Elizabeth A. Eckman

Insulin-degrading enzyme regulates the levels of insulin, amyloid β-protein, and the β-amyloid precursor protein intracellular domain in vivo

Two substrates of insulin-degrading enzyme (IDE), amyloid β-protein (Aβ) and insulin, are critically important in the pathogenesis of Alzheimers disease (AD) and type 2 diabetes mellitus (DM2), respectively. We previously identified IDE as a principal regulator of Aβ 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 Aβ 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 Aβ 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 Aβ, 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 β-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.

Degradation of the Alzheimer's amyloid beta peptide by endothelin-converting enzyme.

Deposition of β-amyloid (Aβ) peptides in the brain is an early and invariant feature of all forms of Alzheimers disease. As with any secreted protein, the extracellular concentration of Aβ is determined not only by its production but also by its catabolism. A major focus of Alzheimers research has been the elucidation of the mechanisms responsible for the generation of Aβ. Much less, however, is known about the mechanisms responsible for Aβ removal in the brain. In this report, we describe the identification of endothelin-converting enzyme-1 (ECE-1) as a novel Aβ-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 Aβ concentration that appears to be due to inhibition of intracellular Aβ degradation. Furthermore, we show that overexpression of ECE-1 in Chinese hamster ovary cells, which lack endogenous ECE activity, reduces extracellular Aβ 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 Aβ40 and Aβ42 in vitro at multiple sites.

Amyloid-β peptide levels in brain are inversely correlated with insulysin activity levels in vivo

Factors that elevate amyloid-β (Aβ) peptide levels are associated with an increased risk for Alzheimers disease. Insulysin has been identified as one of several proteases potentially involved in Aβ degradation based on its hydrolysis of Aβ peptides in vitro. In this study, in vivo levels of brain Aβ40 and Aβ42 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 γ-secretase-generated C-terminal fragment of the Aβ 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 ≈50%, brain Aβ 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 Aβ peptide levels and suggest that modulation of insulysin activity may alter the risk for Alzheimers disease.

Cellular prion protein regulates beta-secretase cleavage of the Alzheimer's amyloid precursor protein

Proteolytic processing of the amyloid precursor protein (APP) by β-secretase, β-site APP cleaving enzyme (BACE1), is the initial step in the production of the amyloid β (Aβ) peptide, which is involved in the pathogenesis of Alzheimers disease. The normal cellular function of the prion protein (PrPC), the causative agent of the transmissible spongiform encephalopathies such as Creutzfeldt–Jakob disease in humans, remains enigmatic. Because both APP and PrPC are subject to proteolytic processing by the same zinc metalloproteases, we tested the involvement of PrPC in the proteolytic processing of APP. Cellular overexpression of PrPC inhibited the β-secretase cleavage of APP and reduced Aβ formation. Conversely, depletion of PrPC in mouse N2a cells by siRNA led to an increase in Aβ peptides secreted into the medium. In the brains of PrP knockout mice and in the brains from two strains of scrapie-infected mice, Aβ levels were significantly increased. Two mutants of PrP, PG14 and A116V, that are associated with familial human prion diseases failed to inhibit the β-secretase cleavage of APP. Using constructs of PrP, we show that this regulatory effect of PrPC on the β-secretase cleavage of APP required the localization of PrPC to cholesterol-rich lipid rafts and was mediated by the N-terminal polybasic region of PrPC via interaction with glycosaminoglycans. In conclusion, this is a mechanism by which the cellular production of the neurotoxic Aβ is regulated by PrPC and may have implications for both Alzheimers and prion diseases.

Partial Loss-of-Function Mutations in Insulin-Degrading Enzyme that Induce Diabetes also Impair Degradation of Amyloid β-Protein

The causes of cerebral accumulation of amyloid beta-protein (Abeta) in most cases of Alzheimers 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.

Regulation of Steady-state β-Amyloid Levels in the Brain by Neprilysin and Endothelin-converting Enzyme but Not Angiotensin-converting Enzyme

The deposition of β-amyloid in the brain is a pathological hallmark of Alzheimer disease (AD). Normally, the accumulation of β-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 β-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 β-amyloid levels for two reasons: first, to determine whether β-amyloid degradation might be the mechanism by which ACE is associated with AD, and second, to determine whether ACE inhibitor drugs might block β-amyloid degradation in the brain and potentially increase the risk for AD. We analyzed β-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 β-amyloid concentration. In contrast, β-amyloid levels are significantly elevated in endothelin-converting enzyme and neprilysin knock-out mice, and inhibitors of these enzymes cause a rapid increase in β-amyloid concentration in the brain. The results of these studies do not support a physiological role for ACE in the degradation of β-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 β-peptide levels.

