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C G Glabe

Publications and source records attributed to C G Glabe.

At least 19 recordsLinked to original sources
PubMed2001

Antibody-mediated phagocytosis of the amyloid beta-peptide in microglia is differentially modulated by C1q.

Microglial ingestion of the amyloid beta-peptide (Abeta) has been viewed as a therapeutic target in Alzheimer's disease, in that approaches that enhance clearance of Abeta relative to its production are predicted to result in decreased senile plaque formation, a proposed contributor to neuropathology. In vitro, scavenger receptors mediate ingestion of fibrillar Abeta (fAbeta) by microglia. However, the finding that cerebral amyloid deposition in a transgenic mouse model of Alzheimer's disease was diminished by inoculation with synthetic Abeta has suggested a possible therapeutic role for anti-Abeta Ab-mediated phagocytosis. Microglia also express C1qR(P), a receptor for complement protein C1q, ligation of which in vitro enhances phagocytosis of immune complexes formed with IgG levels below that required for optimal FcR-mediated phagocytosis. The data presented here demonstrate FcR-dependent ingestion of Abeta-anti-Abeta complexes (IgG-fAbeta) by microglia that is a function of the amount of Ab used to form immune complexes. In addition, C1q incorporated into IgG-fAbeta enhanced microglial uptake of these complexes when they contained suboptimal levels of anti-Abeta Ab. Mannose binding lectin and lung surfactant protein A, other ligands of C1qR(P), also enhanced ingestion of suboptimally opsonized IgG-fAbeta, whereas control proteins did not. Our data suggest that C1qR(P)-mediated events may promote efficient ingestion of Abeta at low Ab titers, and this may be beneficial in paradigms that seek to clear amyloid via FcR-mediated mechanisms by minimizing the potential for destructive Ab-induced complement-mediated processes.

Amyloid beta-Peptides↗
PubMed2000

A rapidly diverging EGF protein regulates species-specific signal transduction in early sea urchin development.

The macromolecules mediating species-specific events during fertilization and early development and their molecular evolution are only beginning to be understood. We screened sea urchin ovary mRNA for species-specific gene products using representational differential analysis to identify unique transcripts in Strongylocentrotus franciscanus that are absent or divergent from a closely related species, S. purpuratus. One of the transcripts identified by this screening process is SfEGF-II, which contains four EGF repeats. SfEGF-II is orthologous to the previously reported genes S. purpuratus SpEGF-II and Anthocidaris crassispina AcEGF-II, encoding exogastrulation-inducing peptides (EGIP). EGF peptides derived from EGIP induce exogastrulation, a classical developmental defect, when added to embryos prior to gastrulation. The first three EGF repeats (EGF1-3) share 50 to 60% identity among the three species, but the fourth repeat (EGF4) is more divergent, displaying only 30% identity. Analysis of the sequence divergence indicates that the EGF-II genes display a relatively high nonsynonymous-to-synonymous ratio, a significant excess of radical compared to conservative amino acid substitutions, and a lack of polymorphism within SfEGF-II, indicating that these genes have been subjected to positive Darwinian selection. Recombinant EGF3 from S. franciscanus induces exogastrulation in both S. franciscanus and S. purpuratus. In contrast, recombinant EGF4 from both S. franciscanus and S. purpuratus induces exogastrula in a species-specific manner. In hybrid embryos, both species of EGF4 induce exogastrulation, suggesting that the receptor for this EGF molecule is expressed from both parental genomes during development. Both EGF3 and EGF4 induce the phosphorylation of membrane proteins of the blastula stage embryos, but EGF4 stimulates phosphorylation of proteins only in membranes prepared from homologous embryos, suggesting that it utilizes a unique pathway involving a species-specific receptor for EGF4. Thus, species-specific events of gastrulation and early development may be controlled by these rapidly diverging EGF molecules, through a novel species-specific signal transduction pathway.

Amino Acid Sequence↗
PubMed2000

A conformation change in the carboxyl terminus of Alzheimer's Abeta (1-40) accompanies the transition from dimer to fibril as revealed by fluorescence quenching analysis.

