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Journal of Neuropathology & Experimental Neurology:
doi: 10.1097/NEN.0b013e3181afe475
Original Articles

Neprilysin and Insulin-Degrading Enzyme Levels Are Increased in Alzheimer Disease in Relation to Disease Severity

Miners, James Scott PhD; Baig, Shabnam PhD; Tayler, Hannah BSc; Kehoe, Patrick Gavin PhD; Love, Seth MBBCh, PhD, FRCP, FRCPath

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Author Information

From Dementia Research Group, Clinical Science at North Bristol, University of Bristol, Frenchay Hospital (JSM, SB, HT, PGK, SL), Frenchay, Bristol, United Kingdom.

Send correspondence and reprint requests to: James Scott Miners, PhD, Clinical Science at North Bristol, University of Bristol, Frenchay Hospital, Frenchay, Bristol BS16 1LE, UK; E-mail: scott.miners@bristol.ac.uk

This study was supported by the Bristol Research into Alzheimer's and Care of the Elderly (BRACE) and by the James Tudor Foundation.

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Abstract

Experimental reduction of neprilysin (NEP) or insulin-degrading enzyme (IDE) in vivo exacerbates β-amyloid accumulation in the brain. The level of these enzymes is reportedly reduced during aging and in postmortem brains of patients with sporadic Alzheimer disease (AD). To distinguish between primary decreases in NEP and IDE activity that might contribute to β-amyloid accumulation and decreases secondary to neurodegenerative changes in AD, we measured NEP and IDE levels by indirect sandwich ELISA and enzyme activities by immunocapture-based fluorogenic assays in postmortem frontal cortex from patients of different ages and at different pathological stages of AD, as indicated by Braak tangle stage. The ELISA measurements of neuron-specific enolase were used to adjust for neuronal loss. Both unadjusted and neuron-specific enolase-adjusted NEP levels and activity were significantly increased in AD and positively correlated with Braak stage but negatively with age in AD patients. Insulin-degrading enzyme activity was higher in AD than controls; this was significant after adjustment for neuron-specific enolase level; unadjusted IDE protein level was decreased in AD but not after adjustment. Our findings suggest that reduction in NEP and IDE activity is not the primary cause of β-amyloid accumulation in AD, but rather a late-stage phenomenon secondary to neurodegeneration.

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INTRODUCTION

The β-amyloid (Aβ) cascade hypothesis posits that the neurodegenerative abnormalities of Alzheimer disease (AD) are initiated by the accumulation of Aβ in the brain (1, 2). Production of Aβ is normally balanced by its removal through perivascular drainage (3), receptor-mediated transport across the blood-brain barrier (4), and enzymatic degradation (5, 6). In familial AD, increased amyloidogenic processing of amyloid precursor protein (APP), as a result of mutations in the APP or presenilin genes (7), causes Aβ to accumulate. In sporadic late-onset AD, in which no gene mutations seem to be causative, higher levels of β-secretase (the rate-limiting enzyme of APP amyloidogenesis) have been detected in human brain tissue (8). In mouse models of AD, however, abnormal accumulation of Aβ itself seems to induce a paradoxical upregulation of β-secretase activity (9). In sporadic AD, it remains unclear as to what extent Aβ accumulation results from the relative contributions of increased production and decreased removal.

Both neprilysin (NEP) and insulin-degrading enzyme (IDE) cleave Aβ in vitro (10). Disruption/deletion of the NEP (11-13) or IDE genes (14, 15) in mice caused Aβ to accumulate in the brain. In contrast, overexpression of human NEP or IDE in mice transgenic for human APP (hAPP) lowered Aβ levels in the brain, reduced plaque formation, and improved cognitive performance (16). The Aβ load was also lowered when human NEP was transferred into the brains of hAPP mice by lentiviral vector (12, 17) or implantation of genetically engineered fibroblasts (18).

Overexpression of NEP was reported to protect hippocampal neurons from Aβ-mediated toxicity in vitro (19), and upregulation of NEP or IDE has been proposed as a potential means of protecting the brain against Aβ accumulation and consequent cognitive decline (18, 20, 21).

