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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Alzheimers Dis. Author manuscript; available in PMC Feb 10, 2010.
Published in final edited form as:
PMCID: PMC2819406
NIHMSID: NIHMS173996
Effects of 4-Hydroxy-Nonenal and Amyloid-β on Expression and Activity of Endothelin Converting Enzyme and Insulin Degrading Enzyme in SH-SY5Y Cells
Rui Wang,1 Suqing Wang,1 James S. Malter, and Deng-Shun Wang*
Department of Pathology and Laboratory Medicine, School of Medicine and Public Health, University of Wisconsin, Madison, WI, USA
* Corresponding author: Deng-Shun Wang, MD, PhD, Department of Pathology and Laboratory Medicine, Madison, WI 53706, USA. Tel.: +1 608262 9825; Fax: +1 608 265 3301; dwang6/at/wisc.edu
1These authors contributed equally to this work.
The cerebral accumulation of amyloid-β (Aβ) is a consistent feature of and likely contributor to the development of Alzheimer’s disease (AD). In addition to dysregulated production, increasing experimental evidence suggests reduced catabolism plays an important role in Aβ accumulation. Although endothelin converting enzyme (ECE) and insulin degrading enzyme (IDE) degrade and thus contribute to regulating the steady-state levels of Aβ, how these enzymes are regulated remain poorly understood. In this study, we investigated the effects of 4-hydroxy-nonenal (HNE) and Aβ on the expression and activity of ECE-1 and IDE in human neuroblastoma SH-SY5Y cells. Treatment with HNE or Aβ upregulated ECE-1 mRNA and protein, while IDE was unchanged. Although both ECE-1 and IDE were oxidized within 24 h of HNE or Aβ treatment, ECE-1 catalytic activity was elevated while IDE specific activity was unchanged. The results demonstrated for the first time that both ECE-1 and IDE are substrates of HNE modification induced by Aβ. In addition, the results suggest complex mechanisms underlying the regulation of their enzymatic activity.
Keywords: Alzheimer’s disease, amyloid-β, degradation, endothelin converting enzyme, 4-hydroxy-nonenal (HNE), insulin degrading enzyme, oxidative stress
Alzheimer’s disease (AD) is a progressive neurode-generative disorder that gradually damages the neocortex and hippocampus, reducing memory, learning, and reasoning [1]. The extensive deposition of amyloid-β (Aβ) is a pathological hallmark of AD. While still controversial, Aβ deposition and AD development is supported by a variety of in vivo and in vitro evidence [25]. Although the mechanisms that contribute to abnormal Aβ accumulation are not fully understood, both increased production and decreased degradation have been observed [6]. Despite its importance, Aβ catabolism remains understudied [7]. In normal brain, rapid proteolysis attenuates Aβ accumulation. The degradation process occurs primarily through the action of a group of metallopeptidases including neprilysin (NEP), insulysin (insulin degrading enzyme, IDE) and endothelin converting enzyme (ECE). While NEP appears to be the dominant Aβ protease [810], other catabolic enzymes such as ECE and IDE likely participate in regulating the steady-state levels of Aβ [10].
ECEs (including ECE-1 and ECE-2) are homologues belonging to the M13 family of zinc metallopeptidases that also includes NEP [7,11], which degrade Aβ in acidic intracellular compartments [12]. ECE-1 is widely expressed in human brain [1214] with the capability to degrade Aβ [15]. Both Aβ40 and Aβ42 levels were significantly higher in knockout mice [16] consistent with an in vitro role for ECE-1 in amyloid catabolism.
Besides NEP and ECE, IDE also contributes to Aβ degradation [17]. Purified rat IDE effectively degraded synthetic Aβ in vitro as did IDE from synaptic membrane fractions from postmortem human and fresh rat brain [18,19] or in cultured cells [20,21]. Overexpression of IDE in cells or mice markedly reduced intracellular and extracellular Aβ40 and Aβ42 [22] while IDE knockout mice demonstrated a clear elevation of brain Aβ [23]. For unknown reasons, presenilin 1 V97L mutants linked to familial Alzheimer’s disease (FAD) related to a functional defect in IDE and demonstrated increased intracellular and extracellular Aβ42 [24]. Thus, IDE is likely another important Aβ-degrading enzyme that may play a role in the pathology of AD.
Oxidative stress has long been recognized as a contributing, early factor in AD [2530]. Aβ induces free radical generation [25,3138] and elevated 4-hydroxy-nonenal (HNE), a marker of lipid peroxidation that is present in plaques [39]. HNE can interact with and inactivate a variety of enzymes including NEP [40]. However HNE adduction and its effects on ECE and IDE activity have not been evaluated. The present study addressed this question by examining the expression and adduction of IDE and ECE-1, in cells treated with Aβ or HNE. Our results indicate that such treatments rapidly induce IDE and ECE-1 oxidation but paradoxically increase ECE activity.
Chemicals and reagents
Synthetic human Aβ peptide 1–42 was purchased from BACHEM. Dithiothreitol (DTT), protease inhibitors cocktail (P8430) were obtained from Sigma-Aldrich. HNE was obtained from A.G. Scientific, Inc. Mca-R-P-P-G-F-S-A-F-K (Dnp)-OH Fluorogenic Peptide Substrate V, RNase-free DNase came from R&D and Promega, respectively. GE nitrocellulose membrane was purchased from ISC BioExpress. An enhanced chemiluminescence (ECL) kit and Seize® Classic (G) Immunoprecipitation Kit were obtained from Pierce. Other general chemicals and reagents were from Fisher Scientific.
Antibodies were from the following sources: Rabbit anti-HNE (Chemicon); goat anti-ECE-1 and goat anti-IDE (Santa Cruz); Alexa Fluor® 594 donkey anti-goat IgG (H+L), Alexa Fluor® 488 chicken anti-rabbit IgG (H+L) were from Invitrogen, goat anti-rabbit and rabbit anti-goat HRP conjugated secondary antibody were purchased from Chemicon. Clean-Blot IP Detection Reagent is the product of Thermo Scientific.
Cell culture
SH-SY5Y neuroblastoma cells, obtained from the American Type Culture Collection (ATCC), were maintained in a humidified atmosphere of 95% air and 5% CO2 at 37°C. Cells were seeded into plates or dishes in DMEM/F12 (1:1) medium, supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin.
