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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↗
PubMed1993

Modifications of transthyretin in amyloid fibrils: analysis of amyloid from homozygous and heterozygous individuals with the Met30 mutation.

The finding of individuals homozygous for FAP I (familial amyloidotic polyneuropathy, transthyretin TTRMet30) with amyloid deposits in the vitreous body, gave us access to a unique material lacking wild type transthyretin and contaminating proteins. Amyloid TTR is modified in several ways. Besides the full-length protein and its dimer form, two smaller bands were identified by SDS-PAGE and protein sequencing. One corresponded to a peptide starting at amino acid Thr49, the other was a mixture of two peptides starting at positions 1 and 3 in a 3:1 ratio. Upon reduction the amount of the TTR dimer decreased, the monomer amount increased, and the resulting monomers became available for carboxymethylation. Moreover, the mobility of the small band, which includes Cys10, increased upon reduction. This cysteine seemed to be involved in an interchain disulfide bridge both between intact TTR molecules and between small fragments. The same pattern was found in heterozygous fibril material although smaller amounts of the truncated peptides were found. Fibrils were formed both from normal and mutated TTR in heterozygotes. The significance of our results for amyloid formation is discussed.

Amino Acid Sequence↗
PubMed2005

Amyloid-beta aggregates formed at polar-nonpolar interfaces differ from amyloid-beta protofibrils produced in aqueous buffers.

The deposition of aggregated amyloid-beta (Abeta) peptides in the brain as senile plaques is a pathological hallmark of Alzheimer's disease (AD). Several lines of evidence indicate that fibrillar and, in particular, soluble aggregates of these 40- and 42-residue peptides are important in the etiology of AD. Recent studies also stress that amyloid aggregates are polymorphic and that a single polypeptide can fold into multiple amyloid conformations. Here we review our recent reports that Abeta(1-40) in vitro can form soluble aggregates with predominant beta-structures that differ in stability and morphology. One class of aggregates involved soluble Abeta protofibrils, prepared by vigorous overnight agitation of monomeric Abeta(1-40) in low ionic strength buffers. These aggregates were quite stable and disaggregated to only a limited extent on dilution. A second class of soluble Abeta aggregates was generated at polar-nonpolar interfaces. Aggregation in a two-phase system of buffer over chloroform occurred more rapidly than in buffer alone. In buffered 2% hexafluoroisopropanol (HFIP), microdroplets of HFIP were formed and the half-time for aggregation was less than 10 minutes. Like Abeta protofibrils, these interfacial aggregates showed increased thioflavin T fluorescence and were rich in beta-structure by circular dichroism. However, electron microscopy and atomic force microscopy revealed very different morphologies. The HFIP aggregates formed initial globular clusters that progressed over several days to soluble fibrous aggregates. When diluted out of HFIP these aggregates initially were very unstable and disaggregated completely within 2 minutes. However, their stability increased as they progressed to fibers. It is important to determine whether similar interfacial Abeta aggregates are produced in vivo.

Amyloid beta-Peptides↗
PubMed1994

Immunolocalization of serum amyloid A and AA amyloid in lysosomes in murine monocytoid cells: confocal and immunogold electron microscopic studies.

Murine AA amyloid (AA) protein represents the amino-terminal two-third portion of SAA2, one of the isoforms of serum amyloid A. Whether plasma membrane-bound or lysosomal enzymes in activated murine monocytoid cells degrade SAA2 to generate amyloidogenic AA-like peptides is not clearly understood, although AA has been localized in the lysosomes. Here we show, using confocal and immunogold microscopy (IEM), that both SAA and AA localize in lysosomes of activated monocytoid cells from amyloidotic mice. Rabbit anti-mouse AA IgG (RAA) and two monoclonal antibodies against murine lysosome-associated membrane proteins (LAMP-1 and LAMP-2) were used to immunolocalize SAA/AA and lysosomes, respectively. Confocal analysis co-localized both anti-RAA and anti-LAMP-1/LAMP-2 reactivities in the perikaryal organelles which by IEM proved to be electron-dense lysosomes. LAMP-1/LAMP-2-specific gold particles were also localized on lysosomal and perikaryal AA. The results suggest sequestration of SAA into the lysosomes. Since monocytoid cells are not known to phagocytose native amyloid fibrils, our results implicate lysosomes in AA formation.

