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PubMed1997

Identification and structure of activated-platelet protein-1, a protein with RNA-binding domain motifs that is expressed by activated platelets.

Beyond their critical role in thrombosis, platelets perform important functions in vascular remodeling, inflammation, and wound repair. Many of these functions are executed by molecules expressed by activated platelets. A novel molecule, activated-platelet protein-1 (APP-1), was identified by a monoclonal antibody against activated rabbit platelets. When platelets were stimulated by thrombin, A23187 or ADP, APP-I was expressed on the platelet surface. APP-1 was also detected in whole cell lysates of platelets, but not on the external surfaces of resting platelets. With maximal activation by thrombin, 15 900 +/- 2800 molecules APP-1 were expressed/platelet. A 2.3-kb cDNA fragment containing a partial coding sequence for APP-1 was isolated from a rabbit bone marrow library by expression cloning with the anti-APP-1 monoclonal antibody. When expressed as a recombinant fusion protein in bacteria, APP-1 bound specifically to poly(A)-Sepharose. The full-length cDNA coding for human APP-1, obtained by DNA hybridization techniques, showed 98.7% amino acid sequence identity with the rabbit protein. Northern analysis with human APP-1 identified a 3.7-kb mRNA transcript in megakaryocytic lines that express transcripts for platelet proteins. Human APP-1 has four ribonucleotide binding domains with ribonucleoprotein 1 and 2 motifs. By virtue of its ribonucleotide binding domains, APP-1 is structurally related to polyadenylate-binding protein, which regulates translation initiation and polyadenylate shortening, and to nucleolysin, a specific effector molecule found in the granules of cytotoxic T lymphocytes.

Amino Acid Sequence↗
PubMed2004

The beta-amyloid precursor protein controls a store-operated Ca2+ entry in cortical neurons.

A polyclonal antibody (APP-Ab) raised against the extracellular domain of the beta-amyloid precursor protein (APP) triggers a marked neuronal cell death preceded by activation of Ca(2+)-dependent enzymes, neurite degeneration, oxidative stress and nuclear condensation [Mbebi et al. (2002) J. Biol. Chem., 277, 20979-20990]. We have investigated whether activation of APP by this antibody could promote cell death through cellular Ca2+ homeostasis alteration. We carried out time-lapse recordings of intracellular Ca2+ signals in cultured mice cortical neurons by means of a scanning confocal microscope. When applied in the presence of 2 mm external Ca2+, APP-Ab elicited a long-lasting elevation of the intracellular concentration of Ca2+ ([Ca2+]i). Experiments performed in the absence of external Ca2+ showed that APP-Ab triggers the release of Ca2+ from intracellular stores. The re-admission of external Ca2+ provides an additional rise of Ca2+ most likely through store-operated channels. A pretreatment of the cells with pertussis toxin, to inhibit the activity of Gi/Go proteins, or with the phospholipase C inhibitor, 3-nitrocoumarin, prevented both the APP-dependent elevation of Ca2+ as well as the APP-Ab-mediated cell death. Similarly, the store-operated channel inhibitors, 2-APB or SKF-96365 block both the APP-Ab-dependent Ca2+ entry and the APP-Ab-mediated cell death. Altogether, our data provide functional evidence that APP can perturb intracellular Ca2+ homeostasis by emptying intracellular Ca2+ stores and triggering Ca2+ entry through store-operated channels. In response to APP activation, the long-lasting elevation of [Ca2+]i due to an entry of Ca2+ via store-operated channels appears as a major event that leads to neuronal cell death.

Amyloid beta-Protein Precursor↗
PubMed2004

Atorvastatin-induced activation of Alzheimer's alpha secretase is resistant to standard inhibitors of protein phosphorylation-regulated ectodomain shedding.