Cell treatments with HNE and Aβ peptide
SH-SY5Y cells were seeded into 100 mm dishes at a density of 2 × 104 cells per ml. Experiments were carried out 24–48 h after cells were seeded. Different concentrations of HNE (dissolved in 3% DMSO) or Aβ (dissolved in 0.4% DMSO) were added to the cultures 24 h before harvest. Final concentrations of DMSO in medium were < 0.003%. Vehicles (with same concentrations of DMSO) were added to the cultures as controls.
Real time RT-PCR
Total RNA from a well of 6-wells plate was isolated using the RNeasy kit (Qiagen Inc., Valencia, CA) according to the manufacturer’s instructions. The concentration of nucleic acids was determined spectrophotometrically at 260 nm and 280 nm, taking into account the dilution factor. RQ1 RNase-free DNase (Promega) was used to remove relict DNA which might interfere the output of PCR. The PCR first strand synthesis was performed using the Omiscript® RT Kit (Qiagen Inc.). The resulting cDNA was then assayed by real time RT-PCR.
Real time RT-PCR was performed in 0.2 ml thin wall PCR plates using the iCycler thermal cycler (Bio-rad) and carried out with iQ SYBR Green supermix (Bio-rad) according to the manufacturer’s instructions. The standard reaction mix consisted of iQ SYBR Green supermix, forward and reverse primers at a final concentration of 500 nM each, 10 pg DNA template, and DNase free water to give final volume of 20 μl. The mixture was heated to 95°C for 3 min followed by 35 cycles with denaturation at 95°C for 30 s, annealing at 60°C for 30 s and extension at 72°C for 30 s. Human S26 (housekeeping gene) [41] was used as reference gene. Primer sequences for real time PCR: S26: 5′-CGC AGC AGT CAG GGA CAT TT-3′ (F), 5′-TTC ACA TAC AGC TTG GGA AGC-3′ (R); ECE-1: 5′-GAC GCC GAT GAG AAG TTC ATG-3′ (F), 5′-GCA AAA CTT CCA GCG AGG AA (R); IDE: 5′-GCC GAA GCC TTG TCT CAA CT (F), 5′-CAA ATA GGC CAT GTT ACA GTG CAA (R). The melting curves were performed to identify the specificity of the primers.
Relative quantification of genes expression was carried out by comparative Ct method according to manufacturer’s protocol (User Bulletin #2: ABI PRISM 7700 Sequence Detection System). Briefly, the genes mRNA level was expressed in cycle threshold (Ct) value; the Ct values for each sample were averaged from duplicate. Differences between the mean Ct values of ECE or IDE and reference gene were calculated as ΔCtsample = CtECE or IDE−Cts26 for different treated groups. The ΔCt for the control groups were set for calibrator (ΔCtcalibrator). Final results, the sample-calibrator ratio, expressed as N-fold differences of ECE-1 or IDE expression in the HNE or Aβ142 groups compared with control, were determined as 2−(ΔCt sample−ΔCt calibrator).
Immunoprecipitation, immunoblotting, and protein quantification
After incubation with HNE (5, 10 and 20 μM) or Aβ (0.5, 1 and 2 μM) for 24 h, cells were harvested and lysed in Passive Lysis Buffer containing protease inhibitors cocktail (Sigma P8430). Although this cocktail of protease inhibitors with broad specificity for the inhibition of aspartic, cysteine, and serine proteases as well as aminopeptidases, it does not interfere with metallopeptidases such as IDE, ECE, and NEP since it is EDTA free and has been used in the previous study [10,42]. Lysates were incubated for 30 min on ice, and then centrifuged at 10,000 X g for 10 min at 4°C. Protein concentration in the supernatant was determined by Coomassie blue protein binding method using protein quantification Kit-rapid (Sigma/Fluka) with bovine serum albumin as standard. The same amount of protein from HNE-treated, Aβ142 and vehicle-treated cells were used for western blotting assays and immunoprecipitation.
Immunoprecipitation with anti-ECE or IDE was performed with Seize® Classic (G) Immunoprecipitation Kit according to the manufacturer’s protocol. For total cell extract preparation, supernatants (with same amount of protein) were incubated with goat anti-ECE or goat anti-IDE polyclonal antibody (Santa Cruz, 60 μg/ml) at 4°C overnight. After washing Immobilized Protein G by adding 0.4 ml of BupH Modified Dulbecco’s PBS twice, the immune complexes were added to the spin cup containing the equilibrated immobilized protein G and incubated for more than 1 h at 4°C. The samples were centrifuged briefly to remove the flow-through solution. The beads were extensively washed with BupH Modified Dulbecco’s PBS. The immunoprecipitated proteins were eluted by adding equal volume of ImmunoPure® Elution Buffer and then analyzed by Western blotting with anti-HNE antibody. Briefly, Eluted samples were denatured in protein sample buffer (100 mM Tris-Cl pH 6.8, 4% SDS, 0.2% bromophenol blue, 20% glycerol, 20% H2O, 200 mM DTT) at 100°C for 10 min and loaded, separated on 10% SDS–polyacrylamide gels and transferred to nitrocellulose membranes in a Bio-rad electrophoresis system. After blocking with TBST (Tris-buffered saline with 0.1% Tween) containing 5% non-fat milk, the membranes were kept at 4°C overnight with HNE, ECE, and IDE primary antibody (1:1000, 1:500, 1:500, respectively), followed by Clean-Blot IP Reagent (HRP) (1:100) at room temperature (RT) for 2 h. The target protein bands were detected using the ECL Western blotting detection system (Pierce) and autoradiography film (Fisher).
For ECE and IDE protein levels detection, regular Western Blotting assay was performed using ECE-1 and IDE antibodies (1:500), followed by HRP-conjugated second antibodies (1:5000 to 1:10000 dilution) at RT for 2 h. The target protein bands were detected using the ECL Western blotting detection system (Pierce) and autoradiography film (Fisher). β-actin (1:5000) was used as reference.
The density of the ECE-1, IDE, and HNE blotting bands were quantified by ImageJ software (1.37v, Wayne Rasband, National Institutes of Health, USA) as described [43,44].
