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Biomedical subjects

C L Masters

Publications and source records attributed to C L Masters.

At least 91 records · Page 5Linked to original sources
PubMed1999

Amyloid precursor protein of Alzheimer's disease: evidence for a stable, full-length, trans-membrane pool in primary neuronal cultures.

We and colleagues have shown that the amyloid protein precursor of Alzheimer's disease (APP) is distributed along the surface of neurites of fixed but nonpermeabilized neurons in primary culture in a segmental pattern, which shows colocalization with some markers of adhesion patches. This is in contrast to the diffuse pattern of immunoreactivity seen after permeabilization. We have also recently demonstrated that the APP in these surface patches is likely to be integral to the membrane rather than secreted and re-adsorbed, based on alkali stripping experiments and on soluble APP adsorption experiments. Total cellular APP has previously been shown to have a short half-life of approximately 30-60 min. We confirm this in neurons in primary culture in pulse-chase experiments using short labelling times. Additionally, we provide evidence that a separate, stable pool of neuronal APP can be demonstrated in pulse-chase experiments using long labelling times. Experiments involving inhibition of protein synthesis suggest that this corresponds with the surface, segmental pool. Metabolic labelling followed by surface biotinylation and two-stage precipitation demonstrates that the surface APP is trans-membrane and full-length (not carboxyl-terminal truncated), and confirms that the surface APP belongs to the stable pool. This two-stage procedure is necessary as the surface APP appears to be present in low copy number, and is difficult to detect by direct labelling. This information is consistent with a role for APP in stable cell-matrix or cell-cell interactions.

Alzheimer Disease↗
PubMed1999

Exacerbation of copper toxicity in primary neuronal cultures depleted of cellular glutathione.

Perturbations to glutathione (GSH) metabolism may play an important role in neurodegenerative disorders such as Alzheimer's, Parkinson's, and prion diseases. A primary function of GSH is to prevent the toxic interaction between free radicals and reactive transition metals such as copper (Cu). Due to the potential role of Cu in neurodegeneration, we examined the effect of GSH depletion on Cu toxicity in murine primary neuronal cultures. Depletion of cellular GSH with L-buthionine-[S,R]-sulfoximine resulted in a dramatic potentiation of Cu toxicity in neurons without effect on iron (Fe) toxicity. Similarly, inhibition of glutathione reductase (GR) activity with 1,3-bis(2-chloroethyl)-1-nitrosurea also increased Cu toxicity in neurons. To determine if the Alzheimer's amyloid-beta (Abeta) peptide can affect neuronal resistance to transition metal toxicity, we exposed cultures to nontoxic concentrations of Abeta25-35 in the presence or absence of Cu or Fe. Abeta25-35 pretreatment was found to deplete neuronal GSH and increase GR activity, confirming the ability of Abeta to perturb neuronal GSH homeostasis. Abeta25-35 pretreatment potently increased Cu toxicity but had no effect on Fe toxicity. These studies demonstrate an important role for neuronal GSH homeostasis in selective protection against Cu toxicity, a finding with widespread implications for neurodegenerative disorders.

Amyloid beta-Peptides↗
PubMed1999

Intracellular accumulation of detergent-soluble amyloidogenic A beta fragment of Alzheimer's disease precursor protein in the hippocampus of aged transgenic mice.

To study amyloid beta-protein (A beta) production and aggregation in vivo, we created two transgenic (Tg) mouse lines expressing the C-terminal 100 amino acids of human amyloid precursor protein (APP): Tg C100.V717F and Tg C100.WT. Western blot analysis showed that human APP-C100 and A beta were produced in brain and some peripheral tissues and A beta was produced in serum. Using antibodies specific for the A beta C terminus we found that Tg C100.V717F produced a 1.6-fold increase in A beta42/A beta40 compared with Tg C100.WT. Approximately 30% of total brain A beta (approximately 122 ng/g of wet tissue) was water-soluble. The remaining 70% of A beta partitioned into the particulate fraction and was completely sodium dodecyl sulfate-soluble. In contrast, human Alzheimer's disease brain has predominantly sodium dodecyl sulfate-insoluble A beta. Immunohistochemistry with an A beta(5-8) antibody showed that A beta or A beta-containing fragments accumulated intracellularly in the hippocampus of aged Tg C100.V717F mice. The soluble A beta levels in Tg brain are similar to those in normal human brain, and this may explain the lack of microscopic amyloid deposits in the Tg mice. However, this mouse model provides a system to study the intracellular processing and accumulation of A beta or A beta-containing fragments and to screen for compounds directed at the gamma-secretase activity.