Studies of metabolism of the Alzheimer amyloid precursor protein (APP) have focused much recent attention on the biology of juxta- and intra-membranous proteases. Release or 'shedding' of the large APP ectodomain can occur via one of two competing pathways, the alpha- and beta-secretase pathways, that are distinguished both by subcellular site of proteolysis and by site of cleavage within APP. The alpha-secretase pathway cleaves within the amyloidogenic Abeta domain of APP, precluding the formation of toxic amyloid aggregates. The relative utilization of the alpha- and beta-secretase pathways is controlled by the activation of certain protein phosphorylation signal transduction pathways including protein kinase C (PKC) and extracellular signal regulated protein kinase [ERK/mitogen-activated protein kinase (MAP kinase)], although the relevant substrates for phosphorylation remain obscure. Because of their apparent ability to decrease the risk for Alzheimer disease, the effects of statins (HMG CoA reductase inhibitors) on APP metabolism were studied. Statin treatment induced an APP processing phenocopy of PKC or ERK activation, raising the possibility that statin effects on APP processing might involve protein phosphorylation. In cultured neuroblastoma cells transfected with human Swedish mutant APP, atorvastatin stimulated the release of alpha-secretase-released, soluble APP (sAPPalpha). However, statin-induced stimulation of sAPPalpha release was not antagonized by inhibitors of either PKC or ERK, or by the co-expression of a dominant negative isoform of ERK (dnERK), indicating that PKC and ERK do not play key roles in mediating the effect of atorvastatin on sAPPalpha secretion. These results suggest that statins may regulate alpha-secretase activity either by altering the biophysical properties of plasma membranes or by modulating the function of as-yet unidentified protein kinases that respond to either cholesterol or to some intermediate in the cholesterol metabolic pathway. A 'phospho-proteomic' analysis of N2a cells with and without statin treatment was performed, revealing changes in the phosphorylation state of several protein kinases plausibly related to APP processing. A systematic evaluation of the possible role of these protein kinases in statin-regulated APP ectodomain shedding is underway.

Amyloid Precursor Protein Secretases↗
PubMed2005

Amyloid precursor protein expression in the rat hippocampal dentate gyrus modulates during memory consolidation.

Despite advances in our understanding of the basic biology of amyloid precursor protein (APP), the normal physiological function(s) of APP in learning and memory remains unclear. Here we show increased APP degradation in the hippocampus to be associated with the consolidation of a passive avoidance response. Neurone-specific APP695 expression became transiently reduced 2-4 h post-training through association with endosomal adaptin proteins and enhanced internalization. By contrast, internalization of glial-associated APP containing a Kunitz protease inhibitor-like domain (APP-KPI) was dependent on the low-density lipoprotein receptor-related protein (LRP). In addition, LRP expression and association with apolipoprotein E increased in the 2-4 h post-training period. The LRP antagonist receptor-associated protein prevented the APP-KPI internalization and LRP-apolipoprotein E association and this resulted in amnesia. Degradation of APP695 and APP-KPI did not appear to be related to alpha-secretase activity, as no learning-associated increase of secreted APP was observed in the CSF. Moreover, as internalization of APP isoforms was observed only in dentate gyrus, it probably relates to the learning-associated restructuring of the perforant path terminals. Memory-associated APP processing in both neuronal and glial compartments points to a role for glial unsheathing of synaptic connections, an event required for the synaptic restructuring that accompanies memory consolidation. These observations may have a direct relevance to understanding the pathophysiology of Alzheimer's disease as beta/gamma-secretase-derived beta-amyloid is formed following internalization of cell surface APP into the endosomal compartment.

Adaptor Protein Complex Subunits↗
PubMed1993

Receptor-coupled amyloid precursor protein processing.

The family of beta-amyloid protein precursors (APP) can be processed via several alternative proteolytic pathways. Some generate potentially amyloidogenic APP derivatives, whereas others preclude the formation of such fragments. The cellular mechanisms regulating the relative activities of these pathways are thus important in determining the factors contributing to the formation of amyloidogenic APP derivatives. In order to investigate whether cell-surface receptor activity can regulate APP processing, HEK 293 cell lines stably expressing human muscarinic acetylcholine receptors (mAChR; subtypes m1, m2, m3, m4) were stimulated with the muscarinic agonist carbachol, and the release of APP derivatives was measured. Carbachol increased the release of large amino-terminal APP-fragments 4- to 6-fold in cell lines expressing the m1 or m3 receptors but not in those expressing m2 or m4 subtypes. This increase was blocked by various protein kinase inhibitors and mimicked by phorbol esters, indicating that it is mediated by protein kinase activation, presumably by protein kinase C (PKC). To determine whether additional cell-surface receptor types linked to this signal transduction pathway could also regulate APP processing, we stimulated differentiated PC-12 cells with bradykinin and found that this neuropeptide also increased the secretion of amino-terminal APP derivatives. We next investigated the possibility that neuronal depolarization might affect APP processing in mammalian brain. Electrically stimulated rat hippocampal slices released two times more amino-terminal APP derivatives than unstimulated control slices. This release increased with increasing stimulation frequencies in the physiological firing range of hippocampal pyramidal cells, and was blocked by tetrodotoxin. These results suggest that, in brain, APP processing is regulated by neuronal activity.