Essential roles for the FE65 amyloid precursor protein-interacting proteins in brain development

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.

Reductions in levels of the Alzheimer’s amyloid β peptide after oral administration of ginsenosides

For millennia, ginseng and some of its components have been used to treat a wide variety of medical conditions, including age‐related memory impairment. Because of its purported effects and apparently low rate of side effects, ginseng remains one of the top selling natural product remedies in the United States. Given its potential role for improving age‐related memory impairments and its common use in China for the treatment of Alzheimers disease‐like symptoms, we analyzed the effects of commercially available preparations of ginseng on the accumulation of the Alzheimers amyloid β peptide (Aβ) in a cell‐based model system. In this model system, ginseng treatment resulted in a significant reduction in the levels of Aβ in the conditioned medium. We next examined the effects of several compounds isolated from ginseng and found that certain ginsenosides lowered Aβ concentration in a dose‐dependent manner with ginsenoside Rg3 having an approximate IC50 of under 25 µM against Aβ42. Furthermore, we found that three of these isolated components, ginsenoside Rgl, Rg3, and RE, resulted in significant reductions in the amount of Aβ detected in the brains of animals after single oral doses of these agents. The results indicate that ginseng itself, or purified ginsenosides, may have similarly useful effects in human disease.—Chen, F., Eckman, E. A., Eckman, C. B. Reductions in levels of the Alzheimers amyloid β peptide after oral administration of ginsenosides. FASEB J. 20, E599‐E604 (2006)

Molecular Characterization of Mutations That Cause Globoid Cell Leukodystrophy and Pharmacological Rescue Using Small Molecule Chemical Chaperones

Globoid cell leukodystrophy (GLD) (Krabbe disease) is an autosomal recessive, degenerative, lysosomal storage disease caused by a severe loss of galactocerebrosidase (GALC) enzymatic activity. Of the >70 disease-causing mutations in the GALC gene, most are located outside of the catalytic domain of the enzyme. To determine how GALC mutations impair enzymatic activity, we investigated the impact of multiple disease-causing mutations on GALC processing, localization, and enzymatic activity. Studies in mammalian cells revealed dramatic decreases in GALC activity and a lack of appropriate protein processing into an N-terminal GALC fragment for each of the mutants examined. Consistent with this, we observed significantly less GALC localized to the lysosome and impairment in either the secretion or reuptake of mutant GALC. Notably, the D528N mutation was found to induce hyperglycosylation and protein misfolding. Reversal of these conditions resulted in an increase in proper processing and GALC activity, suggesting that glycosylation may play a critical role in the disease process in patients with this mutation. Recent studies have shown that enzyme inhibitors can sometimes “chaperone” misfolded polypeptides to their appropriate target organelle, bypassing the normal cellular quality control machinery and resulting in enhanced activity. To determine whether this may also work for GLD, we examined the effect of α-lobeline, an inhibitor of GALC, on D528N mutant cells. After treatment, GALC activity was significantly increased. This study suggests that mutations in GALC can cause GLD by impairing protein processing and/or folding and that pharmacological chaperones may be potential therapeutic agents for patients carrying certain mutations.

Reduction of Aβ accumulation in the Tg2576 animal model of Alzheimer’s disease after oral administration of the phosphatidyl-inositol kinase inhibitor wortmannin 1

The abnormal accumulation of the amyloid β protein (Aβ) has been implicated as an early and critical event in the etiology and pathogenesis of Alzheimers disease (AD). Compounds that reduce Aβ accumulation may therefore be useful therapeutically. In cell‐based screens we detected a significant reduction in Aβ concentration after treatment with the phosphatidylinositol kinase inhibitors wortmannin and LY294002. To determine the effect of this class of compounds on in vivo Aβ accumulation, we administered wortmannin to the Tg2576 mouse model of AD. Oral administration of wortmannin over four months resulted in a significant, non‐overlapping 40%–50% reduction in the number of senile plaques, one of the pathological hallmarks of AD. Sandwich ELISA analysis of formic acid extractable Aβ in the brain of treated animals indicates that both Aβ40 and the longer, more amyloidogenic form of the peptide, Aβ42, were significantly reduced. These data provide the first direct evidence that compounds identified by their ability to reduce Aβ concentration in vitro can reduce Aβ accumulation and deposition in the brain, thus establishing a basic paradigm for the identification and evaluation of additional compounds that lower Aβ accumulation.