• Alzheimer’s disease;
  • Alzheimer’s disease brain;
  • amyloid-degrading enzymes;
  • degradation;
  • endothelin-converting enzyme;
  • insulin-degrading enzyme;
  • neprilysin;
  • β-amyloid


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

J. Neurochem. (2010) 115, 47–57.


The brain steady state level of β-amyloid (Aβ) is determined by the balance between its production and removal, the latter through egress across blood and CSF barriers as well as Aβ degradation. The major Aβ-degrading enzymes are neprilysin (NEP), insulin-degrading enzyme (IDE), and endothelin-converting enzyme (ECE-1). Although evidence suggests that NEP is down-regulated in Alzheimer’s disease (AD), the role of IDE and ECE in the Aβ accumulation in aging and dementia remains less certain. In this study, we examined mRNA and protein expression, as well as biological activity of NEP, IDE, and ECE-1 in human frontal cortex by real-time RT-PCR for mRNA, immunoblotting for protein, and highly sensitive and specific fluorescence assays for activity. The relationships between Aβ-degrading enzymes and pathologic measures and clinical features were also assessed. The results showed that NEP mRNA, protein level, and activity were decreased in AD compared with normal controls with no cognitive impairment (NCI). In contrast, IDE activity was unchanged, but there was higher expression of IDE mRNA, indicating a possible compensatory reaction because of deficits in activity. ECE-1 expression in AD brain showed no significant difference compared with age-matched controls. Correlation analyses suggested that NEP expression was correlated with Aβ accumulation and clinical diagnosis, being lower in AD than in no cognitive impairment. In contrast, neither IDE nor ECE-1 correlated with Aβ or clinical diagnosis. These findings provide additional support for NEP as the major protease involved in Aβ degradation and suggest its possible therapeutic targeting in AD.

Abbreviations used:

Alzheimer’s disease


amyloid precursor protein



endothelin converting enzyme


horseradish peroxidase


insulin-degrading enzyme


mild cognitive impairment


mini-mental status examination


no cognitive impairment




phosphate-buffered saline


phenylmethylsulfonyl fluoride




Tris-Buffered Saline Tween-20

Alzheimer disease is a progressive neurodegenerative disease of the elderly characterized by deposition of β-amyloid (Aβ) in the brain as amyloid plaques and amyloid angiopathy. Aβ is physiologically produced by proteolytic cleavage of amyloid precursor protein (APP) by the concerted action of β- and γ-secretases, and its pathologic accumulation in Alzheimer’s disease (AD) indicates an imbalance between Aβ biosynthesis and clearance (Eckman and Eckman 2005; Higuchi et al. 2005; Wang et al. 2006). Under normal conditions, as revealed in pulse-chase studies in mice, Aβ is rapidly removed from the brain after its synthesis (Cirrito et al. 2003). Deficiencies in Aβ clearance or enzyme-mediated Aβ degradation are thought to lead to accumulation and aggregation of Aβ, which in turn, triggers a complex multistep cascade leading to dementia. Therefore, strategies that lower Aβ levels in the brain may stop or delay onset of AD (Eckman and Eckman 2005; Higuchi et al. 2005; Wang et al. 2006; Miners et al. 2008c; Bates et al. 2009).

In the past decade, multiple proteases have been shown to be capable of cleaving Aβ, including neprilysin (NEP), insulin-degrading enzyme (IDE), and endothelin-converting enzyme-1 (ECE-1), and a number of experimental and epidemiological studies implicate these enzymes in normal homeostasis of Aβ in the brain (Eckman and Eckman 2005; Higuchi et al. 2005; Wang et al. 2006; Miners et al. 2008c; Bates et al. 2009).

Although NEP appears to be the dominant Aβ protease (Vardy et al. 2005; El-Amouri et al. 2008; Hersh and Rodgers 2008; Miners et al. 2008c), other catabolic enzymes, such as ECE and IDE likely participate in regulating the steady-state levels of Aβ (Eckman et al. 2006; Qiu and Folstein 2006; Llovera et al. 2008). NEP is a type-II integral membrane protein, known as zinc metallopeptidase, composed of 750 residues with an active site at its extracellular carboxyl-domain. NEP is capable of degrading monomeric and oligomeric forms of Aβ (El-Amouri et al. 2008). NEP-deficient mice have significantly elevated brain Aβ levels, and treatment with NEP inhibitors can cause a rapid increase of Aβ levels in the brain, associated with memory impairment (Apelt et al. 2003; Farris et al. 2007). In contrast, deposition of Aβ in NEP-deleted mice can be reversed by injection of exogenous NEP (Iwata et al. 2001; Hemming et al. 2007). Over-expression of NEP in brains of APP transgenic mice (Leissring et al. 2003; Marr et al. 2003; Miners et al. 2008c; Meilandt et al. 2009) or over-expression of NEP in the periphery (Guan et al. 2009) is associated with lower brain Aβ levels and amyloid plaque load. In addition to these investigations carried out in animal models, observations in human postmortem tissue suggest that NEP expression is inversely related to the extent of AD pathology (Akiyama et al. 2001; Eckman et al. 2006; Hellstrom-Lindahl et al. 2008).

