional to the secretion efficiencies among APP species, suggesting the localization of alpha-secretase within a compartment late in the constitutive secretion pathway. These secretion mutants which utilize ER targeting signals are useful tools for analyzing the location of secretases and the intracellular degradation system within a constitutive secretion pathway such as ER quality control.

Adenovirus Early Proteins↗
PubMed1999

Cysteine 144 is a key residue in the copper reduction by the beta-amyloid precursor protein.

The beta-amyloid precursor protein (beta-APP) contains a copper-binding site localized between amino acids 135 and 156 (beta-APP(135-156)). We have employed synthetic beta-APP peptides to characterize their capacities to reduce Cu(II) to Cu(I). Analogues of the wild-type beta-APP(135-156) peptide, containing specific amino acid substitutions, were used to establish which residues are specifically involved in the reduction of copper by beta-APP(135-156). We report here that beta-APP's copper-binding domain reduced Cu(II) to Cu(I). The single-mutant beta-APP(His147-->Ala) and the double-mutant beta-APP(His147-->Ala/His149-->Ala) showed a small decrease in copper reduction in relation to the wild-type peptide and the beta-APP(Cys144-->Ser) mutation abolished it, suggesting that Cys144 is the key amino acid in the oxidoreduction reaction. Our results confirm that soluble beta-APP is involved in the reduction of Cu(II) to Cu(I).

Amino Acid Substitution↗
PubMed2001

Brain-derived neurotrophic factor stimulates beta-amyloid gene promoter activity by a Ras-dependent/AP-1-independent mechanism in SH-SY5Y neuroblastoma cells.

The beta-amyloid peptide, the major component of Alzheimer-associated plaques, derives from a larger beta-amyloid precursor protein (APP), that is expressed in both neural and non-neural cells. Overexpression of APP actively contributes to the development of senile plaques and is considered a risk factor for the disease. APP expression is regulated by a variety of cellular mediators, among them ligands of tyrosine kinase receptors. In this study, we present evidence that brain-derived neurotrophic factor (BDNF) modulates, in a dose- and time-dependent fashion, APP promoter activity in SH-SY5Y neuroblastoma cells transiently expressing the receptor TrkB. The APP promoter contains two potential AP-1 sites, and we examined whether or not protein kinase C (PKC) and the AP-1 sites of the promoter mediate the BDNF-induced stimulation of APP. Stimulation of APP promoter activity by BDNF was not affected by the PKC inhibitor bisindolylmaleimide, or by dominant negative mutants of the AP-1 components Fos and Jun, which, however, blocked the response to phorbol esters. These results suggest that activation of the APP promoter by BDNF is largely independent of PKC and AP-1. In contrast, activated Ras increased APP promoter activity in SH-SY5Y cells, and a dominant negative mutant of Ras abolished BDNF-mediated promoter stimulation. Taken together, our results suggest a mechanism that involves activation of the Ras/MAP kinase signaling pathway, and phosphorylation of as yet unidentified effectors which in turn can activate response elements within the APP promoter.

Amyloid beta-Protein Precursor↗
PubMed2006

A peptide zipcode sufficient for anterograde transport within amyloid precursor protein.

Fast anterograde transport of membrane-bound organelles delivers molecules synthesized in the neuronal cell body outward to distant synapses. Identification of the molecular "zipcodes" on organelles that mediate attachment and activation of microtubule-based motors for this directed transport is a major area of inquiry. Here we identify a short peptide sequence (15 aa) from the cytoplasmic C terminus of amyloid precursor protein (APP-C) sufficient to mediate the anterograde transport of peptide-conjugated beads in the squid giant axon. APP-C beads travel at fast axonal transport rates (0.53 mum/s average velocity, 0.9 mum/s maximal velocity) whereas beads coupled to other peptides coinjected into the same axon remain stationary at the injection site. This transport appears physiologic, because it mimics behavior of endogenous squid organelles and of beads conjugated to C99, a polypeptide containing the full-length cytoplasmic domain of amyloid precursor protein (APP). Beads conjugated to APP lacking the APP-C domain are not transported. Coinjection of APP-C peptide reduces C99 bead motility by 75% and abolishes APP-C bead motility, suggesting that the soluble peptide competes with protein-conjugated beads for axoplasmic motor(s). The APP-C domain is conserved (13/15 aa) from squid to human, and peptides from either squid or human APP behave similarly. Thus, we have identified a conserved peptide zipcode sufficient to direct anterograde transport of exogenous cargo and suggest that one of APP's roles may be to recruit and activate axonal machinery for endogenous cargo transport.

