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PubMed2002

Contrasting, species-dependent modulation of copper-mediated neurotoxicity by the Alzheimer's disease amyloid precursor protein.

The amyloid precursor protein (APP) of Alzheimer's disease (AD) has a copper binding domain (CuBD) located in the N-terminal cysteine-rich region that can strongly bind copper(II) and reduce it to Cu(I) in vitro. The CuBD sequence is similar among the APP family paralogs [amyloid precursor-like proteins (APLP1 and APLP2)] and its orthologs (including Drosophila melanogaster, Xenopus laevis, and Caenorhabditis elegans), suggesting an overall conservation in its function or activity. The APP CuBD is involved in modulating Cu homeostasis and amyloid beta peptide production. In this paper, we demonstrate for the first time that Cu-metallated full-length APP ectodomain induces neuronal cell death in vitro. APP Cu neurotoxicity can be induced directly or potentiated through Cu(I)-mediated oxidation of low-density lipoprotein, a finding that may have important implications for the role of lipoproteins and membrane cholesterol composition in AD. Cu toxicity induced by human APP, Xenopus APP, and APLP2 CuBDs is dependent on conservation of histidine residues at positions corresponding to 147 and 151 of human APP. Intriguingly, APP orthologs with different amino acid residues at these positions had dramatically altered Cu phenotypes. The corresponding C. elegans APL-1 CuBD, which has tyrosine and lysine residues at positions 147 and 151, respectively, strongly protected against Cu-mediated lipid peroxidation and neurotoxicity in vitro. Replacement of histidines 147 and 151 with tyrosine and lysine residues conferred this neuroprotective Cu phenotype to human APP, APLP2, and Xenopus APP CuBD peptides. Moreover, we show that the toxic and protective CuBD phenotypes are associated with differences in Cu binding and reduction. These studies identify a significant evolutionary change in the function of the CuBD in modulating Cu metabolism. Our findings also suggest that targeting of inhibitors to histidine residues at positions 147 and 151 of APP could significantly alter the oxidative potential of APP.

Alzheimer Disease↗

PubMed1992

Release of amino-terminal fragments from amyloid precursor protein reporter and mutated derivatives in cultured cells.

Abnormal proteolytic processing of amyloid precursor protein (APP) is thought to be central to the formation and deposition of beta amyloid peptide in Alzheimer's disease. A putative "secretase" activity normally releases an amino-terminal APP fragment by cleaving APP at residues within the beta amyloid peptide thereby precluding amyloidogenesis. In order to better understand the requirements for APP cleavage by secretase, we have expressed a modified cDNA construct representing the 751-amino acid isoform of APP (APP-REP) and mutated APP-REP proteins in cultured cells. Here, we show that: (a) APP-REP is predominantly associated with membranes; (b) intracellular turnover and processing of APP-REP is similar to that reported for the intact APP protein; (c) secretion appears unaltered by introduction of the glutamate to glutamine mutation found in the APP gene of patients suffering from hereditary cerebral hemorrhage with amyloidosis of Dutch origin; (d) a mutation in which the 18 juxtamembranous amino acids encompassing the secretase site are deleted also allows release of an amino-terminal fragment into the conditioned medium; and (e) kinetics of cleavage of APP-REP and its mutated derivatives are similar. These results indicate that the secretory cleavage of the extracellular amino-terminal fragments of APP-REP can occur in the presence of different novel juxtamembranous amino acid sequences.

Amino Acid Sequence↗

PubMed1990

Proteolytic processing of beta-amyloid precursor by calpain I.