Insulin degrading enzyme is also a zinc metalloendopeptidase that is predominantly cytosolic (Duckworth et al. 1994), with less in the plasma membrane (Goldfine et al. 1984); it has a molecular weight of 110 kDa. As demonstrated by in vitro studies, IDE is not only a major soluble protease involved in the degradation of extracellular Aβ in the brain, but it also has the ability to remove the cytoplasmic products of APP (Bernstein et al. 1999; Edbauer et al. 2002). APP transgenic mouse that develop AD-like neuropathology with aging have elevated IDE mRNA and protein, and IDE protein expression is positively correlated with expression of full-length APP in cerebral cortex, indicating that IDE expression responds to Aβ accumulation (Vepsalainen et al. 2008). Mice with a homozygous deletion of IDE have elevated endogenous brain Aβ (Miller et al. 2003; Leal et al. 2006). Conversely, over-expression of IDE attenuates brain plaque formation, secondary pathology, and prevents premature death in a transgenic AD mouse model (Leissring et al. 2003). In AD, in situ hybridization and western blot analyses demonstrate reduced neuronal IDE mRNA and protein levels in hippocampus (Cook et al. 2003), and immunohistochemistry studies show that neuronal IDE expression in hippocampus is also significantly reduced compared with controls (Miners et al. 2008c). Reduced levels of cytosolic IDE have also been reported in AD (Perez et al. 2000). In contrast, Zhao et al. (2007) found reduced hippocampal IDE protein and activity in membrane fraction, but not the cytosol. Studies that focused on IDE in the cortex rather than hippocampus, showed different results; Caccamo et al. (2005) reported that IDE protein levels diminish with of age, whereas cortical IDE expression is elevated in AD. Kim et al. (2007) reported reduced IDE catalytic activity in AD lymphoblast lines without significant changes in IDE expression and suggested that this might involve genetic factors. The inconsistent results for IDE in AD remain to be explained, but may be owing to methodological issues. Clearly, additional studies are needed to better understand the significance of changes in IDE in aging and AD.

As another set of type-II integrated membrane zinc metalloendopeptidases, ECEs are primarily localized in endothelial cells, but have also been detected in neurons and glia (Davenport et al. 1998). Two ECE isoforms, ECE-1 and ECE-2, share 59% sequence homology and have similar catalytic activity; in the brain, the predominant isoform is ECE-1 (Davenport et al. 1998; Miners et al. 2008c). In both animal models and postmortem human brain tissue, the role of ECE-1 in AD is far from established. Eckman et al. (2001) were the first to identify ECE-1 as a novel Aβ-degrading enzyme in cell models. Later, they found that the level of brain Aβ was increased in ECE-1-deficient mice (Eckman et al. 2003), providing the first direct evidence that ECE-1 might play a role in Aβ metabolism in the brain. Over-expression of ECE-1 in APP presenilin 1 bigenic transgenic mice with gene delivery mechanisms decreased Aβ accumulation in the cortex and hippocampus (Carty et al. 2008). Although there is evidence for a role of ECE-1 in Aβ metabolism in experimental models, there is limited information on its role in AD. Only one study has investigated ECE-1 levels in AD brains, which showed increased neuronal expression in cerebral cortex and hippocampus in controls compared with AD (Funalot et al. 2004). The results need to be interpreted with caution because there is 59% identity between ECE-1 and ECE-2 (Barnes and Turner 1997), the possibility of cross-reactivity of antibodies with ECE-2 was not completely excluded.