Amyloid beta-Protein Precursor↗
PubMed1993

The role of extracellular matrix in the processing of the amyloid protein precursor of Alzheimer's disease.

Alzheimer's disease (AD) is characterized by the presence of extracellular amyloid plaques, which contain a protein referred to as the amyloid or beta A4 protein. The beta A4 protein is derived from a larger precursor protein (APP). Studies of autosomal-dominant forms of AD have established the central role of APP in the pathogenesis of the disease. Despite considerable research, the function of APP is unknown. APP can be processed by at least two separate routes. The first route involves a protease known as "APP secretase," which cleaves within the amyloid sequence, thereby mitigating amyloid formation. The second route may result in the production of potentially amyloidogenic fragments. Our studies suggest that following release from the cell membrane, APP interacts with components of the extracellular matrix (ECM) such as the heparan sulfate proteoglycans (HSPG's). The interaction of APP with HSPG's may be important for the function of APP. Substratum-bound APP was found to dramatically increase neurite outgrowth and survival of chick sympathetic neurons in vitro. This effect was dependent upon the presence of substratum-bound HSPG. The results suggest that normally, when bound to the ECM, APP functions to promote neurite outgrowth and/or cell survival. Loss of this normal trophic function might occur in AD, when APP is proteolytically processed via the amyloidogenic pathway.

Alzheimer Disease↗
PubMed2000

Regulation of cytokine secretion and amyloid precursor protein processing by proinflammatory amyloid beta (A beta).

Neurodegenerative processes in Alzheimer's disease (AD) are thought to be driven, in part, by the deposition of amyloid beta (A beta), a 39-43-aminoacid peptide product resulting from an alternative cleavage of amyloid precursor protein (APP). In addition to its neurotoxic properties, A beta may influence neuropathology by stimulating glial cell cytokine and acute phase protein secretion in affected areas of the brain (e.g., cortex, hippocampus). Using an in vitro human astrocyte model (U-373 MG astrocytoma cells), the effects of A beta treatment on acute phase protein (APP and alpha-1-antichymotrypsin [alpha 1-ACT]) and interleukin-8 (IL-8) were examined. U-373 MG cells secreted increased levels of alpha 1-ACT and neurotrophic/neuroprotective alpha-cleaved APP (alpha APP) after exposure to interleukin-1 beta (IL-1 beta) for 24 hours. A beta treatment resulted in a similar, but modest increase in alpha 1-ACT secretion, a two- to threefold stimulation of IL-8 production, and, conversely, a profound reduction in the levels of secreted alpha APPs. A beta inhibited alpha APP secretion by U-373 MG cells in a concentration- and conformation-dependent manner. Moreover, the reduction in alpha APP secretion was accompanied by an increase in cell-associated APP. Another proinflammatory amyloidogenic peptide, human amylin, similarly affected APP processing in U-373 astrocytoma cells. These data suggest that A beta may contribute to Alzheimer's-associated neuropathology by lowering the production of neuroprotective/neurotrophic alpha APPs. Moreover, the concomitant increase in cell-associated APP may provide increased substrate for the generation of amyloidogenic peptides within astrocytes.

Alzheimer Disease↗
PubMed1982

Comparative effects of 4-aminopyrazolopyrimidine, its 2'-deoxyriboside derivative, and allopurinol on in vitro growth of American Leishmania species.