The beta-amyloid peptide is a core component of the neuritic plaques that accumulate in Alzheimer's disease. Since the beta-peptide resides within a family of precursor proteins (APPs), proteolytic processing of APP is required for beta-amyloid deposition into plaques. Here, we have examined the role played by the calcium-dependent cysteine protease calpain I in APP processing. Immunoblotting with a specific APP antiserum was used to assess the in vitro degradation of rat brain APP, which appears as a triplet of polypeptides of Mr 110-130 kDa. Both soluble and membrane-bound APP were extraordinarily sensitive to activated calpain I. APP contains at least 3 distinct calpain I cleavage sites. The most protease-sensitive site was located within the highly acidic structural motif called the PEST domain, a second site was upstream of the putative N-linked glycosylation sites, and a third generated a 16 kDa carboxy-terminal fragment that contains the beta-peptide. Based on light microscopic immunohistochemistry, APP and calpain I were extensively colocalized within large numbers of neurons distributed throughout the rat brain, with especially high levels of each in neocortical layer 5, subiculum, globus pallidus, entopeduncular nucleus, anterodorsal and reticular thalamic nuclei, motor trigeminal nucleus, deep cerebellar nuclei, and Purkinje cells. Both antigens were most prevalent within neuronal perikarya. Intraventricular kainate infusion, which is known to cause rapid activation of hippocampal calpain I, produced a 32% decline in APP levels after 24 hr, suggestive of in vivo degradation of APP by calpain I. Following kainate-induced neuronal loss, both APP and calpain I immunoreactivities appeared in the surrounding reactive astroglia. These results indicate that calpain I may be involved in the normal and, perhaps, pathological processing of APP, and that this processing could occur in either neurons or reactive astrocytes. Calcium influx and calpain I activation may provide a mechanism by which excitatory neurotransmission regulates APP metabolism.

Alzheimer Disease↗

PubMed1989

The beta amyloid protein precursor: mRNAs, membrane-associated forms, and soluble derivatives.

We used in situ hybridization to assess total beta amyloid protein precursor (beta APP) mRNA and the subset of beta APP mRNA containing the Kunitz protease inhibitor (KPI) insert in 11 Alzheimer's disease (AD) brains and 7 control brains well matched for age and postmortem interval. In AD, we observed a significant two-fold increase in total beta APP mRNA in nucleus basalis and locus ceruleus neurons but not in occipital cortical neurons, hippocampal subicular neurons, or neurons of the basis pontis. The increase in total beta APP mRNA in locus ceruleus and nucleus basalis neurons was due exclusively to an increase in KPI-free beta APP mRNA. We conclude that increased production of KPI-free beta APP in nucleus basalis and locus ceruleus neurons may contribute to the deposition of cerebral amyloid that occurs in AD. To identify the beta APP, we prepared antisera to synthetic peptides corresponding to the carboxyl-terminus (anti-C1), to a near amino-terminal domain (anti-beta APP45-62), and to the KPI domain (anti-KPI36-48). We established that these antisera detect the beta APP by showing that they specifically detect proteins that are markedly augmented in cells transfected with beta APP expression constructs. Anti-beta APP45-62 specifically labels (i) a set of approximately 110-135 kDa membrane-associated brain proteins previously detected by antisera to the carboxyl-terminus of the beta APP (anti-C1) and (ii) soluble approximately 105 and approximately 125 kDa proteins not detected by anti-C1. Anti-KPI36-48 specifically labels the two largest membrane-associated forms of the beta APP and the soluble approximately 125 kDa derivative, but does not label the two smallest membrane-associated forms or the approximately 105 kDa soluble derivative. Anti-beta APP45-62 and anti-C1 both specifically stain senile plaques. This finding suggests (i) that full-length beta APP is present in senile plaques and (ii) that proteolytic processing of the beta APP into insoluble amyloid fibrils occurs, at least in part, locally at the sites of amyloid deposition in AD brain. Analysis of cerebrospinal fluid (CSF) showed that the soluble KPI-containing (approximately 125 kDa) and KPI-free (approximately 105 kDa) derivatives present in brain are readily detected in CSF from both AD cases and controls.(ABSTRACT TRUNCATED AT 400 WORDS)

Alzheimer Disease↗

PubMed1995

Characterization of sorting signals in the beta-amyloid precursor protein cytoplasmic domain.