We previously reported that NEP was selectively decreased in AD brain, and that there was an inverse correlation between NEP activity and vulnerability to AD pathology (Wang et al. 2005); however, there is still little comparative data exploring Aβ degrading enzymes simultaneously in AD and controls. To address this issue, in this study, we measured expression and activity levels of three Aβ degrading enzymes (NEP, IDE, and ECE-1) in brain tissue of prospectively studied controls with no cognitive impairment (NCI), as well as those with mild cognitive impairment (MCI) and clinical AD. The findings provide further evidence connecting enzyme activity, Aβ accumulation, and clinical features during pathogenesis of AD.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Case and clinical features data

Frozen brain tissue from frontal cortex was obtained from participants in the Religious Orders Study of the Rush Alzheimer Disease Center (P30AG10161) as described previously (Wang et al. 2007). Included in this study were 10 NCI, 10 MCI, and 10 AD. All cases had undergone a uniform structured clinical evaluation that included a medical history, neurologic examination, neuropsychological performance testing, and review of a brain scan when available. The evaluation included the mini-mental status examination (MMSE) as an overall test of cognition and 20 other cognitive performance tests, 19 of which were used to create a global measure of cognition, as described previously (Bennett et al. 2002; Wilson et al. 2002). Characteristics of the three groups are summarized in Table 1.

Table 1.   Clinical features of cases
 NCI (n = 10)MCI (n = 10)AD (n = 10)
  1. AD, Alzheimer’s disease; MCI, mild cognitive impairment; MMSE, mini-mental status examination; NCI, no cognitive impairment; NFT, neurofibrillary tangle.

  2. *p < 0.05 as compared with NCI and mild cognitive impairment (MCI). †p < 0.05 as compared with NCI.

Age at death (year)80.5 ± 1.984.3 ± 1.687.3 ± 2.1
Sex (M : F) 7 : 3 4 : 6 3 : 7
Postmortem delay (h)6.2 ± 1.25.7 ± 0.977.3 ± 1.1
Education (year)17.9 ± 1.517.2 ± 1.217.2 ± 0.45
Braak NFT stage1.7 ± 0.382.8 ± 0.433.6 ± 0.50†
MMSE27.3 ± 0.4227.2 ± 0.5019.4 ± 1.8*
Global z-score0.49 ± 0.110.24 ± 0.0−1.2 ± 0.20*

Chemicals and reagents

Oligonucleotides were synthesized by Gibco BRL Life Technologies (Gaithersburg, MD, USA). TRIreagent and Omniscript Reverse Transcriptase were acquired from Invitrogen (Cincinnati, OH, USA) and Qiagen (Valencia, CA, USA), respectively. SYBR green PCR master mix was from Applied Biosystems (Foster City, CA, USA). NEP, IDE, and ECE-1 antibody were purchased from Chemicon (Temecula, CA, USA), Abcam (Cambridge, MA, USA), and R&D Systems (Minneapolis, MN, USA), respectively. Synaptophysin monoclonal antibody was a generous gift from Dr William Honer, University of British Columbia, Vancouver, Canada. β-Actin and α-actin antibodies were from Amersham (Piscataway, NJ, USA) and Sigma-Aldrich (St Louis, MO, USA). Anti-rabbit and anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibody and anti-goat HRP-conjugated secondary antibody were from Cell Signaling (Danvers, MA, USA) and Santa Cruz (Santa Cruz, CA, USA). Mca-R-P-P-G-F-S-A-F-K(Dnp)-OH Fluorogenic peptide substrate came from R&D Systems. An enhanced chemiluminescence kit was obtained from Pierce (Rockford, IL, USA). Other general chemicals and reagents were from Sigma-Aldrich.

RNA extraction and RT-PCR

Frontal cortical gray matter was carefully dissected from frozen tissue and analyzed blinded to clinical information. Total RNA was isolated from approximately 50 mg of tissue according to the TRI-Reagent protocol. RNA was quantified and assessed for purity by UV spectrophotometer. Total RNA (1.5 μg) was subjected to reverse transcription with the Omniscript RT in a 20 μL reaction. The RT reaction was diluted fivefolds and 5 μL was amplified by Real-time PCR (quantitative PCR) in 25 μL reaction mixture containing Master Sybr Green (Applied Biosystems).

The primer sets were designed to span the intron–exon borders to distinguish amplified cDNA from genomic DNA by Primer Express software (Applied Biosystems). The sequences of primers used in this study were listed in Table 2. Quantitative PCR was performed on an ABI Prism 7500 real-time PCR system. The thermal cycling conditions comprised an initial denaturation step at 95°C for 2 min, then 40 cycles of two steps PCR including 95°C for 15 s and 60°C for 1 min. Data were collected during the 60°C annealing step. Optimization of primers concentration was performed at Serial dilutions of the template cDNA made from PCRs within the linear range. The authenticity of the PCR products was confirmed with melting curve analysis using the Applied Biosystems software and electrophoreses in 2.0% agarose gel.