Double immunofluorescence staining
Cells were fixed with 4% paraformaldehyde at RT for 20 min and permeabilized with 0.2% Triton X-100 in PBS. After blocking for 1 h at RT with 1% goat serum/PBS, cells were incubated overnight at 4°C in a humidity chamber with primary anti-ECE-1 or IDE (1:100 Santa Cruz). At the end of the incubation, the cells were rinsed three times with PBS-Tween-20 (0.05%) and incubated with Alexa Fluor® 594 donkey anti-goat IgG (H+L) (1:5000) for 60 min at RT. Then the cells were fixed with 4% paraformaldehyde at RT for 15 min. After rinsing with PBS for 3 times, second primary rabbit anti-HNE (1:200) were added and incubated for 2 h at RT or overnight at 4°C. Secondary, Alexa Fluor® 488 chicken anti-rabbit IgG (H+L) (1:5000) was incubated for another 60 min at RT. All primary and secondary antibodies were diluted in PBS with 1% normal goat serum. After a rinse with PBS, the nuclei were counterstained with DAPI/Antifade solution (0.4 μg/mL, Chemicon), and then the cells were photographed and analyzed with a fluorescent microscope (Nikon, E600, Japan). The immunofluorescence staining was done at least three times to assess the reproducibility of the results. In order to confirm the specificity of immunostaining for the second cycle, the primary or secondary antibodies were omitted or replaced with preimmue IgG.
Fluorometric assay of ECE activity
ECE activity was determined by Fluorescence Resonance Energy Transfer (FRET). After extraction with a final concentration of 0.1% Triton X-100 in phosphate-buffered saline (PBS; pH 7.2) for 30 min on ice, ECE activity in cell lysates was analyzed using a synthetic ECE fluorogenic peptide substrate (Mca-RPPGFSAFK[Dnp]-OH; R&D Systems, Inc., Minneapolis, MN) [45] at RT in the presence or absence of the ECE inhibitor, Phosphoramidon (1 μM, Sigma). Phosphoramidon inhibition of ECE-1 is highly pH dependent [46]. The IC50 value of Phosphoramidon is about 50-fold lower at PH 5.8 than at 7.2. In addition, NEP activity is also PH-dependent. There is almost no NEP activity at PH 5.8 [47]. Thus, the inhibition by phosphoramidon at PH 5.8 can be considered to be the inhibition of ECE. Briefly, samples dissolved in 50 mM HEPES buffer [pH 5.8]) were preincubated with 1 μM Phosphoramidon or PBS for 10 min prior to adding fluorogenic peptide substrate (dissolved in HEPES). Fluorescence was read after excitation at 320 nm and emission at 405 nm on a fluorescent ELISA plate reader (Spectra Max Gemini; Molecular Devices, Sunnyvale, CA). For kinetic analysis the cell lysates were incubated with increasing concentrations of substrate (4–20 μM) at RT. Fluorescence over the time was measured for 1 h. The specific ECE activity was determined as the fluorescence difference occurring in the presence or absence of 1 μM Phosphoramidon. Kinetic isotherms (Vmax and Km values) for ECE activity were determined by means of non-linear least squares fitting to the Michaelis-Menten equation using GraphPad Prism software (version 3.0 for Windows, ISI Software San Diego, CA, USA).
To measure the activity of membrane-bound enzyme, cell suspensions were prepared, and then washed with PBS. 5 × 104 cells were resuspended in 50 μl of PBS contained in each well of black microtiter plates. The 50 μl substrate (10 μM in 50 mM HEPES) was added, and incubated at RT for 60 min. As above, Phosphoramidon was used to determine the specific ECE activity in intact cells.
Immunocaptured specific IDE activity assay
Cells treated by vehicles, HNE, or Aβ142 were lysated with Passive Lysis Buffer containing protease inhibitors cocktail (Sigma P8430). After 30 min incubation on ice, the lysates were centrifuged at 1300 rpm for 15 min at 4°C.
IDE specific activity was measured by using immunocapture based fluorometric assay [48]. In brief, Greiner 96-well high binding ELISA plate were coated with capture IDE (Santa Cruz, 50 μg/ml) antibody diluted in carbonate buffer (100 mM, pH 8.6) and left for 18 h at 4°C. The plates were washed with PBS containing 0.5% tween-20 for 3–5 times. After 30 min blocking with PBST containing 2% BSA, the supernatants of cell lysates (50 μl) were added and incubated at 4°C overnight. After 3–5 washes, the fluorogenic peptide (10 μM) diluted in 50 mM HEPES (pH 7.5) was added and incubated at 30°C in dark. Fluorescent readings were taken after 60 min. Control wells included in each plate contained PBS and fluorogenic peptide. Values are the mean ± SEM, presented in the percentage of control.
Statistical analysis
All results are given as mean ± SEM unless otherwise stated. Statistical analyses were performed with One-way ANOVA followed by least significant difference post hoc analysis (multiple comparisons) with threshold of P < 0.05.
HNE and Aβ treatment induce ECE and IDE modification
First we evaluated the effects of HNE and Aβ on intracellular ECE-1 and IDE mRNA and protein expression. Vehicle-, HNE-, or Aβ-treated SH-SY5Y cells were analyzed by real-time RT-PCR for ECE-1 and IDE mRNA. Low dose HNE (5 μM) for 12 h upregulated ECE expression, which was suppressed at higher doses (10 μM) (Fig. 1A). Similar treatments with Aβ142 upregulated ECE-1 mRNA, with the greatest change at 2 μM (Fig. 1B). IDE mRNA showed no change after HNE treatment (Fig. 1A), but was upregulated by Aβ142 at 1 μM, and returned to initial levels after 2μM treatment (Fig. 1B).
Fig. 1
Fig. 1
ECE-1 and IDE mRNA are altered in SH-SY5Y cells after HNE or Aβ1-42 treatment. SH-SY5Y cells were untreated or treated with HNE (5, 10 μM) (Panel A) or Aβ1-42 (1, 2 μM) (Panel B) for 12 h, respectively. Total RNA were analyzed (more ...)
ECE-1 protein levels as determined by western blotting displayed dramatic increases both in HNE and Aβ142-treated cells in a concentration-dependent manner (Fig. 2). The results suggest that ECE-1 has a prolonged in vitro half-life (> 12 h) and that ECE-1 gene expression is sensitive to both HNE and Aβ. Compared to ECE-1, IDE protein changes were insignificant. Only slight decreases of IDE in HNE-treated cells (Fig. 2A, C), while no obvious changes in Aβ-treated cells (Fig. 2B, D) were observed.
