Age-Related Increase of Insulin-Degrading Enzyme Is Inversely Correlated with Cognitive Function in APPswe/PS1dE9 Mice.

BACKGROUND Insulin-degrading enzyme (IDE) is an important regulator for Ab clearance and diabetes. Although it is indispensable in removing plaques related to onset Alzheimer's disease (AD) and in degrading insulin related to diabetes, there have been few studies on the dynamic level of IDE in different stages of AD. MATERIAL AND METHODS The present study explored the level IDE protein in different stages of APPswe/PS1dE9 mice and their correlations with cognitive decline. The 4-month-old, 10-month-old, and 18-month-old mice were used as the different age stages of mice. Cognitive function was evaluated using the Morris water maze test. We also observed the level of Ab plaques in brain regions of different stages. RESULTS The data revealed that the expression of IDE was dramatically higher than in age-matched wild mice at the age of 10 months and 18 months. In terms of distribution, Aß plaques were deposited mostly in the cortex and hippocampus, especially in 10-month-old and 18-month-old APPswe/PS1dE9 mice. The cognitive function of 4-month-old APPswe/PS1dE9 mice was not significantly differ in spatial learning. However, the cognitive function, both spatial learning and spatial memory, was dramatically lower in 10-month-old and 18-month-old groups. CONCLUSIONS There was a positive correlation between the expression of IDE and spatial memory in 10-month-old and 18-month-old APPswe/PS1dE9 mice. The study of this protein may provide reference values for the further study of IDE in Alzheimer's disease.

Multiple functions of insulin-degrading enzyme: a metabolic crosslight?

Insulin-degrading enzyme (IDE) is a ubiquitous zinc peptidase of the inverzincin family, which has been initially discovered as the enzyme responsible for insulin catabolism; therefore, its involvement in the onset of diabetes has been largely investigated. However, further studies on IDE unraveled its ability to degrade several other polypeptides, such as ß-amyloid, amylin, and glucagon, envisaging the possible implication of IDE dys-regulation in the "aggregopathies" and, in particular, in neurodegenerative diseases. Over the last decade, a novel scenario on IDE biology has emerged, pointing out a multi-functional role of this enzyme in several basic cellular processes. In particular, latest advances indicate that IDE behaves as a heat shock protein and modulates the ubiquitin-proteasome system, suggesting a major implication in proteins turnover and cell homeostasis. In addition, recent observations have highlighted that the regulation of glucose metabolism by IDE is not merely based on its largely proposed role in the degradation of insulin in vivo. There is increasing evidence that improper IDE function, regulation, or trafficking might contribute to the etiology of metabolic diseases. In addition, the enzymatic activity of IDE is affected by metals levels, thus suggesting a role also in the metal homeostasis (metallostasis), which is thought to be tightly linked to the malfunction of the "quality control" machinery of the cell. Focusing on the physiological role of IDE, we will address a comprehensive vision of the very complex scenario in which IDE takes part, outlining its crucial role in interconnecting several relevant cellular processes.

Characterization of insulin-degrading enzyme-mediated cleavage of Aß in distinct aggregation states.

To enhance our understanding of the potential therapeutic utility of insulin-degrading enzyme (IDE) in Alzheimer's disease (AD), we studied in vitro IDE-mediated degradation of different amyloid-beta (Aß) peptide aggregation states. Our findings show that IDE activity is driven by the dynamic equilibrium between Aß monomers and higher ordered aggregates. We identify Met(35)-Val(36) as a novel IDE cleavage site in the Aß sequence and show that Aß fragments resulting from IDE cleavage form non-toxic amorphous aggregates. These findings need to be taken into account in therapeutic strategies designed to increase Aß clearance in AD patients by modulating IDE activity.

Metabolism of cryptic peptides derived from neuropeptide FF precursors: the involvement of insulin-degrading enzyme.

The term "cryptome" refers to the subset of cryptic peptides with bioactivities that are often unpredictable and very different from the parent protein. These cryptic peptides are generated by proteolytic cleavage of proteases, whose identification in vivo can be very challenging. In this work, we show that insulin-degrading enzyme (IDE) is able to degrade specific amino acid sequences present in the neuropeptide pro-NPFFA (NPFF precursor), generating some cryptic peptides that are also observed after incubation with rat brain cortex homogenate. The reported experimental findings support the increasingly accredited hypothesis, according to which, due to its wide substrate selectivity, IDE is involved in a wide variety of physiopathological processes.