Table 2.   Primers used in qRT-PCR
GenePrimer set
  1. ECE, endothelin converting enzyme; IDE, insulin-degrading enzyme; NEP, neprilysin.


Relative quantification of gene expression was carried out by comparative Ct method according to manufacture protocol (User Bulletin #2: ABI Prism 7500 Sequence Detection System). Briefly, the mRNA level of the genes was expressed in cycle threshold (Ct) value; the Ct values for each sample were averaged from duplicate. Based on the amplification efficiency, human β-actin for NEP, S26 for ECE and IDE were used as reference genes. Differences between the mean Ct values of NEP and reference genes were calculated as inline image for AD and MCI groups, and that of the ΔCt for the NCI group was set for calibrator (inline image). Final results, the sample–calibrator ratio, expressed as N-fold differences of NEP expression in the AD or MCI groups compared with NCI group, were determined as inline image. Similar method was used in ECE and IDE calculation of relative quantification of gene expression.

Sodium dodecyl sulfate–page acrylamide gel electrophoresis, western blot and protein quantification

Brain tissue homogenates were prepared from dissected samples of unfixed frozen frontal cortex. Protease inhibitor cocktail (Sigma-Aldrich) and 1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma-Aldrich) were added to the lysis buffer (Pierce), 50 μL whole tissue lysates was added to 150 μL 1% sodium dodecyl sulfate that contain protease inhibitor and 1 mM PMSF. After vortexing, the samples were incubated on ice for 30 min. From 1000 μg whole tissue lysates, 4× sample buffer was added and boiled at 95°C for 5 min, cooled on ice, and subsequently loaded to a 12% sodium dodecyl sulfate–page acrylamide gel electrophoresis gel. Proteins were transferred to nitrocellulose membrane at 100 V for 70 min. After blocking with 5% non-fat milk-Tris-Buffered Saline Tween-20 (TBST) for 1 h at room temperature, membranes were incubated in 5% non-fat milk-TBST with mouse anti-Aβ (1 : 1000; Clone Ab9, a gift from Dr T. Golde, Mayo Clinic, Jacksonville, FL, USA), rabbit anti-NEP antibody (1 : 2000; Chemicon, Temecula, CA, USA), mouse anti-IDE (1 : 2000; Abcam, Cambridge, USA), goat anti-ECE-1 (1 : 2000; R&D Systems), mouse anti-synaptophysin (EP10; 1 : 500), mouse monoclonal antibody BAN50 (recognizing the amino terminus of Aβ; 1 : 1000), mouse anti-β-actin (1 : 2000; Amersham, Piscataway, NJ, USA) or rabbit anti-α-actin antibodies (1 : 4000; Sigma-Aldrich), respectively, at 4°C overnight. After washing with TBST, membranes were incubated with HRP-conjugated anti-rabbit (1 : 8000; Cell Signaling, Danvers, MA, USA), or anti-mouse (1 : 8000; Cell Signaling) or anti-goat (1 : 5000; Santa Cruz) secondary antibodies for 1 h at 23–25°C, detected by the enhanced chemiluminescence plus western blotting detection system and autoradiography film (Amersham). The densities of target bands were measured with Image Quant 5.2 (GE Healthcare, Piscataway, NJ, USA) and expressed as relative level with respect to the normal control.

Measurement of enzymatic activity

Neprilysin activity was determined by fluorescence resonance energy transfer (Johnson and Ahn 2000; Takaki et al. 2000; Wang et al. 2005). Human frontal cortex gray matter was homogenized in phosphate-buffered saline (PBS; pH 7.4) containing protease inhibitor cocktail (Sigma; 200 mg/mL), 1 mM PMSF, and solubilized by sonication. Samples were then added to 0.1% Triton-X-100 in PBS to final concentration 2.0 mg/mL, incubated on ice for 30 min to extract membrane proteins. Fluorogenic peptide substrate (50 μL; Mca-RPPGFSAFK-[Dnp]-OH; R&D Systems) dissolved in HEPES buffer (pH 7.4) was added to 50 μL of homogenate from a normal case and then diluted to 2000, 1000, 500, 250, 125, 62.5, and 31.25 mg/mL to produce a standard curve. To determine the specificity of the assay, a set of samples was initially pre-incubated with 100 nM thiorphan (Sigma-Aldrich), a NEP specific inhibitor (Wang et al. 2003; Miners et al. 2008b), for 10 min. Specific NEP activity is optimal at pH 7.4 (Wang et al. 2005). Sample homogenates (1000 μg/mL) were pre-incubated with or without inhibitor prior to adding fluorogenic peptide substrate. Fluorescence was read after 30 min incubation at 23–25°C with excitation at 320 nm and emission at 405 nm on a fluorescent 96-well plate reader (Spectra Max Gemini; Molecular Devices, Sunnyvale, CA, USA).

