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Biomedical subjects

C L Masters

Publications and source records attributed to C L Masters.

At least 109 records · Page 6Linked to original sources
PubMed1998

Relative increase in Alzheimer's disease of soluble forms of cerebral Abeta amyloid protein precursor containing the Kunitz protease inhibitory domain.

Although a number of studies have examined amyloid precursor protein (APP) mRNA levels in Alzheimer's disease (AD), no clear consensus has emerged as to whether the levels of transcripts for isoforms containing a Kunitz protease inhibitory (KPI)-encoded region are increased or decreased in AD. Here we compare AD and control brain for the relative amounts of APP protein containing KPI to APP protein lacking this domain. APP protein was purified from the soluble subcellular fraction and Triton X-100 membrane pellet extract of one hemisphere of AD (n = 10), normal (n = 7), and neurological control (n = 5) brains. The amount of KPI-containing APP in the purified protein samples was determined using two independent assay methods. The first assay exploited the inhibitory action of KPI-containing APP on trypsin. The second assay employed reflectance analysis of Western blots. The proportion of KPI-containing forms of APP in the soluble subcellular fraction of AD brains is significantly elevated (p < 0.01) compared with controls. Species containing a KPI domain comprise 32-41 and 76-77% of purified soluble APP from control and AD brains, respectively. For purified membrane-associated APP, 72-77 and 65-82% of control and AD samples, respectively, contain a KPI domain. Since KPI-containing species of APP may be more amyloidogenic (Ho, L., Fukuchi, K., and Yonkin, S. G. (1996) J. Biol. Chem. 271, 30929-30934), our findings support an imbalance of isoforms as one possible mechanism for amyloid deposition in sporadic AD.

Alternative Splicing↗
PubMed1998

Inhibition of platelet activation by the Alzheimer's disease amyloid precursor protein.

The amyloid precursor protein (APP) of Alzheimer's disease is abundantly expressed in the platelet alpha-granule where its role remains unclear. This study describes a novel function for APP in regulating human platelet activation. Preincubation of platelet-rich plasma with recombinant secreted APP (sAPP) isoforms dose-dependently inhibited platelet aggregation and secretion induced by ADP or adrenaline. Similarly, sAPP potently inhibited low-dose thrombin-induced activation in washed platelet suspensions, indicating that the activity does not require plasma cofactors. There were no functional differences between sAPP forms with or without the Kunitz protease inhibitor domain or derived from either alpha- or beta-secretase cleavage. In fact, the N-terminal cysteine-rich region of APP (residues 18-194) was as effective as the entire sAPP region in the inhibition of platelet activation. The inhibitory activity of sAPP correlated with a significant reduction in the agonist-induced production of the arachidonic acid (AA) metabolites thromboxane B2 and prostaglandin E2. However, sAPP did not affect AA-induced platelet aggregation or secretion, indicating the enzymatic conversion of AA was not inhibited. The addition of a threshold dose of AA reversed the sAPP-inhibition of agonist-induced platelet activation. This suggests that sAPP decreases the availability of free AA, although the mechanism is not yet known. These data provide evidence that the release of sAPP upon platelet degranulation may result in negative feedback regulation during platelet activation.

Adenosine Diphosphate↗
PubMed1998

Acetylcholinesterase is increased in the brains of transgenic mice expressing the C-terminal fragment (CT100) of the beta-amyloid protein precursor of Alzheimer's disease.

Acetylcholinesterase (AChE) expression is markedly affected in Alzheimer's disease (AD). AChE activity is lower in most regions of the AD brain, but it is increased within and around amyloid plaques. We have previously shown that AChE expression in P19 cells is increased by the amyloid beta protein (A beta). The aim of this study was to investigate AChE expression using a transgenic mouse model of A beta overproduction. The beta-actin promoter was used to drive expression of a transgene encoding the 100-amino acid C-terminal fragment of the human amyloid precursor protein (APP CT100). Analysis of extracts from transgenic mice revealed that the human sequences of full-length human APP CT100 and A beta were overexpressed in the brain. Levels of salt-extractable AChE isoforms were increased in the brains of APP CT100 mice. There was also an increase in amphiphilic monomeric form (G1A) of AChE in the APP CT100 mice, whereas other isoforms were not changed. An increase in the proportion of G1A AChE was also detected in samples of frontal cortex from AD patients. Analysis of AChE by lectin binding revealed differences in the glycosylation pattern in APP CT100 mice similar to those observed in frontal cortex samples from AD. The results are consistent with the possibility that changes in AChE isoform levels and glycosylation patterns in the AD brain may be a direct consequence of altered APP metabolism.

