The majority of early-onset cases of familial Alzheimer's disease (FAD) are linked to mutations in two related genes, PS1 and PS2, located on chromosome 14 and 1, respectively. Using two highly specific antibodies against nonoverlapping epitopes of the PS1-encoded polypeptide, termed presenilin 1 (PS1), we document that the preponderant PS1-related species that accumulate in cultured mammalian cells, and in the brains of rodents, primates, and humans are approximately 27-28 kDa N-terminal and approximately 16-17 kDa C-terminal derivatives. Notably, a FAD-linked PS1 variant that lacks exon 9 is not subject to endoproteolytic cleavage. In brains of transgenic mice expressing human PS1, approximately 17 kDa and approximately 27 kDa PS1 derivatives accumulate to saturable levels, and at approximately 1:1 stoichiometry, independent of transgene-derived mRNA. We conclude that PS1 is subject to endoproteolytic processing in vivo.

Polymerization of amyloid ␤-peptide (A␤) into amyloid fibrils is a critical step in the pathogenesis of Alzheimer's disease. Here, we show that peptides incorporating a short A␤ fragment (KLVFF; A␤ 16 -20 ) can bind full-length A␤ and prevent its assembly into amyloid fibrils. Through alanine substitution, it was demonstrated that amino acids Lys 16 , Leu 17 , and Phe 20 are critical for binding to A␤ and inhibition of A␤ fibril formation. A mutant A␤ molecule, in which these residues had been substituted, had a markedly reduced capability of forming amyloid fibrils. The present data suggest that residues A␤ 16 -20 serve as a binding sequence during A␤ polymerization and fibril formation. Moreover, the present KLVFF peptide may serve as a lead compound for the development of peptide and nonpeptide agents aimed at inhibiting A␤ amyloidogenesis in vivo.The preeminent neuropathological feature of Alzheimer's disease is the deposition of amyloid in the brain parenchyma and cerebrovasculature (1, 2). The basic components of the amyloid are thin fibrils of a peptide termed A␤ (3, 4). This peptide is a 40-to 42-amino acid-long proteolytic fragment of the Alzheimer amyloid precursor protein (APP), 1 a protein expressed in most tissues (5). Genetic and neuropathological studies provide strong evidence for a central role of A␤ amyloid in the pathogenesis of Alzheimer's disease (6), but the pathophysiological consequences of the amyloid deposition are unclear. However, it has been suggested that A␤ polymers and amyloid are toxic to neurons, either directly or via induction of radicals, and hence cause neurodegeneration (7-9). Previous studies indicate that A␤ polymerization in vivo and in vitro is a specific process that probably involves interactions between binding sequences in the A␤ peptide (10 -12). A rational pharmacological approach for prevention of amyloid formation would therefore be to use drugs that specifically interfere with A␤-A␤ interaction and polymerization. We hypothesized that ligands capable of binding to and blocking such sequences might inhibit amyloid fibril formation as outlined schematically in Fig. 1. Our strategy in searching for an A␤ ligand was to identify binding sequences in A␤ and then, based on their primary structures, synthesize a peptide ligand. Binding sequences were identified by systematically synthesizing short peptides corresponding to sequences of the A␤ molecule. The minimum length of an identified binding sequence was determined by truncating the peptide. Residues critical for binding were identified by alanine scanning. These critical residues were then substituted in an A␤ fragment (A␤ ) that normally is capable of forming amyloid fibrils (13,14) in order to determine if they indeed are important for A␤ amyloid fibril formation. Finally, it was determined if the identified ligand, in addition to binding to the A␤ molecule, was capable of inhibiting fibril formation of A␤ . EXPERIMENTAL PROCEDURES Materials-Synthetic A␤1-40 and all other soluble peptides were synthesized b...

Polymerization of the amyloid beta (A␤) peptide into protease-resistant fibrils is a significant step in the pathogenesis of Alzheimer's disease. It has not been possible to obtain detailed structural information about this process with conventional techniques because the peptide has limited solubility and does not form crystals. In this work, we present experimental results leading to a molecular level model for fibril formation. Systematically selected A␤-fragments containing the A␤ 16 -20 sequence, previously shown essential for A␤-A␤ binding, were incubated in a physiological buffer. Electron microscopy revealed that the shortest fibril-forming sequence was A␤ 14 -23 . Substitutions in this decapeptide impaired fibril formation and deletion of the decapeptide from A␤ 1-42 inhibited fibril formation completely. All studied peptides that formed fibrils also formed stable dimers and/or tetramers. Molecular modeling of A␤ 14 -23 oligomers in an antiparallel ␤-sheet conformation displayed favorable hydrophobic interactions stabilized by salt bridges between all charged residues. We propose that this decapeptide sequence forms the core of A␤-fibrils, with the hydrophobic C terminus folding over this core. The identification of this fundamental sequence and the implied molecular model could facilitate the design of potential inhibitors of amyloidogenesis.

