Amyloid Deposition In Alzheimer's Disease Research Articles

Chrysamine-G binding to Alzheimer and control brain: Autopsy study of a new amyloid probe

Chrysamine-G (CG) is a carboxylic acid analogue of Congo red, a histologic dye which stains amyloid. CG binds to the beta-amyloid protein of Alzheimer's disease (AD) in vitro and partitions into the brain of normal mice. In this study, we demonstrate increased binding of [ 14C]CG to homogenates of several regions of AD brain as compared to control. The total binding of CG to AD brain was approximately two- to threefold that of control brain. The cerebellum could be used as an internal standard for each brain as CG binding to cerebellum did not differ between AD and control. The binding of [ 14C]CG correlated with numbers of senile plaques and neurofibrillary tangles. In addition, CG could be used to stain cerebrovascular amyloid in tissue sections. These results suggest that CG may prove useful as an in vivo probe of amyloid deposition in AD.

The NACP/synuclein gene: chromosomal assignment and screening for alterations in Alzheimer disease

The major component of the vascular and plaque amyloid deposits in Alzheimer disease is the amyloid β peptide (Aβ). A second intrinsic component of amyloid, the NAC (non-Aβ component of amyloid) peptide, has recently been identified, and its precursor protein was named NACP. A computer homology search allowed us to establish that the human NACP gene was homologous to the rat synuclein gene. We mapped the NACP/ synuclein gene to chromosome 4 and cloned three alternatively spliced transcripts in lymphocytes derived from a normal subject. We analyzed by RT-PCR and direct sequencing the entire coding region of the NACP/synuclein gene in a group of patients with familial early onset Alzheimer disease. No mutation was found in 26 unrelated patients. Further studies are required to investigate the implication of the NACP/ synuclein gene in Alzheimer disease.

Characterization of Apolipoprotein J-Alzheimer's Aβ Interaction

The main component of Alzheimer's amyloid deposits, A beta, has been found also as a soluble (sA beta) normal constituent of biological fluids and cell culture supernatants. Whether or not sA beta is the immediate precursor of A beta, it is clear that peptides with the same amino acid sequence can have both fibrillar and non-fibrillar conformations. The interconversion mechanism from one form to another is presently under intensive investigation. We have previously described that (i) a synthetic peptide A beta 1-40 immobilized on affinity matrices was able to retrieve apolipoprotein J (apoJ) from plasma and cerebrospinal fluid; and (ii) the interaction of sA beta with apoJ occurs in vivo, as demonstrated by the ability of anti-apoJ to co-precipitate sA beta from normal cerebrospinal fluid. We have characterized the binding between A beta 1-40 and apoJ and found that the interaction is saturable, specific, and reversible. The dissociation constant of 2 x 10(-9) M is indicative of high affinity binding. The stoichiometry of the reaction is 1:1; apoJ has five times more affinity for fresh A beta 1-40 than for the aggregated peptide. Competitive inhibition studies carried out with apolipoprotein E (isoforms E2, E3, and E4), transthyretin, vitronectin, and alpha 1-antichymotrypsin indicate that the complex apoJ.A beta 1-40 cannot be dissociated by any of these competitors at physiologic concentrations. The data strongly suggest that apoJ plays an important role as a carrier protein for sA beta.

Apolipoprotein E4 genotype is not a risk factor for systemic AA amyloidosis or familial amyloid polyneuropathy

Apolipoprotein E (apoE) is present in the amyloid deposits in Alzheimer's disease (AD) and in acquired and hereditary systemic amyloidosis, and apoE genotype is an important risk factor for late onset AD. In order to determine whether it is also a risk factor for developing reactive systemic amyloid A protein (AA) amyloidosis or transthyretin (TTR) Met 30 familial amyloid polyneuropathy (FAP), apoE genotype was determined in 32 patients with AA amyloid and 31 patients with FAP. Prevalence of the apoE e4 allele was not significantly different than in a normal population. There was also no correlation between apoE genotype and either the time from presentation of the underlying disease to the diagnosis of AA amyloid or the age of onset of symptoms in FAP. These results suggest that apoE genotype does not affect susceptibility to or expression of AA amyloidosis or TTR Met 30 FAP.

