Excessive cerebral accumulation of the 42-residue amyloid ␤-protein (A␤) is an early and invariant step in the pathogenesis of Alzheimer's disease. Many studies have examined the cellular production of A␤ from its membrane-bound precursor, including the role of the presenilin proteins therein, but almost nothing is known about how A␤ is degraded and cleared following its secretion. We previously screened neuronal and nonneuronal cell lines for the production of proteases capable of degrading naturally secreted A␤ under biologically relevant conditions and concentrations. The major such protease identified was a metalloprotease released particularly by a microglial cell line, BV-2. We have now purified and characterized the protease and find that it is indistinguishable from insulin-degrading enzyme (IDE), a thiol metalloendopeptidase that degrades small peptides such as insulin, glucagon, and atrial natriuretic peptide. Degradation of both endogenous and synthetic A␤ at picomolar to nanomolar concentrations was completely inhibited by the competitive IDE substrate, insulin, and by two other IDE inhibitors. Immunodepletion of conditioned medium with an IDE antibody removed its A␤-degrading activity. IDE was present in BV-2 cytosol, as expected, but was also released into the medium by intact, healthy cells. To confirm the extracellular occurrence of IDE in vivo, we identified intact IDE in human cerebrospinal fluid of both normal and Alzheimer subjects. In addition to its ability to degrade A␤, IDE activity was unexpectedly found be associated with a time-dependent oligomerization of synthetic A␤ at physiological levels in the conditioned media of cultured cells; this process, which may be initiated by IDE-generated proteolytic fragments of A␤, was prevented by three different IDE inhibitors. We conclude that a principal protease capable of down-regulating the levels of secreted A␤ extracellularly is IDE.Converging lines of evidence support the hypothesis that progressive cerebral accumulation of the 40 -42-residue amyloid ␤-proteins (A␤s) 1 is an early, invariant, and necessary step in the pathogenesis of Alzheimer's disease (AD). As a result, there is growing interest in decreasing cerebral A␤ levels as a therapeutic and preventative approach to the disease. A␤ is generated by endoproteolysis of the ␤-amyloid precursor protein (APP) and secreted constitutively by most mammalian cells throughout life. Whereas many studies have examined the proteolytic processing of APP and the mechanisms of A␤ production, almost nothing is known about how A␤ peptides are normally degraded and cleared following their secretion. We recently screened the conditioned media of several different cell lines for A␤-degrading activity and found that the principal such activity was conferred by a nonmatrix metalloprotease that was released by microglial cells and other cells and efficiently degraded both endogenous and synthetic A␤ (1). The release of the protease from microglial cells was augmented by activating the cells with lipopolysa...

Filamentous aggregates of the 40-42-residue amyloid beta-protein (A beta) accumulate progressively in the limbic and cerebral cortex in Alzheimer's disease, where they are intimately associated with neuronal and glial cytopathology. Attempts to model this cytotoxicity in vitro using synthetic peptides have shown that monomeric A beta is relatively inert, whereas aggregated A beta reproducibly exerts a variety of neurotoxic effects. The processes that mediate the conversion of monomeric A beta into a toxic aggregated state are thus of great interest. Previous studies of this conversion have employed high concentrations (10(-5)-10(-3) M) of synthetic A beta peptides under nonbiological conditions. We report here the detection of small amounts (< 10(-9) M) of SDS-stable A beta oligomers in the culture media of Chinese hamster ovary cells expressing endogenous or transfected amyloid beta-protein precursor genes. The identity of these oligomers (primarily dimers and trimers) was established by immunoprecipitation with a panel of A beta antibodies, by electrophoretic comigration with synthetic A beta oligomers, and by amino acid sequencing. The oligomeric A beta species comprised approximately 10-20% of the total immunoprecipitable A beta in these cultures. A truncated A beta species beginning at Arg 5 was enriched in the oligomers, suggesting that amino-terminal heterogeneity can influence A beta oligomerization in this system. Addition of Congo red (10 microM) during metabolic labeling of the cells led to increased monomeric and decreased oligomeric A beta. The ability to detect and quantitate oligomers of secreted A beta peptides in cell culture should facilitate dynamic studies of the critical process of initial A beta aggregation under physiological conditions.