Analysis of the proteolysis of bioactive peptides using a peptidomics approach.

Identifying the peptidases that inactivate bioactive peptides (e.g., peptide hormones and neuropeptides) in mammals is an important unmet challenge. This protocol describes a recent approach that uses liquid chromatography-mass spectrometry (LC-MS) peptidomics to identify endogenous cleavage sites of a bioactive peptide; it also addresses the subsequent biochemical purification of a candidate peptidase on the basis of these cleavage sites and the validation of the candidate peptidase's role in the physiological regulation of the bioactive peptide by examining a peptidase-knockout mouse. We highlight the successful application of this protocol in the discovery that insulin-degrading enzyme (IDE) regulates physiological calcitonin gene-related peptide (CGRP) levels, and we detail the key stages and steps in this approach. This protocol requires 7 d of work; however, the total time for this protocol is highly variable because of its dependence on the availability of biological reagents such as purified enzymes and knockout mice. The protocol is valuable because it expedites the characterization of mammalian peptidases, such as IDE, which in certain instances can be used to develop novel therapeutics.

Insulin-degrading enzyme (IDE): a novel heat shock-like protein.

Insulin-degrading enzyme (IDE) is a highly conserved zinc metallopeptidase that is ubiquitously distributed in human tissues, and particularly abundant in the brain, liver, and muscles. IDE activity has been historically associated with insulin and ß-amyloid catabolism. However, over the last decade, several experimental findings have established that IDE is also involved in a wide variety of physiopathological processes, including ubiquitin clearance and Varicella Zoster Virus infection. In this study, we demonstrate that normal and malignant cells exposed to different stresses markedly up-regulate IDE in a heat shock protein (HSP)-like fashion. Additionally, we focused our attention on tumor cells and report that (i) IDE is overexpressed in vivo in tumors of the central nervous system (CNS); (ii) IDE-silencing inhibits neuroblastoma (SHSY5Y) cell proliferation and triggers cell death; (iii) IDE inhibition is accompanied by a decrease of the poly-ubiquitinated protein content and co-immunoprecipitates with proteasome and ubiquitin in SHSY5Y cells. In this work, we propose a novel role for IDE as a heat shock protein with implications in cell growth regulation and cancer progression, thus opening up an intriguing hypothesis of IDE as an anticancer target.

The association between small vessel infarcts and the activities of amyloid-ß peptide degrading proteases in apolipoprotein E4 allele carriers.

Small vessel (SV) and large vessel (LV) brain infarcts are distinct pathologies. Using a homebound elderly sample, the numbers of either infarct subtypes were similar between those apolipoprotein E4 allele (ApoE4) carriers (n = 80) and noncarriers (n = 243). We found that the higher the number of SV infarcts, but not LV infarcts, a participant had, the higher the activity of substrate V degradation in serum especially among ApoE4 carriers (ß = +0.154, SE = 0.031, P < .0001) after adjusting for the confounders. Since substrate V degradation could be mediated by insulin-degrading enzyme (IDE) or/and angiotensin-converting enzyme (ACE), but no relationship was found between SV infarcts and specific ACE activities, blood IDE may be a useful biomarker to distinguish the brain infarct subtypes. Insulin-degrading enzyme in blood may also imply an important biomarker and a pathological event in Alzheimer disease through SV infarcts in the presence of ApoE4.

Norepinephrine promotes microglia to uptake and degrade amyloid beta peptide through upregulation of mouse formyl peptide receptor 2 and induction of insulin-degrading enzyme.

