Artifactual strain-specific signs of incipient brain amyloidosis in APP transgenic mice.

In an attempt to generate transgenic mice modeling Alzheimer-type amyloidogenesis, the COOH-terminal 103 residue human APP segment was expressed in brain regions known to be vulnerable in AD. Transfected cells overexpressing this transgene were previously shown to develop intracytoplasmic inclusions that were immunoreactive with antibodies to the APP COOH-terminus. Transgenic C57B6/SJL mice produced transgene-coded mRNA in their brains at levels up to sixfold above endogenous APP, most abundantly within cortical and hippocampal pyramidal neurons. Immunocytochemistry with anti-A beta antibodies revealed occasional structures that resembled diffuse amyloid, but which could not be detected on serial sections. Immunolabeling with antibodies to APP regions NH2-terminal to the transgene-coded domain revealed elevated immunoreactivity within perikarya and neurites in regions expressing the highest transgene and endogenous APP mRNA levels, similar to observations previously reported within vulnerable neurons in AD brain. However, subsequent breeding revealed that this phenotype segregated with the B6/SJL background rather than the transgene, thus emphasizing the importance of genetic background to observations of putative AD-type pathology in transgenic animals.

Two-hybrid system as a model to study the interaction of beta-amyloid peptide monomers.

The kinetics of amyloid fibril formation by beta-amyloid peptide (Abeta) are typical of a nucleation-dependent polymerization mechanism. This type of mechanism suggests that the study of the interaction of Abeta with itself can provide some valuable insights into Alzheimer disease amyloidosis. Interaction of Abeta with itself was explored with the yeast two-hybrid system. Fusion proteins were created by linking the Abeta fragment to a LexA DNA-binding domain (bait) and also to a B42 transactivation domain (prey). Protein-protein interactions were measured by expression of these fusion proteins in Saccharomyces cerevisiae harboring lacZ (beta-galactosidase) and LEU2 (leucine utilization) genes under the control of LexA-dependent operators. This approach suggests that the Abeta molecule is capable of interacting with itself in vivo in the yeast cell nucleus. LexA protein fused to the Drosophila protein bicoid (LexA-bicoid) failed to interact with the B42 fragment fused to Abeta, indicating that the observed Abeta-Abeta interaction was specific. Specificity was further shown by the finding that no significant interaction was observed in yeast expressing LexA-Abeta bait when the B42 transactivation domain was fused to an Abeta fragment with Phe-Phe at residues 19 and 20 replaced by Thr-Thr (AbetaTT), a finding that is consistent with in vitro observations made by others. Moreover, when a peptide fragment bearing this substitution was mixed with native Abeta-(1-40), it inhibited formation of fibrils in vitro as examined by electron microscopy. The findings presented in this paper suggest that the two-hybrid system can be used to study the interaction of Abeta monomers and to define the peptide sequences that may be important in nucleation-dependent aggregation.

An interaction between basement membrane and Alzheimer amyloid precursor proteins suggests a role in the pathogenesis of Alzheimer's disease.

BACKGROUND: Extracellular matrix proteins (ECMPs) of the basement membrane type, such as the heparan sulfate proteoglycan perlecan, laminin, entactin, collagen IV, and fibronectin are present in and have been implicated in the genesis of amyloids. As in many forms of amyloid, perlecan, laminin, collagen IV, and fibronectin are present in Alzheimer deposits. We have previously demonstrated high-affinity interactions between Alzheimer amyloid precursor proteins (beta PP-695, -751, and -770), and perlecan or laminin. With a view to examining our hypothesis that beta PP:ECMP interactions are involved in Alzheimer's amyloidogenesis, additional studies have now been performed examining the interactions of the beta PPs with entactin, fibronectin, and collagen IV, the influence each of the ECMPs has on the binding of the others to beta PPs, and the effect of beta PPs on interactions among the various ECMPs. EXPERIMENTAL DESIGN: A modified solid-phase enzyme-linked immunosorbent assay was used to assess the binding of the various ECMPs to the beta PPs. One element was immobilized on plastic, and another element, operationally defined as a ligand, was incubated in solution at various concentrations over the immobilized protein. To evaluate the effect of one ECMP on the binding of other ECMPs to beta PP, the beta PP was immobilized and the binding of the "ligand" ECMP was assessed in the presence of a single concentration of a second "competitor" ECMP. Similarly, in evaluating the effect of beta PPs on the binding of ECMPs to each other, one ECMP was immobilized and the binding of a second ECMP "ligand" was assessed in the presence of a fixed concentration of beta PP "competitor." RESULTS: As in the case of perlecan and laminin, each of the ECMPs bound to the beta PPs with high affinity (Kd values in the nanomolar range). The binding of entactin to beta PPs was stimulated by collagen IV but was markedly inhibited by laminin, perlecan, and fibronectin. Conversely, the presence of entactin inhibited the binding of perlecan, laminin, and fibronectin to beta PPs. Moreover, the presence of beta PPs usually interfered with the binding of ECMPs to each other. Generally, in all binding assays, beta PP-751 and -770, behaved in similar ways, but beta PP-695, the brain-specific form, exhibited unique characteristics. CONCLUSIONS: These binding data may reflect the normal interactions of beta PPs with ECMPs. However, the fact that beta PPs interfere with the normal interactions between ECMPs themselves, a process that spontaneously generates a basement membrane, suggests that aspects of ECMP:beta PP binding may be a pathologic part of the amyloidogenic process in Alzheimer's disease.

