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At least 127 records · Page 7Linked to original sources
PubMed2001

The amyloid precursor protein (APP)-cytoplasmic fragment generated by gamma-secretase is rapidly degraded but distributes partially in a nuclear fraction of neurones in culture.

The gamma-secretase cleavage is the last step in the generation of the beta-amyloid peptide (Abeta) from the amyloid precursor protein (APP). The Abeta precipitates in the amyloid plaques in the brain of Alzheimer's disease patients. The fate of the intracellular APP carboxy-terminal stub generated together with Abeta has been, in contrast, only poorly documented. The analogies between the processing of APP and other transmembrane proteins like SREBP and Notch suggests that this intracellular fragment could have important signalling functions. We demonstrate here that APP-C59 is rapidly degraded (half-life approximately 5 min) when overexpressed in baby hamster kidney cells or primary cultures of neurones by a mechanism that is not inhibited by endosomal/lysosomal or proteasome inhibitors. Furthermore, APP-C59 binds to the DNA binding protein Fe65, although this does not increase the half-life of APP-C59. Finally, we demonstrate that a fraction of APP-C59 becomes redistributed to the nuclear detergent-insoluble pellet, in which the transcription factor SP1 is also present. Overall our results reinforce the analogy between Notch and APP processing, and suggest that the APP intracellular domain, like the Notch intracellular domain, could have a role in signalling events from the plasma membrane to the nucleus.

Amyloid Precursor Protein Secretases↗
PubMed2001

The beta-amyloid precursor protein APP is tyrosine-phosphorylated in cells expressing a constitutively active form of the Abl protoncogene.

The cytosolic domain of the beta-amyloid precursor protein APP interacts with three PTB (phosphotyrosine binding domain)-containing adaptor proteins, Fe65, X11, and mDab1. Through these adaptors, other molecules can be recruited at the cytodomain of APP; one of them is Mena, that binds to the WW domain (a protein module with two conserved tryptophans) of Fe65. The enabled and disabled genes of Drosophila, homologues of the mammalian Mena and mDab1 genes, respectively, are genetic modulators of the phenotype observed in flies null for the Abl tyrosine kinase gene. The involvement of Mena and mDab1 in the APP-centered protein-protein interaction network suggests the possibility that Abl plays a role in APP biology. We show that Fe65, through its WW domain, binds in vitro and in vivo the active form of Abl. Furthermore, in cells expressing the active form of Abl, APP is tyrosine-phosphorylated. Phosphopeptide analysis and site-directed mutagenesis support the hypothesis that Tyr(682) of APP(695) is the target of this phosphorylation. Co-immunoprecipitation experiments demonstrate that active Abl and tyrosine-phosphorylated APP also form a stable complex, which could result from the interaction of the pYENP motif of the APP cytodomain with the SH2 domain of Abl. These results suggest that Abl, Mena, and mDab1 are involved in a common molecular machinery and that APP can play a role in tyrosine kinase-mediated signaling.

Amino Acid Sequence↗
PubMed2004

Downregulation of amyloid precursor protein (APP) expression following post-traumatic cyclosporin-A administration.

The aim of these studies was to assess and quantitate the effects of cyclosporin-A (CyA) on brain APP messenger RNA and neuronal perikaryal APP antigen expression following controlled focal head impact in sheep. Impact results in a significant increase in both APP mRNA and neuronal perikaryal APP antigen expression. Post-traumatic administration of CyA (intrathecal 10 mg/kg) resulted in a reduction in APP mRNA and neuronal perikaryal antigen expression. At 2 h postinjury, CyA treatment caused a statistically significant (p < 0.05) 1.3 +/- 0.1-fold decrease in APP mRNA in the central gray matter of impacted sheep compared to untreated impacted sheep. A more profound reduction in APP mRNA synthesis (1.6 +/- 0.2 fold) was evident at 6 h (p < 0.05). The mean percentage brain area with APP immunoreactive neuronal perikarya at 6 h post-injury was 94.5% in untreated impacted animals, 10.0% in CyA-treated impacted animals, 5.5% in untreated nonimpacted animals, and 6% in CyA-treated non-impacted controls. These results demonstrate that CyA has a downregulatory effect on increased APP expression caused by TBI.