Amyloidosis↗
PubMed1999

Amyloid phenotype characterization of transgenic mice overexpressing both mutant amyloid precursor protein and mutant presenilin 1 transgenes.

Doubly transgenic mice (PSAPP) overexpressing mutant APP and PS1 transgenes were examined using antibodies to Abeta subtypes and glial fibrillary acidic protein (GFAP). Visible Abeta deposition began primarily in the cingulate cortex of PSAPP mice at approximately 10 weeks of age. By 6 months, the mice had extensive amyloid deposition throughout the hippocampus and cortex as well as other regions of the brain. Highly congophilic deposits consisting of N-terminal normal and modified forms of Abeta were identified, reminiscent of those found in human AD brain. Both immunohistochemistry and mass spectrometry showed that Abeta42 forms were underrepresented relative to Abeta40, and Abeta43 was undetectable. Deposits were associated with prominent gliosis which increased with age, but in 14-month-old PSAPP mice, GFAP immunoreactivity in the vicinity of amyloid deposits was substantially reduced compared to APP littermates. These mice have considerable utility in the study of the amyloid phenotype of AD.

Age Factors↗
PubMed1986

Hereditary cerebral amyloid angiopathy: the amyloid fibrils contain a protein which is a variant of cystatin C, an inhibitor of lysosomal cysteine proteases.

Hereditary Cerebral Hemorrhage With Amyloidosis is an autosomal dominant form of amyloidosis restricted to the cerebral vasculature. We have previously demonstrated that the amyloid protein subunit is similar to Cystatin C (or gamma-trace), an inhibitor of lysosomal cysteine proteinases, and homologous to kininogens. High pressure liquid chromatography tryptic fingerprint analysis was developed to distinguish Cystatin C from the amyloid protein. Moreover, we isolated and sequenced tryptic peptides in which the differences were detected. The data prove that the amyloid protein is 10 residues shorter than Cystatin C and has one amino acid substitution at residue 58.

Amino Acid Sequence↗
PubMed1991

Sequencing of exons 16 and 17 of the beta-amyloid precursor protein gene in 14 families with early onset Alzheimer's disease fails to reveal mutations in the beta-amyloid sequence.

A mutation within exon 17 at codon 717 of the beta-amyloid protein precursor (APP) gene is one cause of early onset familial Alzheimer's disease. Direct sequencing of exons 16 and 17 of the beta-amyloid precursor protein gene in 14 families with familial early onset Alzheimer's disease without the known pathogenic mutation (APP717) failed to reveal other mutations within the beta-amyloid sequence in this form of the disorder.

Adult↗
PubMed1995

In vitro production of beta-amyloid in smooth muscle cells isolated from amyloid angiopathy-affected vessels.

Our recent results indicate that in Alzheimer's disease (AD) amyloid angiopathy, smooth muscle cells are responsible for beta-amyloid deposition in the vascular wall. Aged dogs have been shown to develop beta-amyloid angiopathy similar to that in AD. Thus, we used brain and peripheral vessels from aged and young dogs to isolate cells of the vascular wall: smooth muscle cells, fibroblasts, and endothelial cells, and to study their ability to produce beta-protein. We demonstrate that only myocytes from aged animals cultured for up to 4 weeks accumulate beta-protein-immunoreactive material intracellularly, in the form of fibrillar and amorphous deposits.

Aging↗
PubMed1995

Amyloid beta-peptide in cerebrospinal fluid in individuals with the Swedish Alzheimer amyloid precursor protein mutation.