Characterization of high affinity binding between laminin and Alzheimer's disease amyloid precursor proteins.

BACKGROUND: In vivo amyloid formation apparently involves several extracellular matrix components that are usually found associated with basement membranes. These include laminin, heparan sulfate proteoglycan, collagen type IV, and entactin. These components have also been found in neuritic plaques. We have therefore been examining interactions between extracellular matrix components and the Alzheimer's amyloid precursors (AAPs). EXPERIMENTAL DESIGN: Binding interactions of laminin with AAP-695, -751, and -770 were examined using a solid phase enzyme-linked immunosorbent assay technique. RESULTS: Objective, quantitative analyses of the laminin AAP-695, -751, and -770 binding data reveal two binding sites for laminin, with Kd values of 1 x 10(-10) M and 1 x 10(-8) M. Zinc and dithiothreitol profoundly stimulate laminin binding to AAPs. Furthermore, zinc fingers were found in the laminin amino acid sequences. Previous binding studies of AAPs with the basement membrane heparan sulfate proteoglycan revealed similar affinities. A particular order of addition of laminin and heparan sulfate proteoglycan to AAPs can be demonstrated. CONCLUSIONS: These avid interactions with extracellular matrix proteins likely reflect normal functions of the AAPs and may be involved in nucleation events in Alzheimer-type amyloid formation.

Alzheimer's disease. Beta-amyloid precursor protein expression in the nucleus basalis of Meynert.

The nucleus basalis of Meynert (nbM) was examined using immunocytochemistry for beta-amyloid precursor protein (beta APP) expression in Alzheimer's disease (AD). In mild AD cases, light labeling of the cell body and proximal processes was observed, and small intracellular structures were labeled rarely. In the more severe cases, intense cytoplasmic beta APP labeling was seen, often along with small beta APP-positive structures. Double-labeling experiments demonstrated that in the more severe cases these small structures were also decorated by a neurofibrillary tangle (NFT) antiserum. Other neurons in the severe cases showed incorporation of beta APP into large inclusions, which were also labeled with the NFT antiserum. However, some large inclusions in the severe cases were labeled by the NFT antiserum but contained no beta APP. Extraneuronal NFTs did not show beta APP labeling and did not react with an antibody to the beta-amyloid peptide. These results suggest that increased expression of beta APP coincides with intracellular NFT formation in the nbM, but that the formation of extraneuronal NFTs results in a loss of beta APP immunoreactivity.

Subcellular localization of amyloid precursor protein in senile plaques of Alzheimer's disease.