Acetylcholinesterase↗
PubMed1998

Comparative neuropathology of Kuru with the new variant of Creutzfeldt-Jakob disease: evidence for strain of agent predominating over genotype of host.

The three major influences on the phenotype of the transmissible spongiform encephalopathies are believed to be strain of agent, route of infection and host genotype. We have compared the pathologic profiles and genotypes of the new variant of Creutzfeldt-Jakob disease (vCJD) and kuru. The comparison reveals that there are distinct lesional differences particularly in the prion protein (PrP) load and distribution as seen by immunohistochemistry. The clinico-pathologic phenotypes and the genotypes of these two diseases are sufficiently different to suggest that the strain of agent may play a greater role than any presumptive common route of peripherally acquired infection.

Adolescent↗
PubMed1998

Subcellular localization of the Alzheimer's disease amyloid precursor protein and derived polypeptides expressed in a recombinant yeast system.

Different isoforms and derived polypeptides of the Alzheimer's disease amyloid protein precursor (A beta PP) have been expressed in the yeast Pichia pastoris. The expression characteristics of the different A beta PP polypeptides were studied by post-embedding immunogold electron microscopy with various A beta PP antibodies. The site of intracellular expression could be readily identified with specific antibodies. Full length A beta PP was expressed in association with the nuclear membrane and the endoplasmic reticulum. Secretory derivatives of A beta PP were localized in membrane-bound secretory vesicles. A construct encoding two copies of beta A4[1-42] linked head-to-tail (beta A4duplex) accumulated as irregular dense cytoplasmic and intranuclear inclusions which reacted with all beta A4 antibodies tested. A beta A4-C-terminal construct accumulated into membranous structures in the cytoplasm and nucleus and reacted with most antibodies to beta A4 and the cytoplasmic domain of A beta PP. The two shorter constructs containing the beta A4 sequence formed similar intranuclear aggregates to those reported for intranuclear inclusions of polyglutamine peptides from huntingtin (in Huntington's disease) and ataxin protein fragments (in spinocerebellar ataxia). This is of interest because intracellular aggregation of the polyglutamine and beta A4 peptides may affect cells by similar toxic mechanisms. These studies demonstrate clear differences in the expression properties of different A beta PP polypeptides.

Alzheimer Disease↗
PubMed1998

Secretion of Alzheimer's disease Abeta amyloid peptide by activated human platelets.

Alzheimer's disease (AD) is characterized by the deposition of Abeta (betaA4) peptides of 39 to 43 amino acid residues, which are normal cellular metabolic products derived by proteolysis of the amyloid precursor protein (APP). The physiologic function of Abeta/APP in vivo is poorly understood. We analyzed human platelets for Abeta production by immunoprecipitation coupled to immunoblotting. A 4-kd Abeta fragment that comigrates with an Abeta40 synthetic peptide and reacts with several antibodies specific for the N- and C-termini of Abeta is detected. The majority of platelet Abeta appears to end at residue 40, as determined by immunoreactivity with an Abeta40-specific antibody. Furthermore, Abeta is secreted upon platelet stimulation with the physiologic agonists thrombin and collagen, together with secretion of soluble APP (sAPP). A comparison between serum and plasma shows a 1.6-fold increase in Abeta levels and a 2.4-fold increase in sAPP levels in serum. This is consistent with the view that platelets are the primary source of circulating Abeta and APP. The release of platelet Abeta by physiologic stimuli suggests that it may play a role in platelet aggregation and coagulation or in the repair mechanisms associated with injury.

Amyloid beta-Peptides↗
PubMed1997

Mutations in the transmembrane domain of APP altering gamma-secretase specificity.

Alzheimer's disease (AD) beta-amyloid peptide (Abeta and betaA4) is derived from the amyloid precursor protein (APP) by the subsequent action of the so-far unidentified beta- and gamma-secretases. gamma-secretase, which generates the C-terminus of Abeta, cleaves within the transmembrane domain of APP, preferentially after Abeta-residue 40 (Abeta 40) but also after residue 42 (Abeta 42). This Abeta 42 represents the major subunit of the plaques in AD. Since the position of gamma-secretase cleavage is crucial for understanding the pathogenic pathway, we investigated the effect of different point mutations at Thr43 on gamma-secretase specificity in SPA4CT (SPC99)-expressing COS7 cells. These constructs only require gamma-cleavage for Abeta release. We observed that all Thr43 mutations altered the specificity of gamma-secretase. Small hydrophobic residues favored the generation of Abeta 42, leading to an increase in the 42/40 ratio of Abeta (1.6-2.8-fold). The increase was even stronger (5.6-5.8-fold) when combined with the familial mutation Val46Phe. Thus, these constructs might be highly valuable for the generation of animal models for AD. Processing of full-length APP or SPA4CT yielded the same 42/40 ratio of Abeta (4. 7%). Both constructs, bearing the familial AD mutation Val46Phe, led to a similar increase in the 42/40 ratio (3.3- versus 3.6-fold). The p3 fragment, produced by alpha- and gamma-secretase, showed 42/40 ratios similar to Abeta when derived from wild-type and mutant proteins. These results suggest that the different Abeta- and p3-species are generated by gamma-cleavage activities with a similar enzymatic mechanism.