When assayed on promastigotes of 10 American Leishmania isolates (including Leishmania brasiliensis and mexicana species), 4-aminopyrazolopyrimidine (AAP) was severalfold more active than allopurinol as a leishmanistatic drug. There were some intraspecific and interspecific differences among the isolates in their susceptibility to the inhibitory effects of APP and allopurinol. APP-2'-deoxyriboside did not affect the 10 isolates tested. This was surprising, because allopurinol riboside has previously been shown to be more active than allopurinol. In all of the American Leishmania isolates tested, the metabolism of [14C]6-APP resulted in a high level of HPP-ribose-5'-P and lower levels of APP-ribose-5'-P, APP-ribose-5'-PP, and APP-ribose-5'-PPP. MP, LR, LBY, and JAP isolates strongly converted APP into APP derivatives, thus perhaps explaining their greater susceptibility to the inhibitory effects of APP. With the 10 American Leishmania isolates tested, several purines reversed the inhibitory effects of allopurinol, but only adenine countered the inhibitory effects of APP. This suggests biochemical differences in the mechanisms of action of allopurinol and APP. Finally, and contrary to previous claims, the reversal by purines of the allopurinol-induced growth inhibition was not a Leishmania species-specific effect.

Adenine↗
PubMed2006

Subcellular trafficking of the amyloid precursor protein gene family and its pathogenic role in Alzheimer's disease.

Changes in the intracellular transport of amyloid precursor protein (APP) affect the extent to which APP is exposed to alpha- or beta-secretase in a common subcellular compartment and therefore directly influence the degree to which APP undergoes the amyloidogenic pathway leading to generation of beta-amyloid. As the presynaptic regions of neurons are thought to be the main source of beta-amyloid in the brain, attention has been focused on axonal APP trafficking. APP is transported along axons by a fast, kinesin-dependent anterograde transport mechanism. Despite the wealth of in vivo and in vitro data that have accumulated regarding the connection of APP to kinesin transport, it is not yet clear if APP is coupled to its specific motor protein via an intracellular interaction partner, such as the c-Jun N-terminal kinase-interacting protein, or by yet another unknown molecular mechanism. The cargo proteins that form a functional complex with APP are also unknown. Due to the long lifespan, and vast extent, of neurons, in particular axons, neurons are highly sensitive to changes in subcellular transport. Recent in vitro and in vivo studies have shown that variations in APP or tau affect mitochondrial and synaptic vesicle transport. Further, it was shown that this axonal dysfunction might lead to impaired synaptic plasticity, which is crucial for neuronal viability and function. Thus, changes in APP and tau expression may cause perturbed axonal transport and changes in APP processing, contributing to cognitive decline and neurodegeneration in Alzheimer's disease.

Alzheimer Disease↗
PubMed2006

Ectodomain shedding of the amyloid precursor protein: cellular control mechanisms and novel modifiers.

Proteolytic cleavage in the ectodomain of the amyloid precursor protein (APP) is a key regulatory step in the generation of the Alzheimer's disease amyloid-beta (Abeta) peptide and occurs through two different protease activities termed alpha- and beta-secretase. Both proteases compete for APP cleavage, but have opposite effects on Abeta generation. At present, little is known about the cellular pathways that control APP alpha- or beta-secretase cleavage and thus Abeta generation. To explore the contributory pathways in more detail we have recently employed an expression cloning screen and identified several activators of APP cleavage by alpha- or beta-secretase. Among them were known activators of APP cleavage, for example protein kinase A, and novel activators, such as endophilin and the APP homolog amyloid precursor-like protein 1 (APLP1). Mechanistic analysis revealed that both endophilin and APLP1 reduce the rate of APP endocytosis and strongly increase APP cleavage by alpha-secretase. This review summarizes the results of the expression cloning screen in the context of recent developments in our understanding of the cellular regulation of APP alpha-secretase cleavage. Moreover, it highlights the particular importance of endocytic APP trafficking as a prime modulator of APP shedding.

Amyloid Precursor Protein Secretases↗
PubMed1992

Solubilization of the receptors for avian pancreatic polypeptide in chicken, canine, and pig brains.