Amyloid beta-Protein Precursor↗
PubMed1993

Beta APP gene expression is increased in the rat brain after motor neuron axotomy.

The response of the beta APP gene to neuronal injury was studied in the facial and hypoglossal nerve nuclei of the rat after corresponding nerve axotomy. Increased levels of beta APP 695, 714, 751 and 770 mRNAs were observed after either facial or hypoglossal nerve axotomy in the parent ipsilateral motor neurons. The increase was gradual, with maximal values 7 days after axotomy. beta APP mRNA expression returned to normal values 60 days after the lesion. Increased beta APP immunostaining was also detected in ipsilateral chromatolytic motor neurons. No change in beta APP immunoreactivity was observed in oligodendrocytes, another cell type expressing beta APP under normal conditions. A rapid increase in the expression of the GFAP gene was observed in reactive astrocytes surrounding chromatolytic neurons in the ipsilateral facial or hypoglossal nuclei. Thus, in contrast with other models of neuronal injury, where only the Kunitz protease inhibitor-containing beta APP mRNA isoforms are increased, all beta APP mRNAs are increased in the axotomy model. Furthermore, although beta APP expression has been shown to be increased in reactive astrocytes following neuronal injury, in the present study the increase was essentially found in the motor neurons reacting to axotomy.

Amyloid beta-Protein Precursor↗
PubMed1993

The rat central nervous system expresses Alzheimer's amyloid precursor protein APP695, but not APP677 (L-APP form).

A novel splicing form of beta A4 amyloid precursor protein (APP) lacking exon 15, corresponding to 18 residues, was first reported in leukocytes and then in ubiquitous organs. To determine which APP molecules (APP695, APP751, or APP770) either with (N-APP) or without (L-APP; leukocyte-derived APP) exon 15 were expressed in various organs, we investigated the alternative splicing at exon 15 in the rat brain, kidney, heart, and testis by a PCR analysis of reverse-transcribed RNA and Southern blot analysis. Regarding APP695 without exons 7 and 8, L-APP was either seldom or never expressed in the brain, whereas both N- and L-APP were expressed in other organs. On the other hand, regarding APP751/770 containing exon 7, which codes for the Kunitz-type serine protease inhibitor domain, both N- and L-APP were expressed in all the organs examined, including the brain. These results suggest that a particular alternative regulation system related to exon 15 might be present in only APP695 of the brain and influence the proteolytic processing of APP.

Alzheimer Disease↗
PubMed1996

The role of APP processing and trafficking pathways in the formation of amyloid beta-protein.

The amyloid beta-protein (A beta) is a proteolytic fragment of the beta-amyloid precursor protein (beta APP). We previously reported the constitutive secretion of A beta peptides from a variety of cells expressing beta APP under normal culture conditions. These endogenously produced A beta peptides have heterogeneous N- and C-termini that vary as a function of beta APP missense mutations. Treatment of A beta-secreting cells with agents that alter intravesicular pH showed that an acidic compartment is required for proper A beta generation. One such compartment appears to be the endosome. Immunolabeling of cell-surface beta APP in living neurons and non-neuronal cells directly demonstrated the endocytosis of the protein and its rapid recycling (within 5-10 minutes) to the cell surface, as well as the trafficking of some beta APP to lysosomes. Expression of beta APP with various deletions of the cytoplasmic domain, including the NPTY motif, leads to decreased internalization and an associated decrease in the production of A beta peptides that begin at the usual asp1 start site. These and other data suggest that A beta production begins with cleavage of beta APP by a still unknown protease(s) (beta-secretase[s]) at the met-asp bond proceeding the A beta N-terminus and that this occurs in part in early endosomes. To characterize the substrate requirements of beta-secretase, beta APP was mutagenized by placing stop codons within or at the end of the transmembrane domain or substituting other amino acids for the wild-type met and asp at the P1 and P1' positions. These experiments showed that proper beta-secretase cleavage requires the precursor to be membrane-anchored and is highly sequence specific; most substitutions at met or asp substantially decrease A beta production. Analogous mutagenesis experiments around the A beta C-terminus revealed that the unknown protease(s) cleaving here ("gamma-secretase[s]") does not show such specificity. Cells secreting A beta may also be useful for examining the critical issue of the aggregation of A beta into its neurotoxic polymeric form under physiological conditions. In this regard, we have found that beta APP-expressing CHO cells show aggregation of > or = 10-20% of their secreted A beta peptides into SDS-stable dimers, trimers and sometimes tetramers under normal culture conditions. The identity of these small multimers was confirmed by extensive immunochemical characterization and radiosequencing. They are present at approximately 100-500 pM levels in conditioned medium of CHO transfectants. Using this endogenous A beta aggregating system, we have begun to examine variables that influence aggregation and compounds which may retard it. In conclusion, studies of the regulation of A beta production and aggregation in cell culture can provide information under physiological conditions that can complement analyses of these processes in vivo.