Fig. 2
Fig. 2
ECE-1 and IDE protein changes in HNE or Aβ1-42 treated SH-SY5Y cells. After incubation with vehicle, HNE (5, 10 and 20 μM) or Aβ (0.5, 1 and 2 μM) for 24 h, SH-SY5Y cells were harvested and equal amounts of protein western (more ...)
In order to determine if HNE-modification occurred in the cultured SH-SY5Y cells treated with HNE or Aβ142, ECE-1 and IDE were immunoprecipitated followed by western blotting. As shown in Fig. 3, ECE-1 or IDE were variably upregulated and modified after treatment with HNE or Aβ. While both proteins showed adduction, only IDE was significantly more modified after HNE than control (Fig. 3A–D).
Fig. 3
Fig. 3
ECE-1 and IDE show HNE adduction after HNE or Aβ1-42 treatment. (A) SH-SY5Y cells were incubated with HNE and Aβ1-42 for 24 h. Cell lysates were prepared and immunoprecipitated with monoclonal anti-ECE-1 and anti-IDE antibodies, respectively. (more ...)
To determine the subcellular location of modified proteins, we performed double fluorescence staining followed by confocal analysis of oxidatively stressed SH-SY5Y cells (Fig. 4). ECE-1 was detected both in cytosol and membrane, which is consistent with previous reports [4953]. HNE was almost undetectable in controls (Fig. 4b) but positive in a punctal pattern both in HNE- and Aβ-treated cells (Fig. 4f, j). Merged images revealed ECE-1 and HNE partly colocalized in cells treated with HNE or Aβ(Fig. 4h, l) consistent with the results observed after western blotting and immunoprecipitation. However, ECE-1, which did not colocalize with HNE, was mainly located on the cell surface. Therefore, ECE-1 is partially modified by exogenously added or endogenously produced HNE induced by Aβ.
Fig. 4
Fig. 4
ECE-1 and HNE colocalize in oxidatively stressed SH-SY5Y cells. Cells were untreated (a–d), treated with HNE (e–h) or Aβ1-42 (i–l) for 24 h, prior to fixation and staining with anti-ECE-1 (red) (a, e, i) or anti-HNE (green) (more ...)
As previous reports [52,54], IDE staining was mostly cytosolic (Fig. 5a, e, i). As for ECE-1, (Fig. 5f, j), HNE and IDE were both colocalized and independent after HNE or Aβ treatment (Fig. 5h, l). These data suggested that HNE adduction develops both after exogenous HNE and endogenous HNE induced by Aβ protein.
Fig. 5
Fig. 5
IDE and HNE colocalize in oxidatively stressed SH-SY5Y cells. Cells were untreated (a–d), treated with HNE (e–h) or Aβ1-42 (i–l) for 24 h, prior to fixation and staining with anti-IDE (red) (a, e, i) or anti-HNE (green) (more ...)
ECE, but not IDE, activity is altered after HNE and Aβ treatment
ECE activity in cell lysates or intact SH-SY5Y cells treated by HNE or Aβ for 24 h was determined with a commercially available fluorogenic substrate. The activity assay was highly sensitive, linear over-time (up to 60 min examined, data not shown), and sensitive to the ECE inhibitor, phosphoramidon (data not shown). Treatment of cells with HNE led to a concentration dependent increase of ECE activity in cell lysates (Fig. 6a). In intact cells, HNE at low concentration (5 μM) significantly increased ECE activity (p < 0.01), which returned to normal at higher concentrations (10 and 20 μM) (Fig. 6b). Specific ECE activity in cell lysates was increased ~300% (p < 0.05) while intact cells showed up to a 70% increase. Despite adduction, samples treated with Aβ showed increased ECE activity with similar trends in cell lysates and intact cells (Fig. 6d and e). Specific IDE activity in SH-SY5Y cells treated by HNE or Aβ for 24 h was determined by immunocapture based activity assay with a commercially available fluorogenic substrate. Consistent with the protein level, IDE activity treated by HNE or Aβ did not show significant differences compared to control (Fig. 7).
Fig. 6
Fig. 6
HNE or Aβ increases ECE specific activity in SH-SY5Y cells. After 24 h incubation with vehicle, HNE (5, 10 and 20 μM) (a, b and c) or Aβ (0.5, 1, and 2 μM) (d, e and f), cells were harvested, ECE activity in the cell lysates (more ...)
Fig. 7
Fig. 7
Effect of HNE or Aβ on IDE specific activity. After 24 h incubation with vehicle, HNE (10 μM) or Aβ1-42 (1 μM), cells were harvested, specific IDE activity in the cell lysates was analyzed by the assay of immunocapture (more ...)
To further analyze enzymatic kinetics, ECE activity was compared over a range of substrate concentrations (4–20 μM) in SH-SY5Y cells. Activity was saturable, and followed Michealis-Menten kinetics in all cell lysates treated with vehicle, HNE and Aβ peptide. Lineweaver-Burk double-reciprocal plots of reaction velocities and substrate concentrations allowed calculation of the apparent Michaelis Constant (Km) for the enzyme in all samples (Fig. 6c and f, Table 1). After HNE treatment, the Km was slightly decreased compared to control (with no statistic significance) which implies that the enzyme affinity for substrate tended to be increased (Table 1, Fig. 6c). Similar results were observed in the Aβ treatment. There was no significant effect on the Km at any of the three concentrations of Aβ (Table 1, VFig. 6f). The change of max was not significant after either HNE or Aβ treatment (Table 1). These kinetics features are consistent with the alteration of ECE activity. Together with the Western blotting data and immunofluorescence staining, these results suggest that HNE adduction induced either by HNE or Aβ treatment does not play a substantial role in suppressing ECE or IDE activity.