When n-octyl-beta-D-glucoside was used in several detergents to extract active avian pancreatic polypeptide (APP) receptors, a specific binding of [125I]APP to the solubilized chicken cerebellar and porcine hippocampal membranes was found. The binding of [125I]APP to the solubilized receptors was dependent on incubation time, temperature, and protein concentrations and appeared to have a slightly acidic optimal pH. APP binding to chicken and porcine brain extracts showed a high specificity for APP, although the chicken receptors do not discriminate well between APP and its related peptides, neuropeptide Y and peptide YY. Scatchard analyses of competitive binding data indicated the presence of two classes of binding sites in the brain extracts as in membrane-bound receptors; however, the high affinity component of the chicken receptor showed a decreased affinity after extraction. APP receptors in chicken and porcine brain extracts retained their insensitivity to the nonhydrolyzable GTP analog guanosine 5'-O-(3-thiotriphosphate). Cross-linking studies were performed with the homobifunctional cross-linker disuccinimidyl suberate and brain membrane receptors solubilized with n-octyl-beta-D-glucoside. An APP receptor species with a M(r) of 67,000, the same size as that of the labeled protein in native membrane homogenates of chicken and pig brains, was identified. However, in the canine brain we observed a M(r) 85,000 receptor protein, suggesting that species differences exist among the structures of brain APP receptors. The solubilized cross-linked APP receptors in these species were adsorbed by wheat germ agglutinin-agarose and by concanavalin A, indicating that they are glycoprotein in nature. The availability of the solubilized receptors from vertebrate brains with n-octyl-beta-D-glucoside represents an important step toward the purification and molecular characterization of the APP receptors.

Animals↗
PubMed2004

Amyloid precursor protein compartmentalization restricts beta-amyloid production: therapeutic targets based on BACE compartmentalization.

Alzheimer's disease (AD) is defined by deposits of the 42-residue amyloid-beta peptide (Abeta42) in the brain. Abeta42 is a minor metabolite of the amyloid precursor protein (APP), but its relative levels are increased by mutations on APP and presenilins 1 and 2 linked to familial AD. beta-secretase (BACE-1), an aspartyl protease, cleaves approx 10% of the APP in neuronal cells on the N-terminal side of Abeta to produce the C-terminal fragment (CTFbeta), which is cleaved by gamma-secretase to produce mostly Abeta of 40 residues (90%) and approx10% Abeta42. A third enzyme, alpha-secretase, cleaves APP after Abeta16 to secrete sAPPalpha and CTFalpha, the major metabolites of APP. Moreover, previous studies have demonstrated that phorbol esters stimulate processing of APP by alpha-secretase. Because alpha-secretase and BACE-1 cleave APP within the secretory pathway, it is likely that the two enzymes compete for the APP substrate. This type of competition can explain the failure to saturate the minor BACE-1 pathway by overexpressing APP in the cell. In this study, we demonstrate that inhibition of constitutive alpha-secretase processing in a human neuroblastoma cell line does not increase the yield of Abeta, suggesting that the APP substrate targeted for alpha-secretase processing is not diverted to the BACE-1 pathway. However, when phorbol ester-induced alpha-secretase was similarly inhibited, we detected an increase in BACE-1 processing and AB yield. We explain these results compartmentalization of BACE-1 and alpha-secretase with processing depending on sorting of APP to the two compartments. The simplest explanation for the detection of competition between the two pathways upon phorbol ester stimulation is the partial failure of this compartmentalization by phorbol ester-induced release of secretory vesicles.

ADAM Proteins↗
PubMed2006

Vascular effects of ambient pollutant particles and metals.

Exposure to ambient pollutant particle (APP) is associated with increased cardiovascular morbidity and mortality. Recent evidence indicates that APP-induced vasoconstriction may be an important mechanism. APP constricts systemic arteries and increases blood pressure in human. APP decreases the diameter of pulmonary arterioles in animals. Intratracheal instillation of APP increases pulmonary artery resistance in isolated buffer-perfused lungs, and APP constricts isolated arterial rings. APP-induced vasoconstriction may be secondary to the release of inflammatory mediators from lung cells, which then activate vascular endothelial and smooth muscle cells. The vasoconstriction may also be caused by alterations in autonomic nervous system balance. Some soluble metals (e.g., vanadium) can produce acute vasoconstriction in in vitro and in vivo systems, and contribute to the systemic health effects of APP since they can more easily permeate the alveolar-capillary membrane than the whole particle. Both APP and its associated metals have been shown to enhance the release of endothelin 1 and reactive oxygen species, activate epithelial growth factor receptor and mitogen-activated protein kinases, and inhibit nitric oxide vasodilator activity. The vasoactive properties of APP and metals raised the possibility that patients with vascular diseases may be more susceptible to APP-induced adverse health effects, and that people who are regularly exposed to high amount of metals, e.g., vanadium contained in certain dietary and muscle-building regimens or in the air of boiler making plants, may have increased risk for vascular diseases. Understanding how metals induce vasoconstriction may lead to the development of novel vasodilator therapies for vascular diseases.