Endothelin converting enzyme activity was also analyzed by fluorogenic peptide substrate Mca-RPPGFSAFK(Dnp)-OH at 23–25°C in the presence or absence of the ECE inhibitor, phosphoramidon (1 μM; Sigma; Wang et al. 2003; Miners et al. 2008b). Phosphoramidon inhibition of ECE-1 is highly pH dependent (Ahn et al. 1998). The IC50 value of phosphoramidon is about 50-fold lower at pH 5.8 than at pH 7.2. In addition, NEP activity is also pH-dependent. There is almost no NEP activity at pH5.8 (Fahnoe et al. 2000). Thus, the inhibition by phosphoramidon at pH 5.8 can be considered the inhibition of ECE.

Insulin-degrading enzyme-specific activity was measured by using immunocapture-based fluorometric assay (Miners et al. 2008a; Wang et al. 2009a). Frontal cortex brain tissue homogenates were mixed with nine times volumes of sample buffer [0.5% Triton-X-100, 20 mM Tris pH 7.4, 10% sucrose (w/v)] containing protease inhibitor cocktail (Sigma-Aldrich) for 30 min, ultrasonic homogenized and then centrifuged at 100 g for 15 min at 4°C. Greiner 96-well high binding ELISA plate were coated with capture IDE antibody (sc27266, 50 mg/mL; Santa Cruz) diluted in carbonate buffer (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 PBS-Tween containing 2% bovine serum albumin, 50 μL of brain homogenate was added and incubated at 4°C overnight. After three to five washes, 10 μM fluorogenic peptide 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.

All the assays were triplicates, and the mean values were calculated for each sample.

Statistical analyses

All data were expressed as mean ± standard error of the mean. Statistical analyses were performed using the GraphPad Prism 4.0 software (GraphPad Software, Inc., San Diego, CA, USA). One-way anova analysis was used to evaluate differences in mean values among three groups, followed by post hoc tests of multiple comparisons when anova were significant. Pearson correlation analysis was used to study relations between variables. Statistical significance level was set at p < 0.05.


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Real-time RT-PCR

In an effort to understand the gene transcription of Aβ-degrading enzyme participation in the regulation of Aβ accumulation in AD development, we examined NEP, IDE, and ECE-1 levels in frontal cortex from NCI, MCI, and AD patients using quantitative RT-PCR. NEP mRNA levels were decreased in MCI and AD groups with significance in AD compared with NCI (Fig. 1a). In contrast to NEP, both ECE-1 and IDE mRNA increased in MCI and AD brains, and for IDE this reached significance in AD compared with NCI.


Figure 1.  Neprilysin (NEP), insulin-degrading enzyme (IDE) and endothelin converting enzyme (ECE) levels in frontal cortex of no cognitive impairment (NCI), mild cognitive impairment (MCI), and AD patients. (a) Alterations of NEP, IDE, and ECE mRNA expression in frontal cortex of NCI, MCI, and AD patients. Relative NEP, IDE, and ECE mRNA levels in three groups were detected by qRT-PCR using the comparative cycle threshold method, assigning a value of 1 to the NCI group. NEP mRNA levels were normalized to β-actin in each sample, and IDE and ECE levels were normalized to S26 in each sample. Bars show mean values and standard error of mean (n = 10 in each group). Decreased NEP mRNA level was observed in AD group compared with NCI group, whereas increased IDE and ECE mRNA level in AD group compared with NCI group. (b) NEP protein levels are significantly decreased in AD patient, whereas IDE and ECE protein expression stay steady in three groups. Western blot analysis on brain frontal cortex was performed, the actin bands confirm an equal loading of all samples. Data shown are representative of all brain biopsies studied. The densitometry of western blots showed the down-regulation of NEP protein in AD group compared with NCI (p < 0.05). NEP protein level in MCI group tend to decrease, but not statistically significant. IDE and ECE protein level in three groups did not changed significantly. (c) NEP activity in AD frontal cortex is significantly decreased, whereas IDE and ECE activity remains unchanged. Specific NEP and ECE activity in brain homogenate from all cases were measured based on high sensitive fluorogenic analysis; specific IDE activity is measured by using immunocapture fluorometric assay. Assays were repeated on three separate occasions. Bar show mean values and standard error of mean of 9 cases in AD group and 10 cases in NCI and MCI groups, respectively. *p < 0.05 as compared with NCI group.