Alzheimer's disease is characterized by the presence of insoluble, fibrous deposits composed principally of amyloid beta (Abeta) peptide. A number of studies have provided information on the fibril structure and on the factors affecting fiber formation, but the details of the fibril structure are not known. We used fluorescence quenching to investigate the solvent accessibility and surface charge of the soluble Abeta(1-40) dimer and amyloid fibrils. Analogs of Abeta(1-40) containing a single tryptophan were synthesized by substituting residues at positions 4, 10, 34, and 40 with tryptophan. Quenching measurements in the dimeric state indicate that the amino-terminal analogs (AbetaF4W and AbetaY10W) are accessible to polar quenchers, and the more carboxyl-terminal analog AbetaV34W is less accessible. AbetaV40W, on the other hand, exhibits a low degree of quenching, indicating that this residue is highly shielded from the solvent in the dimeric state. Correcting for the effect of reduced translational and rotational diffusion, fibril formation was associated with a selective increase in solvent exposure of residues 34 and 40, suggesting that a conformation change may take place in the carboxyl-terminal region coincident with the dimer to fibril transition.

Alzheimer Disease↗
PubMed2000

Complement component C1q modulates the phagocytosis of Abeta by microglia.

Recent studies showing that microglia internalize the amyloid beta-peptide (Abeta) suggest that these cells have the potential for clearing Abeta deposits in Alzheimer's disease, and mechanisms that regulate the removal of Abeta may therefore be of clinical interest. Previous studies from this laboratory showing that C1q enhances phagocytosis of cellular targets by rat microglia prompted the current investigations characterizing the effects of C1q on microglial phagocytosis of Abeta. Microglia were shown to phagocytose Abeta1-42, in agreement with observations of other investigators. Uptake of Abeta1-42 was observed for concentrations of 5-50 microM, and phagocytosis of peptides containing (14)C or fluorescein (FM) labels was not affected by the interaction of microglia with C1q-coated surfaces. However, inclusion of C1q (125 nM-1.4 microM) in solutions of 50 microM Abeta1-42 inhibited the uptake of (14)C-Abeta1-42 and FM-Abeta1-42, suggesting that C1q blocks the interaction of Abeta with microglia. Uptake of Abeta was partially blocked by the scavenger receptor ligands polyinosinic acid and maleylated BSA. Inhibition of Abeta uptake by C1q may contribute to the accumulation of fibrillar, C1q-containing plaques that occurs in parallel with disease progression. These data suggest that mechanisms which interfere with the binding of C1q to Abeta may be of therapeutic value both through inhibition of the inflammatory events resulting from complement activation and via altered access of Abeta sites necessary for ingestion by microglia.

Alzheimer Disease↗
PubMed1999

Binding of Zn(II), Cu(II), and Fe(II) ions to Alzheimer's A beta peptide studied by fluorescence.

Binding of Zn(II), Cu(II) and Fe(II) ions to A beta1-40, A beta1-42 and a single tryptophan mutant of Abeta 1-40 in solution at pH 7.4 was studied by fluorescent titration. Job plots and fitting of titration curves revealed formation of 1:1 and 1:2 peptide-metal complexes. For dimeric peptides A beta1-40 and A betaF4W the order of metal to peptide affinities is Fe < Cu > Zn, which is in agreement with the Irving-Williams series of complex stability. The affinity of A beta1-42 for Fe increases dramatically upon aggregation: K(D) changes from ca. 100 to ca. 0.2 microM.

Alzheimer Disease↗
PubMed1999

Intracellular accumulation of insoluble, newly synthesized abetan-42 in amyloid precursor protein-transfected cells that have been treated with Abeta1-42.

Our early study indicates that intracellular Abeta1-42 aggregates are resistant to degradation and accumulate as an insoluble residue in lysosomes, where they alter the normal catabolism of amyloid precursor protein (APP) to cause the accumulation of insoluble APP and amyloidogenic fragments. In this study, we examined whether the addition of exogenous Abeta1-42 also leads to the accumulation of newly synthesized intracellular Abeta. Here we describe that newly synthesized Abeta, especially Abetan-42, is generated from metabolically labeled APP and accumulates in the insoluble fraction of cell lysates after Abeta1-42 treatment. These results suggest that intracellular Abeta may derive from a solid phase, intracellular pathway. In contrast to the pathway that primarily produces secreted Abeta1-40, the solid-phase intracellular pathway preferentially produces Abetan-42 with ragged amino termini. Biochemical studies and amino acid sequencing analyses indicate that these intracellular Abeta also share the same types of Abeta structures that accumulate in the brain of Alzheimer's disease patients, suggesting that a significant fraction of the amyloid deposits in Alzheimer's disease may arise by this solid-phase pathway.