Locus ceruleus (LC) is the main subcortical site of norepinephrine synthesis. In Alzheimer's disease (AD) patients and rodent models, degeneration of LC neurons and reduced levels of norepinephrine in LC projection areas are significantly correlated with the increase in amyloid plaques, neurofibrillary tangles, and severity of dementia. Activated microglia play a pivotal role in the progression of AD by either clearing amyloid beta peptide (Abeta) deposits through uptake of Abeta or releasing cytotoxic substances and proinflammatory cytokines. Here, we investigated the effect of norepinephrine on Abeta uptake and clearance by murine microglia and explored the underlying mechanisms. We found that murine microglia cell line N9 and primary microglia expressed beta(2) adrenergic receptor (AR) but not beta(1) and beta(3)AR. Norepinephrine and isoproterenol upregulated the expression of Abeta receptor mFPR2, a mouse homolog of human formyl peptide receptor FPR2, through activation of beta(2)AR in microglia. Norepinephrine also induced mFPR2 expression in mouse brain. Activation of beta(2)AR in microglia promoted Abeta(42) uptake through upregulation of mFPR2 and enhanced spontaneous cell migration but had no effect on cell migration in response to mFPR2 agonists. Furthermore, activation of beta(2)AR on microglia induced the expression of insulin-degrading enzyme and increased the degradation of Abeta(42). Mechanistic studies showed that isoproterenol induced mFPR2 expression through ERK1/2-NF-kappaB and p38-NF-kappaB signaling pathways. These findings suggest that noradrenergic innervation from LC is needed to maintain adequate Abeta uptake and clearance by microglia, and norepinephrine is a link between neuron and microglia to orchestrate the host response to Abeta in AD.

Inulin hydrolysis and citric acid production from inulin using the surface-engineered Yarrowia lipolytica displaying inulinase.

The INU1 gene encoding exo-inulinase cloned from Kluyveromyces marxianus CBS 6556 was ligated into the surface display plasmid and expressed in the cells of the marine-derived yeast Yarrowia lipolytica which can produce citric acid. The expressed inulinase was immobilized on the yeast cells. The activity of the immobilized inulinase with 6 x His tag was found to be 22.6 U mg(-1) of cell dry weight after cell growth for 96 h. The optimal pH and temperature of the displayed inulinase were 4.5 and 50 degrees C, respectively and the inulinase was stable in the pH range of 3-8 and in the temperature range of 0-50 degrees C. During the inulin hydrolysis, the optimal inulin concentration was 12.0% and the optimal amount of added inulinase was 181.6 U g(-1) of inulin. Under such conditions, over 77.9% of inulin was hydrolyzed within 10h and the hydrolysate contained main monosaccharides and disaccharides, and minor trisaccharides. During the citric acid production in the flask level, the recombinant yeast could produce 77.9 g L(-1) citric acid and 5.3 g L(-1) iso-citric acid from inulin while 68.9 g L(-1) of citric acid and 4.1 g L(-1) iso-citric acid in the fermented medium were attained within 312 h of the 2-L fermentation, respectively.

Protective role of Cys-178 against the inactivation and oligomerization of human insulin-degrading enzyme by oxidation and nitrosylation.

Insulin-degrading enzyme (IDE), a 110-kDa metalloendopeptidase, hydrolyzes several physiologically relevant peptides, including insulin and amyloid-beta (Abeta). Human IDE has 13 cysteines and is inhibited by hydrogen peroxide and S-nitrosoglutathione (GSNO), donors of reactive oxygen and nitrogen species, respectively. Here, we report that the oxidative burst of BV-2 microglial cells leads to oxidation or nitrosylation of secreted IDE, leading to the reduced activity. Hydrogen peroxide and GSNO treatment of IDE reduces the V(max) for Abeta degradation, increases IDE oligomerization, and decreases IDE thermostability. Additionally, this inhibitory response of IDE is substrate-dependent, biphasic for Abeta degradation but monophasic for a shorter bradykinin-mimetic substrate. Our mutational analysis of IDE and peptide mass fingerprinting of GSNO-treated IDE using Fourier transform-ion cyclotron resonance mass spectrometer reveal a surprising interplay of Cys-178 with Cys-110 and Cys-819 for catalytic activity and with Cys-789 and Cys-966 for oligomerization. Cys-110 is near the zinc-binding catalytic center and is normally buried. The oxidation and nitrosylation of Cys-819 allow Cys-110 to be oxidized or nitrosylated, leading to complete inactivation of IDE. Cys-789 is spatially adjacent to Cys-966, and their nitrosylation and oxidation together trigger the oligomerization and inhibition of IDE. Interestingly, the Cys-178 modification buffers the inhibition caused by Cys-819 modification and prevents the oxidation or nitrosylation of Cys-110. The Cys-178 modification can also prevent the oligomerization-mediated inhibition. Thus, IDE can be intricately regulated by reactive oxygen or nitrogen species. The structure of IDE reveals the molecular basis for the long distance interactions of these cysteines and how they regulate IDE function.

Nitric oxide inhibits insulin-degrading enzyme activity and function through S-nitrosylation.