Download figure to PowerPoint

Western blots

Many studies suggest that post-transcriptional regulation of Aβ-degrading enzymes may play a role in Aβ degradation and clearance (Wang et al. 2006; Miners et al. 2008c). To study the protein expression of these enzymes, immunoblotting was performed in postmortem brain samples. As shown in Fig. 1(b), NEP had a double band at 80- and 100-kDa molecular mass. Densitometric analyses of blots revealed a significant decrease in NEP in AD compared with NCI (p < 0.05), in agreement with previous reports (Higuchi et al. 2005). Despite increases in mRNA observed for IDE and ECE-1, their protein levels did not show significant change.

Activity assays

Neprilysin enzymatic activity was measured in frontal cortex with a fluorometric assay. As shown in Fig. 1(c), NEP catalytic activity was significantly decreased in AD compared with NCI and MCI. On the other hand, IDE and ECE activity tended to increase in AD compared with MCI and NCI, but this did not reach statistic significance. It is reasonable to speculate that deficits in NEP expression and activity in AD may lead to compensatory increases in IDE and ECE-1 expression and possibly to activity.

Synaptophysin, a pre-synaptic marker, is not altered with AD development

Neprilysin and ECE-1 are integral transmembrane proteases that are primarily expressed in pre-synaptic terminals of neurons (Fukami et al. 2002), where IDE is expressed in neurons and other cell types. Neuron loss should potentially affect the expression of these enzymes. To clarify that decreased NEP level in AD is not merely because of neuronal or synaptic loss, western blots of a pre-synaptic marker, synaptophysin, were performed in the same frontal cortex homogenates used for western blots of Aβ degrading enzymes. As shown in Fig. 2, synaptophysin level was less in MCI and AD, but the difference did not reach significance compared with NCI, probably because of the location of the tissue (frontal cortex, which has neuronal loss later than medial temporal lobe in AD pathogenesis), the fact that AD and MCI had relatively mild cognitive deficits, and the relatively small sample size. After adjusted by synaptophysin, NEP protein in AD cases remained significantly lower in AD compared to NCI, indicating that NEP reduction is not merely because of neuronal and synaptic loss.


Figure 2.  Neuronal loss makes limited contribution to neprilysin (NEP) protein down-regulation. Synaptophysin (Synapt), pre-synaptic marker in neuron, was measured by western blot (a). AD and mild cognitive impairment (MCI) group have very slightly declined synaptophysin expression compared to no cognitive impairment (NCI), the difference did not reach statistic significance. NEP protein levels were significantly decreased in AD cases by normalizing with synaptophysin (b). *p < 0.05 as compared with NCI.

Download figure to PowerPoint

NEP protein level, but not ECE-1 or IDE, is closely correlated with Aβ accumulation

To understand the relationship between Aβ degrading enzymes and Aβ accumulation, we conducted western blot to measure the content of Aβ by full-length Aβ antibody in human postmortem brain tissue. As expected, we observed a marked increase in Aβ in AD compared with NCI (Fig. 3). Further exploration of the functional association between the Aβ-degrading enzyme and Aβ accumulation indicated that NEP immunoreactivity was inversely correlated with Aβ in all cases (Fig. 4; R2 = −0.37, p = 0.0004). Similar analyses performed in subsets showed negative correlation in NCI (R2 = −0.32, p = 0.08) and MCI (R2 = −0.49, p = 0.02) group. However, there was no association in the AD group (R2 = 0.26, p = 0.13; Fig. 4). No significant correlations between IDE or ECE immunoreactivity and Aβ content were observed (data not shown).


Figure 3.  Western blot analysis conform β-amyloid (Aβ) deposition in AD patients. Total Aβ protein content in frontal cortex homogenate was detected by full-length Aβ antibody in three groups. As a reference protein, β-actin indicates equal loading of all samples. Results show Aβ increases dramatically in AD group compared with no cognitive impairment (NCI). **p < 0.01 as compared with NCI group.

Download figure to PowerPoint


Figure 4.  Correlation of neprilysin (NEP) protein level and β-amyloid (Aβ) accumulation. The negative correlation between NEP protein and Aβ accumulation was observed (R2 = 0.37, p = 0.0004) when combined all the three groups together suggesting that NEP is a key factor in Aβ clearance during AD development. However, when correlation analysis were performed in sub-group, the positive correlation between NEP protein level and Aβ content was found in AD group (R2 = 0.26, p = 0.13). These results implied that the capacity of NEP clearance in AD development is not unlimited.