Amino Acid Sequence↗
PubMed1989

Interleukin 1 regulates synthesis of amyloid beta-protein precursor mRNA in human endothelial cells.

We have analyzed the modulation of amyloid beta-protein precursor (APP) gene expression in human umbilical vein endothelial cells (HUVEC). The level of the APP mRNA transcripts increased as HUVEC reached confluency. In confluent culture the half-life of the APP mRNA was 4 hr. Treatment of the cells with human-recombinant interleukin 1 (IL-1), phorbol 12-myristate 13-acetate, or heparin-binding growth factor 1 enhanced the expression of APP gene in these cells, but calcium ionophore A23187 and dexamethasone did not. The protein kinase C inhibitor 1-(isoquinolinsulfonyl)-2-methylpiperazine (H7) inhibited IL-1-mediated increase of the level of APP transcripts. To map IL-1-responsive elements of the APP promoter, truncated portions of the APP promoter were fused to the human growth hormone reporter gene. The recombinant plasmids were transfected into mouse neuroblastoma cells, and the cell medium was assayed for the human growth hormone. A 180-base-pair region of the APP promoter located between position -485 and -305 upstream from the transcription start site was necessary for IL-1-mediated induction of the reporter gene. This region contains the upstream transcription factor AP-1 binding site. These results suggest that IL-1 upregulates APP gene expression in HUVEC through a pathway mediated by protein kinase C, utilizing the upstream AP-1 binding site of the APP promoter.

Amyloid↗
PubMed1992

Chloroquine inhibits intracellular degradation but not secretion of Alzheimer beta/A4 amyloid precursor protein.

The metabolic fate of the Alzheimer beta/A4 amyloid precursor protein (APP) includes intraamyloid proteolysis that leads to the production of secreted N-terminal and cell-associated C-terminal fragments. The cellular sites at which this processing occurs are not known. We have examined the route of APP processing in metabolically labeled PC12 cells. The lysosomotropic drug chloroquine exerted inhibitory effects on the degradation of mature APP holoprotein. In addition, recovery of a C-terminal fragment resulting from normal intraamyloid cleavage was significantly increased in the presence of chloroquine, suggesting that further degradation of the C-terminal fragment was inhibited. Chloroquine had virtually no effect on APP maturation (N- and O-glycosylation and tyrosine sulfation) or secretion. Treatment with either monensin (which inhibits distal Golgi function) or brefeldin A (which causes resorption of the Golgi into the endoplasmic reticulum and fusion of the trans-Golgi network with the endosomal system) prevented normal APP maturation and abolished APP secretion and recovery of C-terminal fragments, indicating that intact Golgi function is necessary for APP maturation and processing. Our results suggest that a substantial proportion of APP is degraded in an intracellular acidic compartment but that the coupled APP cleavage/secretion event occurs in a chloroquine-insensitive compartment. The observations are consistent with the existence of multiple cellular routes for the trafficking and proteolysis of APP.

Ammonium Chloride↗
PubMed1998

Transgenic Drosophila expressing human amyloid precursor protein show gamma-secretase activity and a blistered-wing phenotype.