Alzheimer Disease↗
PubMed1999

Endogenous presenilin 1 redistributes to the surface of lamellipodia upon adhesion of Jurkat cells to a collagen matrix.

Most familial early-onset Alzheimer's disease cases are caused by mutations in the presenilin 1 (PS1) gene. Subcellular localization of the endogenous PS1 is essential for understanding its function, interactions with proteins, and role in Alzheimer's disease. Although numerous studies revealed predominant localization of PS1 to endoplasmic reticulum and Golgi, there are conflicting reports on the localization of PS1 to the cell surface. We found that endogenous PS1 is highly expressed in T lymphocytes (Jurkat cells). Using a variety of methods, we present evidence that endogenous PS1 is localized to the cell surface in addition to intracellular membrane compartments. Moreover, PS1 appeared in high levels on the surface of lamellipodia upon adhesion of the cells to a collagen matrix. The redistribution of PS1 in adhered cells was strikingly similar to that of the well characterized adhesion protein CD44. Cell surface PS1 formed complexes in vivo with actin-binding protein filamin (ABP-280), which is known to form bridges between cell surface receptors and cytoskeleton and mediate cell adhesion and cell motility. Taken together, our results suggest a role of PS1 in cell adhesion and/or cell-matrix interaction.

Algorithms↗
PubMed1998

gamma-secretase cleavage is distinct from endoplasmic reticulum degradation of the transmembrane domain of the amyloid precursor protein.

One of the critical cleavage events that generates Alzheimer's amyloid Abeta peptide occurs within the transmembrane domain (TMD) of the amyloid precursor protein (APP) and is carried out by a poorly understood enzyme activity known as gamma-secretase. To investigate this processing, a probe molecule, H26-57C, was constructed containing the TMD of APP flanked immediately on each side by unique epitope tags. H26-57C-transfected cells secrete a approximately 2.9-kDa fragment, indicating that the lumenal and cytosolic domains of APP are not required for gamma-secretase processing. Pulse-chase experiments indicate that the probe turns over with a half-life of 8 min. No degradation intermediates are detected during the chase period, indicating that TMD turnover is a highly processive mechanism. The protease inhibitors, ALLN and MG132, cause a dramatic (50-fold) increase in the steady-state amount of the probe. All of the inhibitors that prevent degradation of the probe in the rough endoplasmic reticulum increase the amount of the approximately 2.9-kDa fragment that is secreted into the media and also causes a similar increase the secretion of 4 kDa Abeta from APP-transfected cells. These results indicate that the system responsible for the degradation of the probe in the rough endoplasmic reticulum and the intramembrane cleavage by gamma-secretase that produces soluble, secreted Abeta are distinct and opposing processes.

Amino Acid Sequence↗
PubMed1998

Amyloid beta protein is internalized selectively by hippocampal field CA1 and causes neurons to accumulate amyloidogenic carboxyterminal fragments of the amyloid precursor protein.

A critical issue concerning Alzheimer's disease is its selectivity, which leads to cellular degeneration in certain brain areas but not in others, and whether this pathogenic selectivity involves products of the amyloid precursor protein (APP). Here, we show that the amyloid beta protein Abeta1-42 is accumulated gradually and is retained intact by field CA1, but not by other subdivisions, of organotypic hippocampal slice cultures. In contrast, the slightly shorter Abeta1-40 peptide was not sequestered selectively. Sequestration of Abeta1-42 was followed by the build-up of carboxyterminal fragments of the endogenous precursor protein that were identified by immunoprecipitation. Unlike the peptide uptake, this induction appeared to be stochastic at the cellular level. In addition, the APP fragments were distributed more broadly within the CA1 pyramidal neurons than the sequestered Abeta1-42, and they appeared to be localized to synaptic terminals in the molecular layer of the dentate gyrus and in the stratum lacunosum-moleculare of the subfield CA3. Concentrations of synaptophysin, a presynaptic marker, decreased as the number of neurons producing amyloidogenic species increased. These results indicate that exogenous Abeta1-42 sets into motion a sequence that involves 1) selective uptake of the peptide by vulnerable cells at risk in Alzheimer's disease, 2) markedly enhanced production of amyloidogenic precursor material, and 3) slow deterioration of central synapses.