Insulin-degrading enzyme (IDE) is responsible for the degradation of a number of hormones and peptides, including insulin and amyloid beta (Abeta). Genetic studies have linked IDE to both type 2 diabetes and Alzheimer's disease. Despite its potential importance in these diseases, relatively little is known about the factors that regulate the activity and function of IDE. Protein S-nitrosylation is now recognized as a redox-dependent, cGMP-independent signaling component that mediates a variety of actions of nitric oxide (NO). Here we describe a mechanism of inactivation of IDE by NO. NO donors decreased both insulin and Abeta degrading activities of IDE. Insulin-degrading activity appeared more sensitive to NO inhibition than Abeta degrading activity. IDE-mediated regulation of proteasome activity was affected similarly to insulin-degrading activity. We found IDE to be nitrosylated in the presence of NO donors compared to that of untreated enzyme and the control compound. S-nitrosylation of IDE enzyme did not affect the insulin degradation products produced by the enzyme, nor did NO affect insulin binding to IDE as determined by cross-linking studies. Kinetic analysis of NO inhibition of IDE confirmed that the inhibition was noncompetitive. These data suggest a possible reversible mechanism by which inhibition of IDE under conditions of nitrosative stress could contribute to pathological disease conditions such as Alzheimer's disease and type 2 diabetes.

Evidence that the gene encoding insulin degrading enzyme influences human lifespan.

Studies in model organisms have demonstrated that components of insulin and insulin-like signaling pathways are involved in the regulation of lifespan but the relevance of those findings to humans has remained obscure. Here we provide evidence suggesting that variants of the gene encoding insulin-degrading enzyme (IDE) may be influencing human lifespan. We have employed a variety of models and diverse samples that reproducibly indicate the relative change in IDE genotype frequency across the age spectrum as well as allow the detection of association with age-at-death. A tenable molecular basis of this is suggested by the observation of genetic association with both fasting plasma insulin levels and IDE mRNA expression. Across populations the emergent genetic model is indicative of over-dominance, where heterozygotes of critical markers have increased IDE mRNA expression and insulin levels, and this is reflected in diminished heterozygosity at advanced age. A critical and replicating feature of this study is that change in IDE genotype frequency with advancing age appears to be occurring only in men, and this is supported in that insulin levels are only associated with IDE in men. Results suggest a relationship between a gene that is intimately involved in insulin metabolism and the determination of lifespan in humans, but over-dominance and gender specificity will be important parameters to consider clarifying the biological importance of these findings.

The role of insulin, insulin growth factor, and insulin-degrading enzyme in brain aging and Alzheimer's disease.

Most brain insulin comes from the pancreas and is taken up by the brain by what appears to be a receptor-based carrier. Type 2 diabetes animal models associated with insulin resistance show reduced insulin brain uptake and content. Recent data point to changes in the insulin receptor cascade in obesity-related insulin resistance, suggesting that brain insulin receptors also become less sensitive to insulin, which could reduce synaptic plasticity. Insulin transport to the brain is reduced in aging and in some animal models of type 2 diabetes; brain insulin resistance may be present as well. Studies examining the effect of the hyperinsulinic clamp or intranasal insulin on cognitive function have found a small but consistent improvement in memory and changes in brain neuroelectric parameters in evoked brain potentials consistent with improved attention or memory processing. These effects appear to be due to raised brain insulin levels. Peripheral levels of Insulin Growth Factor-1 (IGF-I) are associated with glucose regulation and influence glucose disposal. There is some indication that reduced sensitivity to insulin or IGF-I in the brain, as observed in aging, obesity, and diabetes, decreases the clearance of Abeta amyloid. Such a decrease involves the insulin receptor cascade and can also increase amyloid toxicity. Insulin and IGF-I may modulate brain levels of insulin degrading enzyme, which would also lead to an accumulation of Abeta amyloid.

Insulin-degrading enzyme in brain microvessels: proteolysis of amyloid {beta} vasculotropic variants and reduced activity in cerebral amyloid angiopathy.