Elevated Aβ levels in Alzheimer's disease brain are associated with selective accumulation of Aβ42 in parenchymal amyloid plaques and both Aβ40 and Aβ42 in cerebrovascular deposits

Amyloid deposits in Alzheimer's disease (AD) brains are composed primarily of the protein Aβ, a proteolytic fragment of the β-amyloid precursor protein. Antibodies were generated to peptides corresponding to the five COOH-terminal residues of Aβ proteins ending either at residue 40 (anti-Aβ36–40) or 42 (anti-Aβ 38-42). The selectivity of these antibodies for their respective peptides was determined by antibody preabsorption followed by ELISA or immunohistochemistry. Anti-Aβ38–42 labeled core-containing and diffuse plaques in both frozen and paraffin sections. Anti-Aβ36–40 labeled a smaller number of core-containing plaques and no diffuse plaques. Vascular and perivascular amyloid contained Aβ proteins ending both at residue 40 and 42. Forms ending at residue 40 comprised a larger fraction of both vascular and perivascular amyloid compared to parenchymal amyloid, suggesting that parenchymal amyloid and vascular/perivascular amyloid are derived by two distinct mechanisms. In addition, Aβ proteins immunoaffinity purified from plaque-enriched human brains were resolved by a novel electrophoretic method into two predominant forms, co-migrating with synthetic Aβ1–40and Aβ1–42. The quantity of Aβ protein solubilized from AD brains was greater than that from age-matched controls, demonstrating a preferential accumulation of this soluble Aβ in association with amyloid deposition. Our results demonstrate immunohistochemical and electrophoretic methods for defining further the processes contributing to Aβ amyloidogenesis.

Metabolism of the “Swedish” amyloid precursor protein variant in Madin-Darby canine kidney cells.

The 4-kDa beta-amyloid peptide (A beta), a major constituent of parenchymal amyloid deposits in Alzheimer's disease, is derived from larger amyloid precursor proteins (APP). We have examined the metabolism of APP in Madin-Darby canine kidney cells stably transfected with cDNA encoding either wild-type human APP-695 or human APP-695 that harbors the Swedish double mutation associated with familial early-onset Alzheimer's disease. Although approximately 90% of total soluble APP secreted from wild-type cells is secreted basolaterally following cleavage at the alpha-secretase site, soluble derivatives cleaved near or at the amino terminus of A beta (presumably the "beta-secretase" site) are preferentially secreted into the apical compartment of SWE cells. Concomitantly, levels of a specific A beta-containing carboxyl-terminal fragment are elevated in SWE cell lysates. Using domain-specific biotinylation and release assays, we failed to detect a beta-secretase-generated soluble derivative (APPs beta) released from the surface of SWE cells. However, APPs beta can be detected in SWE cell lysates, consistent with "beta-secretase" cleavage occurring in an intracellular compartment. Finally, we demonstrate that A beta is secreted into the basolateral compartment of SWE cells and that the majority of these A beta-related species contains an amino-terminal aspartate residue (+1).

The Alzheimer A beta peptide develops protease resistance in association with its polymerization into fibrils.

An intriguing property of the polypeptide constituents of amyloid is that they apparently can escape the proteolytic mechanisms that normally catalyze turnover and prevent abnormal tissue accumulation of polypeptides. Here, we demonstrate that the A beta peptide, the principal component of cerebrovascular amyloid deposits in Alzheimer's disease, becomes resistant to an array of proteases as a result of structural changes associated with its polymerization into amyloid fibrils. It is further demonstrated that fibril formation per se does not lead to protease resistance but probably structural changes associated with polymerization. The results suggest that higher order structural changes, regulated by the primary structure, enable amyloidogenic polypeptides to escape proteolytic degradation and accumulate in tissues.

Interleukin-1β dissociates β-amyloid precursor protein and β-amyloid peptide secretion

A heightened production of interleukin 1β(IL-1β) has been reported in microglial-associated amyloid deposits in Alzheimer's disease (AD) brains. These plaques are composed predominantly of β/A4 peptide derived from β-amyloid precursor protein (βAPP). We demonstrate that short-term (1 h) IL-1β-treatment increases βAPP s secretion and concomitantly decreases cell-associated βAPP in human H4 neuroglioma cells. Long-term (5 h) IL-1β treatment did not alter secreted or cell-associated βAPP content. In contrast, the secretion of β/A4-containing epitope was not affected by short-term IL-1β stimulation; however, long-term IL-1β treatment decreased the amount of β/A4-containing epitope secreted from the cells. These results show that IL-1β modifies the processing and secretion of βAPP to exacerbate perhaps the neuropathology of AD.

Amyloid-associated proteins alpha 1-antichymotrypsin and apolipoprotein E promote assembly of Alzheimer beta-protein into filaments.