Amyloid beta-Peptides↗
PubMed1997

Expression of the APP gene family in brain cells, brain development and aging.

The Alzheimer's beta A4-amyloid protein precursor (APP) and the APP-like proteins (APLPs) are transmembrane glycoproteins with a similar modular domain structure. Alternatively spliced exons found in both genes comprise a Kunitz protease inhibitor domain encoding exon, and another exon within the divergent regions adjacent to the transmembrane domain, i.e. exon 15 of the APP gene and an exon encoding 12 residues in APLP2. Omission of the latter exons in L-APP and L-APLP2 isoforms, respectively, generates a functional recognition sequence for xylosyltransferase-mediated addition of glycosaminoglycans and proteoglycan formation. In this paper, we summarize our analyses of the regulated expression of these alternatively spliced exons in APP and APLP2 in primary cultured rat brain cells, rat brain development and aging. In conjunction with additional data for the human brain, these data provide important clues for understanding the functional significance of alternative splicing and glycosylation in APP biology. On the basis of recent results showing a higher amyloidogenicity of exon 15 encoding APP than L-APP isoforms, we further discuss the potential significance of the low levels of L-APP in neurons for the susceptibility of the brain towards Alzheimer's disease.

Aging↗
PubMed2004

The APP intracellular domain forms nuclear multiprotein complexes and regulates the transcription of its own precursor.

The physiological functions of the beta-amyloid precursor protein (APP) may include nuclear signaling. To characterize the role of the APP adaptor proteins Fe65, Jip1b, X11alpha (MINT1) and the chromatin-associated protein Tip60, we analyzed their interactions by confocal microscopy and co-immunoprecipitations. AICD corresponding to S3-cleaved APP bound to Fe65 that transported it to nuclei and docked it to Tip60. These proteins formed AICD-Fe65-Tip60 (AFT) complexes that were concentrated in spherical nuclear spots. gamma-Secretase inhibitors prevented AFT-complex formation with AICD derived from full-length APP. The APP adaptor protein Jip1b also transported AICD to nuclei and docked it to Tip60, but AICD-Jip1b-Tip60 (AJT) complexes had different, speckle-like morphology. By contrast, X11alpha trapped AICD in the cytosol. Induced AICD expression identified the APP-effector genes APP, BACE, Tip60, GSK3beta and KAI1, but not the Notch-effector gene Hes1 as transcriptional targets. These data establish a role for APP in nuclear signaling, and they suggest that therapeutic strategies designed to modulate the cleavage of APP affect AICD-dependent signaling.

Acetyltransferases↗
PubMed1999

Amyloid precursor protein metabolism in fibroblasts from individuals with one, two or three copies of the amyloid precursor protein (APP) gene.

Protein kinase C (PKC)-activated modulation of amyloid precursor protein (APP) metabolism has been investigated in natural models of altered APP expression due to the presence of one, two or three copies of the APP gene. We show that levels of APP present in human skin fibroblasts strongly influence the effect of PKC activation of soluble APP (sAPP) release. Thus fibroblasts derived from a patient with a deletion in chromosome 21 including the APP locus (Delta21) had lower levels of both APP mRNA and cell-associated APP, and showed an exaggerated phorbol-ester-induced sAPP release, when compared with fibroblasts from control individuals. In contrast, fibroblasts from chromosome 21 trisomic Down's syndrome patients failed to show a concentration-dependent response to phorbol ester treatment. These results suggest that the levels of APP expression can affect the degree of response to PKC-mediated modulation of the metabolism of this protein.