The neuropathological hallmarks of Alzheimer's disease (AD) are amyloid-containing plaques and neurofibrillary tangles. The main constituent of senile plaques is amyloid beta-peptide (A beta) and in recent years, pathogenic mutations in the amyloid precursor protein (APP) gene have been discovered in some AD families. The APP670/671 mutation, found in a Swedish AD family, has revealed over-production of A beta as one pathogenic mechanism for the development of AD. In the present study we have used an immunoassay to measure A beta levels in cerebrospinal fluid (CSF) from APP670/671 mutation-carriers and non-carriers. A correlation was seen between decrease in A beta levels and duration of disease although no difference was found in levels of A beta between the groups (14.5 +/- 3.3 ng/ml versus 14.9 +/- 2.3 ng/ml).

Adult↗
PubMed2005

Inhibition of amyloid fibril formation of beta-amyloid peptides via the amphiphilic surfactants.

Beta-amyloid peptide (A beta) is the major proteinacious constituent of senile plaques in Alzheimer's disease and is believed to be responsible for the neurodegeneration process associated with the disease. While the actual size of the aggregated species responsible for A beta neurotoxicity and fibrillogenesis mechanism(s) remain unknown, retardation of A beta aggregation still holds assurance as an effective strategy in reducing A beta-elicited toxicity. The research presented here is aimed at examining the inhibitory effect of two amphiphilic surfactants, di-C6-PC and di-C7-PC, on the in vitro fibrillogenesis process of A beta(1--40) peptides at physiological pH (pH 7.2). Using ThT-induced fluorescence, turbidity, Congo red binding, and circular dichroism spectroscopy studies, our research demonstrated that the inhibition of A beta(1--40) fibril formation was di-C6-PC and di-C7-PC concentration-dependent. The best inhibitory action on fibril formation was observed when A beta was incubated with di-C7-PC at 100 microM over time. We believe that the outcome from this work will aid in the development and/or design of potential inhibitory agents against amyloid formation associated with Alzheimer's and other amyloid diseases.

Amyloid beta-Peptides↗
PubMed2005

Decreased serum amyloid beta(1-42) autoantibody levels in Alzheimer's disease, determined by a newly developed immuno-precipitation assay with radiolabeled amyloid beta(1-42) peptide.

BACKGROUND: Autoantibodies against amyloid beta (A beta) peptide found in patients with Alzheimer's disease (AD) also occur naturally in the general population independently of the cognitive status. METHODS: We compared serum A beta(1-42) autoantibody levels (A beta(1-42)-AL) of 96 AD patients and 30 healthy elderly control subjects (HC), assessing their diagnostic value for AD with a newly developed immunoprecipitation assay with radiolabeled A beta(1-42) peptide. RESULTS: We found a highly significant decrease of A beta(1-42)-AL in AD patients (p = .001) independently of age, cognitive status, and apolipoprotein E epsilon4 carrier status. Amyloid beta(1-42) autoantibody levels were correlated with gender in AD, with a higher level occurring in women. When A beta(1-42) autoantibody sensitivity (specificity) was set >80%, specificity (sensitivity) was below 50% to correctly allocate patients and healthy control subjects. CONCLUSIONS: Our data indicate a potentially pathophysiologic decrease of serum A beta(1-42) antibodies in AD. Amyloid beta(1-42) antibodies in the serum alone, however, seem not to be useful as a diagnostic marker of AD.

Aged↗
PubMed1992

Complement inhibitor C4-binding protein in amyloid deposits containing serum amyloid P in Alzheimer's disease.

Complement C4-binding protein (C4BP) is a plasma protein that complexes with serum amyloid P and is involved in inhibition of the complement cascade. We used immunochemical methods to demonstrate C4BP in brains of subjects with Alzheimer's disease and normal controls. C4BP antibodies stained cerebral amyloid deposits that were indistinguishable from those recognized by antibodies to serum amyloid P or by thioflavin S. Immunoblots of solubilized proteins from cerebral cortex, cerebral microvessels and cerebrospinal fluid showed specific antibody reactivity to 70-90 kDa bands. Our results suggest the involvement of complement regulatory proteins in the pathogenesis of Alzheimer's disease.

Aged↗
PubMed1997

Isoform-specific binding of human apolipoprotein E to the non-amyloid beta component of Alzheimer's disease amyloid.