Table 1
Table 1
ECE Km and Vmax values alteration after treated by HNE and Aβ peptide in SH-SY5Y cells
Although many factors such as abnormal protein cross-linking [5557], trauma [58], abnormal phosphorylation of multiple proteins [59], synaptic loss [60], white matter changes [61], and some imbalanced metabolism [6264] have been implicated in the AD pathogenesis, excessive Aβ deposition is still considered to play a major role in AD. ECE expression and activity correlates well with Aβ deposition and the onset of AD [16,6568], and data from cultured cells and animal model indicated that Aβ is a physiologically relevant substrate of ECE [16]. Animals lacking ECE-2 are deficient in learning and memory [69]. Homozygous carriers of the A allele, which is associated with increased ECE-1 mRNA expression in human neocortex, had a reduced risk of AD [13] while the upregulation of ECE and IDE in mice was associated with decreased Aβ [68]. However, there are little data from studies on human tissue and the responses of ECE to HNE or Aβ. As A D typically develops over decades, the data from postmortem samples provides limited mechanistic insights into how AD-related factors such as Aβ peptides and oxidative stress alter ECE. In our study, ECE-1 mRNA was upregulated after the treatment of HNE and Aβ. Consistent with mRNA level, the changes of ECE-1 were more obvious in protein level after 24 h. ECE-1 protein was dramatically increased both in HNE- and Aβ-treated cells in a dose-dependent manner. These results agree with our study in human brain [70]. Moreover, we showed previously that HNE could enhance Aβ accumulation in culture media [70]. Thus increased ECE-1 levels, either mRNA or protein, may be the compensatory response to the increased endogenous or exogenous Aβ. In our previous study, cell viability after the treatments with increasing concentrations of HNE or Aβ showed a concentration dependent decrease (~25% with 20 μM HNE and ~15% in 2 μM Aβ) [70]. The evidence also indicated the cell death induced by HNE and Aβ was not dominant on mRNA depression.
Considerable evidence exists that Aβ can increase the production of free radicals by neurons [6,7173]. The formation of HNE is initiated by oxidative stress and culminates in lipid, DNA, and protein adduction. The HNE-macromolecular adducts are abundant in the brain of patients with AD suggesting a role for oxidative damage in disease pathogenesis [36,39,74]. Consistent with previous reports [75], we also show Aβ-induced HNE-adductions determined by western blotting and immunofluorescence staining. Among Aβ degrading enzymes, NEP is clearly one of the targets of HNE-mediated modification in AD brain and cultured neurons [76]. HNE adduction could inactivate endogenous and recombinant NEP [70,77], which likely contributed to Aβ accumulation [76]. Since ECE and IDE are also capable of degrading Aβ, we explored whether HNE adduction on ECE-1 or IDE occurred in HNE-or Aβ-treated cells and whether these changes altered their activities in cultured cells. The data in this study show that both exogenous HNE and endogenous HNE induced by Aβ mediate ECE-1 and IDE modification. However, the extent of modification was significantly different. The ratio of HNE to ECE-1 was moderately higher to control both in HNE- and Aβ-treated cells. The ratio of HNE to IDE was higher after HNE than Aβ treatments although both treatments induced adduction. These results, along with prior analysis of human brain tissue and culture cells, suggest that although multiple Aβ degrading enzymes are oxidatively modified, the biological consequences from the oxidization of different enzymes may vary.
Real-time RT-PCR indicated that IDE mRNA was upregulated after cell treatment with 1 μM Aβ, but western blotting did not show statistically significant changes in IDE protein levels. These data imply that IDE mRNA expression is more sensitive than protein and may reflect compensation to reduced IDE (protein levels and catalytic activity) induced by Aβ modifications.
We also evaluated if HNE adduction altered ECE activity. The specific activity of ECE increased significantly in cell lysates and intact cells after HNE or Aβ treatment. Vmax, and Km revealed that ECE activity in SH-SY5Y cells followed Michealis-Menten kinetics, and demonstrated a hyperbolic dependence of v (velocity) on substrate concentration. The ECE kinetic features are similar in HNE- and Aβ-treated cells. The Km of ECE after HNE or Aβ treatment was slightly decreased when compared to control (Table 1), which implies that substrate affinity was increased but not significant. The Vmax was unchanged in HNE and Aβ incubated cells compared to controls. Overall, these kinetic parameters suggest some changes in affinity for substrate after HNE and Aβ treatment. However, together with the result of co-localization exam, HNE adduction induced either by HNE or Aβ treatment is not to be crucial to ECE activity.
Unlike ECE, the alteration of IDE specific activity was not significant after HNE or Aβ treatments although a downward trend was observed in A β-treated cells. Shinall and colleagues reported that IDE was inactivated by reaction with HNE with the concomitant formation of protein adducts in a cell-free system [77]. In this study, there was marked adduction without significant decrease of activity after HNE treatment, while Aβ treatment showed more inactivation of IDE activity but less adduction. These results suggest that the HNE adduction may not be the predominant means of inactivating IDE and imply that the mechanism of inactivation is more complicated. Alternatively, Aβ-induced HNE modifies IDE at a different intracellular location than exogenous HNE, accounting for dissimilar changes in enzymatic activity.
The experiments on cell viability after the treatments with increasing concentrations of HNE or Aβ performed along with other experiments reported in this manuscripts showed that there was a concentration dependent decrease (~25% with 20 μM HNE and ~15% in 2 μM Aβ) in cell viability as determined by MTT reduction (data not shown). Although both HNE and Aβ are known to be toxic to cultured neuronal cells, it is possible that other mechanisms also involved in neuronal cell death induced by Aβ in addition to the induction of HNE.
Acknowledgments
This work is supported by NIH grants AG025722 and AG029972 (to DSW), and an Alzheimer’s Association Grant IIRG-08-90524 (to DSW), and the start fund from the Department of Pathology and Laboratory Medicine, University of Wisconsin and Public Health, Madison, Wisconsin (to DSW).