Download figure to PowerPoint

NEP, not ECE-1, or IDE, activity is directly related with clinical manifestations

Two clinical neuropsychological parameters, MMSE and global neuropsychological z-scores were examined with respect to Aβ degrading enzymes (Fig. 5). The MMSE is widely used to assess the severity of cognitive impairment, and there was a significant correlation between MMSE and NEP activity (p = 0.016), and an even more significant correlation between NEP activity and z-score (p = 0.006). In contrast, there was no correlation between IDE and ECE-1 in either mental state scores or the neuropsychologic summary measure of cognitive function.


Figure 5.  Correlation of neprilysin (NEP) enzymatic activity and AD clinical parameters. The positive correlation between NEP activity and mini-mental status examination (MMSE), NEP activity, and global z-score were observed when all cases were pooled together, indicating that NEP activity may be an essential factor in AD clinical physiological feature development.

Download figure to PowerPoint


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Although the physiological functions of Aβ remain to be elucidated, imbalance in its production and clearance contributes to its accumulation in brain, which is one of the most characteristic features of AD. Considerable evidence has emerged indicating that proteolytic degradation of Aβ is a major process involved in Aβ clearance. To date, most attention has been focused on NEP and its homologs IDE and ECE-1. A range of different studies has produced conflicting results for NEP and IDE in AD (El-Amouri et al. 2008; Miners et al. 2008c; Bates et al. 2009). Except for experimental models, little is known about ECE-1 especially in human tissue (Weeraratna et al. 2007; Miners et al. 2008c). In this study, we measured mRNA, protein, and enzyme activity for these Aβ-degrading enzymes in frontal cortex of a series of well-characterized patients and correlated these findings to Aβ levels and clinical features.

Although some previous studies have shown increases in NEP and IDE levels in AD that correlated with disease severity (Dorfman et al. 2008; Miners et al. 2009), in this study we found mRNA, protein, and activity of NEP were decreased in AD, which is in agreement with our previous studies (Wang et al. 2003, 2005) as well findings of independent investigators using comparable methods (Akiyama et al. 2001; Miners et al. 2006). Previous experiments in cultured neuronal cells indicated that NEP mRNA, protein, and activity are up-regulated by treatment with Aβ (Wang et al. 2009b). In the context of the present findings, it suggests that any increase in NEP in the human brain in response to Aβ may be relatively early in the disease process. With the advent of amyloid imaging, it is becoming clear that Aβ deposition in the frontal cortex is an early pathologic process (Jack et al. 2009). Compensatory increases in NEP would be expected in clinically normal individuals with cortical amyloid deposits, a process referred to as pathological aging, and this is what was found in our previous study (Wang et al. 2005).

Neprilysin is known as an integral membrane protein and to be expressed predominantly in axonal and synaptic membranes (Fukami et al. 2002). Given this localization, it is important that any decreases in NEP mRNA, protein, or catalytic activity, be assessed in light of neuron or synaptic loss to exclude the possibility that decreases are merely secondary to synaptic loss. In previous studies, neuron-specific enolase has been used (Miners et al. 2009), while we used the pre-synaptic marker synaptophysin in this and previous studies (Wang et al. 2005). We found a trend for decreasing synaptophysin immunoreactivity in AD and MCI compared with NCI, but this did not reach statistic significance. Synaptophysin loss in AD shows regional anatomical differences (Masliah et al. 1994), with frontal cortex involved later than medial temporal lobe, which may explain the subtle changes we observed. NEP protein level, after adjusting for synaptophysin level, still showed significant decreases in AD compared with NCI. The results suggest that neuron or synaptic loss cannot explain the decreases we observed in NEP in AD.

Experimental studies have shown that ECE-1 can degrade Aβ (Eckman et al. 2001, 2003, 2006), but the relative contribution of ECE-1 to Aβ degradation in the human brain is unknown. This study is the first systematic investigation of ECE-1 levels and catalytic activity in AD. A previous study reported on ECE-1 protein expression in cortex and hippocampus, but did not measure enzyme activity (Funalot et al. 2004). We found a trend for increases in ECE-1 mRNA, protein and activity in AD compared with NCI, although none of these reached statistical significance. Our findings support the hypothesis that increased ECE-1 in AD is compensatory to increases in Aβ and fit with our in vitro findings that SH-SY5Y treated with Aβ had significantly increased ECE-1 expression and activity (Wang et al. 2009a).