We and others previously found that NEP was expressed by pyramidal neurons in the cerebral neocortex and hippocampus, and that neuronal immunopositivity for NEP was reduced in AD (22, 23). The IDE is also expressed by neurons and in AD; neuronal labeling for IDE is reduced in the CA2/3 and CA4 regions of the hippocampus (24). Reduced IDE protein levels or enzyme activity have been reported in the cytosolic (25) and membrane (26) fractions of postmortem brain tissue from patients with mild cognitive impairment or AD, whereas levels of both NEP and IDE have been found to be decreased with age in postmortem brain tissue from both AD patients and controls (27) and in hAPP transgenic mice (28, 29). APOE ϵ4, a genetic risk factor for AD, has been associated with reduced levels of NEP (23) and IDE (30) in AD.

These collective findings are consistent with the hypothesis that accumulation of Aβ in sporadic late-onset AD may result from reduced activity of NEP and/or IDE. The previous studies on human tissue have, however, mostly focused on relatively small cohorts of patients with end-stage disease and have rarely adjusted for reduction in NEP and IDE levels secondary to neuronal loss or damage. Furthermore, it has generally been assumed that measurements of NEP and IDE mRNA and protein levels provide a reliable indicator of the activity of these enzymes. This assumption may not be valid, however, particularly if there are disease-specific modifications to these proteins that might affect their activities.

Our aim in the present study was to determine whether sporadic AD results from decreased NEP and/or IDE activity or whether decreased NEP and/or IDE activity in sporadic AD is secondary to neurodegenerative changes. We examined NEP and IDE levels and their corresponding specific activities at different pathological stages of disease (as defined by Braak tangle stages) and in relation to APOE genotype in a large cohort of postmortem brains. We also measured the levels of neuron-specific enolase (NSE) as a marker of neuronal integrity (31) and used them to adjust measurements of NEP and IDE levels and enzymatic activities to account for neuronal loss or damage. Our data indicate that when adjusted for neuronal degeneration (as defined by reduction in NSE levels), NEP and IDE levels and activity in frontal cortex increase with progression of AD and are not influenced by APOE genotype.

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MATERIALS AND METHODS

Brain Tissues

Brain tissue was obtained from the South West Dementia Brain Bank (Human Tissue Authority licence no. 12273), University of Bristol, United Kingdom, with the local research ethics committee approval. The brains had been subdivided midsagittally at autopsy; the right halves were fixed in formalin, and multiple blocks were taken for paraffin histology and detailed neuropathologic examination. The left halves were sliced, and the slices were frozen at −80°C for subsequent biochemical studies. To ensure that we examined the full spectrum of disease progression, cases of definite AD (assessed according to the criteria of the Consortium to Establish a Registry for Alzheimer's Disease [CERAD]) and controls (showing the absence of AD or other neuropathologic abnormalities) and cases with AD pathology of intermediate severity (CERAD possible and probable AD) were examined. All cases were categorized according to the Braak tangle stage and grouped for analysis into 3 categories: Braak Stages 0 to II, III to IV, and V to VI. For analyses in which the comparison was between AD and control brains, cases of possible, probable, and definite AD (as defined by CERAD) were included in the AD group. The AD and control groups were matched in terms of age, sex, and postmortem delay (Table).

TABLE. Summary of Pa...
TABLE. Summary of Pa...
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Brain tissue homogenates and subcellular fractions were prepared from dissected samples of unfixed frozen cortex from the left midfrontal region (Brodmann area 6). Protein concentrations were determined using Total Protein Kit (Sigma Aldrich, Dorset, UK) according to manufacturer's instructions. Tissue (200 mg) allowed to thaw to 4°C was homogenized for 30 seconds in a Precellys 24 automated tissue homogenizer (Stretton Scientific, Derbyshire, UK) with 2.3-mm silica beads (Biospec, Thistle Scientific, Glasgow, UK). For ELISA, the tissue was homogenized in 1% sodium dodecyl sulfate buffer containing 5 mol/L NaCl, 1 mol/L Tris pH 7.6. For enzyme activity assays, the tissue was homogenized in 0.5% triton X-100, 20 mmol/L Tris pH 7.4, 10% sucrose (wt/vol) (all reagents from Sigma Aldrich). Both sets of lysis buffers contained aprotinin (1 μg/mL; Sigma Aldrich) and phenylmethane sulfonyl fluoride (10 μmol/L; Sigma Aldrich). The homogenates were centrifuged at 13,000 rpm for 15 minutes at 4°C, and the supernatants were aliquoted and stored at −80°C until used.