The non-A beta component (NAC) of Alzheimer's disease amyloid is a newly discovered 35 amino acid peptide found to be closely linked to the beta-amyloid fibrils in senile plaques. Apolipoprotein E (apoE) is another prominent constituent of senile plaques. In vitro studies have shown that apoE binds beta-amyloid (A beta) with high avidity, but it is unknown to what extent apoE interacts with NAC. We examined the interactions between apoE and NAC and found that apoE bound synthetic NAC, forming a complex that resisted reducing agents and separation on SDS-PAGE. The complex could be formed using apoE from either purified human very low density lipoprotein (VLDL) particles, unfractionated human cerebrospinal fluid (CSF), or recombinant protein. The binding was established within 15 min upon mixing, and the interaction between NAC and apoE was dose-dependent and specific as revealed by competition experiments. The NAC-apoE complex was affected by non-physiological pH, but not by reducing agents such as DTT or beta-mercaptoethanol. ApoE exists in different isoforms of which the apoE3 genotype is the most frequent. Notably, the apoE4 genotype has been linked to late-onset Alzheimer's disease. This study presents evidence that apoE3 as well as apoE4 bind NAC, but the binding to apoE4 is about twice as strong as to apoE3. The isoform-specific binding of NAC to apoE may thus play an important role in amyloidogenesis and in the sequestering of apoE in senile plaques during the progress of Alzheimer's disease.

Alzheimer Disease↗
PubMed1991

Amyloid plaques, neurofibrillary tangles and neuronal loss in brains of transgenic mice overexpressing a C-terminal fragment of human amyloid precursor protein.

Alzheimer's disease (AD) affects more than 30% of people over 80 years of age. The aetiology and pathogenesis of this progressive dementia is poorly understood, but symptomatic disease is associated histopathologically with amyloid plaques, neurofibrillary tangles and neuronal loss primarily in the temporal lobe and neocortex of the brain. The core of the extracellular plaque is a derivative of the amyloid precursor protein (APP), referred to as beta/A4, and contains the amino-acid residues 29-42 that are normally embedded in the membrane-spanning region of the precursor. The cellular source of APP and the relationship of its deposition to the neuropathology of AD is unknown. To investigate the relationship between APP overexpression and amyloidogenesis, we have developed a vector to drive expression specifically in neurons of a C-terminal fragment of APP that contains the beta/A4 region, and have used a transgenic mouse system to insert and express this construct. We report here that overexpression of this APP transgene in neurons is sufficient to produce extracellular dense-core amyloid plaques, neurofibrillary tangles and neuronal degeneration similar to that in the AD brain.

Alzheimer Disease↗
PubMed2002

Assembling amyloid fibrils from designed structures containing a significant amyloid beta-peptide fragment.

The amyloid plaque, consisting of amyloid beta-peptide (Abeta) fibrils surrounded by dystrophic neurites, is an invariable feature of Alzheimer's disease. The determination of the molecular structure of Abeta fibrils is a significant goal that may lead to the structure-based design of effective therapeutics for Alzheimer's disease. Technical challenges have thus far rendered this goal impossible. In the present study, we develop an alternative methodology. Rather than determining the structure directly, we design conformationally constrained peptides and demonstrate that only certain 'bricks' can aggregate into fibrils morphologically identical to Abeta fibrils. The designed peptides include variants of a decapeptide fragment of Abeta, previously shown to be one of the smallest peptides that (1) includes a pentapeptide sequence necessary for Abeta-Abeta binding and aggregation and (2) can form fibrils indistinguishable from those formed by full-length Abeta. The secondary structure of these bricks is monitored by CD spectroscopy, and electron microscopy is used to study the morphology of the aggregates formed. We then made various residue deletions and substitutions to determine which structural features are essential for fibril formation. From the constraints, statistical analysis of side-chain pair correlations in beta-sheets and experimental data, we deduce a detailed model of the peptide strand alignment in fibrils formed by these bricks. Our results show that the constrained decapeptide dimers rapidly form an intramolecular, antiparallel beta-sheet and polymerize into amyloid fibrils at low concentrations. We suggest that the formation of an exposed beta-sheet (e.g. an Abeta dimer formed by interaction in the decapeptide region) could be a rate-limiting step in fibril formation. A theoretical framework that explains the results is presented in parallel with the data.