Adult↗
PubMed2000

Presenilin 2 mutation does not influence expression and concentration of APP forms in human platelets.

BACKGROUND: The pattern of platelet amyloid precursor protein (APP) forms is altered in sporadic Alzheimer's disease patients, compared with both control subjects and non-Alzheimer's disease-demented patients. The aims of this study were to evaluate in platelets of symptomatic and presymptomatic patients carrying the mutation Met239Val in presenilin 2 (PS2) whether: i) PS2 and presenilin 3 (PS1) were expressed in platelets; ii) an altered expression of different APP isoforms mRNAs could be related to the presence of the mutation; and iii) an abnormal pattern of APP forms was associated to the mutation. MATERIALS AND METHODS: Reverse transcriptionpolymerase chain reaction (RT-PCR) of APP isoforms, PS1 and PS2 was performed on RNA extracted from platelets of three PS2 Met239Val mutated subjects, seven sporadic Alzheimer's patients and nine control subjects. The pattern of platelet APP forms at protein level was evaluated in the same population of subjects by means of Western blots analysis with specific antibody. RESULTS: We found that PS1 and PS2 were expressed correctly in human platelets. When the relative amount of expression of mRNA coding for APP 771/ 751-695 was measured, a similar ratio of expression was found in PS2-mutated subjects, compared with both sporadic Alzheimer's patients and to control subjects. Furthermore, when APP forms were evaluated in platelet homogenates by means of Western blots analysis with appropriate antibody, no difference was found in the pattern of APP forms in presence of PS2 mutation in platelets, compared with control subjects. CONCLUSIONS: These results indicated that PS2 was expressed in human platelets and that PS2 mutation did not affect APP forms pattern, thus, suggesting that in this peripheral cell the pathological effect of PS2 mutation might occur upstream of the amyloid cascade.

Aged↗
PubMed1992

Identification and differential expression of a novel alternative splice isoform of the beta A4 amyloid precursor protein (APP) mRNA in leukocytes and brain microglial cells.

The gene for the beta A4-amyloid precursor protein (APP) consists of 19 exons which code for a typical N- and O-glycosylated transmembrane protein with four extracellular domains followed by the transmembrane domain and a short cytoplasmic domain. The beta A4-amyloid sequence is part of exons 16 and 17. Several APP isoforms can be generated by alternative splicing of exons 7 and 8, encoding domains with homologies to Kunitz-type protease inhibitors and the MRC OX-2 antigen, respectively. The mechanism by which the pathological beta A4 is generated is unknown, it is however a critical event in Alzheimer's disease and is distinct from the normally occurring cleavage and secretion of APPs within the beta A4 sequence. We report here for the first time considerable APP mRNA expression by rat brain microglial cells. In addition we showed by S1 nuclease protection and polymerase chain reaction analysis of reverse transcribed RNA (RT-PCR) that T-lymphocytes, macrophages, and microglial cells expressed a new APP isoform by selection of a novel alternative splice site and exclusion of exon 15 of the APP gene. This leads to a transmembrane, beta A4 sequence containing APP variant, lacking 18 amino acid residues close to the amyloidogenic region. The use of this novel alternative splice site alters the structure of APP in close proximity to the beta A4 region and thus may determine a variant, potentially pathogenic processing of leukocyte-derived APP in brain.

Amyloid beta-Protein Precursor↗
PubMed1994

Expression of a ubiquitous, cross-reactive homologue of the mouse beta-amyloid precursor protein (APP).

Alzheimer's disease is characterized by the presence of senile plaques comprised primarily of deposits of the beta-amyloid protein (A beta) derived from larger amyloid precursor proteins (APP). We have identified a cDNA that encodes a 751-amino acid APP-like protein (designated APLP2) from the mouse that, with exception of the A beta region, is highly homologous to APP. In situ hybridization and quantitative polymerase chain reaction reveal that APLP2 and APP mRNA are expressed in similar, if not identical, neuronal populations and at similar levels. APLP2 appears to mature through the same unusual secretory/cleavage pathway as APP. Furthermore, widely utilized antibodies generated against non-overlapping epitopes of APP do not discriminate between APP and APLP2. Although APLP2 cannot give rise to A beta, its near identity to APP outside the A beta domain confounds the interpretation of previous immunocytochemical and biochemical characterizations of APP biosynthesis and metabolism.