The beta-amyloid precursor protein (APP) is proteolytically processed to generate beta-amyloid protein, the principal protein component of neuropathological lesions characteristic of Alzheimer's disease. To investigate potential sorting signals in the cytoplasmic tail of APP, we transplanted APP cytoplasmic tail sequences into the cytoplasmic tail of the human transferrin receptor (TR) and showed that two sequence motifs from the APP cytoplasmic tail promote TR internalization. One sequence, GYENPTY, is related to the low density lipoprotein receptor internalization signal, FDNPVY, but also involves a critical glycine residue; the other, YTSI, conforms to the 4-residue tyrosine-based internalization signal consensus sequence. Furthermore, a chimeric molecule (APP-TR) consisting of the cytoplasmic domain of APP and the transmembrane and external domains of TR was rapidly internalized enabling the transport of iron into the cell at approximately 50% the rate of wild-type TR. Alanine scanning mutations indicated that the two sequences identified in transplantation experiments were required for internalization of the chimera. Metabolic pulse-chase experiments showed that the APP-TR chimeras were degraded in a post-Golgi membrane compartment within 2-4 h following normal glycosylation. Degradation was partially dependent upon the two internalization signals and was inhibited by ammonium chloride. A fraction of APP-TR chimeras traffic to a degradative endocytic compartment after appearing on the cell surface. Comparison of soluble APP released from cells expressing either full-length human APP or mutant APP with the sequence YENPTY deleted indicated that this sequence is required for sorting of full-length APP along similar trafficking pathways as the APP-TR chimera.

Amino Acid Sequence↗

PubMed1995

Cell-surface beta-amyloid precursor protein stimulates neurite outgrowth of hippocampal neurons in an isoform-dependent manner.

beta-Amyloid precursor protein (beta APP) is an integral membrane polypeptide expressed in many neural and non-neural cells. beta APP occurs in part at the cell surface and undergoes proteolytic processing to release the large soluble ectodomain (APPs) and the amyloid beta-peptide (A beta), both of which have apparent trophic activity in vitro. Despite intense interest in beta APP expression and metabolism, there is limited knowledge about the function mediated by beta APP inserted at the cell-surface. We established a coculture system in which beta APP-transfected CHO cells serve as a substrate for the growth of primary rat hippocampal neurons. Compared to nontransfected CHO cells, the increased surface beta APP of the transfectants stimulated short-term neuronal adhesion and longer-term neurite outgrowth, whereas the increased amount of secreted APPs and A beta in conditioned medium produced no such effects when neurons were grown either on untransfected CHO cells or on a polylysine substrate. Moreover, a peptide which has been shown to block the trophic effects of secreted APPs (Ninomiya et al., 1993) failed to interrupt the neurite promoting activity mediated by the surface-expressed beta APP. Surface-expressed beta APP751 or beta APP770 isoforms mediated more neurite outgrowth than did the beta APP695 isoform. Antibody blocking and regional deletion experiments indicated that the mid-region of the beta APP ectodomain (residues 361-648) is involved in promoting neurite outgrowth. We conclude that surface-expressed cellular beta APP has a neurite-promoting function which is distinct from the trophic function of the secreted beta APP derivatives and may have special significance during brain development.

Amino Acid Sequence↗

PubMed1994

Peptides containing the RERMS sequence of amyloid beta/A4 protein precursor bind cell surface and promote neurite extension.