The progressive aggregation and deposition of amyloid beta-protein (Abeta) in brain regions subserving memory and cognition is an early and invariant feature of Alzheimer's disease, the most common cause of cognitive failure in aged humans. Inhibiting Abeta aggregation is therapeutically attractive because this process is believed to be an exclusively pathological event. Whereas many studies have examined the aggregation of synthetic Abeta peptides under nonphysiological conditions and concentrations, we have detected and characterized the oligomerization of naturally secreted Abeta at nanomolar levels in cultures of APP-expressing CHO cells [Podlisny, M. B., Ostaszewski, B. L., Squazzo, S. L., Koo, E. H., Rydell, R. E., Teplow, D. B., and Selkoe, D. J. (1995) J. Biol. Chem. 270, 9564-9570 (1); Podlisny, M. B., Walsh, D. M., Amarante, P., Ostaszewski, B. L., Stimson, E. R., Maggio, J. E., Teplow, D. B., and Selkoe, D. J. (1998) Biochemistry 37, 3602-3611 (2)]. To determine whether similar species occur in vivo, we probed samples of human cerebrospinal fluid (CSF) and detected SDS-stable dimers of Abeta in some subjects. Incubation of CSF or of CHO conditioned medium at 37 degrees C did not lead to new oligomer formation. This inability to induce oligomers extracellularly as well as the detection of oligomers in cell medium very early during the course of pulse-chase experiments suggested that natural Abeta oligomers might first form intracellularly. We therefore searched for and detected intracellular Abeta oligomers, principally dimers, in primary human neurons and in neuronal and nonneural cell lines. These dimers arose intracellularly rather than being derived from the medium by reuptake. The dimers were particularly detectable in neural cells: the ratio of intracellular to extracellular oligomers was much higher in brain-derived than nonbrain cells. We conclude that the pathogenically critical process of Abeta oligomerization begins intraneuronally.

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Progressive cerebral deposition of extracellu- (15,16). A related issue concerns the mechanism whereby the normal, age-related deposition of (AP in selected brain regions and vessels is augmented in AD. The local proteolytic processing of the precursor and the role of amyloidassociated proteins, such as the serine protease inhibitor a1-antichymotrypsin (24), require elucidation.Although molecular studies of 8APP have advanced rapidly, the native precursor protein in human brain and other tissues has not been detected and characterized. To begin to address some of the questions posed above, we produced antibodies to synthetic peptides with sequences predicted from amyloidogenic and nonamyloidogenic regions of J3APP and have identified forms of the precursor molecule in brain, nonneural tissues, and cultured cells of several species. METHODSPreparation of Antibodies to Synthetic Peptides. Several peptides were synthesized according to the sequence deduced from a P3APP cDNA (14). Those used in this study were: amino acid residues 597-624 (peptide (31-28), comprising the first 28 residues of PAP; residues 676-695 (peptide C1), comprising the 20 C-terminal amino acids of PAPP; and residues 681-695, a second C-terminal peptide (C2), comprising the last 15 residues. The latter peptide and an antiserum to it were the gifts of Tsuyoshi Ishii (Psychiatric Research Institute, Tokyo). Peptide C1 was HPLC-purified and coupled to edestin. Peptide C2 had been coupled to keyhole limpet hemocyanin. Peptide p1-28 was injected uncoupled. Antisera were assayed by serial dilutions on dot blots of unconjugated peptide. Previously characterized antibodies used in this study included two antisera (A and C) to the -4-kDa P3AP purified from amyloid-rich fractions of AD cortex (12,25), two antisera (Fl and Ph) to the -4-kDa ,8AP purified from AD meningovascular amyloid (10), a paired helical filament-specific antiserum having no reaction with amyloid deposits (26), and an antiserum to heat-stable microtubule-associated proteins (principally X and MAP 2) purified from fetal human brain (25).Abbreviations: AD, Alzheimer disease; flAP, f8-amyloid protein; f3APP, 13-amyloid precursor polypeptide. 7341The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Mutations in a gene encoding a multitransmembrane protein, termed presenilin 1 (PS1), are causative in the majority of early-onset cases of AD. To determine the topology of PS1, we utilized two strategies: first, we tested whether putative transmembranes are sufficient to export a protease-sensitive substrate across a lipid bilayer; and second, we examined the binding of antibodies to specific PS1 epitopes in cultured cells selectively permeabilized with the pore-forming toxin, streptolysin-O. We document that the "loop," N-terminal, and C-terminal domains of PS1 are oriented toward the cytoplasm.