Alzheimer Disease↗
PubMed1991

Expression patterns of beta-amyloid precursor protein (beta-APP) in neural and nonneural human tissues from Alzheimer's disease and control subjects.

Both neural and nonneural human tissues from patients with or without Alzheimer's disease (AD) were surveyed to detect the presence of the beta-amyloid protein and its precursors. This was accomplished using polyclonal and monoclonal antibodies to epitopes in the 695 amino acid long beta-APP (i.e., beta-APP695), as well as in related beta-APPs. Immunoreactivity in beta-APP in brain was prominent in senile plaques, extraneuronal tangles, and neurons. Outside the brain, beta-APP staining was seen in neurons and satellite glial cells of the dorsal root, enteric and trigeminal ganglia, the adeno- and neurohypophysis, megakaryocytes, and adrenal gland in samples from patients with AD and those without AD. Western blots of neocortex revealed three major proteins with apparent molecular masses of 105, 115, and 125 kDa in the insoluble membrane-associated fractions, while two broad bands with a molecular weight centered at about 100 and 120 kDa were detected in soluble fractions. In addition, the pituitary and adrenal glands as well as cardiac muscle revealed prominent immunobands in membrane-associated fractions. Notably, other nonneural tissues were devoid of beta-APP immunoreactivity. Thus, the beta-APPs are detectable only in a limited number of nonneural tissues. Taken together, these data suggest that beta-APPs produced in the brain are sources of beta-APP peptides that accumulate as senile plaques in AD.

Alzheimer Disease↗
PubMed1991

Regulatory region of human amyloid precursor protein (APP) gene promotes neuron-specific gene expression in the CNS of transgenic mice.

The accumulation of beta-amyloid protein in specific brain regions is a central pathological feature of Alzheimer's disease (AD). The 4 kd beta-amyloid protein derives from a larger amyloid precursor protein (APP) by as yet unknown mechanisms. In the absence of a laboratory animal model of AD, transgenic mice expressing various APP gene products may provide new insights into the relationship between APP and beta-amyloid formation and the pathogenesis of AD. beta-amyloid accumulation in AD brain may result from interactions between APP and other molecules. Such interactions are likely to be developmentally regulated and tissue-specific. A transgenic mouse model of AD, therefore, would aim for APP transgene expression that mimics the endogenous APP gene. As an initial step in developing an animal model, we have identified a 4.5 kb DNA fragment from the 5' end of the human APP gene, which mediates neuron-specific gene expression in the CNS of transgenic mice, using E. coli lacZ as a reporter gene. Detectable levels of transgene expression are found in most neurons but not in glial and vascular endothelial cells. The expression pattern of this reporter gene closely resembles the distribution of endogenous APP mRNA in both the human and mouse CNS.

Alzheimer Disease↗
PubMed2006

Effects of huperzine A on amyloid precursor protein processing and beta-amyloid generation in human embryonic kidney 293 APP Swedish mutant cells.

The amyloid precursor protein (APP) is cleaved enzymatically by nonamyloidogenic and amyloidogenic pathways. alpha-Secretase (alpha-secretase), cleaves APP within the beta-amyloid (Abeta) sequence, resulting in the release of a secreted fragment of APP (alphaAPPs) and precluding Abeta generation. In this study, we investigated the effects of an acetylcholinesterase inhibitor, huperzine A (Hup A), on APP processing and Abeta generation in human embryonic kidney 293 cells transfected with human APP bearing the Swedish mutation (HEK293 APPsw). Hup A dose dependently (0-10 microM) increased alphaAPPs release and membrane-coupled APP CTF-C83, suggesting increased APP metabolism toward the nonamyloidogenic alpha-secretase pathway. The metalloprotease inhibitor TAPI-2 inhibited the Hup A-induced increase in alphaAPPs release, further suggesting a modulatory effect of Hup A on alpha-secretase activity. The synthesis of full-length APP and cell viability were unchanged after Hup A incubation, whereas the level of Abeta(Total) was significantly decreased, suggesting an inhibitory effect of Hup A on Abeta production. Hup A-induced alphaAPPs release was significantly reduced by the protein kinase C (PKC) inhibitors GF109203X and Calphostin C. These data, together with the finding that the PKCalpha level was enhanced prior to the increase of alphaAPPs secretion, indicate that PKC may be involved in Hup A-induced alphaAPPs secretion by HEK293 APPsw cells. Our data suggest alternative pharmacological mechanisms of Hup A relevant to the treatment of Alzheimer's disease.