Amyloid beta/A4 protein precursor (APP) is secreted into medium by most cultured cells and can function as an autocrine factor. To study the biological function of secreted forms of APP (sAPP) on neurons, we used a clonal CNS neuronal line, B103, which does not synthesize detectable levels of APP. B103 cells transfected with APP construct developed neurites faster than the parent B103 cells when plated in a serum-free defined medium. Neurite outgrowth of B103 cells was promoted by the conditioned medium of APP-695-over-producing cells or by the bacteria-produced sAPP-695 (named KB75). A series of peptides having sequences between Ala-319 and Met-335 of APP-695 also stimulated neurite outgrowth of B103 cells. The sequence of five amino acids, RERMS (APP 328-332), within this stretch of sequence, was the shortest active peptide, although the concentration required for the neuritotropic activity was higher than that of KB75. Binding assay using 125I-labeled APP 17-mer peptide corresponding to Ala-319 to Met-335 of APP-695 as a ligand demonstrated specific and saturable cell-surface binding sites. The predicted KD value was 20 +/- 5 nM and the Bmax value was 80 +/- 8 fmol/10(6) cells. The binding could be displaced with KB75. A 17-mer peptide with reverse sequence neither induced neurite outgrowth nor competed for the binding. A bacteria-produced sAPP fragment lacking the active 17-mer sequence (named KB75 delta) did not compete with 125I-labeled 17-mer for binding or stimulate neurite extension. A peptide of sequence RMSQ (APP 330-333), which partially overlaps the active sequence RERMS, could block the neuritotropic effects of both KB75 and the 17-mer at higher concentrations. APP 17-mer was also found to induce the accumulation of inositol polyphosphates, suggesting that the APP 17-mer effects involve activation of inositol phospholipid signal transduction systems. These data indicate that sAPP induces neurite extension through cell-surface binding and that the domain containing the RERMS sequence (APP 328-332) represents the active site responsible for this function.

Amino Acid Sequence↗

PubMed1995

Inhibition of protein phosphatase 1 stimulates secretion of Alzheimer amyloid precursor protein.

BACKGROUND: Aberrant metabolism of the Alzheimer amyloid precursor protein (APP) or its amyloidogenic A beta fragment is thought to be centrally involved in Alzheimer's disease. Nonamyloidogenic processing of APP involves its cleavage within the A beta domain by a protease, termed alpha-secretase, and release of the large extracellular domain, termed APPS. Secretion of APPS can be stimulated by phorbol esters, activators of protein kinase C, with concurrent inhibition of A beta production. While the role of protein kinases of APP metabolism has been investigated, considerably less effort has been devoted to elucidating the role played by protein phosphatases. Okadaic acid, a protein phosphatase inhibitor, has been shown to stimulate secretion of APPS, but the identity of the phosphatase involved has not been investigated. MATERIALS AND METHODS: The secretion of APPS from COS-1 cells was measured in the absence or presence of various doses of serine/threonine-specific phosphatase inhibitors. Quantitation of the derived IC50 values was used to determine the identity of the phosphatase involved in the control of APP secretion. RESULTS: The availability of protein phosphatase inhibitors with different relative potencies against the different types of serine/threonine-specific protein phosphatase allowed us to examine which of the four known types of protein phosphatase might be involved in the regulation of APP secretion. Both okadaic acid and calyculin A stimulated the secretion of APP from COS-1 cells in a dose-dependent manner. The half-maximal dose for stimulation of APP secretion was approximately 100-fold higher with okadaic acid than with calyculin A. CONCLUSIONS: The nearly 100-fold difference in the observed IC50 values for okadaic acid and calyculin A implicates a type 1 protein phosphatase in the control of APPS production. Protein phosphatase 1 (PP1) is known to be highly expressed in adult mammalian brain, both in neurons and glia. The identification of a specific phosphatase type in the control of APP secretion opens new avenues to the development of rational therapeutic intervention strategies aimed at the prevention and/or treatment of Alzheimer's Disease.

Aged↗

PubMed1997

Acute postoperative pancreatitis after laparoscopic cholecystectomy. Results of the Prospective Swiss Association of Laparoscopic and Thoracoscopic Surgery Study.