Aging↗
PubMed1999

Alzheimer's disease: correlation of the suppression of beta-amyloid peptide secretion from cultured cells with inhibition of the chymotrypsin-like activity of the proteasome.

Peptide aldehyde inhibitors of the chymotrypsin-like activity of the proteasome (CLIP) such as N-acetyl-Leu-Leu-Nle-H (or ALLN) have been shown previously to inhibit the secretion of beta-amyloid peptide (A beta) from cells. To evaluate more fully the role of the proteasome in this process, we have tested the effects on A beta formation of a much wider range of peptide-based inhibitors of CLIP than published previously. The inhibitors tested included several peptide boronates, some of which proved to be the most potent peptide-based inhibitors of beta-amyloid production reported so far. We found that the ability of the peptide aldehyde and boronate inhibitors to suppress A beta formation from cells correlated extremely well with their potency as CLIP inhibitors. Thus, we conclude that the proteasome may be involved either directly or indirectly in A beta formation.

Aldehydes↗
PubMed1999

The hydrophobic core sequence modulates the neurotoxic and secondary structure properties of the prion peptide 106-126.

The neurodegeneration seen in spongiform encephalopathies is believed to be mediated by protease-resistant forms of the prion protein (PrP). A peptide encompassing residues 106-126 of human PrP has been shown to be neurotoxic in vitro. The neurotoxicity of PrP106-126 appears to be dependent upon its adoption of an aggregated fibril structure. To examine the role of the hydrophobic core, AGAAAAGA, on PrP106-126 toxicity, we performed structure-activity analyses by substituting two or more hydrophobic residues for the hydrophilic serine residue to decrease its hydrophobicity. A peptide with a deleted alanine was also synthesized. We found all the peptides except the deletion mutant were no longer toxic on mouse cerebellar neuronal cultures. Circular dichroism analysis showed that the nontoxic PrP peptides had a marked decrease in beta-sheet structure. In addition, the mutants had alterations in aggregability as measured by turbidity, Congo red binding, and fibril staining using electron microscopy. These data show that the hydrophobic core sequence is important for PrP106-126 toxicity probably by influencing its assembly into a neurotoxic structure. The hydrophobic sequence may similarly affect aggregation and toxicity observed in prion diseases.

Alanine↗
PubMed1999

Proteolytic fragments of Alzheimer's disease-associated presenilin 1 are present in synaptic organelles and growth cone membranes of rat brain.

Previous studies have demonstrated the molecular linkage of three causative genes for early-onset Alzheimer's disease: the presenilin 1 gene on chromosome 14, the presenilin 2 gene on chromosome 1, and the amyloid precursor protein gene on chromosome 21. In the present study, we have investigated the distributions of the approximately 20-kDa C-terminal and approximately 30-kDa N-terminal fragments of presenilin 1 and the amyloid precursor protein in rat brain and compared them with the distribution of several marker proteins. The fragments of presenilin 1 are present in synaptic plasma membranes, neurite growth cone membranes, and small synaptic vesicles of rat brain. Both proteolytic fragments are coenriched in the corresponding tissue fractions. Based on this observation, it seems likely that N- and C-terminal presenilin 1 fragments form a functional unit while remaining associated. In contrast to a predominant subcellular localization of presenilin 1 to the endoplasmic reticulum and Golgi apparatus in different cell lines, our results indicate that rat brain presenilin 1 fragments exit from these biosynthetic compartments to reach synaptic organelles in neurons.

Alzheimer Disease↗
PubMed1999

Neurite-outgrowth regulating functions of the amyloid protein precursor of Alzheimer's disease.