Air Pollutants↗
PubMed1999

The Alzheimer's disease amyloid precursor protein modulates copper-induced toxicity and oxidative stress in primary neuronal cultures.

The amyloid precursor protein (APP) of Alzheimer's disease can reduce copper (II) to copper (I) in a cell-free system potentially leading to increased oxidative stress in neurons. We used neuronal cultures derived from APP knock-out (APP(-/-)) and wild-type (WT) mice to examine the role of APP in copper neurotoxicity. WT cortical, cerebellar, and hippocampal neurons were significantly more susceptible than their respective APP(-/-) neurons to toxicity induced by physiological concentrations of copper but not by zinc or iron. There was no difference in copper toxicity between APLP2(-/-) and WT neurons, demonstrating specificity for APP-associated copper toxicity. Copper uptake was the same in WT and APP(-/-) neurons, suggesting APP may interact with copper to induce a localized increase in oxidative stress through copper (I) production. This was supported by significantly higher levels of copper-induced lipid peroxidation in WT neurons. Treatment of neuronal cultures with a peptide corresponding to the human APP copper-binding domain (APP142-166) potentiated copper but not iron or zinc toxicity. Incubation of APP142-166 with low-density lipoprotein (LDL) and copper resulted in significantly increased lipid peroxidation compared to copper and LDL alone. Substitution of the copper coordinating histidine residues with asparagines (APP142-166(H147N, H149N, H151N)) abrogated the toxic effects. A peptide corresponding to the zinc-binding domain (APP181-208) failed to induce copper or zinc toxicity in neuronal cultures. These data support a role for the APP copper-binding domain in APP-mediated copper (I) generation and toxicity in primary neurons, a process that has important implications for Alzheimer's disease and other neurodegenerative disorders.

Amino Acid Sequence↗
PubMed2001

c-Jun N-terminal kinase (JNK)-interacting protein-1b/islet-brain-1 scaffolds Alzheimer's amyloid precursor protein with JNK.

Using a yeast two-hybrid method, we searched for amyloid precursor protein (APP)-interacting molecules by screening mouse and human brain libraries. In addition to known interacting proteins containing a phosphotyrosine-interaction-domain (PID)-Fe65, Fe65L, Fe65L2, X11, and mDab1, we identified, as a novel APP-interacting molecule, a PID-containing isoform of mouse JNK-interacting protein-1 (JIP-1b) and its human homolog IB1, the established scaffold proteins for JNK. The APP amino acids Tyr(682), Asn(684), and Tyr(687) in the G(681)YENPTY(687) region were all essential for APP/JIP-1b interaction, but neither Tyr(653) nor Thr(668) was necessary. APP-interacting ability was specific for this additional isoform containing PID and was shared by both human and mouse homologs. JIP-1b expressed by mammalian cells was efficiently precipitated by the cytoplasmic domain of APP in the extreme Gly(681)-Asn(695) domain-dependent manner. Reciprocally, both full-length wild-type and familial Alzheimer's disease mutant APPs were precipitated by PID-containing JIP constructs. Antibodies raised against the N and C termini of JIP-1b coprecipitated JIP-1b and wild-type or mutant APP in non-neuronal and neuronal cells. Moreover, human JNK1beta1 formed a complex with APP in a JIP-1b-dependent manner. Confocal microscopic examination demonstrated that APP and JIP-1b share similar subcellular localization in transfected cells. These data indicate that JIP-1b/IB1 scaffolds APP with JNK, providing a novel insight into the role of the JNK scaffold protein as an interface of APP with intracellular functional molecules.

Adaptor Proteins, Signal Transducing↗
PubMed1992

[Alzheimer's dementia and amyloid precursor-protein].