For preparation of tissue for separate analysis of plasma membrane and cytosolic fractions, 400-mg samples of cortex were homogenized in 1 mL ice-cold PBS with a handheld homogenizer. Fractionation was then performed using the Fraction-Prep system (BioVision, Cambridge Biosciences, Cambridge, UK) according to the manufacturer's guidelines. All fractions were stored at −80°C until used.

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NEP ELISA

The Neprilysin Duoset ELISA kit (R&D Systems Europe, Oxford, UK) was used according to the manufacturer's guidelines with minor modifications. Goat anti-human NEP (1.6 μg/mL) diluted in PBS (pH 7.4) was coated on a high-binding Costar 96-well plate (R&D Systems Europe) for 18 hours at room temperature (RT). The plates were washed 6 times in PBS containing 0.5% Tween 20 (Sigma Aldrich). Nonspecific binding of antibody was blocked by addition of 1% PBS-bovine serum albumin (BSA) (Sigma Aldrich) for 3 hours at RT, after which the plates were washed a further 6 times. Serial dilutions of recombinant human NEP or crude homogenates (250 μg total protein) diluted in 1% PBS-BSA, or 1% PBS-BSA alone as a control, were incubated for 2 hours with continuous shaking at RT. After a further 6 washes, biotinylated anti-NEP (1.6 μg/mL) was added for 2 hours before another wash and incubation with streptavidin-peroxidase (1:100) for 20 minutes in the dark. Substrate solution (tetramethyl benzidine; R&D Systems Europe) was added for 30 minutes, and the optical density for each well was read at 450 nm in a FLUOstar plate reader (BMG Labtech, Aylesbury, UK). The NEP protein levels were interpolated from the standard curve generated from serial dilutions of recombinant human NEP (R&D Systems Europe). Each measurement was repeated on 2 separate occasions, and the average value was calculated. According to the manufacturers, the ELISA demonstrated minimal cross reactivity with recombinant human NEP-2, endothelin-converting enzyme 1, or endothelin-converting enzyme 2.

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IDE ELISA

An indirect sandwich ELISA was developed and optimized to measure IDE levels in crude tissue homogenates. Anti-human IDE (2.5 μg/mL) (Abcam, Cambridge, UK) diluted in PBS was coated on high-binding Costar 96-well plates and incubated overnight at RT. The plates were washed 6 times in PBS containing 0.5% Tween 20, and nonspecific binding of antibody was blocked by addition of 1% PBS-BSA (Sigma Aldrich) for 3 hours at RT, after which the plates were washed a further 6 times. Recombinant human IDE (R&D Systems Europe) and crude tissue homogenates (total protein, 100 μg) were incubated for 3 hours at RT with shaking. After 6 washes, anti-IDE ([2 μg/mL] R&D Systems) was added to the plate for 2 hours at RT. After a further wash, biotinylated anti-mouse antibody (1:500) (Vector Labs, Peterborough, UK) was added for 20 minutes, followed by a wash step and incubation with streptavidin-peroxidase ([1:100] R&D Systems Europe) for 20 minutes in the dark. Tetramethyl benzidine was added for 30 minutes and the optical density for each well was read at 450 nm. The IDE protein levels were interpolated from a standard curve generated by serial dilution of recombinant human IDE (R&D Systems Europe). The assay was linear in range 0 to 1,000 ng/mL.

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NEP Enzyme Activity Assay

The enzyme activity assay has been described in detail (32). Goat anti-human NEP (1.6 μg/mL) diluted in PBS (pH 7.4) was coated on Nunc maxi-sorp 96-well plates (Fisher Scientific, Loughborough, UK). Nonspecific binding of antibody was blocked by addition of 1% PBS-BSA. After washing, crude brain tissue homogenate or subcellular fractions (250 μg total protein), diluted in PBS (pH 7.4), were incubated together with serial dilutions of recombinant human NEP (R&D Systems Europe) at RT for 2 hours with continuous shaking. After washing, thiorphan (200 nmol/L) (Sigma Aldrich) prepared in 100 mmol/L Tris-HCl pH 7.5, 50 mmol/L NaCl, 10 μmol/L ZnCl2, or buffer alone, was added to the wells for 10 minutes at RT. The fluorogenic peptide Mca-RPPGFSAFK(Dnp)-OH (10 μmol/L) (R&D Systems Europe) diluted in 100 mmol/L Tris-HCl pH 7.5, 50 mmol/L NaCl, and 10 μmol/L ZnCl2 was added to each well and incubated at 37°C for 3 hours in the dark. Fluorescence was measured with excitation at 320 nm and emission at 405 nm.

