Two substrates of insulin-degrading enzyme (IDE), amyloid ␤-protein (A␤) and insulin, are critically important in the pathogenesis of Alzheimer's disease (AD) and type 2 diabetes mellitus (DM2), respectively. We previously identified IDE as a principal regulator of A␤ levels in neuronal and microglial cells. A small chromosomal region containing a mutant IDE allele has been associated with hyperinsulinemia and glucose intolerance in a rat model of DM2. Human genetic studies have implicated the IDE region of chromosome 10 in both AD and DM2. To establish whether IDE hypofunction decreases A␤ and insulin degradation in vivo and chronically increases their levels, we characterized mice with homozygous deletions of the IDE gene (IDE ؊͞؊). IDE deficiency resulted in a >50% decrease in A␤ degradation in both brain membrane fractions and primary neuronal cultures and a similar deficit in insulin degradation in liver. The IDE ؊͞؊ mice showed increased cerebral accumulation of endogenous A␤, a hallmark of AD, and had hyperinsulinemia and glucose intolerance, hallmarks of DM2. Moreover, the mice had elevated levels of the intracellular signaling domain of the ␤-amyloid precursor protein, which was recently found to be degraded by IDE in vitro. Together with emerging genetic evidence, our in vivo findings suggest that IDE hypofunction may underlie or contribute to some forms of AD and DM2 and provide a mechanism for the recently recognized association among hyperinsulinemia, diabetes, and AD. I nsulin-degrading enzyme (IDE, insulysin) is an Ϸ110-kDa thiol zinc-metalloendopeptidase located in cytosol, peroxisomes, endosomes, and on the cell surface (1-4) that cleaves small proteins of diverse sequence, many of which share a propensity to form ␤-pleated sheet-rich amyloid fibrils under certain conditions [e.g., amyloid ␤-protein (A␤), insulin, glucagon, amylin, atrial natriuretic factor, and calcitonin] (5, 6). IDE is the major enzyme responsible for insulin degradation in vitro (1), but the extent to which it mediates insulin catabolism in vivo has been controversial, with doubts expressed that IDE has any physiological role in insulin catabolism (7). Insulin, which is critical for glucose, lipid, and protein metabolism, as well as for cell growth and differentiation, is cleared mainly by the liver and kidney, but most other tissues also degrade the hormone. It was recently shown that transferring an Ϸ3.7-cM chromosomal region containing the IDE gene from an inbred rat model of type 2 diabetes mellitus (DM2) (the GK rat) to a normoglycemic rat recapitulated several features of the diabetic phenotype, including hyperinsulinemia and postprandial hyperglycemia (8). The GK allele of IDE in this chromosomal region was found to bear two missense mutations that, when transfected into COS-1 cells, resulted in 31% less insulin degradation compared with cells transfected with the WT allele. Furthermore, the IDE region of chromosome 10q has been genetically linked to DM2 (9, 10) and to elevated fasting glucose levels [20-year means (1...

Proteolytic processing of the amyloid precursor protein (APP) by ␤-secretase, ␤-site APP cleaving enzyme (BACE1), is the initial step in the production of the amyloid ␤ (A␤) peptide, which is involved in the pathogenesis of Alzheimer's disease. The normal cellular function of the prion protein (PrP C ), the causative agent of the transmissible spongiform encephalopathies such as CreutzfeldtJakob disease in humans, remains enigmatic. Because both APP and PrP C are subject to proteolytic processing by the same zinc metalloproteases, we tested the involvement of PrP C in the proteolytic processing of APP. Cellular overexpression of PrP C inhibited the ␤-secretase cleavage of APP and reduced A␤ formation. Conversely, depletion of PrP C in mouse N2a cells by siRNA led to an increase in A␤ peptides secreted into the medium. In the brains of PrP knockout mice and in the brains from two strains of scrapieinfected mice, A␤ levels were significantly increased. Two mutants of PrP, PG14 and A116V, that are associated with familial human prion diseases failed to inhibit the ␤-secretase cleavage of APP. Using constructs of PrP, we show that this regulatory effect of PrP C on the ␤-secretase cleavage of APP required the localization of PrP C to cholesterol-rich lipid rafts and was mediated by the N-terminal polybasic region of PrP C via interaction with glycosaminoglycans. In conclusion, this is a mechanism by which the cellular production of the neurotoxic A␤ is regulated by PrP C and may have implications for both Alzheimer's and prion diseases.lipid raft ͉ proteolysis ͉ scrapie ͉ glycosaminoglycan A lzheimer's disease (AD) is characterized by the presence of extracellular senile plaques and intracellular neurofibrillary tangles within the afflicted brain. The major constituents of senile plaques are the amyloid ␤ (A␤) peptides, which are derived from the proteolytic processing of the amyloid precursor protein (APP) (1). In the amyloidogenic pathway, ␤-secretase cleavage of APP yields a soluble N-terminal fragment sAPP␤, along with a short membrane-bound C-terminal fragment that is subsequently cleaved by ␥-secretase to release the A␤ peptides. In the alternative, nonamyloidogenic pathway, ␣-secretase cleaves APP within the A␤ sequence, thus precluding the formation of A␤, and releases a soluble N-terminal fragment sAPP␣. The transmembrane aspartyl protease, ␤-site APP cleaving enzyme (BACE1), has been identified as ␤-secretase (2), members of the ADAM (a disintegrin and metalloprotease) family, particularly ADAM10 and ADAM17, are responsible for ␣-secretase cleavage (3), while a complex of at least four proteins, the presenilins, nicastrin, Aph-1, and Pen-2, constitutes the ␥-secretase (2).The prion protein (PrP) is the causative agent of the transmissible spongiform encephalopathies (TSEs) that include CreutzfeldtJakob disease (CJD), Gerstmann-Scheinker-Straussler (GSS) disease, kuru and fatal familial insomnia in humans, bovine spongiform encephalopathy in cattle, and scrapie in sheep (4). In these diseases, the normal ce...