1. Selkoe DJ. Alzheimer’s disease: genes, proteins, and therapy. Physiol Rev. 2001;81:741–766. [PubMed]
2. Bard F, Cannon C, Barbour R, Burke RL, Games D, Grajeda H, Guido T, Hu K, Huang J, Johnson-Wood K, Khan K, Kholodenko D, Lee M, Lieberburg I, Motter R, Nguyen M, Soriano F, Vasquez N, Weiss K, Welch B, Seubert P, Schenk D, Yednock T. Peripherally administered antibodies against amyloid beta-peptide enter the central nervous system and reduce pathology in a mouse model of Alzheimer disease. Nat Med. 2000;6:916–919. [PubMed]
3. Forman MS, Trojanowski JQ, Lee VM. Neurodegenerative diseases: a decade of discoveries paves the way for therapeutic breakthroughs. Nat Med. 2004;10:1055–1063. [PubMed]
4. Hardy J. Amyloid, the presenilins and Alzheimer’s disease. Trends Neurosci. 1997;20:154–159. [PubMed]
5. Hardy J, Selkoe DJ. The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics. Science. 2002;297:353–356. [PubMed]
6. Crouch PJ, Harding SM, White AR, Camakaris J, Bush AI, Masters CL. Mechanisms of A beta mediated neurodegeneration in Alzheimer’s disease. Int J Biochem Cell Biol. 2008;40:181–198. [PubMed]
7. Saido TC, Iwata N. Metabolism of amyloid beta peptide and pathogenesis of Alzheimer’s disease. Towards presymptomatic diagnosis, prevention and therapy. Neurosci Res. 2006;54:235–253. [PubMed]
8. Carson JA, Turner AJ. Beta-amyloid catabolism: roles for neprilysin (NEP) and other metallopeptidases? J Neurochem. 2002;81:1–8. [PubMed]
9. Vardy ER, Catto AJ, Hooper NM. Proteolytic mechanisms in amyloid-beta metabolism: therapeutic implications for Alzheimer’s disease. Trends Mol Med. 2005;11:464–472. [PubMed]
10. Wang DS, Dickson DW, Malter JS. beta-Amyloid Degradation and Alzheimer’s Disease. J Biomed Biotechnol. 2006;2006:58406. [PMC free article] [PubMed]
11. Turner AJ, Murphy LJ. Molecular pharmacology of endothelin converting enzymes. Biochem Pharmacol. 1996;51:91–102. [PubMed]
12. Eckman EA, Eckman CB. Abeta-degrading enzymes: modulators of Alzheimer’s disease pathogenesis and targets for therapeutic intervention. Biochem Soc Trans. 2005;33:1101–1105. [PubMed]
13. Funalot B, Ouimet T, Claperon A, Fallet C, Delacourte A, Epelbaum J, Subkowski T, Leonard N, Codron V, David JP, Amouyel P, Schwartz JC, Helbecque N. Endothelin-converting enzyme-1 is expressed in human cerebral cortex and protects against Alzheimer’s disease. Mol Psychiatry. 2004;9:1122–1128. 1059. [PubMed]
14. Naidoo V, Naidoo S, Raidoo DM. Immunolocalisation of endothelin-1 in human brain. J Chem Neuroanat. 2004;27:193–200. [PubMed]
15. Eckman EA, Reed DK, Eckman CB. Degradation of the Alzheimer’s amyloid beta peptide by endothelin-converting enzyme. J Biol Chem. 2001;276:24540–24548. [PubMed]
16. Eckman EA, Watson M, Marlow L, Sambamurti K, Eckman CB. Alzheimer’s disease beta-amyloid peptide is increased in mice deficient in endothelin-converting enzyme. J Biol Chem. 2003;278:2081–2084. [PubMed]
17. Leissring MA, Farris W, Chang AY, Walsh DM, Wu X, Sun X, Frosch MP, Selkoe DJ. Enhanced proteolysis of beta-amyloid in APP transgenic mice prevents plaque formation, secondary pathology, and premature death. Neuron. 2003;40:1087–1093. [PubMed]
18. McDermott JR, Gibson AM. Degradation of Alzheimer’s beta-amyloid protein by human and rat brain peptidases: involvement of insulin-degrading enzyme. Neurochem Res. 1997;22:49–56. [PubMed]
19. Perez A, Morelli L, Cresto JC, Castano EM. Degradation of soluble amyloid beta-peptides 1-40, 1-42, and the Dutch variant 1-40Q by insulin degrading enzyme from Alzheimer disease and control brains. Neurochem Res. 2000;25:247–255. [PubMed]
20. Qiu WQ, Walsh DM, Ye Z, Vekrellis K, Zhang J, Podlisny MB, Rosner MR, Safavi A, Hersh LB, Selkoe DJ. Insulin-degrading enzyme regulates extracellular levels of amyloid beta-protein by degradation. J Biol Chem. 1998;273:32730–32738. [PubMed]
21. Qiu WQ, Ye Z, Kholodenko D, Seubert P, Selkoe DJ. Degradation of amyloid beta-protein by a metalloprotease secreted by microglia and other neural and non-neural cells. J Biol Chem. 1997;272:6641–6646. [PubMed]
22. Sudoh S, Frosch MP, Wolf BA. Differential effects of proteases involved in intracellular degradation of amyloid beta-protein between detergent-soluble and -insoluble pools in CHO-695 cells. Biochemistry. 2002;41:1091–1099. [PubMed]
23. Farris W, Mansourian S, Chang Y, Lindsley L, Eckman EA, Frosch MP, Eckman CB, Tanzi RE, Selkoe DJ, Guenette S. Insulin-degrading enzyme regulates the levels of insulin, amyloid beta-protein, and the beta-amyloid precursor protein intracellular domain in vivo. Proc Natl Acad Sci U S A. 2003;100:4162–4167. [PubMed]
24. Qin W, Jia J. Down-regulation of insulin-degrading enzyme by presenilin 1 V97L mutant potentially underlies increased levels of amyloid beta 42. Eur J Neurosci. 2008;27:2425–2432. [PubMed]
25. Smith CD, Carney JM, Starke-Reed PE, Oliver CN, Stadtman ER, Floyd RA, Markesbery WR. Excess brain protein oxidation and enzyme dysfunction in normal aging and in Alzheimer disease. Proc Natl Acad Sci U S A. 1991;88:10540–10543. [PubMed]
26. Smith MA, Sayre LM, Monnier VM, Perry G. Radical AGEing in Alzheimer’s disease. Trends Neurosci. 1995;18:172–176. [PubMed]
27. Smith MA, Rottkamp CA, Nunomura A, Raina AK, Perry G. Oxidative stress in Alzheimer’s disease. Biochim Biophys Acta. 2000;1502:139–144. [PubMed]