Each homogenate and subcellular fraction were assayed in duplicate in the presence and absence of the NEP inhibitor, thiorphan. Specific NEP activity levels were calculated after subtraction of fluorescence in the presence of thiorphan. The NEP levels were interpolated from a standard curve generated from serial dilutions of recombinant full-length NEP. Average NEP enzyme was calculated for each homogenate from measurements made on 2 separate occasions.

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IDE Activity Assay

The assay method was similar to that for NEP and has been described in detail (32, 33). Briefly, anti-IDE antibodies raised against the inactive domain of IDE (2.5 μg/mL) (Abcam) were used to capture IDE in crude brain tissue homogenates or subcellular fractions (100 μg total protein). Enzyme activity was calculated by measuring fluorescence after the addition of the fluorogenic peptide substrate Mca-RPPGFSAFK(Dnp)-OH (10 μmol/L) (R&D Systems Europe); the assay was calibrated against serial dilutions of full-length rat IDE (Calbiochem, Nottingham, UK).

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Measurement of NSE

Neuron-specific enolase levels in crude brain homogenates were measured by a direct ELISA. Costar EIA microplate wells were coated with 10 μL of brain tissue homogenate in 90 µL PBS for 2 hours, washed 5 times with PBS containing 0.5% Tween 20 and incubated with anti-NSE (1:1000) for 2 hours. After further washes, mouse-horseradish peroxidase (1:100) was added for 20 minutes in the dark, washed, and tetramethyl benzidine was added for 10 minutes. Serial dilutions of recombinant human NSE (Biomol, Exeter, UK) were used to construct a best-fit curve, and NSE concentrations were calculated by interpolation. Each sample was run in duplicate, and the mean was determined. The NSE concentration was used to provide a proxy measurement of the number of neurons in tissue samples. Measurements of NEP and IDE levels and activity were adjusted for neuronal loss or damage according to the formula: NEP/IDEadjusted = NEP/IDEsample × NSEmean control/NSEsample, where NEP/IDEadjusted corresponds to the level or activity of NEP or IDE after correcting for neuronal loss in the sample, NEP/IDEsample was the unadjusted level or activity of NEP or IDE in the sample, NSEmean control was the mean level of NSE in the control cohort, and NSEsample was the measured level of NSE in individual test samples.

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NEP and IDE Activities and Postmortem Stability

To test the postmortem stability of NEP and IDE activity, we used 1) several immediately adjacent samples of frontal cortex from 2 control brains with relatively short postmortem delays (6-10 hours) that were incubated for 6, 12, 18, 24, 48, and 72 hours at RT, and 24, 48, and 72 hours at 4°C before homogenization; 2) cold (4°C) saline-perfused mouse brains (n = 3) that were finely chopped, mixed and incubated for 10, 30, 60, 120, and 360 minutes before homogenization; and 3) recombinant human standards (500 ng/mL) that were diluted in PBS and incubated at 4°C or RT for 10, 30, 60, and 120 minutes before measurement. Enzyme activities were measured as previously described.

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Statistical Analysis

Data were analyzed using an independent-samples t-test, 1-way analysis of variance (ANOVA) with Bonferroni posttesting, and Spearman or Pearson correlation analysis, as appropriate, using the Statistical Package for Social Science software (12.0.1). Values of p < 0.05 were considered statistically significant. To normalize the data (which were right-skewed in both the AD and control groups), all NSE-adjusted NEP and IDE protein and activity measurements were logarithmically transformed for statistical analysis.

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RESULTS

NSE Level in Relation to Braak Stage

The NSE level was significantly reduced in AD compared with controls (p = 0.0018) (Fig. 1A). The NSE levels differed significantly between the Braak Stages 0 to II, III to IV, and V to VI subgroups (p = 0.003, ANOVA). Post hoc comparisons showed that the level in Braak Stages V to VI was significantly lower than in Braak Stages 0 to II (p = 0.020) and Stages III to IV (p = 0.013) (Fig. 1B).