Alzheimer Disease↗
PubMed1997

Rifampicin inhibits the toxicity of pre-aggregated amyloid peptides by binding to peptide fibrils and preventing amyloid-cell interaction.

Rifampicin and its analogues, p-benzoquinone and hydroquinone, inhibited the toxicity of preformed aggregates of human islet amyloid polypeptide, amylin, to rat pheochromocytoma PC12 cells, when preincubated with the aggregated peptide before addition to cell cultures. Immunofluorescence microscopy showed that they prevented the adhesion of amylin aggregates to the cell surface, and this effect was induced probably by their binding to peptide fibrils during preincubation. Other quinone derivatives, i.e., p-methoxyphenol, AA-861 and idebenone, failed to inhibit the toxicity and cell-surface adhesion of amylin aggregates. Rifampicin analogues also inhibited the toxicity of pre-aggregated amyloid beta1-42 peptides, suggesting a common toxic mechanism of different amyloid peptides and their therapeutic potential for several amyloidoses.

Amyloid↗
PubMed1995

Full-length and truncated Alzheimer amyloid precursors in chromaffin granules: solubilization of membrane amyloid precursor is mediated by an enzymatic mechanism.

The amyloid beta peptide (A beta) of Alzheimer disease is derived from the proteolytic processing of the amyloid precursor proteins (APPs), which are considered type I transmembrane proteins. Here we report that the soluble fraction of isolated adrenal medullary chromaffin granules (CG), a model neuronal secretory vesicle system, contains an antigen that immunochemically and on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was indistinguishable from full-length APP. A truncated APP fragment with intact A beta sequence was also detected in the soluble fraction of CG. In vitro experiments showed that full-length APP was solubilized from CG membranes at 37 degrees C as a function of pH, with a peak of activity between pH 8.5 and pH 9.0. Solubilization of full-length APP was inhibited by several protease inhibitors, including aprotinin, cystatin, and iodoacetamide, by the divalent cations Ca2+ and Zn2+, and by preheating of the membranes. These results are consistent with and suggest the involvement of an enzymatic mechanism in the solubilization of potentially amyloidogenic full-length APP. Production of A beta from a transmembrane APP predicts a proteolytic cleavage within the lipid bilayer, a site relatively inaccessible to proteases. Thus, the detected soluble, potentially amyloidogenic, full-length APP may be a substrate for the proteases producing A beta. The detection of soluble APP with intact A beta sequence in secretory vesicles is consistent with the extracellular topology of amyloid depositions.

Adrenal Medulla↗
PubMed1996

High-resolution separation of amyloid beta-peptides: structural variants present in Alzheimer's disease amyloid.

In Alzheimer's disease (AD), one of the cardinal neuropathological signs is deposition of amyloid, primarily consisting of the amyloid beta-peptide (Abeta). Structural variants of AD-associated Abeta peptides have been difficult to purify by high-resolution chromatographic techniques. We therefore developed a novel chromatographic protocol, enabling high-resolution reverse-phase liquid chromatography (RPLC) purification of Abeta variants displaying very small structural differences. By using a combination of size-exclusion chromatography and the novel RPLC protocol, Abeta peptides extracted from AD amyloid were purified and subsequently characterized. Structural analysis by microsequencing and electrospray-ionization mass spectrometry revealed that the RPLC system resolved a complex mixture of Abeta variants terminating at either residue 40 or 42. Abeta variants differing by as little as one amino acid residue could be purified rapidly to apparent homogeneity. The resolution of the system was further illustrated by its ability to separate the structural isomers of Abeta1-40. The present chromatography system might provide further insight into the role of N-terminally and posttranslationally modified Abeta variants, because each variant can now be studied individually.

Alzheimer Disease↗