BACKGROUND: The introduction of laparoscopic cholecystectomy (LC) changed the treatment strategies for patients undergoing biliary surgery. There is a lack of data about acute postoperative pancreatitis (APP) as a complication of LC. OBJECTIVES: To determine the incidence, morbidity, and mortality of APP after LC and to analyze the possible intraoperative and pathogenic factors associated with APP. DESIGN: A prospective cohort study of 10174 patients who underwent LC. The data for 32 patients with APP after the completion of LC and 8 patients with APP after an attempted LC that was converted to an open cholecystectomy were analyzed. SETTING: A multi-institutional study of the Swiss Association of Laparoscopic and Thoracoscopic Surgery. Eighty-one surgical services or surgeons in private practice participated. RESULTS: The incidence of APP after a completed LC was 0.34%. In comparison, the incidence of APP after conversion to an open procedure (0.96%) was significantly (P = .02) increased. A biliary origin of APP could be established in 4 (12.5%) of the 32 patients. No evidence for a causative role of intraoperative cholangiography or trauma to the pancreas was found. Factors shown to be associated with APP were the surgeon's experience level and a high morbidity of 31.3%. The mortality was 3%. CONCLUSIONS: The incidence of APP after LC is low; the risk increases after conversion to an open procedure. In the rare event of an APP after LC, a biliary cause should be suspected. The mortality of patients with APP did decrease substantially compared with those undergoing open biliary surgery.

Acute Disease↗

PubMed2000

A beta peptides and calcium influence secretion of the amyloid protein precursor from chick sympathetic neurons in culture.

The major constituent of amyloid plaques in the Alzheimer disease (AD) brain is the amyloid protein (A beta). A beta has been shown to be neurotoxic to cells, but the exact mechanism of its effects are still not known. Most studies have focussed on A beta neurotoxicity, but little is known about the effect of A beta peptides on cellular protein metabolism and secretion. To examine the effect of A beta peptides on APP secretion, chick sympathetic neurons were metabolically labeled with [(35)S]methionine and the amounts of radiolabeled APP and A beta quantitated. Several A beta peptides (A beta(25-35), [pyroglu(3)]A beta(3-40), and [pyroglu(11)]A beta(11-40)) inhibited secretion of [(35)S]APP and increased cell-associated [(35)S]APP. There was also a 2-2.5-fold increase in secretion of several other proteins when cells were incubated with A beta(25-35). However, the amount of A beta secreted into the medium was decreased. Treatment of cells with the calcium ionophore A23187 caused a 1.5-fold increase in secreted [(35)S]APP and a decrease in cell-associated [(35)S]APP. Although L-type voltage-dependent calcium channels (VDCC) have been implicated in A beta toxicity, the effect of L-type VDCC on APP secretion has not previously been examined. The L-type VDCC antagonists nifedipine and diltiazem both increased [(35)S]APP secretion into the medium but did not influence the effect of A beta on [(35)S]APP secretion. These studies suggest that A beta interferes with the secretory pathway of APP. Insofar as secreted APP has been proposed to have a neuroprotective function, the accumulation of A beta in the AD brain could decrease secreted APP and thereby indirectly increase A beta toxicity.

Amyloid beta-Peptides↗

PubMed2004

Increased expression of amyloid precursor protein in oral squamous cell carcinoma.

In our previous study, we identified amyloid precursor protein (APP) in an oral squamous cell carcinoma (OSCC)-enriching subtractive hybridization library. Our present study attempts to define the significance of APP expression in the genesis of OSCC. RT-PCR analysis showed increase in APP mRNA expression for more than 2-fold in 76% of OSCC (n = 55) relative to corresponding non-cancerous matched tissues (NCMT). The majority of esophageal SCCs also had increase in APP mRNA expression. OSCC patients exhibiting increase in APP mRNA expression had significantly lower survival rate compared to patients exhibiting the opposite status. Western blotting analysis identified APP751 and APP770 as the major APP isoforms in oral keratinocytes. A high correlation between mRNA and protein expressions of APP was noted in OSCC/NCMT pairs. Immunohistochemistry further showed a remarkable increase of APP in OSCC tissue relative to NCMT. Treatment with an antisense oligonucleotide against APP reduced cellular and secreted APP as well as growth in an OSCC cell line. Our study provides novel clues that APP expression is involved in the proliferation and carcinogenesis of OSCC. Correlated with such pathogenesis was the survival of its victims. The degree of APP expression could serve as an invaluable marker for oral carcinogenesis.