Figure 1
Figure 1
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NEP Level in Relation to AD Diagnosis and Braak Stage

Unadjusted NEP level did not significantly differ with respect to diagnosis (Fig. 2A) or Braak stage (Fig. 2B), although there was a trend toward increased NEP level in AD and with Braak stage. After adjustment for NSE, NEP level was significantly higher in AD cases than controls (p = 0.007) (Fig. 2C) and showed a highly significant correlation with Braak stage (p = 0.001, rs = 0.301) (Fig. 2D). Adjusted NEP level significantly differed between the Braak Stages 0 to II, III to IV, and V to VI subgroups (p = 0.004, ANOVA); post hoc comparison between subgroups indicated that the level was significantly higher in Braak Stages V to VI than Braak Stages 0 to II brains (p = 0.006) (Fig. 2D).

Figure 2
Figure 2
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NEP Level in Relation to Age

In a combined cohort including all cases, there was a trend toward an inverse correlation between NEP level and age, but this was not significant for either unadjusted or adjusted levels. In the AD cohort alone, unadjusted (p = 0.066, Pearson r = −0.194) and adjusted (p = 0.023, r = −0.238) NEP level showed an inverse correlation with age (Figs. 2E, F). In the control cohort alone, unadjusted and adjusted NEP level did not correlate with age (Figs. 2E, F). When analyzed according to Braak tangle stage, unadjusted and adjusted NEP levels did not correlate with age in the Braak Stages 0 to II or III to IV subgroups. In Braak Stages V to VI, however, both unadjusted (p = 0.036, r = −0.259) and adjusted (p = 0.027, r = −0.272) NEP levels negatively correlated with age.

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NEP Activity in Relation to AD Diagnosis and Braak Stage

Unadjusted NEP activity was significantly higher in the AD cohort (p = 0.035) (Fig. 3A) but did not correlate significantly with Braak stage (Fig. 3B). Adjusted NEP activity was significantly higher in the AD cohort (p = 0.001) (Fig. 3C), positively correlated with Braak stage (p = 0.008, rs = 0.232) (Fig. 3D), and significantly differed between the Braak Stages 0 to II, III to IV, and V to VI subgroups (p = 0.009, ANOVA) (Fig. 3D). Further post hoc testing showed the increased activity in Braak Stages V to VI to approach significance compared with Stages 0 to II (p = 0.051) and to be significantly greater than in the Stages III to IV subgroup (p = 0.027).

Figure 3
Figure 3
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NEP Activity in Relation to Age

In the combined cohort, unadjusted NEP activity inversely correlated with age (p = 0.021, r = −0.188), but the correlation disappeared after correction for NSE. Within the AD cohort alone, both unadjusted (p = 0.004, r = −0.291) and adjusted (p = 0.001, r = −0.337) NEP activity showed a significant inverse correlation with age (Figs. 3E, F). In the control group, neither unadjusted nor adjusted NEP levels correlated with age. When stratified according to Braak stage subgroups, unadjusted and adjusted NEP activity did not correlate with age in the Stages 0 to II or III to IV subgroups. In Braak Stages V to VI, both unadjusted (p = 0.001, r = −0.388) and adjusted (p = 0.0007, r = −0.398) NEP activity showed significant inverse correlations with age.

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IDE Level in Relation to AD Diagnosis and Braak Stage

Unadjusted IDE level was significantly decreased in the AD cohort compared with controls (p = 0.003) (Fig. 4A) and showed a significant inverse correlation with Braak stage (p = 0.028, rs = −0.197) (Fig. 4B). Analysis of variance revealed significantly different IDE level in Braak Stages 0 to II, III to IV, and V to VI subgroups (p = 0.024), and post hoc testing indicated that the level was significantly lower in the Braak Stages V to VI than the 0 to II subgroup (p = 0.032) (Fig. 4B). After adjustment for NSE, however, neuronal IDE level did not correlate with AD or Braak stage or differ significantly between Braak subgroups (Figs. 4C, D).