The importance of the amyloid precursor protein (APP) in the pathogenesis of Alzheimer's disease (AD) became apparent through the identification of distinct mutations in the APP gene, causing early onset familial AD with the accumulation of a 4-kDa peptide fragment (betaA4) in amyloid plaques and vascular deposits. However, the physiological role of APP is still unclear. In this work, Drosophila melanogaster is used as a model system to analyze the function of APP by expressing wild-type and various mutant forms of human APP in fly tissue culture cells as well as in transgenic fly lines. After expression of full-length APP forms, secretion of APP but not of betaA4 was observed in both systems. By using SPA4CT, a short APP form in which the signal peptide was fused directly to the betaA4 region, transmembrane domain, and cytoplasmic tail, we observed betaA4 release in flies and fly-tissue culture cells. Consequently, we showed a gamma-secretase activity in flies. Interestingly, transgenic flies expressing full-length forms of APP have a blistered-wing phenotype. As the wing is composed of interacting dorsal and ventral epithelial cell layers, this phenotype suggests that human APP expression interferes with cell adhesion/signaling pathways in Drosophila, independently of betaA4 generation.

Amyloid Precursor Protein Secretases↗
PubMed1995

The chondroitin sulfate attachment site of appican is formed by splicing out exon 15 of the amyloid precursor gene.

Appicans are secreted and cell-associated chondroitin sulfate proteoglycans containing Alzheimer amyloid precursor (APP) as their core protein. Appicans are found in brain tissue, and in cell cultures their expression depends on both cell type and growth conditions. Here we report that the core protein of appicans derives from an APP mRNA lacking exon 15. Splicing out of this exon creates a new consensus sequence for the attachment of a chondroitin sulfate chain in the resulting APP product. Transfection of C6 glioma or 293 kidney fibroblast cells with APP cDNAs containing exon 15 produced no appican, while transfection with an APP cDNA lacking this exon induced high levels of appican production. Polymerase chain reactions indicated that appican-producing cells contained an APP mRNA species without exon 15, whereas cells without this mRNA produced no appican. Site-directed mutagenesis combined with immunoreactivity experiments showed that the chondroitin sulfate chain is attached to a serine residue 16 amino acids upstream of the amino terminus of the A beta sequence of APP. The attachment of a glycosaminoglycan chain close to the A beta sequence of APP may affect the proteolytic processing of APP and production of A beta. The proteoglycan nature of APP suggests that addition of the chondroitin sulfate glycosaminoglycan is important for the implementation of the biological function of these proteins.

Alternative Splicing↗
PubMed1995

Cell-type and amyloid precursor protein-type specific inhibition of A beta release by bafilomycin A1, a selective inhibitor of vacuolar ATPases.

Treatment of human 293 cells transfected with amyloid precursor protein (APP)K595N,M596L (the "Swedish" mutation) with a specific inhibitor of the vacuolar H(+)-ATPases, bafilomycin A1 (baf A), leads to a potent inhibition of the release of the A beta peptide. This is accompanied by a selective inhibition of beta-secretase activity. Surprisingly, baf A did not inhibit the production of A beta from either wild-type APP (WT APP) or from APPv7171 (the "Hardy" mutation), expressed in the same cell type. In contrast, the robust production of A beta from a human neuroglioma-derived cell line (HS683) transfected with WT APP, or from primary human mixed brain cultures (HMBC) expressing genomic WT APP, were also effectively inhibited by baf A. The inhibition of A beta production from the HMBC was also accompanied by the inhibition of beta-s-APP release. No inhibition of alpha-s-APP release was seen in any of the cell types tested. These results indicate that intracellular acidic processes are rate-limiting for beta-secretase cleavage and A beta production from SW APP, but not WT APP, in the peripheral 293 cell line. Furthermore, such acidic processes also play a rate-limiting role in A beta release from human central nervous system-derived cells, including HMBC. Differential trafficking of the SW APP into an acidic compartment conducive to beta-secretase cleavage and A beta release could be one explanation for the increased production of A beta observed on expression of this mutation.

Adenosine Triphosphatases↗