Amyloid beta-Peptides↗
PubMed1998

Membrane fusion is induced by a distinct peptide sequence of the sea urchin fertilization protein bindin.

Fertilization in the sea urchin is mediated by the membrane-associated acrosomal protein bindin, which plays a key role in the adhesion and fusion between sperm and egg. We have investigated the structure/function relationship of an 18-amino acid peptide fragment "B18," which represents the minimal membrane binding motif of the protein and resembles a putative fusion peptide. The peptide was found to mimic the behavior of its parent protein bindin with respect to (a) its high affinity for lipid bilayers, (b) the ability to aggregate and fuse vesicles, (c) the binding of Zn2+ by a histidine-rich motif, (d) the tendency to self-assemble, and (e), as indicated earlier, the adhesion to cell surface polysaccharides. Fluorescence and light scattering assays were used here to monitor peptide-induced lipid mixing, leakage, and aggregation of large unilamellar sphingomyelin/cholesterol vesicles. For these activities, B18 requires the presence of Zn2+ ions, with which it forms oligomeric complexes and assumes a partially alpha-helical conformation, as observed by circular dichroism. We conclude that aggregation and fusion involves a "trans-complex" between peptides on apposing vesicles that are connected by Zn2+ bridges.

Amino Acid Sequence↗
PubMed1998

Loss of endosomal/lysosomal membrane impermeability is an early event in amyloid Abeta1-42 pathogenesis.

Previous studies have implicated the failure to degrade aggregated Abeta1-42 in late endosomes or secondary lysosomes as a mechanism for the accumulation of beta-amyloid in Alzheimer's disease. We examined the consequences of intracellular accumulation of Abeta1-42 on the integrity of the endosomal/lysosomal compartment by monitoring Lucifer Yellow fluorescence and the release of lysosomal hydrolases into the soluble, cytosolic fraction. In control cells, the Lucifer Yellow fluorescence is observed as punctate staining in a perinuclear distribution with no apparent cytoplasmic fluorescence, consistent with its localization in late endosomes or secondary lysosomes. After incubation with Abeta1-42 for 6 hr, a loss of lysosomal membrane impermeability is observed as evidenced by redistribution of the fluorescence to a diffuse, cytoplasmic pattern. The loss of lysosomal membrane impermeability is correlated with Abeta1-42 accumulation, since incubation of the cells with the nonaccumulating isoform of amyloid, Abeta1-40, does not induce leakage. The same results were obtained using the release of soluble lysosomal hydrolases, cathepsin D and beta-hexosaminidase, into the cytosol as an assay for the leakage of lysosomal contents. Together, our results suggest that the loss of lysosomal membrane impermeability may be an early event in Abeta pathogenesis, and provide an explanation for the miscompartmentalization of extracellular and cytoplasmic components observed in Alzheimer's disease (AD). The release of hydrolases may further cause the breakdown of the cytoskeleton and the blebbing of the plasma membrane, and the leakage of heparan sulfate glycosaminoglycans from the lysosome may ultimately promote the assembly of tau into neurofibrillary tangles (NFT).

Amyloid beta-Peptides↗
PubMed1997

Complement activation by cross-linked truncated and chimeric full-length beta-amyloid.

The activation of complement by beta-amyloid (A beta) has been implicated in the local inflammatory response in Alzheimer's disease. To assess the structural parameters required for this activation, beta-sheet-containing fibrils of A beta1-28 were induced by low pH and then chemically cross-linked to constrain the beta-sheet conformation. Chimeric A beta peptides with a substituted C-terminal sequence derived from two different transmembrane proteins were also assessed for the ability to form fibrils rich in beta-sheet structure and to activate complement. Both the cross-linked A beta1-28 and the chimeric A beta peptides were strong activators of the classical complement pathway. These results suggest that the C-terminal residues (29-42) of A beta facilitate fibril assembly required for complement activation but do not contain the interaction sites required for complement activation, further supporting the hypothesis that C1q binds to the N-terminal hydrophilic domain of A beta, and that a fibrillar beta-sheet-rich conformation is required for effective binding and activation of C1.