Adult↗

PubMed2006

Cerebrolysin decreases amyloid-beta production by regulating amyloid protein precursor maturation in a transgenic model of Alzheimer's disease.

Cerebrolysin is a peptide mixture with neurotrophic effects that might reduce the neurodegenerative pathology in Alzheimer's disease (AD). We have previously shown in an amyloid protein precursor (APP) transgenic (tg) mouse model of AD-like neuropathology that Cerebrolysin ameliorates behavioral deficits, is neuroprotective, and decreases amyloid burden; however, the mechanisms involved are not completely clear. Cerebrolysin might reduce amyloid deposition by regulating amyloid-beta (Abeta) degradation or by modulating APP expression, maturation, or processing. To investigate these possibilities, APP tg mice were treated for 6 months with Cerebrolysin and analyzed in the water maze, followed by RNA, immunoblot, and confocal microscopy analysis of full-length (FL) APP and its fragments, beta-secretase (BACE1), and Abeta-degrading enzymes [neprilysin (Nep) and insulin-degrading enzyme (IDE)]. Consistent with previous studies, Cerebrolysin ameliorated the performance deficits in the spatial learning portion of the water maze and reduced the synaptic pathology and amyloid burden in the brains of APP tg mice. These effects were associated with reduced levels of FL APP and APP C-terminal fragments, but levels of BACE1, Notch1, Nep, and IDE were unchanged. In contrast, levels of active cyclin-dependent kinase-5 (CDK5) and glycogen synthase kinase-3beta [GSK-3beta; but not stress-activated protein kinase-1 (SAPK1)], kinases that phosphorylate APP, were reduced. Furthermore, Cerebrolysin reduced the levels of phosphorylated APP and the accumulation of APP in the neuritic processes. Taken together, these results suggest that Cerebrolysin might reduce AD-like pathology in the APP tg mice by regulating APP maturation and transport to sites where Abeta protein is generated. This study clarifies the mechanisms through which Cerebrolysin might reduce Abeta production and deposition in AD and further supports the importance of this compound in the potential treatment of early AD.

Alzheimer Disease↗

PubMed1994

Tacrine alters the secretion of the beta-amyloid precursor protein in cell lines.

The characteristic features of Alzheimer's disease (AD) include a high density of beta-amyloid-containing plaques in the cerebral cortex and the loss of basal forebrain cholinergic neurons. Amyloid beta-protein (A beta, Mr. approximately 4.5 kDa) is derived from a family of large (Mr. approximately 110-140 kDa) beta-amyloid precursor proteins (APP) which are integral membrane glycoproteins consisting of a large extracytoplasmic domain, a transmembrane domain, and a short cytoplasmic tail. Secreted derivatives of APP lacking the cytoplasmic tail, transmembrane domain, and a small portion of the extracellular domain are generated by the proteolytic processing of full length APP by a family of proteolytic enzymes known as APP secretases. Using cell cultures, we investigated the possibility that APP processing can be regulated by a centrally active cholinesterase inhibitor, tacrine, which has recently been shown to improve memory and cognitive functions in patients with AD. We analyzed the level of APP in glial, fibroblast, pheochromocytoma (PC12), and neuroblastoma cells by immunoblotting cell lysates and conditioned media. Normal levels of secretion of soluble APP derivatives by cells into conditioned media were severely inhibited by treating cells with tacrine. A similar decrease after treatment with tacrine was observed when neuroblastoma and PC12 cells were pretreated with either growth factors, phorbol ester, or retinoic acid. To determine whether the effect of tacrine on APP levels was specific or a more general phenomenon affecting other proteins, we measured the level of heat shock protein-70 (HSP-70) and another secretory protein, protease nexin-1 (PN-1). Tacrine treatment did not alter the level of HSP-70 in cell extracts and tacrine affected mildly the secretion of PN-1. Thus, the processing of HSP and PN-1, unlike APP, was not severely affected by treating cells with tacrine. Our results suggest that tacrine may inhibit an acetylcholinesterase-associated proteolytic activity involved in the secretion of APP, which results in less secretion of soluble APP into the conditioned media from tacrine treated cells. These results demonstrate that tacrine regulates APP secretion in cell cultures and suggest the possibility that tacrine therapy of Alzheimer's disease may, in the longer term, have effects on the process of A beta deposition.