The amyloid-β lowering capacity of anti-Aβ antibodies has been demonstrated in transgenic models of Alzheimer's disease (AD) and in AD patients. While the mechanism of immunotherapeutic amyloid-β removal is controversial, antibody-mediated sequestration of peripheral Aβ versus microglial phagocytic activity and disassembly of cerebral amyloid (or a combination thereof) has been proposed. For successful Aβ immunotherapy, we hypothesized that high affinity antibody binding to amyloid-β plaques and recruitment of brain effector cells is required for most efficient amyloid clearance. Here we report the generation of a novel fully human anti-Aβ antibody, gantenerumab, optimized in vitro for binding with sub-nanomolar affinity to a conformational epitope expressed on amyloid-β fibrils using HuCAL(®) phage display technologies. In peptide maps, both N-terminal and central portions of Aβ were recognized by gantenerumab. Remarkably, a novel orientation of N-terminal Aβ bound to the complementarity determining regions was identified by x-ray analysis of a gantenerumab Fab-Aβ(1-11) complex. In functional assays gantenerumab induced cellular phagocytosis of human amyloid-β deposits in AD brain slices when co-cultured with primary human macrophages and neutralized oligomeric Aβ42-mediated inhibitory effects on long-term potentiation in rat brain. In APP751(swedish)xPS2(N141I) transgenic mice, gantenerumab showed sustained binding to cerebral amyloid-β and, upon chronic treatment, significantly reduced small amyloid-β plaques by recruiting microglia and prevented new plaque formation. Unlike other Aβ antibodies, gantenerumab did not alter plasma Aβ suggesting undisturbed systemic clearance of soluble Aβ. These studies demonstrated that gantenerumab preferentially interacts with aggregated Aβ in the brain and lowers amyloid-β by eliciting effector cell-mediated clearance.

We have previously shown that short peptides incorporating the sequence KLVFF can bind to the ϳ40-amino acid residue Alzheimer amyloid ␤-peptide (

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Aortic medial amyloid is a form of localized amyloid that occurs in virtually all individuals older than 60 years. The importance and impact of the amyloid deposits are unknown. In this study we have purified a 5.5-kDa aortic medial amyloid component, by size-exclusion chromatography and RP-HPLC, from three individuals, and we have shown by amino acid sequence analysis that the amyloid is derived from an integral proteolytic fragment of lactadherin. Lactadherin is a 364-aa glycoprotein, previously known to be expressed by mammary epithelial cells as a cell surface protein and secreted as part of the milk fat globule membrane. The multidomain protein has a C-terminal domain showing homology to blood coagulation factors V and VIII. We found that the main constituent of aortic medial amyloid is a 50-aa-long peptide, here called medin, that is positioned within the coagulation factor-like domain of lactadherin. Our result is supported by the specific labeling of aortic medial amyloid in light and electron microscopy with two rabbit antisera raised against two synthetic peptides corresponding to different parts of medin. By using in situ hybridization we have shown that lactadherin is expressed by aortic medial smooth muscle cells. Furthermore, one of the synthetic peptides forms amyloid-like fibrils in vitro. Lactadherin was not previously known to be an amyloid precursor protein or to be expressed in aortic tissue. The structure of lactadherin may implicate an important regulatory function in the aorta.

Genetic evidence suggests a role for apolipoprotein E (apoE) in Alzheimer's disease (AD) amyloidogenesis. Here, amyloid-associated apoE from 32 AD patients was purified and characterized. We found that brain amyloid-associated apoE apparently exists not as free molecules but as complexes with polymers of the amyloid beta peptide (A beta). Brain A beta-apoE complexes were detected irrespective of the apoE genotype, and similar complexes could be mimicked in vitro. The fine structure of purified A beta-apoE complexes was fibrillar, and immunogold labeling revealed apoE immunoreactivity along the fibrils. Thus, we conclude that A beta-apoE complexes are principal components of AD-associated brain amyloid and that the data presented here support a role for apoE in the pathogenesis of AD.