Figure 4
Figure 4
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IDE Level in Relation to Age

In the combined cohort, unadjusted and adjusted IDE protein did not correlate with age. In the AD group, adjusted (p = 0.004, r = −0.332) but not unadjusted IDE level inversely correlated with age (Figs. 4E, F). There was no correlation of either adjusted or unadjusted IDE level with age in the control group. In Braak Stages 0 to II and II to IV, IDE level did not correlate with age. In Braak Stages V to VI, both unadjusted (p = 0.032, r = −0.259) and adjusted (p = 0.0061, r = −0.332) IDE levels were significantly inversely correlated with age.

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IDE Activity in Relation to AD Diagnosis and Braak Stage

Unadjusted IDE activity did not vary according to diagnosis (Fig. 5A) or Braak tangle stage (Fig. 5B). Adjusted IDE activity was higher in AD than control brains (p = 0.016) (Fig. 5C) and positively correlated with Braak stage (p = 0.031, rs = 0.191) (Fig. 5D), ANOVA did not reveal significant differences between the Braak Stages 0 to II, III to IV, and V to VI subgroups.

Figure 5
Figure 5
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IDE Activity in Relation to Age

In the combined cohort, neither unadjusted nor adjusted IDE activity correlated with age. No effect of age was seen on comparison of IDE activity when examined in either the AD or control groups (Figs. 5E, F) or when stratifying according to Braak stage.

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NEP and IDE Levels and Activity in Relation to APOE

In the cohort as a whole, there was no significant association between APOE and NEP level or activity (either unadjusted or adjusted). Similarly, no significant association was demonstrated between APOE and IDE levels or activity.

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NEP and IDE Activity in Subcellular Fractions

Cytosolic NEP activity was significantly increased in AD (p = 0.008) (Fig. 6A) and significantly correlated with Braak stage (p = 0.029, rs = 0.301) (Fig. 6B). The NEP activity in the plasma membrane fraction did not significantly differ with diagnosis or Braak stage (Figs. 6C, D). The IDE activity did not differ with either diagnosis or correlate with Braak stage in either the cytosolic (Figs. 6E, F) or the plasma membrane (Figs. 6F, G) fraction.

Figure 6
Figure 6
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NEP and IDE Activity in Relation to Postmortem Delay and Tissue Storage

The NEP and IDE levels and activities did not correlate with sex, age, or postmortem delay. Incubation of human brain tissue at RT or 4°C for up to 72 hours did not result in significant changes in NEP or IDE activity (Figs. 7A-D). Incubation of recombinant human standards (Figs. 7E, F) and mouse brain tissue homogenates (Figs. 7G, H) at RT or 4°C for shorter incubation times (10, 30, 60, 120, and 360 minutes) did not result in an appreciable loss of enzyme activity.

Figure 7
Figure 7
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DISCUSSION

We have performed the most comprehensive analysis to date of NEP and IDE levels and activity in AD and control brain tissue. Our findings do not support the hypothesis that a reduction in the activity of either enzyme is a primary cause of Aβ accumulation in AD. After adjustment for NSE levels, the amount and activity of NEP were found to rise with progression of disease, as indicated by Braak tangle stage. Although unadjusted IDE levels fell with progression of disease, adjusted IDE levels did not significantly change, and IDE activity increased in AD. NEP levels and activity declined more steeply in relation to age at death in AD than control brains, but this reflected elevated NEP levels in younger AD patients rather than reduction below control levels in the older patients.

In previous studies, we (23) and others (22, 34) showed reduced immunolabeling of neuronal NEP in AD. NEP mRNA levels have also been reported to be reduced in AD (34, 35), particularly within regions vulnerable to neurofibrillary tangles and neuritic plaque formation. Western blot analysis of NEP in human brain tissue homogenates has yielded inconsistent findings; some studies showed reduced levels of NEP in AD (35, 36), and others showed no significant changes (23, 27, 37). We also noted that hippocampal IDE (24) labeling was reduced in AD; others have reported reduced IDE mRNA and protein as well as reduced IDE enzyme activity in AD (25, 26, 30).