Amyloid beta-Protein Precursor↗

PubMed1994

Human IMR-32 neuroblastoma cells as a model cell line in Alzheimer's disease research.

The present study investigated expression and processing of amyloid precursor protein by neuronally differentiated IMR-32 neuroblastoma cells. APP mRNA in these cells was found to consist of approximately 58% APP695, 38% APP751, and < 4% APP770. APP-immunoreactive bands detected in western blots of cellular protein extracts were only detected by anti-APP antibodies to peptides with strong homology to APLP2, suggesting that these bands represent APP-like proteins and not APP itself. This result suggests that previous studies claiming immunodetection of cellular forms of APP may have to be re-evaluated. Four main species of C-terminal truncated, secreted APP were detected in blots of protein extracts from medium conditioned by these cells. The immunoreactive profile of these bands suggested a cleavage site N-terminal to the Lys16-Leu17 bond of alpha-secretase. This, together with differences in number and molecular mass of APP-immunoreactive bands between secreted APP from IMR-32 cells and that from the commonly used PC-12 cells, suggests differences in APP processing between these two neuronally differentiated cell lines. In theory, IMR-32 cells being of human neuronal origin may be a more appropriate cell line to study APP-processing in relation to Alzheimer's disease than the rat phaeochromocytoma PC-12 cell line. Therefore, these detected differences warrant further investigation. Additionally IMR-32 cells under certain tissue culture conditions can form intracellular fibrillary material that reacts with anti-PHF specific antibodies. Neuronally differentiated IMR-32 cells could therefore be used as a model system to investigate possible interactions between APP-processing and PHF formation.

Alzheimer Disease↗

PubMed1993

Interaction between the beta-amyloid peptide precursor and histones.

The function of the beta-amyloid peptide precursors (beta-APPs) remains undefined. In a search for ligands of beta-APP we found that the most prominent beta-APP-binding proteins were histones. To measure the specificity of binding the secreted or extracellular form of beta-APP(751), beta-APP-S, was purified from recombinant baculovirus-infected Sf-9 cells and was labeled with iodine-125. The labeled beta-APP-S bound to each of the histones, which had previously been adsorbed to nitrocellulose membranes. Differences in their apparent affinities were small. beta-APP-S did not bind to histones incorporated into chromatin. beta-APP-S bound relatively weakly to fibronectin, laminin, collagen type I, or collagen type IV. It did not bind to other basic proteins tested, myelin basic protein, or cytochrome c. The beta-APP-S-histone complex adhered to most filters and gel media, which precluded a direct determination of its stability. The apparent dissociation constant of beta-APP-S bound to histone-4-Sepharose was about 30 nM. Although beta-APP normally does not contact histones, it may bind those that are released from damaged cells. A function of beta-APP may be to bind and recycle substances such as histones, proteases, and matrix proteins from the extracellular medium.

Amyloid beta-Protein Precursor↗

PubMed1994

Production of Alzheimer 4kDa beta-amyloid peptide requires the C-terminal cytosolic domain of the amyloid precursor protein.