The protease inhibitor alpha 1-antichymotrypsin and the lipid transport protein apolipoprotein E (apoE) are intimately associated with the 42-amino-acid beta-peptide (A beta) in the filamentous amyloid deposits of Alzheimer's disease. We report here that these two amyloid-associated proteins serve a strong stimulatory role in the polymerization of A beta into amyloid filaments. Addition of either alpha 1-anti-chymotrypsin or apoE to the A beta peptide promoted a 10- to 20-fold increase in filament formation, with apoE-4, the isoform recently linked to the development of late-onset Alzheimer's disease, showing the highest catalytic activity. These and other experiments suggest that Alzheimer amyloid deposits arise when A beta is induced to form filaments by amyloid-promoting factors (pathological chaperones) expressed in certain brain regions.

Structural Determinants of the Alzheimer's Amyloid β‐Peptide

The hallmark event of Alzheimer's disease (AD) is the deposition of amyloid as insoluble fiber masses in extracellular neuritic plaques and around the walls of cerebral blood vessels. The main component of amyloid is a hydrophobic peptide, named amyloid beta-peptide (beta A4), which results from the processing of a much longer membrane amyloid precursor protein (APP). This review focuses on the structural features of beta A4 and the factors that determine beta A4 insolubilization. Theoretical and experimental studies of the primary structure of beta A4 have shown that it is composed of a completely hydrophobic C-terminal domain, which adopts beta-strand structure, and an N-terminal region, whose sequence permits different secondary structures. In fact, this region can exist as an alpha-helical or beta-strand conformation depending on the environmental condition (pH and hydrophobicity surrounding the molecule). The effects of pH and hydrophobicity on beta A4 structure may elucidate the mechanisms determining its aggregation and amyloid deposition in AD.

Effect of acid predissolution on fibril size and fibril flexibility of synthetic beta-amyloid peptide

beta-amyloid peptide (A beta) is the major protein component of senile plaques and cerebrovascular amyloid deposits in Alzheimer's patients. Several researchers have demonstrated that A beta is neurotoxic in in vitro and in vivo systems. Peptide aggregation state and/or conformation might play a significant role in determining the toxicity of the peptide. The size and flexibility of fibrils formed from the synthetic peptide beta (1-39), corresponding to the first 39 residues of A beta, were determined. Samples were prepared either directly from lyophilized peptide or diluted from a 10 mg/ml stock solution in 0.1% trifluoroacetic acid (TFA). All samples had a final peptide concentration of 0.5 mg/ml, a final pH of 7.4, and a final NaCl concentration of 0.14 M. The molecular weight and linear density of the fibrils increased with increasing pre-incubation time in TFA, based on static light scattering measurements. Analysis of the angular dependence of the intensity of scattered light indicated that the fibrils were semi-flexible chains and that the fibril flexibility decreased with increasing pre-incubation time in TFA. There was a concomitant change in phase behavior from precipitation to gelation with the decrease in fibril flexibility.

Solution structure of residues 1-28 of the amyloid beta-peptide.

The three-dimensional solution structure of residues 1-28 of the amyloid beta-peptide was determined using nuclear magnetic resonance spectroscopy, distance geometry, and molecular dynamic techniques. The nuclear magnetic resonance data used to derive the structure consisted of nuclear Overhauser enhancements, vicinal coupling constants, and temperature coefficients of the amide-NH chemical shifts. The beta-peptide is the major proteinaceous component of amyloid deposits in Alzheimer's disease. In membrane-like media, the peptide folds to form a predominately alpha-helical structure with a bend centered at residue 12. The side chains of histidine-13 and lysine-16 are close, residing on the same face of the helix. Their proximity may constitute a binding motif with the heparan sulfate proteoglycans. The molecular details of the structure shown here could facilitate the design of rational treatments to curtail the binding of heparan sulfate proteoglycans or to prevent an alpha-helix-->beta-sheet conversion that may occur during the early stages of amyloid formation in Alzheimer's disease.

Concentration of Serum Amyloid P Component in the CSF as a Possible Marker of Cerebral Amyloid Deposits in Alzheimer′s Disease

Serum amyloid P component (SAP) is a normal plasma protein produced in the liver and co-deposited with amyloid fibrils in all types of amyloidosis, including cerebral β-protein amyloid deposits associated with Alzheimer′s disease (AD). We have measured its concentration and those of α 2-macroglobulin, IgG and albumin in the CSF of 51 patients with AD and 50 healthy and disease control subjects. The mean levels of SAP were 12.8 ng/ml in AD and 8.5 ng/ml in controls (P<0.0125); there was no difference in the levels of the other proteins studied. The observed concentrations of SAP were much lower than expected for a protein of molecular weight 254620. The difference between AD and controls suggests that the concentration of SAP in the CSF may be affected by the presence of cerebral amyloidosis.