Mutations in the presenilin 1 (PS1) and presenilin 2 (PS2) genes cause the most common and aggressive form of early onset familial Alzheimer's disease. To elucidate their pathogenic mechanism, wild-type (wt) or mutant (M146L, C410Y) PS1 and wt or mutant (M239V) PS2 genes were stably transfected into Chinese hamster ovary cells that overexpress the ␤-amyloid precursor protein (APP). The identity of the 43-45-kDa PS1 holoproteins was confirmed by N-terminal radiosequencing. PS1 was rapidly processed (t 1/2 ‫؍‬ 40 min) in the endoplasmic reticulum into stable fragments. Wild-type and mutant PS2 holoproteins exhibited similar half lives (1.5 h); however, their endoproteolytic fragments showed both mutation-specific and cell type-specific differences. Mutant PS1 or PS2 consistently induced a 1.4 -2.5-fold increase (p < 0.001) in the relative production of the highly amyloidogenic 42-residue form of amyloid ␤-protein (A␤ 42 ) as determined by quantitative immunoprecipitation and by enzyme-linked immunosorbent assay. In mutant PS1 and PS2 cell lines with high increases in A␤ 42 /A␤ total ratios, spontaneous formation of low molecular weight oligomers of A␤ 42 was observed in media, suggesting enhanced A␤ aggregation from the elevation of A␤ 42 . We conclude that mutant PS1 and PS2 proteins enhance the proteolysis of ␤-amyloid precursor protein by the ␥-secretase cleaving at A␤ residue 42, thereby promoting amyloidogenesis. All patients with Alzheimer's disease (AD)1 develop extracellular amyloid deposits composed of the 40-and 42-residue amyloid ␤-proteins (A␤) in brain areas subserving memory and cognition. Many of the deposits are intimately associated with degenerating axons and dendrites, activated microglial cells, and reactive astrocytes. Mutations or polymorphisms in four genes that strongly predispose individuals to the premature development of AD have been identified to date. First, missense mutations in the APP gene (1-5), which encodes the precursor of A␤, increase the production of A␤ peptides, particularly A␤ 42 , in vitro and in vivo (6 -10). Second, inheritance of the ⑀4 polymorphism of the apolipoprotein E gene increases the number and density of A␤ deposits in the brain (11-15). The third and fourth genes to be linked to AD, presenilin 1 (PS1) and presenilin 2 (PS2), cause the most common form of early onset familial AD (16 -18). These genes encode highly homologous proteins predicted to span the membrane 7-8 times (19). Missense mutations in PS1 and PS2, more than 30 of which have already been identified (20), result in markedly accelerated clinical and neuropathological features of AD.A clue to the mechanism of the presenilins has come from the recent report of selective elevations in A␤ 42 levels in plasma and skin fibroblast media of subjects harboring PS1 or PS2 mutations (10). Because primary fibroblasts expressing different PS1 or PS2 mutations show very low A␤ secretion that cannot be easily studied mechanistically, we examined stably transfected Chinese hamster ovary (CHO) cell lines in which ...

Recent studies support the hypothesis that soluble oligomers of amyloid ␤-peptide (A␤) rather than mature amyloid fibrils are the earliest effectors of synaptic compromise in Alzheimer's disease. We took advantage of an amyloid precursor protein-overexpressing cell line that secretes SDS-stable A␤ oligomers to search for inhibitors of the pathobiological effects of natural human A␤ oligomers. Here, we identify small molecules that inhibit formation of soluble A␤ oligomers and thus abrogate their block of long-term potentiation (LTP). Furthermore, we show that cell-derived A␤ oligomers can be separated from monomers by size exclusion chromatography under nondenaturing conditions and that the isolated, soluble oligomers, but not monomers, block LTP. The identification of small molecules that inhibit early A␤ oligomer formation and rescue LTP inhibition offers a rational approach for therapeutic intervention in Alzheimer's disease and highlights the utility of our cell-culture paradigm as a useful secondary screen for compounds designed to inhibit early steps in A␤ oligomerization under biologically relevant conditions.