Amyloid precursor protein (APP) is metabolized through at least three pathways: the constitutive secretory pathway, the endosomal-lysosomal pathway and the 4-kDa A beta-producing pathway. The 4-kDa A beta (4 kDa A beta)-producing pathway which may play a primary role in the pathogenesis of Alzheimer's disease (AD) is presently unknown. In the present paper, we examine the production of the 4 kDa A beta in K562 lymphoid cells transfected with a truncated APP695 construct (APP delta 652-695) encoding an APP which lacks the cytosolic C-terminal domain except the four N-terminal amino acids, KKKQ. The APP delta 652-695-transfected cells (APP delta 652-695 cells) do not secrete 4 kDa A beta compared to APP 695-transfected cells (APP695 cells). Moreover, while the APP delta 652-695 and APP695 cells secrete equivalent levels of sAPP, the APP delta 652-695 cells accumulate less APP intracellularly than the APP695 cells. These results reveal that in the K562 cells (1), the last 43 amino acid residues at the C-terminus of APP are important in targeting APP through the 4 kDa A beta producing pathway and (2) processing of APP into 4 kDa A beta is independent of the known secretase pathway.

Alzheimer Disease↗

PubMed1999

Upregulation of amyloid precursor protein messenger RNA in response to traumatic brain injury: an ovine head impact model.

There is evidence that the amyloid precursor protein (APP) plays an important role in neuronal growth and synaptic plasticity and that its increased expression following traumatic brain injury represents an acute phase response to trauma. We hypothesized that the previously described increased APP expression in response to injury (Van den Heuvel et al., Acta Neurochir. Suppl. 71, 209-211) is due to increased mRNA expression and addressed this by examining the expression of APP mRNA and APP within neuronal cell bodies over time in an ovine head impact model. Twenty-five anesthetized and ventilated 2-year-old Merino ewes sustained a left temporal head impact using a humane stunner and 9 normal sheep were used as nonimpact controls. Following postimpact survival periods of 15, 30, 45, 60, and 120 min, brains were perfusion fixed in 4% paraformaldehyde and examined according to standard neuropathological protocol. APP mRNA and antigen expression were examined in 5-microm sections by nonisotopic in situ hybridization and APP immunocytochemistry. The percentage of brain area with APP immunoreactivity within neuronal cell bodies in the impacted animals increased with time from a mean of 7.5% at 15 min to 54.5% at 2 h. Control brains showed only very small numbers of weakly APP-positive neuronal cell bodies ranging from 2 to 14% (mean 7%). Increased expression of APP mRNA was first evident in impacted hemispheres at 30 min after impact and progressively increased over time to involve neurons in all sampled regions of the brain, suggesting increased transcription of APP. In contrast, APP mRNA was undetectable in tissue from nonimpacted sheep. These data show that APP mRNA and antigen expression are sensitive early indicators of neuronal injury with widespread upregulation occurring as early as 30 min after head impact.

Amyloid beta-Protein Precursor↗

PubMed1998

Diffuse neuronal perikaryon amyloid precursor protein immunoreactivity in a focal head impact model.

Amyloid precursor protein (APP) has been shown to accumulate in traumatically injured axons as early as 1 hour after injury. This accumulation may be due to interruption of fast axoplasmic transport and/or upregulation of APP synthesis. The aim of this study was to examine the neuronal cell body response to head impact using APP immunostaining in a focal non-missile head impact model. Ten anaesthetised and ventilated 2 year old Merino ewes were subjected to graded impact in the left temporal region by captive bolt. 2 hours after impact the brain was perfused fixed with formaldehyde. The tissue was mounted in paraffin, sectioned and stained with a monoclonal antibody to APP and standard H&E stain. APP positivity was semi-quantitated using a modification of our previously described sector scoring system [1]. Widespread neuronal APP positivity was found in the cerebral hemispheres and brain stem distant from the site of focal injury in all 10 animals. The most prominent APP positivity was found in the nerve cell bodies of the impacted left cerebral hemisphere. APP positive neurons were also found within regions which were structurally normal when stained with H&E. These results demonstrate diffuse neuronal perikaryon APP immunoreactivity following a focal head impact injury. The expression of APP within the neuronal cell body may be due to upregulation of APP synthesis or alterations in the availability of epitopes of APP. Further studies are in progress to address these hypotheses.

Amyloid beta-Protein Precursor↗