The authors have previously shown that amyloid precursor protein (APP) accumulates in neurites present in senile plaques of Alzheimer's disease (AD). In this ultrastructural immunocytochemical study, we describe the subcellular site of APP accumulation. Vibratome sections of glutaraldehyde-paraformaldehyde fixed hippocampi from five cases of AD were pretreated with methanol and immunostained with an antibody raised against recombinant APP 770 by using either indirect immunogold or peroxidase methods. Immunolabeling was localized in cell processes filled with amorphous, irregular-shaped materials, which were identified as dense bodies deformed by postmortem autolysis and methanol treatment, as well as multilamellar membranous bodies. Identification of these bodies was obtained with comparative ultrastructural examination of biopsy and autopsy tissue fixed with and without methanol treatment. These electron-dense organellae were positive for the lysosomal marker, acid phosphatase. At light microscopy, acid phosphatase and APP colocalized to the same cell processes in senile plaques. Many of those cell processes contained abnormal straight or paired helical filaments supporting their neuritic nature. These results suggest that APP accumulates in the lysosomal system of the dystrophic neurites present in senile plaques and are consistent with a neuronal origin of the APP forming the amyloid in senile plaques.

Alzheimer's amyloid precursor protein produced by recombinant baculovirus expression. Proteolytic processing and protease inhibitory properties.

The baculovirus expression system was used to generate recombinant Alzheimer's amyloid precursor (AAP) proteins. Recombinant baculoviruses were constructed, designed to express full-length 695-, 751-, and 770-amino acid forms. Recombinant baculoviruses designed for constitutive secretion were engineered by placing a termination codon between the beta-protein domain and cytoplasmic anchor of the full-length forms. Insect cells infected with each of these baculoviruses produced both secreted and cell-associated AAPs. Full-length constructs produced secreted derivatives which were COOH-terminally cleaved within the beta-protein domain at Gln15 or Lys16, essentially identical to previous reports utilizing mammalian cell systems. Rare secreted forms (less than 5%) appeared to extend to Lys28. Secretion constructs produced these same forms, but in different ratios. Most (approximately 60%) terminated at Gln15 or Lys16, while the remainder apparently extended to Lys28. AAPs containing the Kunitz-type serine protease inhibitory domain (AAP-751 and -770) were shown to be active inhibitors. No differences were observed in the inhibitors activities of these two forms. The similarities in AAP processing by insect and mammalian systems, together with the large amounts of recombinant protein produced by baculovirus expression, make this an attractive system for studies of AAP processing and biochemical properties.

Alzheimer's disease: beta-amyloid precursor protein expression in plaques varies among cytoarchitectonic areas of the medial temporal lobe.

The anatomic distributions of beta-amyloid peptide (beta AP) and beta-amyloid precursor protein (beta APP) in the medial temporal lobe were examined with immunocytochemistry in Alzheimer's disease. beta AP-containing plaques were found most frequently in the cortical and basal regions of the amygdala, and in the hippocampal CA1, subiculum, and dentate molecular layer. beta APP expression in plaques was found in a similar distribution, with some, but not all beta AP plaques also showing beta APP. In the cortical and basal amygdala, some cases showed beta APP in the centers of plaques, whereas in the hippocampus, all cases displayed beta APP mainly in plaque neurites. The lateral regions of the amygdala contained mainly diffuse beta AP plaques which had little beta APP. These findings suggest that although beta APP expression and beta AP deposition generally colocalize, processing of beta APP may vary among closely interconnected anatomic regions.

Senile plaque neurites in Alzheimer disease accumulate amyloid precursor protein.

Senile plaques are polymorphous beta-amyloid protein deposits found in the brain in Alzheimer disease and normal aging. This beta-amyloid protein is derived from a larger precursor molecule of which neurons are the principal producers in brain. We found that amyloid precursor protein (APP)-immunoreactive neurites were involved in senile plaques and that only a subset of these neurites showed markers for the abnormal filaments characteristic of neurofibrillary pathology. In the neocortex of nondemented individuals with senile plaques but spared of neurofibrillary pathology, dystrophic neurites in senile plaques showed only APP accumulation. In contrast, in the brains of Alzheimer patients, virtually all APP-immunoreactive neurites also showed immunoreactivity with ubiquitin, tau, and phosphorylated neurofilaments. The presence of tau and neurofilament epitopes in dystrophic neurites in senile plaques was correlated with the extent of neurofibrillary pathology in the surrounding brain tissue. Accumulation of APP and the formation of neurofibrillary pathology in senile plaque neurites are therefore distinct phenomena. Our findings suggest that APP accumulation in senile plaque neurites occurs prior to tau accumulation and is therefore more closely related to appearance of neuritic dystrophy.

High affinity interactions between the Alzheimer's beta-amyloid precursor proteins and the basement membrane form of heparan sulfate proteoglycan.

