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...

Cerebral deposition of beta-amyloid (Abeta) peptides is an invariant pathological hallmark in brains of patients with Alzheimer's disease (AD) and transgenic mice coexpressing familial AD-linked APP and PS1 variants. We now report that exposure of transgenic mice to an "enriched environment" results in pronounced reductions in cerebral Abeta levels and amyloid deposits, compared to animals raised under "standard housing" conditions. The enzymatic activity of an Abeta-degrading endopeptidase, neprilysin, is elevated in the brains of "enriched" mice and inversely correlated with amyloid burden. Moreover, DNA microarray analysis revealed selective upregulation in levels of transcripts encoded by genes associated with learning and memory, vasculogenesis, neurogenesis, cell survival pathways, Abeta sequestration, and prostaglandin synthesis. These studies provide evidence that environmental enrichment leads to reductions in steady-state levels of cerebral Abeta peptides and amyloid deposition and selective upregulation in levels of specific transcripts in brains of transgenic mice.

The degenerative process of Alzheimer's disease is linked to a shift in the balance between amyloid-␤ (A␤) production, clearance, and degradation. Neprilysin has recently been implicated as a major extracellular A␤ degrading enzyme in the brain. However, there has been no direct demonstration that neprilysin antagonizes the deposition of amyloid-␤ in vivo. To address this issue, a lentiviral vector expressing human neprilysin (Lenti-Nep) was tested in transgenic mouse models of amyloidosis. We show that unilateral intracerebral injection of Lenti-Nep reduced amyloid-␤ deposits by half relative to the untreated side. Furthermore, Lenti-Nep ameliorated neurodegenerative alterations in the frontal cortex and hippocampus of these transgenic mice. These data further support a role for neprilysin in regulating cerebral amyloid deposition and suggest that gene transfer approaches might have potential for the development of alternative therapies for Alzheimer's disease.

Factors that elevate amyloid-␤ (A␤) peptide levels are associated with an increased risk for Alzheimer's disease. Insulysin has been identified as one of several proteases potentially involved in A␤ degradation based on its hydrolysis of A␤ peptides in vitro. In this study, in vivo levels of brain A␤40 and A␤42 peptides were found to be increased significantly (1.6-and 1.4-fold, respectively) in an insulysin-deficient gene-trap mouse model. A 6-fold increase in the level of the ␥-secretase-generated C-terminal fragment of the A␤ precursor protein in the insulysin-deficient mouse also was found. In mice heterozygous for the insulysin gene trap, in which insulysin activity levels were decreased Ϸ50%, brain A␤ peptides were increased to levels intermediate between those in wild-type mice and homozygous insulysin gene-trap mice that had no detectable insulysin activity. These findings indicate that there is an inverse correlation between in vivo insulysin activity levels and brain A␤ peptide levels and suggest that modulation of insulysin activity may alter the risk for Alzheimer's disease.A myloid-␤ (A␤) peptide-containing senile plaques are a prominent feature of the pathology of Alzheimer's disease (AD) and occur consistently in AD of all etiology, from earlyonset, familial-linked AD to late-onset AD of indeterminate origin (1). A␤ is formed from the amyloid precursor protein (APP) by sequential enzymatic processing. A ␤-secretase cleavage first yields the 99-aa C-terminal fragment (CTF) of APP, CTF␤, which then is cleaved by ␥-secretase to release A␤ peptides, predominately A␤40 and A␤42, and the CTF␥ peptides of 49-51 residues (2).The proteolysis of APP to yield A␤ peptides is a normal physiologic process observed in multiple cell types, although the endogenous function of APP processing and its products is still not well defined (3). To date, all of the genetic mutations linked to AD result in increased A␤ accumulation, albeit by distinct mechanisms.Although considerable effort has been directed toward generating specific inhibitors of the ␤-and ␥-secretases as a means of preventing A␤ formation (4), the mechanism of A␤ clearance also is of considerable interest because the steady-state concentrations of A␤ peptides are dependent on both their rates of synthesis and their rates of clearance. Recent studies suggest that an important route of A␤ clearance is through hydrolytic cleavage by proteases and peptidases (recently reviewed in refs. 36-38). The peptidase insulysin (EC 3.4.24.56) is one of the enzymes that has been suggested as a candidate A␤-degrading enzyme primarily based on its ability to degrade A␤ peptides in vitro (5-7).Insulysin is a zinc metalloprotease, originally identified as an insulin-degrading enzyme (8), that migrates with reported molecular masses of 110-115 kDa on SDS͞polyacrylamide gels and has no demonstrated posttranslational modifications. The observed molecular mass of insulysin is consistent with the use of the second of its two potential translation initiation sites, although N-te...