Many studies have shown that breakdown of the amyloid protein precursor (APP) to produce the amyloid protein is an important step in the pathogenic mechanism which causes Alzheimer's disease (AD). However, little is known about the normal function of APP. Developmental studies show that APP expression increases during the period of brain development when neurite outgrowth and synaptogenesis is maximal. APP is expressed highly within growing neurites and in growth cones, and purified APP has been shown to stimulate neurite outgrowth from cells in culture. Thus APP may regulate neurite outgrowth or synaptogenesis in vivo. APP is actively secreted from many cells, and the C-terminally secreted APP has been shown to associate with components of the extracellular matrix, such as the heparan sulphate proteoglycans (HSPGs). Two putative heparin-binding domains on APP have been reported. Binding of HSPGs to an N-terminal heparin-binding domain (HBD-1) stimulates the effect of substrate-bound APP on neurite outgrowth. In the mature nervous system, APP may play an important role in the regulation of wound repair. It is highly likely that studies on the normal functions of APP will shed further light on aspects of the pathogenesis of AD.

Journal Article↗
PubMed1999

Irregular distribution of cytochrome c oxidase protein subunits in aging and Alzheimer's disease.

This study aims to investigate the cellular distribution of human cytochrome c oxidase (COX) subunit II (CII) and COX subunit IV (CIV) in Alzheimer's disease relative to control brains. The levels of CIV and CII proteins in the cerebellar Purkinje cells were reduced in age-matched controls relative to young controls and in the Alzheimer's disease group relative to both age-matched and young controls. Results suggest that these age-associated changes are more marked in Alzheimer's disease.

Aging↗
PubMed1998

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

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

Amyloid Precursor Protein Secretases↗
PubMed1998

Processing of the Alzheimer's disease amyloid precursor protein in Pichia pastoris: immunodetection of alpha-, beta-, and gamma-secretase products.

betaA4 (Abeta) amyloid peptide, a major component of Alzheimer's disease (AD) plaques, is a proteolytic product of the amyloid precursor protein (APP). Endoproteases, termed beta- and gamma-secretase, release respectively the N- and C-termini of the peptide. APP default secretion involves cleavage within the betaA4 domain by alpha-secretase. To study the conservation of APP processing in lower eukaryotes, the yeast Pichia pastoris was transfected with human APP695 cDNA. In addition to the full-length integral transmembrane protein found in the cell lysate, soluble/secreted APP (sAPP) was detected in the culture medium. Most sAPP comprised the N-terminal moiety of betaA4 and corresponds to sAPPalpha, the product of alpha-secretase. The culture medium also contained minor secreted forms detected by a monoclonal antibody specific for sAPPbeta (the ectodomain released by beta-secretase cleavage). Analysis of the cell lysates with specific antibodies also detected membrane-associated C-terminal fragments corresponding to the products of alpha and beta cleavages. Moreover, immunoprecipitation of the culture medium with three antibodies directed at distinct epitopes of the betaA4 domain yielded a 4 kDa product with the same electrophoretic mobility as betaA4 synthetic peptide. These results suggest that the alpha-, beta-, and gamma-secretase cleavages are conserved in yeast and that P. pastoris may offer an alternative to mammalian cells to identify the proteases involved in the generation of AD betaA4 amyloid.

Alzheimer Disease↗
PubMed1998

Two novel presenilin-1 mutations (Ser169Leu and Pro436Gln) associated with very early onset Alzheimer's disease.

Mutations in the presenilin-1 (PS-1) gene account for the majority of early onset autosomal-dominant familial Alzheimer's disease (FAD) cases. We identified three missense mutations in the coding sequence of the PS-1 gene in three early onset (EO), FAD pedigrees. Alzheimer's disease was confirmed in one pedigree by autopsy. Mutation analysis of PCR products amplified from genomic DNA templates of affected individuals showed two novel mutations resulting in Ser169Leu and Pro436Gln and one known mutation resulting in Glu318Gly. The two new mutations are located within predicted transmembrane domains three (TM-3) and seven (TM-7), and are associated with a very early age of onset which is consistent with a marked loss of function of the protein. The age of onset in the pedigree with Glu318Gly mutation was similar to that reported previously in a separate pedigree with this mutation.

Adult↗
PubMed1998

Survival of cultured neurons from amyloid precursor protein knock-out mice against Alzheimer's amyloid-beta toxicity and oxidative stress.