Amyloid Precursor Protein Secretases↗
PubMed1997

Human amyloid precursor-like protein 1--cDNA cloning, ectopic expression in COS-7 cells and identification of soluble forms in the cerebrospinal fluid.

Amyloid precursor-like protein 1 (APLP1) represents an integral membrane type 1 protein of unknown function which was originally cloned from a mouse cDNA library on the basis of sequence similarity with the Alzheimer's amyloid precursor protein (APP). Here we report on the molecular cloning and expression of the human APLP1 (hAPLP1). hAPLP1 consists of 650 amino acids, displays 89% identity on the amino acid level to its mouse homologue and has a calculated molecular mass of 72 kDa. hAPLP1 synthesized in a cell-free system displays an apparent molecular mass of approximately 80 kDa in SDS-containing gels and becomes N-glycosylated when the in vitro translation is performed in the presence of microsomes. The hAPLP1 cDNA was also expressed ectopically in COS-7 cells and the protein expression was analyzed by immunoprecipitation and western blotting. We have demonstrated that hAPLP1 represents a novel glycoprotein which carries both N- and O-linked glycans. Moreover, hAPLP1 undergoes limited proteolysis which results in the secretion of the carboxy-terminal truncated molecule into the cells conditioned medium. Examination of cells transfected with hAPLP1 cDNA by confocal laser microscopy reveals an intense perinuclear and Golgi staining, a pattern resembling the subcellular distribution of APP. Using a novel hAPLP1-specific antiserum, we identified soluble hAPLP1 in the human cerebrospinal fluid, which suggests that secretion of hAPLP1 from brain cells also takes place in vivo.

Amino Acid Sequence↗
PubMed1997

Late compartments of amyloid precursor protein transport in SY5Y cells are involved in beta-amyloid secretion.

Amyloid plaques, composed mainly of the 39-43 amino acid betaA4 peptide, are a characteristic feature of Alzheimer's disease. Generation of betaA4 by proteolytic processing of the amyloid precursor protein (APP) is thought to occur in a pathway that includes the activity of two as yet unknown proteases, with beta-secretase cleaving at the N terminus and gamma-secretase releasing the C terminus of betaA4. Inhibition studies and the finding that cell surface APP can serve as a direct precursor of betaA4 suggest that the endosomal/lysosomal compartment is involved in the proteolysis of APP into betaA4. In this study we targeted APP695 chimeric proteins directly into the endosomal/lysosomal compartment. This decreased the amount of released betaA4, while the generation of the betaA4 N terminus continued. APP695 proteins were constructed also, which carried sorting signals responsible for recycling between the trans-Golgi network (TGN) and the cell surface. These proteins were processed into secreted betaA4 at even higher levels than wild-type APP695. Moreover, retention of APP695 proteins in the endoplasmic reticulum led to neither betaA4 secretion nor to processing by beta-secretase in human SH-SY5Y neuroblastoma cells. These data suggest that a beta-cleavage activity resides in a late endosomal compartment and that a gamma-cleavage occurs in early endosomes, resulting in the generation of betaA4 peptides with the majority ending at residue 40.

Amino Acid Sequence↗
PubMed1997

Expression and analysis of heparin-binding regions of the amyloid precursor protein of Alzheimer's disease.

Deletion mutagenesis studies have suggested that there are two domains within APP which bind heparan sulphate. These domains have been cloned and expressed in the yeast Pichia pastoris. Both recombinant proteins bound to heparin. One domain (APP316-447) was further characterised by binding studies with peptides encompassing this region. Peptides homologous to APP316-346 and APP416-447 were found to bind heparin. Circular dichroism studies show that APP416-447 shifted towards an alpha-helical conformation in the presence of heparin. This study suggests that heparin-binding domains may lie within regions high in alpha-helical structure.

Alzheimer Disease↗
PubMed1997

Down-regulation of the amyloid protein precursor of Alzheimer's disease by antisense oligonucleotides reduces neuronal adhesion to specific substrata.