Amino Acid Sequence↗
PubMed1997

Acceleration of amyloid fibril formation by specific binding of Abeta-(1-40) peptide to ganglioside-containing membrane vesicles.

The interaction of Alzheimer's Abeta peptide and its fluorescent analogue with membrane vesicles was studied by spectrofluorometry, Congo Red binding, and electron microscopy. The peptide binds selectively to the membranes containing gangliosides with a binding affinity ranging from 10(-6) to 10(-7) M depending on the type of ganglioside sugar moiety. This interaction appears to be ganglioside-specific as under our experimental conditions (neutral pH, physiologically relevant ionic strength), no Abeta binding was observed to ganglioside-free membranes containing zwitterionic or acidic phospholipids. Importantly, the addition of ganglioside-containing vesicles to the peptide solution dramatically accelerates the rate of fibril formation as compared with that of the peptide alone. The present results strongly suggest that the membrane-bound form of the peptide may act as a specific "template" (seed) that catalyzes the fibrillogenesis process in vivo.

Alzheimer Disease↗
PubMed1997

Soluble amyloid Abeta-(1-40) exists as a stable dimer at low concentrations.

Recent studies have implicated the amyloid Abeta peptide and its ability to self-assemble as key factors in the pathogenesis of Alzheimer's disease. Relatively little is known about the structure of soluble Abeta or its oligomeric state, and the existing data are often contradictory. In this study, we used intrinsic fluorescence of wild type Abeta-(1-40), fluorescence resonance energy transfer (FRET), and gel filtration chromatography to examine the structure of Abeta-(1-40) in solution. We synthesized a series of mono-substituted fluorescent Abeta-(1-40) derivatives to use as donors and acceptors in FRET experiments. We selected fluorescent peptides that exhibit aggregation properties comparable to wild type Abeta for analysis in donor-acceptor pairs; two labeled with 5-(2-((iodoacetyl)amino)ethyl)aminonaphthylene-1-sulfonic acid at Cys-25 or Cys-34 and fluorescein maleimide at Cys-4 or Cys-7. Another peptide containing a Trp substitution at position 10 was used as an acceptor for the intrinsic Tyr fluorescence of wild type Abeta-(1-40). Equilibrium studies of the denaturation of Abeta-(1-40) by increasing concentrations of dimethyl sulfoxide (Me2SO) were conducted by monitoring fluorescence, with a midpoint value for the unfolding transition of both the substituted and wild type peptides at among 40 and 50% Me2SO. Abeta-(1-40) is well solvated and largely monomeric in Me2SO as evidenced by a lack of FRET. When donor and acceptor Abeta derivatives are mixed together in Me2SO and then diluted 10-fold into aqueous Tris-HCl buffer at pH 7.4, efficient FRET is observed immediately for all pairs of fluorescent peptides, indicating that donor-acceptor dimers exist in solution. FRET is abolished by the addition of an excess of unlabeled Abeta-(1-40), demonstrating that the fluorescent peptides interact with wild type Abeta-(1-40) to form heterodimers that do not exhibit FRET. The Abeta-(1-40) dimers appear to be very stable, because no subunit exchange is observed after 24 h between fluorescent homodimers. Gel filtration confirms that nanomolar concentrations of 14C-labeled Abeta-(1-40) and fluorescein-labeled Abeta-(1-40) elute at the same dimeric position as wild type Abeta-(1-40), suggesting that soluble Abeta-(1-40) is also dimeric at more physiologically plausible concentrations.

Amyloid beta-Peptides↗
PubMed1997

Identification of a 97-kDa heat shock protein from S. franciscanus ovaries with 94% amino acid identity to the S. purpuratus egg surface receptor for sperm.