28. Harman D. Free radical involvement in aging. Pathophysiology and therapeutic implications. Drugs Aging. 1993;3:60–80. [PubMed]
29. Olanow CW. A radical hypothesis for neurodegeneration. Trends Neurosci. 1993;16:439–444. [PubMed]
30. Varadarajan S, Yatin S, Aksenova M, Butterfield DA. Review: Alzheimer’s amyloid beta-peptide-associated free radical oxidative stress and neurotoxicity. J Struct Biol. 2000;130:184–208. [PubMed]
31. David DC, Hauptmann S, Scherping I, Schuessel K, Keil U, Rizzu P, Ravid R, Drose S, Brandt U, Muller WE, Eckert A, Gotz J. Proteomic and functional analyses reveal a mitochondrial dysfunction in P301L tau transgenic mice. J Biol Chem. 2005;280:23802–23814. [PubMed]
32. Montine TJ, Amarnath V, Martin ME, Strittmatter WJ, Graham DG. E-4-hydroxy-2-nonenal is cytotoxic and cross-links cytoskeletal proteins in P19 neuroglial cultures. Am J Pathol. 1996;148:89–93. [PubMed]
33. Nunomura A, Chiba S, Lippa CF, Cras P, Kalaria RN, Takeda A, Honda K, Smith MA, Perry G. Neuronal RNA oxidation is a prominent feature of familial Alzheimer’s disease. Neurobiol Dis. 2004;17:108–113. [PubMed]
34. Nunomura A, Perry G, Aliev G, Hirai K, Takeda A, Balraj EK, Jones PK, Ghanbari H, Wataya T, Shimohama S, Chiba S, Atwood CS, Petersen RB, Smith MA. Oxidative damage is the earliest event in Alzheimer disease. J Neuropathol Exp Neurol. 2001;60:759–767. [PubMed]
35. Pratico D, VMYL, Trojanowski JQ, Rokach J, Fitzgerald GA. Increased F2-isoprostanes in Alzheimer’s disease: evidence for enhanced lipid peroxidation in vivo. FASEB J. 1998;12:1777–1783. [PubMed]
36. Sayre LM, Zelasko DA, Harris PL, Perry G, Salomon RG, Smith MA. 4-Hydroxynonenal-derived advanced lipid peroxidation end products are increased in Alzheimer’s disease. J Neurochem. 1997;68:2092–2097. [PubMed]
37. Schuessel K, Schafer S, Bayer TA, Czech C, Pradier L, Muller-Spahn F, Muller WE, Eckert A. Impaired Cu/Zn-SOD activity contributes to increased oxidative damage in APP transgenic mice. Neurobiol Dis. 2005;18:89–99. [PubMed]
38. Shi Q, Gibson GE. Oxidative stress and transcriptional regulation in Alzheimer disease. Alzheimer Dis Assoc Disord. 2007;21:276–291. [PMC free article] [PubMed]
39. Ando Y, Brannstrom T, Uchida K, Nyhlin N, Nasman B, Suhr O, Yamashita T, Olsson T, El Salhy M, Uchino M, Ando M. Histochemical detection of 4-hydroxynonenal protein in Alzheimer amyloid. J Neurol Sci. 1998;156:172–176. [PubMed]
40. Yasojima K, McGeer EG, McGeer PL. Relationship between beta amyloid peptide generating molecules and neprilysin in Alzheimer disease and normal brain. Brain Res. 2001;919:115–121. [PubMed]
41. Jerde TJ, Mellon WS, Fischer SM, Liebert M, Bjorling DE, Nakada SY. Suppression of 15-hydroxyprostaglandin dehydrogenase messenger RNA concentration, protein expression, and enzymatic activity during human ureteral obstruction. J Pharmacol Exp Ther. 2004;309:398–403. [PubMed]
42. Wang DS, Lipton RB, Katz MJ, Davies P, Buschke H, Kuslansky G, Verghese J, Younkin SG, Eckman C, Dickson DW. Decreased neprilysin immunoreactivity in Alzheimer disease, but not in pathological aging. J Neuropathol Exp Neurol. 2005;64:378–385. [PubMed]
43. Farrer M, Kachergus J, Forno L, Lincoln S, Wang DS, Hulihan M, Maraganore D, Gwinn-Hardy K, Wszolek Z, Dickson D, Langston JW. Comparison of kindreds with parkinsonism and alpha-synuclein genomic multiplications. Ann Neurol. 2004;55:174–179. [PubMed]
44. Wang S, Simon BP, Bennett DA, Schneider JA, Malter JS, Wang DS. The significance of pin1 in the development of Alzheimer’s disease. J Alzheimers Dis. 2007;11:13–23. [PubMed]
45. Johnson GD, Ahn K. Development of an internally quenched fluorescent substrate selective for endothelin-converting enzyme-1. Anal Biochem. 2000;286:112–118. [PubMed]
46. Ahn K, Herman SB, Fahnoe DC. Soluble human endothelin-converting enzyme-1: expression, purification, and demonstration of pronounced pH sensitivity. Arch Biochem Biophys. 1998;359:258–268. [PubMed]
47. Fahnoe DC, Knapp J, Johnson GD, Ahn K. Inhibitor potencies and substrate preference for endothelin-converting enzyme-1 are dramatically affected by pH. J Cardiovasc Pharmacol. 2000;36:S22–25. [PubMed]
48. Miners JS, Verbeek MM, Rikkert MO, Kehoe PG, Love S. Immunocapture-based fluorometric assay for the measurement of neprilysin-specific enzyme activity in brain tissue homogenates and cerebrospinal fluid. J Neurosci Methods. 2008;167:229–236. [PubMed]
49. Barnes K, Turner AJ. The endothelin system and endothelin-converting enzyme in the brain: molecular and cellular studies. Neurochem Res. 1997;22:1033–1040. [PubMed]
50. Hoang MV, Turner AJ. Novel activity of endothelin-converting enzyme: hydrolysis of bradykinin. Biochem J. 1997;327 (Pt 1):23–26. [PubMed]
51. Schweizer A, Valdenaire O, Nelbock P, Deuschle U, Dumas Milne Edwards JB, Stumpf JG, Loffler BM. Human endothelin-converting enzyme (ECE-1): three isoforms with distinct subcellular localizations. Biochem J. 1997;328 (Pt 3):871–877. [PubMed]
52. Turner AJ, Fisk L, Nalivaeva NN. Targeting amyloid-degrading enzymes as therapeutic strategies in neurodegeneration. Ann N Y Acad Sci. 2004;1035:1–20. [PubMed]
53. Valdenaire O, Rohrbacher E, Mattei MG. Organization of the gene encoding the human endothelin-converting enzyme (ECE-1) J Biol Chem. 1995;270:29794–29798. [PubMed]