Deposition of ␤-amyloid (A␤) peptides in the brain is an early and invariant feature of all forms of Alzheimer's disease. As with any secreted protein, the extracellular concentration of A␤ is determined not only by its production but also by its catabolism. A major focus of Alzheimer's research has been the elucidation of the mechanisms responsible for the generation of A␤. Much less, however, is known about the mechanisms responsible for A␤ removal in the brain. In this report, we describe the identification of endothelin-converting enzyme-1 (ECE-1) as a novel A␤-degrading enzyme. We show that treatment of endogenous ECE-expressing cell lines with the metalloprotease inhibitor phosphoramidon causes a 2-3-fold elevation in extracellular A␤ concentration that appears to be due to inhibition of intracellular A␤ degradation. Furthermore, we show that overexpression of ECE-1 in Chinese hamster ovary cells, which lack endogenous ECE activity, reduces extracellular A␤ concentration by up to 90% and that this effect is completely reversed by treatment of the cells with phosphoramidon. Finally, we show that recombinant soluble ECE-1 is capable of hydrolyzing synthetic A␤40 and A␤42 in vitro at multiple sites.

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...

The abnormal accumulation of ␤-amyloid (A␤) in the brain is an early and invariant feature in Alzheimer's disease (AD) and is believed to play a pivotal role in the etiology and pathogenesis of the disease. As such, a major focus of AD research has been the elucidation of the mechanisms responsible for the generation of A␤. As with any peptide, however, the degree of A␤ accumulation is dependent not only on its production but also on its removal. In cell-based and in vitro models we have previously characterized endothelin-converting enzyme-1 (ECE-1) as an A␤-degrading enzyme that appears to act intracellularly, thus limiting the amount of A␤ available for secretion. To determine the physiological significance of this activity, we analyzed A␤ levels in the brains of mice deficient for ECE-1 and a closely related enzyme, ECE-2. Significant increases in the levels of both A␤40 and A␤42 were found in the brains of these animals when compared with age-matched littermate controls. The increase in A␤ levels in the ECE-deficient mice provides the first direct evidence for a physiological role for both ECE-1 and ECE-2 in limiting A␤ accumulation in the brain and also provides further insight into the factors involved in A␤ clearance in vivo.Alzheimer's disease (AD), 1 the most common cause of dementia in the elderly, is characterized pathologically by the accumulation of ␤-amyloid peptides (A␤40 and A␤42) in the brain. Although considerable attention has been focused on understanding the enzymes and processes involved in the production of A␤, very little is known regarding the processes by which A␤ is normally removed. This removal can be in the form of transport of the peptide into the cerebrospinal fluid for peripheral clearance, by binding to proteins that sequester the peptide in a nonreactive form, or by direct catabolism of A␤. A significant role for A␤ catabolism has been highlighted in a recent report by Saido and colleagues (1) in which they showed that infusion of the metalloprotease inhibitor thiorphan into the hippocampus of rats resulted in localized A␤ deposition, reportedly through the inhibition of A␤ degradation by neprilysin (NEP).A␤ catabolism appears complex. Recent reports suggest significant roles for insulin-degrading enzyme, NEP, and the plasmin system in the catabolism of A␤ (1-10). Angiotensin-converting enzyme, matrix metalloproteinase-9, EC 3.4.24.15, and ␣-2 macroglobulin complexes have also been reported to play a role in A␤ degradation on the basis of in vitro and cell-based assays (11)(12)(13)(14). To date, however, only neprilysin has been reported to influence A␤ levels in the brains of knock-out mice (1,5,15).Using pharmacological, molecular, and biochemical approaches, we have previously characterized endothelin-converting enzyme-1 (ECE-1) as a novel A␤-degrading enzyme that appears to act intracellularly to reduce the amount of A␤ available for secretion (16). Overexpression of ECE-1 in cells that lack endogenous ECE activity results in a pronounced decrease in A␤ accumulation in the...