Alzheimer's dementia is a severe form of dementia characterised by three neuropathological changes: amyloid plaques, neurofibrillary tangles and neurodegeneration. Several laboratories are involved in studies of the molecular mechanisms underlying development of Alzheimer's disease. As a result of recent research, the main component of amyloid plaques, the beta A4-peptide, is focused upon in hypotheses of the pathogenesis of the disease. The beta A4-peptide is produced by cleavage of the larger amyloid precursor protein (APP). During normal processing of APP, production of beta A4 is hindered and the peptide is therefore presumably a result of alternative cleavage mechanisms. Several factors may increase alternative processing of APP. One possibility is that conformational changes in APP, e.g. induced by mutations in the APP gene, may facilitate alternative cleavage of APP and thereby production of beta A4. This seems to be the case in certain families with familial Alzheimer's disease. Another possibility for increased beta A4 production is excessive expression of APP. This seems to be the case in persons with trisomy 21 who all have neuropathological signs of Alzheimer's disease after the age of 40 years. The gene for APP is localised to the human chromosome 21 and these persons have 1.5 times the normal expression of APP. Furthermore, excessive APP-expression may be induced by an acute phase response in the brain. In vitro studies and neuropathological examinations indicate that alpha 2-macroglobulin in neurons may be induced by cytokines. The protease inhibitor activity of this protein may prevent normal cleavage of APP, thereby resulting in increased alternative cleavage and deposition of beta A4.(ABSTRACT TRUNCATED AT 250 WORDS)

Alzheimer Disease↗
PubMed1991

Processing of beta-amyloid precursor protein in microglia and astrocytes favors an internal localization over constitutive secretion.

Microglial cells and astrocytes are closely associated with nearly all compact deposits of the amyloid beta-protein found in the senile plaques characteristic of Alzheimer's disease and trisomy 21. The biosynthesis and metabolic fate of the beta-amyloid precursor protein (beta APP) in astrocytes has not been characterized, and its identification in microglia has not been described. Here, we report the expression of beta APP by astrocytes and microglia in primary cultures of cerebral cortex from newborn rats. Using metabolic labeling followed by immunoprecipitation, we show that both astrocytes and microglia express substantial amounts of the major isoforms of beta APP. This is confirmed by PCR-mediated amplification of the corresponding mRNAs, showing that all three major transcripts (beta APP695, beta APP751, and beta APP770) are present in relatively equal amounts. Despite rapid turnover of the precursor, astrocytes and microglia show a reduced production of soluble fragments of beta APP compared to cells transfected with beta APP cDNAs. The relative amount of soluble beta APP molecules generated is both cell type and isoform specific. Immunocytochemistry reveals that full-length beta APP is located in internal membranous vesicles, with only very little insertion at the cell surface. The latter data are in agreement with the reduced ability of microglia and astrocytes to cleave the beta APP into soluble derivatives. Our findings indicate that both astrocytes and microglia strongly express all three major forms of beta APP but apparently process these molecules by an alternative pathway that generates very small amounts of soluble beta APP. The immunocytochemical localization and the biochemical data lead to the suggestion that beta APP may not function principally as a cell surface or secreted protein in vivo but may have an important intracellular function.

Amyloid beta-Protein Precursor↗
PubMed1994

A heparin-binding domain in the amyloid protein precursor of Alzheimer's disease is involved in the regulation of neurite outgrowth.

The amyloid protein precursor (APP) of Alzheimer's disease is synthesized as an integral transmembrane protein that is released from cells in culture following proteolytic cleavage. The function of released APP is not known, although there is evidence that the protein may bind to components of the extracellular matrix (ECM). In the present study, substratum-bound APP stimulated neurite outgrowth in cultures of chick sympathetic and mouse hippocampal neurons. This effect was dependent upon the presence of substratum-bound heparan sulfate proteoglycans (HSPG). The effect of APP on neurite outgrowth was comparable to that of laminin. A 14 K N-terminal fragment of APP was found to bind heparin and a region close to the N-terminus of APP (residues 96-110) identified as a potential heparin-binding domain based on secondary structure predictions and molecular modeling. Mutagenesis of three basic residues (lysine-99, arginine-100, and arginine-102) resulted in a recombinant protein (APPhep) with decreased heparin-binding capacity. A peptide homologous to the heparin-binding domain was synthesized and found to bind strongly to heparin and to inhibit binding of 125I-labeled APP to heparin (IC50 approximately 10(-7) M). The peptide blocked the effect of APP on neurite outgrowth (IC50 approximately 10(-7) M), whereas two other peptides homologous to other domains in APP had no effect. The results indicate that the binding of APP to HSPG in the ECM may stimulate the effects of APP on neurite outgrowth.

Amino Acid Sequence↗