To identify species-specific regions of the sea urchin egg surface receptor for sperm, we cloned and sequenced the cDNA from S. franciscanus (Sf) ovary mRNA that is homologous to the S. purpuratus (Sp) sperm receptor sequence. The Sf cDNA contains an 886-amino-acid open reading frame (ORF) that is 96% identical at the nucleotide level to the Sp sperm receptor sequence over 2.9 kb. In contrast to the published Sp sequence, the Sf sequence does not encode a signal peptide or transmembrane domain. However, like the Sp sperm receptor sequence, the Sf protein has substantial similarity to the 70-kDa heat shock family of proteins and appears to encode a member of a newly identified subfamily of hsp70-related proteins designated hsp110/SSE. A BLAST search using the 5' end of the published Sp cDNA sequence as a query indicates that a segment of approximately 400 bp, which encodes the putative signal sequence, is 95% identical to Sp mitochondrial DNA. Resequencing of the Sp cDNA clone failed to confirm the presence of the published transmembrane and cytoplasmic domains. These results suggest that the published sequence of the Sp egg receptor for sperm may contain errors in the critical regions that were believed to encode an amino-terminal signal sequence, transmembrane domain, and cytoplasmic tail and that the protein products encoded by these cDNAs are highly related to mammalian cytoplasmic hsp110s.

Amino Acid Sequence↗
PubMed1997

Preferential adsorption, internalization and resistance to degradation of the major isoform of the Alzheimer's amyloid peptide, A beta 1-42, in differentiated PC12 cells.

A central question in Alzheimer's disease (AD) is the role of amyloid in pathogenesis. Recent discoveries implicating the longer A beta 1-42 form of amyloid in pathogenesis led us to characterize the interaction of A beta with cells to elucidate differences that might account for these observations. We characterized the adsorption, internalization and degradation of radiolabeled A beta in NGF-differentiated PC12 cells under conditions that are not acutely toxic. All A beta peptides examined absorb to the surface of PC12 cells and are internalized; however the adsorption and internalization of A beta 1-42 is significantly greater than that of A beta 1-40 and A beta 1-28. The adsorption of A beta 1-42 is decreased by treatment of the cells with neuraminidase, but not heparitinase. The fate of the internalized A beta 1-42 is also very different than shorter A beta peptides; a fraction of the internalized A beta 1-42 accumulates intracellularly and is resistant to degradation for at least 3 days while A beta 1-40 and shorter peptides are eliminated with a half life of about 1 h. A beta 1-42 does not appear to inhibit lysosomal hydrolases, since A beta 1-28 is degraded at the same rate in the presence or absence of A beta 1-42. The intracellular A beta 1-42 is located in a dense organellar compartment and colocalizes with the lysosomal markers Lucifer Yellow and horseradish peroxidase. These data indicate that there are significant differences in the cell surface adsorption, internalization and catabolism of A beta 1-42 compared to A beta 1-40 and A beta 1-28. These differences may be important for the preferential accumulation of the longer A beta 1-42 isoform and its association with AD pathogenesis.

Adsorption↗
PubMed1997

Presenilin-1 immunoreactivity is localized intracellularly in Alzheimer's disease brain, but not detected in amyloid plaques.

The identification of the cellular and subcellular regions of the Alzheimer's disease brain to which the presenilin-1 (PS-1) protein localizes is expected to contribute to an understanding of its pathophysiological role. Toward this end, we have derived an affinity-purified antibody to a synthetic PS-1 peptide. In this report, we demonstrate that this antibody, called SW2, specifically recognizes full-length, 47-kDa PS-1 protein from rat primary cortical neurons, from a human neuronal cell line, and from human brain extracts on Western immunoblots. Immunohistochemical analysis of postmortem brain tissue from control and Alzheimer's disease patients using this SW2 antibody indicates an intracellular localization of PS-1 immunoreactivity with prominent perinuclear characteristics in neurons, with staining also detected in neuritic processes. Despite various treatments of the tissue sections, no PS-1 immunoreactivity was observed in neuritic plaques, the hallmark pathological lesions of Alzheimer's disease. In addition, confocal microscopic analysis of immunostained cultured primary neurons revealed a prominent perinuclear pattern of PS-1 immunoreactivity consistent with vesicular localization, as well as punctate staining in neuritic processes.

Alzheimer Disease↗