54. Duckworth WC, Bennett RG, Hamel FG. Insulin degradation: progress and potential. Endocr Rev. 1998;19:608–624. [PubMed]
55. Ishizawa T, Mattila P, Davies P, Wang D, Dickson DW. Colocalization of tau and alpha-synuclein epitopes in Lewy bodies. J Neuropathol Exp Neurol. 2003;62:389–397. [PubMed]
56. Wang DS. From Lewy body disease to Alzheimer’s disease: hypothesis and evidence. Front Biosci. 2003;8:s223–227. [PubMed]
57. Wang DS, Dickson DW, Malter JS. Tissue Transglutaminase, Protein Cross-linking and Alzheimer’s Disease: Review and Views. Int J Clin Exp Pathol. 2008;1:5–18. [PMC free article] [PubMed]
58. Payette DJ, Xie J, Shirwany N, Guo Q. Exacerbation of apoptosis of cortical neurons following traumatic brain injury in par-4 transgenic mice. Int J Clin Exp Pathol. 2008;1:44–56. [PMC free article] [PubMed]
59. Thakur A, Siedlak SL, James SL, Bonda DJ, Rao A, Webber KM, Camins A, Pallas M, Casadesus G, Lee HG, Bowser R, Raina AK, Perry G, Smith MA, Zhu X. Retinoblastoma protein phosphorylation at multiple sites is associated with neurofibrillary pathology in Alzheimer disease. Int J Clin Exp Pathol. 2008;1:134–146. [PMC free article] [PubMed]
60. Wang DS, Bennett DA, Mufson E, Cochran E, Dickson DW. Decreases in soluble alpha-synuclein in frontal cortex correlate with cognitive decline in the elderly. Neurosci Lett. 2004;359:104–108. [PubMed]
61. Wang DS, Bennett DA, Mufson EJ, Mattila P, Cochran E, Dickson DW. Contribution of changes in ubiquitin and myelin basic protein to age-related cognitive decline. Neurosci Res. 2004;48:93–100. [PubMed]
62. Chan HW, Liu T, Verdile G, Bishop G, Haasl RJ, Smith MA, Perry G, Martins RN, Atwood CS. Copper induces apoptosis of neuroblastoma cells via post-translational regulation of the expression of Bcl-2-family proteins and the tx mouse is a better model of hepatic than brain Cu toxicity. Int J Clin Exp Med. 2008;1:76–88. [PMC free article] [PubMed]
63. Patel AK, Rogers JT, Huang X. Flavanols, mild cognitive impairment, and Alzheimer’s dementia. Int J Clin Exp Med. 2008;1:181–191. [PMC free article] [PubMed]
64. Yi J, Horky LL, Friedlich AL, Shi Y, Rogers JT, Huang X. L-arginine and Alzheimer’s disease. Int J Clin Exp Pathol. 2009;2:211–238. [PMC free article] [PubMed]
65. Carty NC, Nash K, Lee D, Mercer M, Gottschall PE, Meyers C, Muzyczka N, Gordon MN, Morgan D. Adeno-associated viral (AAV) serotype 5 vector mediated gene delivery of endothelin-converting enzyme reduces Abeta deposits in APP + PS1 transgenic mice. Mol Ther. 2008;16:1580–1586. [PMC free article] [PubMed]
66. Choi DS, Wang D, Yu GQ, Zhu G, Kharazia VN, Paredes JP, Chang WS, Deitchman JK, Mucke L, Messing RO. PKCepsilon increases endothelin converting enzyme activity and reduces amyloid plaque pathology in transgenic mice. Proc Natl Acad Sci U S A. 2006;103:8215–8220. [PubMed]
67. Eckman EA, Adams SK, Troendle FJ, Stodola BA, Kahn MA, Fauq AH, Xiao HD, Bernstein KE, Eckman CB. Regulation of steady-state beta-amyloid levels in the brain by neprilysin and endothelin-converting enzyme but not angiotensin-converting enzyme. J Biol Chem. 2006;281:30471–30478. [PubMed]
68. Qu HY, Zhang T, Li XL, Zhou JP, Zhao BQ, Li Q, Sun MJ. Transducible P11-CNTF rescues the learning and memory impairments induced by amyloid-beta peptide in mice. Eur J Pharmacol. 2008;594:93–100. [PubMed]
69. Rodriguiz RM, Gadnidze K, Ragnauth A, Dorr N, Yanagisawa M, Wetsel WC, Devi LA. Animals lacking endothelin-converting enzyme-2 are deficient in learning and memory. Genes Brain Behav. 2009 in press. [PMC free article] [PubMed]
70. Wang R, Wang S, Malter JS, Wang DS. Effects of HNE-modification induced by beta-amyloid on neprilysin expression and activity in SH-SY5Y cells. J Neurochem. 2009;108:1072–1082. [PMC free article] [PubMed]
71. Butterfield DA, Drake J, Pocernich C, Castegna A. Evidence of oxidative damage in Alzheimer’s disease brain: central role for amyloid beta-peptide. Trends Mol Med. 2001;7:548–554. [PubMed]
72. Butterfield DA, Lauderback CM. Lipid peroxidation and protein oxidation in Alzheimer’s disease brain: potential causes and consequences involving amyloid beta-peptide-associated free radical oxidative stress. Free Radic Biol Med. 2002;32:1050–1060. [PubMed]
73. Canevari L, Abramov AY, Duchen MR. Toxicity of amyloid beta peptide: tales of calcium, mitochondria, and oxidative stress. Neurochem Res. 2004;29:637–650. [PubMed]
74. Takeda A, Smith MA, Avila J, Nunomura A, Siedlak SL, Zhu X, Perry G, Sayre LM. In Alzheimer’s disease, heme oxygenase is coincident with Alz50, an epitope of tau induced by 4-hydroxy-2-nonenal modification. J Neurochem. 2000;75:1234–1241. [PubMed]
75. Mark RJ, Lovell MA, Markesbery WR, Uchida K, Mattson MP. A role for 4-hydroxynonenal, an aldehydic product of lipid peroxidation, in disruption of ion homeostasis and neuronal death induced by amyloid beta-peptide. J Neurochem. 1997;68:255–264. [PubMed]
76. Wang DS, Iwata N, Hama E, Saido TC, Dickson DW. Oxidized neprilysin in aging and Alzheimer’s disease brains. Biochem Biophys Res Commun. 2003;310:236–241. [PubMed]
77. Shinall H, Song ES, Hersh LB. Susceptibility of amyloid beta peptide degrading enzymes to oxidative damage: a potential Alzheimer’s disease spiral. Biochemistry. 2005;44:15345–15350. [PubMed]