The causes of cerebral accumulation of amyloid beta-protein (Abeta) in most cases of Alzheimer's disease (AD) remain unknown. We recently found that homozygous deletion of the insulin-degrading enzyme (IDE) gene in mice results in an early and marked elevation of cerebral Abeta. Both genetic linkage and allelic association in the IDE region of chromosome 10 have been reported in families with late-onset AD. For IDE to remain a valid candidate gene for late-onset AD on functional grounds, it must be shown that partial loss of function of IDE can still alter Abeta degradation, but without causing early, severe elevation of brain Abeta. Here, we show that naturally occurring IDE missense mutations in a well-characterized rat model of type 2 diabetes mellitus (DM2) result in decreased catalytic efficiency and a significant approximately 15 to 30% deficit in the degradation of both insulin and Abeta. Endogenously secreted Abeta(40) and Abeta(42) are significantly elevated in primary neuronal cultures from animals with the IDE mutations, but there is no increase in steady-state levels of rodent Abeta in the brain up to age 14 months. We conclude that naturally occurring, partial loss-of-function mutations in IDE sufficient to cause DM2 also impair neuronal regulation of Abeta levels, but the brain can apparently compensate for the partial deficit during the life span of the rat. Our findings have relevance for the emerging genetic evidence suggesting that IDE may be a late-onset AD-risk gene, and for the epidemiological relationships among hyperinsulinemia, DM2, and AD.

The deposition of ␤-amyloid in the brain is a pathological hallmark of Alzheimer disease (AD). Normally, the accumulation of ␤-amyloid is prevented in part by the activities of several degradative enzymes, including the endothelin-converting enzymes, neprilysin, insulin-degrading enzyme, and plasmin. Recent reports indicate that another metalloprotease, angiotensin-converting enzyme (ACE), can degrade ␤-amyloid in vitro and in cellular overexpression experiments. In addition, ACE gene variants are linked to AD risk in several populations. Angiotensin-converting enzyme, neprilysin and endothelin-converting enzyme function as vasopeptidases and are the targets of drugs designed to treat cardiovascular disorders, and ACE inhibitors are commonly prescribed. We investigated the potential physiological role of ACE in regulating endogenous brain ␤-amyloid levels for two reasons: first, to determine whether ␤-amyloid degradation might be the mechanism by which ACE is associated with AD, and second, to determine whether ACE inhibitor drugs might block ␤-amyloid degradation in the brain and potentially increase the risk for AD. We analyzed ␤-amyloid accumulation in brains from ACE-deficient mice and in mice treated with ACE inhibitors and found that ACE deficiency did not alter steady-state ␤-amyloid concentration. In contrast, ␤-amyloid levels are significantly elevated in endothelin-converting enzyme and neprilysin knock-out mice, and inhibitors of these enzymes cause a rapid increase in ␤-amyloid concentration in the brain. The results of these studies do not support a physiological role for ACE in the degradation of ␤-amyloid in the brain but confirm roles for endothelin-converting enzyme and neprilysin and indicate that reductions in these enzymes result in additive increases in brain amyloid ␤-peptide levels.

Targeted deletion of two members of the FE65 family of adaptor proteins, FE65 and FE65L1, results in cortical dysplasia. Heterotopias resembling those found in cobblestone lissencephalies in which neuroepithelial cells migrate into superficial layers of the developing cortex, aberrant cortical projections and loss of infrapyramidal mossy fibers arise in FE65/FE65L1 compound null animals, but not in single gene knockouts. The disruption of pial basal membranes underlying the heterotopias and poor organization of fibrillar laminin by isolated meningeal fibroblasts from double knockouts suggests that FE65 proteins are involved in basement membrane assembly. A similar phenotype is observed in triple mutant mice lacking the APP family members APP, APLP1 and APLP2, all of which interact with FE65 proteins, suggesting that this phenotype may be caused by decreased transmission of an APP-dependent signal through the FE65 proteins. The defects observed in the double knockout may also involve the family of Ena/Vasp proteins, which participate in actin cytoskeleton remodeling and interact with the WW domains of FE65 proteins.