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 (As) 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...
Progressive cerebral accumulation of amyloid beta-protein (Abeta) is an early and invariant feature of Alzheimer's disease. Little is known about how Abeta, after being secreted, is degraded and cleared from the extracellular space of the brain. Defective Abeta degradation could be a risk factor for the development of Alzheimer's disease in some subjects. We reported previously that microglial cells release substantial amounts of an Abeta-degrading protease that, after purification, is indistinguishable from insulin-degrading enzyme (IDE). Here we searched for and characterized a role for IDE in Abeta degradation by neurons, the principal cell type that produces Abeta. Whole cultures of differentiated pheochromocytoma (PC12) cells and primary rat cortical neurons actively degraded endogenously secreted Abeta via IDE. However, unlike that in microglia, IDE in differentiated neurons was not released but localized to the cell surface, as demonstrated by biotinylation. Undifferentiated PC12 cells released IDE into their medium, whereas after differentiation, IDE was cell associated but still degraded Abeta in the medium. Overexpression of IDE in mammalian cells markedly reduced the steady-state levels of extracellular Abeta(40) and Abeta(42), and the catalytic site mutation (E111Q) abolished this effect. We observed a novel membrane-associated form of IDE that is approximately 5 kDa larger than the known cytosolic form in a variety of cells, including differentiated PC12 cells. Our results support a principal role for membrane-associated and secreted IDE isoforms in the degradation and clearance of naturally secreted Abeta by neurons and microglia.
Recent studies suggest that insulin-degrading enzyme (IDE) in neurons and microglia degrades Abeta, the principal component of beta-amyloid and one of the neuropathological hallmarks of Alzheimer's disease (AD). We performed parametric and nonparametric linkage analyses of seven genetic markers on chromosome 10q, six of which map near the IDE gene, in 435 multiplex AD families. These analyses revealed significant evidence of linkage for adjacent markers (D10S1671, D10S583, D10S1710, and D10S566), which was most pronounced in late-onset families. Furthermore, we found evidence for allele-specific association between the putative disease locus and marker D10S583, which has recently been located within 195 kilobases of the IDE gene.
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