Cathepsin G: Localization in human cerebral cortex and generation of amyloidogenic fragments from the β-amyloid precursor protein

Amyloid deposits in Alzheimer's disease, Down's syndrome and aged brain are composed largely of Aβ protein, which is generated by proteolytic processing of β-amyloid precursor protein. Proteases responsible for liberating the Aβ protein from the precursor have not yet been identified. Here, we examined the ability of cathepsin G, a chymotrypsin-like protease, to cleave two protease substrates: (i) a fluorogenic hexapeptide, whose sequence spans the cleavage site in the precursor for generating the Aβ NH 2-terminus, and (ii) recombinant human β-amyloid precursor protein purified from a baculovirus expression system. Unlike two other members of the chymotrypsin family, cathepsin G readily degraded the hexapeptide. Furthermore, cathepsin G cleaved the β-amyloid precursor protein to generate several breakdown products, including a prominent 11,500 mol. wt fragment immunoreactive with antibodies directed against the COOH-terminus of the protein. This COOH-terminal fragment co-migrated using two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis with C-100, a recombinant COOH-terminal segment of the β -amyloid precursor, whose NH 2-terminus is one residue upstream of the NH 2-terminus of the Aβ domain. We also examined the localization of cathepsin G in human brain. The distribution of cathepsin G-containing cells was examined by immunohistochemistry in the temporal cortex of both Alzheimer's and aged control samples. Cathepsin G-like immunoreactivity was contained specifically within neutrophils. As visualized by double-labeling with antibodies to cathepsin G and Factor VIII, neutrophils were most frequently found within meningeal or cortical blood vessels. In addition, occasional neutrophils could be identified without an apparent vascular surround, in the brain parenchyma. By simultaneous labeling with antibodies to cathepsin G and Aβ protein, neutrophils were also sometimes found associated with both parenchymal and vessel amyloid deposits; however, these associations were rare. These findings indicate that cathepsin G is capable of cleaving the β-amyloid precursor protein to liberate the free NH 2-terminus of the Aβ protein and may have access to areas where this material is deposited in Alzheimer's disease. However, since there is no physical association between neutrophils and deposited amyloid and no increase in the number of neutrophils in an Alzheimer's brain, cathepsin G seems to be an unlikely mediator of amyloid deposition in this disease.

Apolipoprotein-E immunoreactivity in amyloid deposits in Alzheimer's disease, but not in pathological aging or diffuse lewy body disease (DLBD) : D. W. Dickson, A. Ivanushkin, S.-H. Yen and P. Davies. Department of Pathology (Neuropathology), Albert Einstein College of Medicine, Bronx, New York 10461, USA

Apolipoprotein-E immunoreactivity in amyloid deposits in Alzheimer's disease, but not in pathological aging or diffuse lewy body disease (DLBD) : D. W. Dickson, A. Ivanushkin, S.-H. Yen and P. Davies. Department of Pathology (Neuropathology), Albert Einstein College of Medicine, Bronx, New York 10461, USA

The amyloid precursor protein in ischemic brain injury and chronic hypoperfusion.

We studied changes in the spatial and temporal distribution of the beta amyloid precursor protein (APP) of Alzheimer's disease (AD) in experimental ischemic brain injury. Rats with repeated reversible occlusions of one middle cerebral artery showed striking APP reactivity in astrocytic processes in perifocal regions and adjacent white matter. APP reactive dystrophic axons and neurons were also evident in the cortex and hippocampus ipsilateral to the MCA occlusion. Such changes were similarly apparent in animals subjected to partial forebrain ischemia induced by bilateral occlusion of the carotid arteries. Our studies suggest that focal ischemic insults or chronic hypoperfusion leads to increased accumulation or induction of APP in surviving cellular elements that may relate to the processes involved in beta amyloid deposition in AD.

Transgenic mice expressing human beta-APP751, but not mice expressing beta-APP695, display early Alzheimer's disease-like histopathology.