Studies on the amyloid precursor protein (APP) have suggested that it may be neuroprotective against amyloid-beta (Abeta) toxicity and oxidative stress. However, these findings have been obtained from either transfection of cell lines and mice that overexpress human APP isoforms or pretreatment of APP-expressing primary neurons with exogenous soluble APP. The neuroprotective role of endogenously expressed APP in neurons exposed to Abeta or oxidative stress has not been determined. This was investigated using primary cortical and cerebellar neuronal cultures established from APP knock-out (APP-/-) and wild-type (APP+/+) mice. Differences in susceptibility to Abeta toxicity or oxidative stress were not found between APP-/- and APP+/+ neurons. This observation may reflect the expression of the amyloid precursor-like proteins 1 and 2 (APLP1 and APLP2) molecules and supports the theory that APP and the APLPs may have similar functional activities. Increased expression of cell-associated APLP2, but not APLP1, was detected in Abeta-treated APP-/- and APP+/+ cultures but not in H2O2-treated cultures. This suggests that the Abeta toxicity pathway differs from other general forms of oxidative stress. These findings show that Abeta toxicity does not require an interaction of the Abeta peptide with the parental molecule (APP) and is therefore distinct from prion protein neurotoxicity that is dependent on the expression of the parental cellular prion protein.

Alzheimer Disease↗
PubMed1998

Effects of the amyloid protein precursor of Alzheimer's disease and other ligands of the LDL receptor-related protein on neurite outgrowth from sympathetic neurons in culture.

The amyloid protein precursor (APP) of Alzheimer's disease can stimulate neurite outgrowth in vitro. The receptor responsible for this effect has not been identified. Kunitz protease inhibitor (KPI)-containing forms of APP bind to the low-density lipoprotein receptor-related protein (LRP). As LRP may regulate neurite outgrowth, we examined whether the effects of APP are mediated by LRP. Inhibitors of LRP decreased neurite outgrowth from chick sympathetic neurons. Most LRP ligands (alpha2-macroglobulin, lactoferrin, and lipoprotein lipase) stimulated outgrowth. However, in soluble form, the KPI-containing APP751 was a weak inhibitor of outgrowth. In substrate-bound form, both APP751 and APP695 (which does not bind to LRP) stimulated outgrowth. Thus the effect of substrate-bound APP on neurite outgrowth is not mediated by LRP.

Alzheimer Disease↗
PubMed1998

Copper-binding amyloid precursor protein undergoes a site-specific fragmentation in the reduction of hydrogen peroxide.

The extracellular domain of transmembrane Abeta amyloid precursor protein (APP) has a Cu(II) reducing activity upon Cu(II) binding associated with the formation of a new disulfide bridge. The complete assignment of the disulfide bond revealed the involvement of cysteines 144 and 158 around copper-binding histidine residues. The vulnerability of APP-Cu(I) complexes to reactive oxygen species was elaborated as a site-specific and random fragmentation of APP in a time-dependent manner and at low concentrations of H2O2. Analysis of the specific reaction revealed the generation of C-terminal polypeptides, containing the Abeta domain. APP catalyzed the reduction of H2O2 and oxidation of Cu(I) to Cu(II) in a "peroxidative" reaction in vitro. The resulting bound copper-hydroxyl radical intermediate [APP-Cu(II)(.OH)] then likely participated in a Fenton type of reaction with radical formation as a prerequisite for protein degradation. Evidence from two observations suggests that the reaction takes place in two phases. Bathocuproine, a trapping agent for Cu(I), abolished the initial fragmentation, and chelation of Cu(II) by DTPA (diethylenetriaminepentaacetic acid) interrupted the reaction cascade induced by H2O2 at later stages. Consequently, the results suggest that a cytotoxic gain-of-function of APP-Cu(I) complexes might result in a perturbation of free radical homeostasis. What significance such a perturbation may have for the pathogenesis of Alzheimer's disease remains to be determined.

Amino Acid Sequence↗
PubMed1998

No association of presenilin-1 intronic polymorphism and Alzheimer's disease in Australia.

We screened 703 Australian subjects for an intronic polymorphism in the presenilin-1 (PS-1) gene. PS-1 intronic allele 1 homozygosity was not associated with individuals with early- or late-onset sporadic Alzheimer's disease (EOAD or LOAD). Carriers for the PS-1 intronic allele 1 were also not associated with significantly increased risk for AD regardless of gender. Our results for the Australian population are consistent with those of recent reports for other populations and do not support the conclusion that the PS-1 intronic polymorphism is associated with AD.

Adult↗