With very few exceptions (36), previous studies of NEP and IDE expression have been based on small sample sizes, a general lack of adjustment of measurements for the effects of neuronal loss or damage (e.g. as a result of tau hyperphosphorylation) and an almost exclusive focus on end-stage AD (i.e. on patients with Braak Tangle Stage V or VI disease and a diagnosis of "definite AD" according to CERAD criteria). In contrast to immunolabeling of NEP in human sections or semiquantitative Western blot analysis, we used indirect sandwich ELISAs and immunocapture-based fluorogenic activity assays to measure protein levels and enzyme activity, respectively, in brain tissue homogenates. These methodological differences, the size and disease characteristics of selected cohorts, the adjustment for neuronal loss, the preparation of tissue samples in the presence of a nonionic detergent, and the area studied (Brodmann Area 6) are likely to account for differences between the present and previous findings. We also note that although NEP and IDE are predominantly neuronal (23, 24), expression in other cell types such as astrocytes and microglia and the presence of soluble forms of the enzymes may have contributed to measurements made on brain tissue homogenates.

There was considerable case-by-case variation in our cohorts. One potential cause and limitation of the present study might be the effect of postmortem delay on enzyme stability. We did not find any association between postmortem delay and NEP or IDE protein level or activity, however. Furthermore, we assessed this by simulating postmortem conditions in human brain tissue during a long period (up to 72 hours) and in mouse brain tissue during a shorter period (10-120 minutes) at both RT and 4°C, as well as by measuring the activity of recombinant human protein. These results suggest that postmortem delay was not a significant contributor to case-by-case variation.

The findings presented here are consistent with those of other studies. In human CSF, NEP increased with progression from mild cognitive impairment to AD (38). The levels of both NEP and IDE were found to be greater in aging hAPP mice than in wild-type littermates, the increase predominantly occurring after the appearance of plaques (39-41). Furthermore, IDE mRNA levels strongly correlated with those of Aβ1-40 and Aβ1-42 in hAPP transgenic mice (41). These data suggest that upregulation of NEP and IDE may be induced by Aβ; in keeping with this is the sharp rise in numbers of NEP- and IDE-positive astrocytes in close proximity to Aβ plaques in hAPP mice at 16 months of age (39, 40).

Other supportive work comes from a variety of in vitro studies. Incubation of neuronal, glial, or smooth muscle cells with fibrillar Aβ1-40 or Aβ1-42 induced expression of a number of Aβ-degrading enzymes, including IDE (40), matrix metalloproteinase 2, 3, and 9 (42-44), and plasminogen activators (45). Enhancement of NEP was found to be neuroprotective in vitro (19) and also ameliorated extracellular amyloid pathology, synaptic dysfunction, and memory defects in both mouse models of Aβ amyloidosis (20) and in transgenic Drosophila (46). It seems likely that any perturbation in Aβ production or removal that results in Aβ accumulation is likely to induce expression of Aβ-degrading enzymes and is a physiological response that minimizes further Aβ accumulation.

The possible transcriptional regulation of NEP expression by APP-FL or its derivatives such as APP intracellular domain has been explored. Pardossi-Piquard et al (47) demonstrated presenilin-dependant transcriptional control of NEP by APP intracellular domain and Aβ precursor-like protein, but another recent study did not find evidence of APP intracellular domain-mediated regulation (48). Posttranslational modification may provide another means of increasing enzyme activity. NEP undergoes posttranslational modification by N-glycosylation and has also been shown to be phosphorylated by casein kinase 2 (49). The activity of several kinases that can potentially phosphorylate NEP and IDE are increased in AD, such as glycogen synthase kinase 3 (50). The effects of phosphorylation and glycosylation on NEP and IDE enzyme activities are currently unknown.

Several studies have found that brain NEP protein and mRNA levels decline with age in humans (27, 37), transgenic hAPP and wild-type mice, (28, 29, 41) and in Drosophila (46). It may be of relevance that the level of somatostatin, a neuropeptide that has been shown to stimulate NEP activity, also falls with age (51) and is lower in people positive for APOE &epsiv;4 (52). We found that the decline in NEP activity with age at death was significant only in AD, reflecting relative elevation of NEP activity in younger AD patients and a convergence of NEP activity to control levels with increasing age. In contrast, IDE activity did not change with age at death. This is in keeping with studies in hAPP mice (40, 41).

In summary, our findings suggest that reduction in NEP and IDE activity is not the primary cause of Aβ accumulation in AD, but rather a late-stage phenomenon secondary to neurodegeneration.

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Keywords:

Alzheimer disease; Amyloid-β peptide; Apolipoprotein E; Insulin-degrading enzyme; Neprilysin; Neuron-specific enolase

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