Alkaloids↗
PubMedDate not supplied

Induction of gene expression of amyloid precursor protein (APP) in activated human lymphoblastoid cells and lymphocytes.

To understand the possible role of amyloid precursor protein (APP) in human lymphocytes, and the regulation of APP gene expression in this cell type, we determined levels of cellular APP protein and of mRNA in human T-cell-derived Jurkat cells that were treated with lectin, phorbol ester, and calcium ionophore. We also related these levels to cell aggregation and adhesion. Cell-cell aggregation and cell-plastic adhesion were observed over a 24-h period after incubating cells for 2 h with phytohemagglutinin or phorbol myristate acetate. Cells treated with a calcium ionophore showed no aggregation or adhesion. Western blots indicated no obvious alteration in the level of cellular APP with different treatments. Northern blots showed a significant transient increase of APP mRNA after incubation with the calcium ionophore, whereas phorbol ester treatment showed a slight increase of APP mRNA. We analyzed the level of APP mRNA in human peripheral T cells which had been separated from peripheral lymphocytes. The level increased transiently by up to threefold after treatment with calcium ionophore plus phorbol esters. These data suggest that cell-cell aggregation and cell-matrix adhesion by human lymphocytes are not associated with an increased level of cellular APP protein or of mRNA.

Amyloid beta-Protein Precursor↗
PubMedDate not supplied

APP-collagen interaction is mediated by a heparin bridge mechanism.

The amyloid precursor protein (APP) is a glycoprotein consisting of at least four isoforms derived from a single gene by a process of alternative splicing. The membrane-bound forms of APP have been suggested to have adhesive properties and to mediate neural cell adhesion. Previous studies have demonstrated the ability of Fab' fragments of antibodies to extracellular domains of APP to inhibit neural cell binding to a collagen substrate, suggesting a physiological role for the collagen-binding properties of APP. The binding of APP has been demonstrated to be specific for type IV collagen, and no binding to other extracellular matrix components, including fibronectin and laminin, was detected. The APP-collagen binding appeared to be mediated by a heparin-bridge mechanism, since the binding was abolished by the addition of excess heparan or heparinase. These results were observed by both a homogenate-collagen binding assay and a cell-surface adhesion assay, thus providing further evidence for the adhesion role of APP. They also pose the question of the possible role of the heparin-binding properties of APP in the genesis of the neuritic plaques characteristic of Alzheimer's disease.

Alzheimer Disease↗
PubMed2005

NSE-controlled carboxyl-terminus of APP gene over-expressing in transgenic mice induces altered expressions in behavior, Abeta-42, and GSK3beta binding proteins.

The amyloid protein precursor (APP) is cleaved in its intramembranous domain by gamma-secrease to generate amyloid beta and a free carboxyl-terminal intracellular fragment. The carboxyl-terminal of 105 amino acids of APP (APP-C105) plays a crucial role in the neuropathology of Alzheimer's disease (AD), but it is incompletely understand how APP-C105 overexpression interacts and regulates the brain function and Abeta-42 levels, and whether or not it is associated with the expressions of GSK3beta-binding proteins. To test this, transgenic mice expressing NSE-controlled APP-C105 were produced and tested for their above phenotypes. A behavioral deficit was observed in the 9 months old transgenic mice, and western blot indicated that there was a predominant expression of APP-C105 in transgenic brains compared with those of non-transgenic brains. In parallel, APP-C105 overexpression resulted in the modulation of the Abeta-42 level, gamma-secretase activity, GSK3beta-binding proteins including PS1, tau, and beta-catenin in the brains of the transgenic mice relative to the non-transgenic mice. Thus, altered expressions of these neuropathological phenotypes in APP-C105 transgenic mice could be useful targets in developing new therapeutic treatments.

Alzheimer Disease↗