High affinity interactions were studied between the basement membrane form of heparan sulfate proteoglycan (HSPG) and the 695-, 751-, and 770-amino acid Alzheimer amyloid precursor (AAP) proteins. Based on quantitative analyses of binding data, we identified single binding sites for the HSPG on AAP-695 (Kd = 9 x 10(-10) M), AAP-751 (Kd = 10 x 10(-9) M), and AAP-770 (Kd = 9 x 10(-9) M). It is postulated that the "Kunitz" protease inhibitor domain which is present in AAP-751 and -770 reduces the affinity of AAPs for the HSPG through steric hindrance and/or conformational alteration. HSPG binding was inhibited by heparin and dextran sulfate, but not by dermatan or chondroitin sulfate. HSPG protein core, obtained by heparitinase digestion, also bound to the beta-amyloid precursor proteins with high affinity, indicating that the high affinity binding site is constituted by the polypeptide chain rather than the carbohydrate moiety. The effects of various cations on these interactions were also studied. Our results suggest that specific interactions between the AAP proteins and the extracellular matrix may be involved in the nucleation stages of Alzheimer's disease type amyloidogenesis.

Neuronal and microglial involvement in beta-amyloid protein deposition in Alzheimer's disease.

This study was undertaken to localize amyloid precursor protein (APP) and to determine how APP might be released and proteolyzed to yield the beta-amyloid protein deposits found in senile plaques in the brains of Alzheimer's disease patients. We found that antibodies to recombinantly expressed APP labeled many normal neurons and neurites. In addition, dystrophic neurites in different types of senile plaques and degenerating neurons in the temporal cortex and hippocampus of Alzheimer's disease patients were immunostained. We also detected small clusters of dystrophic APP immunoreactive neurites that were not associated with beta-amyloid protein deposits. Microglia was involved in different types of senile plaques and often were associated closely with APP immunoreactive neurites and neurons. The greatest concurrence of APP immunoreactivity and reactive microglia was seen in the subiculum and area CA1, regions with a high density of congophilic plaques and subject to intense Alzheimer's pathology. Our findings suggest that neuronally derived APP is the source for senile plaque beta-amyloid protein, while microglia may act as processing cells.

Association study between Alzheimer's disease and restriction fragment length polymorphisms at the human amyloid beta protein gene locus.

Alzheimer's disease, an autosomal dominant disorder, is characterized by the presence of neurofibrillary tangles and senile extracellular plaques in the brain of affected individuals. An amyloid beta protein has been isolated from the core of these plaques, and the gene encoding this protein has been mapped to region q11.2 to q22.2 of chromosome 21. Independent linkage studies have shown that the locus responsible for familial Alzheimer's disease also maps to the long arm of chromosome 21. It is thus very tempting to speculate that a defect (or defects) of the amyloid beta protein gene is the cause of Alzheimer's disease. For this reason, we have done association studies between Alzheimer's disease and restriction fragment length polymorphisms of the amyloid beta protein gene locus. We report a study of six restriction fragment length polymorphisms at the human amyloid beta protein gene locus. Several haplotypes constitute very informative marker systems for this region of chromosome 21. One of the six polymorphisms, a 6.6/7.3 kb (kb = 10(3) base-pairs) EcoRI restriction fragment length polymorphism, is loosely associated with the presence of Alzheimer's disease in a population of 34 subjects.

A new A4 amyloid mRNA contains a domain homologous to serine proteinase inhibitors.

The amyloid proteins isolated from neuritic plaques and the cerebrovasculature of Alzheimer's disease are self-aggregating moieties termed A4 protein and beta-protein, respectively. A putative A4 amyloid precursor (herein termed A4(695] has been characterized by analysis of a human brain complementary DNA. We report here the sequence of a closely related amyloid cDNA, A4(751), distinguished from A4(695) by the presence of a 168 base-pair (bp) sequence which adds 57 amino acids to, and removes one residue from, the predicted A4(695) protein. The peptide predicted from this insert is very similar to the Kunitz family of serine proteinase inhibitors. The two A4-specific messenger RNAs are differentially expressed: in a limited survey, A4(751) mRNA appears to be ubiquitous, whereas A4(695) mRNA has a restricted pattern of expression which includes cells from neuronal tissue. These data may have significant implications for understanding amyloid deposition in Alzheimer's disease.