The accumulation of amyloid beta (Abeta) in the walls of small vessels in the cerebral cortex is associated with diseases characterized by dementia or stroke. These include Alzheimer's disease, Down syndrome, and sporadic and hereditary cerebral amyloid angiopathies (CAAs) related to mutations within the Abeta sequence. A higher tendency of Abeta to aggregate, a defective clearance to the systemic circulation, and insufficient proteolytic removal have been proposed as mechanisms that lead to Abeta accumulation in the brain. By using immunoprecipitation and mass spectrometry, we show that insulin-degrading enzyme (IDE) from isolated human brain microvessels was capable of degrading (125)I-insulin and cleaved Abeta-(1-40) wild type and the genetic variants Abeta A21G (Flemish), Abeta E22Q (Dutch), and Abeta E22K (Italian) at the predicted sites. In microvessels from Alzheimer's disease cases with CAA, IDE protein levels showed a 44% increase as determined by sandwich enzyme-linked immunosorbent assay and Western blot. However, the activity of IDE upon radiolabeled insulin was significantly reduced in CAA as compared with age-matched controls. These results support the notion that a defect in Abeta proteolysis by IDE contributes to the accumulation of this peptide in the cortical microvasculature. Moreover they raise the possibility that IDE inhibition or inactivation is a pathogenic mechanism that may open novel strategies for the treatment of cerebrovascular Abeta amyloidoses.

Alternative translation initiation generates a novel isoform of insulin-degrading enzyme targeted to mitochondria.

IDE (insulin-degrading enzyme) is a widely expressed zinc-metallopeptidase that has been shown to regulate both cerebral amyloid beta-peptide and plasma insulin levels in vivo. Genetic linkage and allelic association have been reported between the IDE gene locus and both late-onset Alzheimer's disease and Type II diabetes mellitus, suggesting that altered IDE function may contribute to some cases of these highly prevalent disorders. Despite the potentially great importance of this peptidase to health and disease, many fundamental aspects of IDE biology remain unresolved. Here we identify a previously undescribed mitochondrial isoform of IDE generated by translation at an in-frame initiation codon 123 nucleotides upstream of the canonical translation start site, which results in the addition of a 41-amino-acid N-terminal mitochondrial targeting sequence. Fusion of this sequence to the N-terminus of green fluorescent protein directed this normally cytosolic protein to mitochondria, and full-length IDE constructs containing this sequence were also directed to mitochondria, as revealed by immuno-electron microscopy. Endogenous IDE protein was detected in purified mitochondria, where it was protected from digestion by trypsin and migrated at a size consistent with the predicted removal of the N-terminal targeting sequence upon transport into the mitochondrion. Functionally, we provide evidence that IDE can degrade cleaved mitochondrial targeting sequences. Our results identify new mechanisms regulating the subcellular localization of IDE and suggest previously unrecognized roles for IDE within mitochondria.

Kinetics of amyloid beta-protein degradation determined by novel fluorescence- and fluorescence polarization-based assays.

Proteases that degrade the amyloid beta-protein (Abeta) are important regulators of brain Abeta levels in health and in Alzheimer's disease, yet few practical methods exist to study their detailed kinetics. Here, we describe robust and quantitative Abeta degradation assays based on the novel substrate, fluorescein-Abeta-(1-40)-Lys-biotin (FAbetaB). Liquid chromatography/mass spectrometric analysis shows that FAbetaB is hydrolyzed at closely similar sites as wild-type Abeta by neprilysin and insulin-degrading enzyme, the two most widely studied Abeta-degrading proteases. The derivatized peptide is an avid substrate and is suitable for use with biological samples and in high throughput compound screening. The assays we have developed are easily implemented and are particularly useful for the generation of quantitative kinetic data, as we demonstrate by determining the kinetic parameters of FAbetaB degradation by several Abeta-degrading proteases, including plasmin, which has not previously been characterized. The use of these assays should yield additional new insights into the biology of Abeta-degrading proteases and facilitate the identification of activators and inhibitors of such enzymes.

Does diabetes protect or provoke Alzheimer's disease? Insights into the pathobiology and future treatment of Alzheimer's disease.

Diabetes mellitus has long been considered a risk factor for the development of vascular dementia. Epidemiologic evidence has suggested that diabetes mellitus significantly increases risk for the development of Alzheimer's disease, independent of vascular risk factors. As insulin's role as a neuromodulator in the brain has been described, its significance for AD has also emerged. Insulin dysregulation may contribute to AD pathology through several mechanisms including decreased cortical glucose utilization particularly in the hippocampus and entorhinal cortex; increased oxidative stress through the formation of advanced glycation end-products; increased Tau phosphorylation and neurofibrillary tangle formation; increased b-amyloid aggregation through inhibition of insulin-degrading enzyme. Future treatment of AD might involve pharmacologic and dietary manipulations of insulin and glucose regulation.