The hallmark of Alzheimer's disease is the cerebral deposition of amyloid which is derived from the amyloid precursor protein (APP). The function of APP is unknown but there is increasing evidence for the role of APP in cell-cell and/or cell-matrix interactions. Primary cultures of murine neurons were treated with antisense oligonucleotides to down-regulate APP. This paper presents evidence that APP mediates a substrate-specific interaction between neurons and extracellular matrix components collagen type I, laminin and heparan sulphate proteoglycan but not fibronectin or poly-L-lysine. It remains to be determined whether this effect is the direct result of APP-matrix interactions, or whether an intermediatry pathway is involved.

Alzheimer Disease↗
PubMed1997

Alzheimer's disease-associated presenilin 1 in neuronal cells: evidence for localization to the endoplasmic reticulum-Golgi intermediate compartment.

The recently identified Alzheimer's disease-associated presenilin 1 and 2 (PS1 and PS2) genes encode two homologous multi membrane-spanning proteins. Rabbit antibodies to the N-terminal domain of PS1 detected PS1 in human neuroblastoma SH-SY5Y wild type and PS1 transfectants (SY5Y-PS1) as well as in mouse P19, in CHO-K1 and CHO-APP770 transfected cells, in rat cerebellar granule and hippocampal neurons, and astrocytes. Immunoblotting detected full-length protein of 50 kDa, and a major presumptive cleavage product of 30 kDa. The immunofluorescence pattern resembled labeling of the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) marker protein ERGIC-53. PS1 distribution showed slight condensation after brefeldin A and more marked condensation after incubation of cells at 16 degrees C, characteristic of the ERGIC compartment. Double labeling showed colocalization of ERGIC-53 with PS1 in the SY5Y-PS1 cells. PS1 labeling of SY5Y-PS1 and P19 cells showed overlap of the cis-Golgi marker p210 and colocalization with p210 after brefeldin A which causes redistribution of p210 to the ERGIC. Expression of PS1 did not change in level or cellular distribution during development of neurons in culture. Double labeling for the amyloid precursor protein (APP) and PS1 on SY5Y-PS1 cells and CHO-APP770 cells showed some overlap under control conditions. These results indicate that PS1 is a resident protein of the ERGIC and could be involved in trafficking of proteins, including APP, between the ER and Golgi compartments.

Alzheimer Disease↗
PubMed1997

Reactive oxygen species and Alzheimer's disease.

Although a consensus that Alzheimer's disease (AD) is a single disease has not been reached yet, the involvement of the amyloid precursor protein (APP) and betaA4 (A beta) in the pathologic changes advances our understanding of the underlying molecular alterations. Increasing evidence implicates oxidative stress in the neurodegenerative process of AD. This hypothesis is based on the toxicity of betaA4 in cell cultures, and the findings that aggregation of betaA4 can be induced by metal-catalyzed oxidation and that free oxygen radicals may be involved in APP metabolism. Another neurological disorder, familial amyotrophic lateral sclerosis (FALS), supports our view that AD and FALS may be linked through a common mechanism. In FALS, SOD-Cu(I) complexes are affected by hydrogen peroxide and free radicals are produced. In AD, the reduction of Cu(II) to Cu(I) by APP involves an electron-transfer reaction and could also lead to a production of hydroxyl radicals. Thus, copper-mediated toxicity of APP-Cu(II)/(I) complexes may contribute to neurodegeneration in AD.

Aging↗
PubMed1997

Intracellular and secreted Alzheimer beta-amyloid species are generated by distinct mechanisms in cultured hippocampal neurons.

Cerebral plaques containing beta-amyloid (beta A4) represent an invariant pathological feature of Alzheimer disease (AD). beta A4 is proteolytically generated from its parent molecule, amyloid precursor protein (APP). In non-neuronal cells beta A4 has been shown to be secreted via a pH-sensitive and endocytosis-dependent pathway, and this process, when occurring in the brain, is considered to play an important role in AD. In neurons the mechanisms of beta A4 production are not known. Here we have analyzed these mechanisms by expressing human APP and its mutant versions in hippocampal neurons using the Semliki forest virus system. We show that these cells initially generate two pools of beta A4, an extracellular and an intracellular, and only the extracellular pool is produced via a pH-sensitive and endocytosis-dependent pathway. Thus, hippocampal neurons are able to utilize an alternate pathway to produce intracellular beta A4. We also show that a common feature of two types of APP mutations ("Swedish" and "London") implicated in early-onset AD is their increased production of C-terminally elongated beta A4 (beta 42), both intra- and extracellularly. Since neurons are the only cells that produce substantial levels of intracellular beta A4 and also the main victims in AD, these findings may provide an important link between beta A4 and neurodegeneration.

Amyloid beta-Peptides↗