Mice transgenic for the 751 amino acid isoform of the human beta-amyloid precursor protein (beta-APP) driven by the rat neuron specific enolase (NSE) promoter (NSE:beta-APP751) show features of early Alzheimer's disease (AD) pathology. These features, which were evident in multiple pedigrees, include: 1) preamyloid deposits which stain with antibodies that are specific for the beta-amyloid peptide and stain AD amyloid deposits and plaques, and 2) neuronal soma and processes which stain with an antibody (Alz50) that detects abnormal isoforms of tau which are characteristic of AD. The quality and distribution of both types of immunoreactivity revealed in the NSE:beta-APP751 mouse brains most closely resemble those seen in brains of young adults with Down's syndrome. Both structures are rarely, if ever, observed in brains from mice transgenic for the 695 amino acid isoform of beta-APP (NSE:beta-APP695) or in wild type mice.

Heat shock partially protects rat pheochromocytoma PC12 cells from amyloid β peptide toxicity

Rat pheochromocytoma PC12 cells are killed by amyloid β protein (ABP), a component of plaques and other amyloid deposits in Alzheimer's disease. To investigate the possibility of protection from ABP toxicity by induced heat shock proteins (hsps), PC12 cells were heat treated for 60 min at 42°C. The stress response of the PC12 cells was examined at the protein level using hsp72-specific monoclonal antibodies. Hsp72 immunoreactivity was highly induced following heat treatment. The toxic effect of the β 25–35 ABP peptide fragment, the major cytotoxic sequence within ABP, was reduced in heat pretreated PC12 cultures. These data indicate a protective role of hsps in neuronal cells exposed to ABP.

Synthetic Alzheimer amyloid β/A4 peptides enhance production of complement C3 component by cultured microglial cells

Primary microglial cultures prepared from newborn mice showed the production and release of the third component of complement (C3). Newly synthesized [35S]methionine-labelled C3 was purified by immunoprecipitation using anti-C3-antibody. C3 was detected by SDS-PAGE and fluoroaraphy of the immunoprecipitated protein from cell lysates as a 195 kDa band, and from the supernatants of cultures as two major bands corresponding to the C3 alpha-chain (125 kDa) and beta-chain (75 kDa), consistent with known C3 characteristics. Increased biosynthesis of C3 was elicited by endotoxin lipopolysaccharide (LPS). Further, the synthesis of C3 was increased 5-10-fold in response to various synthetic peptides corresponding to the amyloid beta/A4 protein, which is the main constituent of extracellular amyloid deposits in Alzheimer's disease (AD). The increased synthesis of C3 was shown to be dose dependent at concentrations of beta/A4 peptide ranging from 10 micrograms/ml to 50 micrograms/ml. These results suggest that complement components found previously in amyloid deposits may be partly derived from reactive microglia preferentially associated with senile plaques in AD brain.

A 109-amino-acid C-terminal fragment of Alzheimer's-disease amyloid precursor protein contains a sequence, -RHDS-, that promotes cell adhesion.

Amyloid beta (A beta), the major constituent of the fibrils composing senile plaques and vascular amyloid deposits in Alzheimer's disease (AD) and related disorders, is a 39-42-residue self-aggregating degradation peptide of a larger multidomain membrane glycoprotein designated amyloid precursor protein (APP). An array of biological functions has been assigned to different APP domains, including growth regulation, neurotoxicity, inhibitory activity of serine proteinases and promotion of cell-cell and cell-matrix interactions. A beta is generated through an as-yet-unknown catabolic pathway that by-passes or inhibits the cleavage of APP within the A beta sequence. We have identified a 16 kDa intermediate APP C-terminal fragment containing A beta in leptomeningeal vessels of aged normal individuals and AD patients by means of its immunoreactivity with a panel of four different anti-(APP C-terminal) antibodies, indicating a different pathway of APP processing. Previous studies have indicated that the APP C-terminal domain is the most likely to be involved in cell-matrix interactions. A 109-amino-acid construct C109 with a sequence analogous to the C-terminal of APP (positions 587-695 of APP695), similar in length and immunoreactivity to the 16 kDa fragment, was found to promote cell adhesion. By use of synthetic peptides, this activity was initially located to the extracellular 28 residues of A beta. Inhibition studies demonstrated that the sequence RHDS (amino acids 5-8 of A beta, corresponding to residues 601-604 of APP695 was responsible for the adhesion-promoting activity. The interaction is dependent on bivalent cations and can be blocked either by the tetrapeptides RHDS and RGDS or by an anti-(beta 1 integrin) antibody. Thus, through integrin-like surface receptors, APP or its derivative proteolytic fragments containing the sequence RHDS may modulate cell-cell or cell-matrix interactions.