Previous studies of IDE regulation in AD brain have yielded inconsistent results; some studies have shown reduction of IDE in AD (Perez et al. 2000; Cook et al. 2003; Caccamo et al. 2005; Zhao et al. 2007), whereas others have shown significant increases (Caccamo et al. 2005; Dorfman et al. 2008). The studies were not directly comparable, given that different cellular components (cytosolic vs. membrane) and different brain regions (hippocampus, neocortex, or cerebellum) were studied. In this studies, we found higher IDE mRNA in AD compared with NCI, with IDE protein and catalytic activity also tending to be higher in AD frontal cortex, but not reaching statistical significance, consistent with findings from the original report (Bernstein et al. 1999). Our in vitro data also support our observation in AD cases (Wang et al. 2009a) and provide support for the hypothesis that IDE is up-regulated in response to increasing levels of brain Aβ, similar to ECE-1.

From the above, we speculate that IDE and ECE-1 might be second line defense to increasing Aβ related to decreases in NEP. On the other hand, decreased catalytic capacity of NEP is not adequately compensated by the ECE-1 and IDE, as Aβ progressively accumulates in the brain. Moreover, these enzymes most likely act independently with respect to Aβ removal from the brain, in agreement with experimental data from mice (Qiu and Folstein 2006).

In the brain, NEP, IDE, and ECE show a distinct pattern of expression. NEP is mainly expressed in pre-synaptic terminals of neurons (Fukami et al. 2002), IDE is predominately a cytosolic protein (Yfanti et al. 2008) and ECE exhibits a dual localization in the cell (Barnes and Turner 1997). The region and subcellular expression of these enzymes may explain their functions during AD development.

There has been strong evidence that Aβ accumulation is central to the pathogenesis of AD. There is, however, little evidence for increased Aβ production in the majority of late onset AD. Deficiencies in Aβ clearance or enzyme-mediated Aβ degradation are increasingly thought to be responsible for Aβ accumulation, particularly in late onset AD (Wang et al. 2006; Miners et al. 2008c). Postmortem AD brain showed NEP mRNA and protein reduced in high plaque areas (Yasojima et al. 2001a,b; Wang et al. 2003, 2005; Caccamo et al. 2005). Reductions were most prominent in most vulnerable regions to AD pathology, such as hippocampus, but not in other brain areas such as cerebellum or in peripheral organs (Yasojima et al. 2001b; Caccamo et al. 2005). Similar to findings in AD, NEP expression is reduced in the hippocampus and cortex of aged mice compared with young mice (Iwata et al. 2002; Apelt et al. 2003). Conversely, over-expression of NEP reduces Aβ levels in a dose-dependent manner (Iwata et al. 2002; Marr et al. 2004), and protects neuronal cells from Aβ toxicity in vitro (El-Amouri et al. 2007). Consistent with the previous studies by our laboratory (Wang et al. 2005) and abovementioned reports by others, present data showed that a decrease in the overall level of NEP mRNA, protein, and activity in AD brains, was observed. Importantly, NEP protein expression was inversely correlated with Aβ levels. A strong correlation was also found between NEP enzymatic activity and clinical cognitive scores, MMSE and Global82 (Fig. 5), regardless of whether or not there were cortical amyloid deposits, which is in agreement with previous findings (Funato et al. 1998; Morishima-Kawashima et al. 2000; Fukumoto et al. 2004; Wang et al. 2005). These data support the hypothesis that decreased NEP contributes to Aβ deposition in AD.

Interestingly, negative correlation between NEP protein and Aβ level was found in MCI (R2 = 0.49, p = 0.02) and NCI (R2 = 0.32, p = 0.08) in this study, which is reasonable and in agreement with previous findings. In contrast to NCI and MCI, however, we observed no significant correlation between NEP levels and Aβ in AD brains. The levels of NEP were uniformly low and the Aβ levels were high providing a narrow dynamic range for correlations. In addition, pathological induction and modification of NEP may occur in AD. In AD transgenic mice (TgCRND8), over-expressing human APP, relative NEP levels are greater in the transgenic than in control mice, and NEP immunoreactivity is localized to a subpopulation of plaques. Aβ deposition correlated with total NEP immunoreactivity. This suggests that NEP may have been pathologically induced because of Aβ deposition (Sato et al. 1991). Our previous study indicated acutely administrated Aβ decreased NEP catalytic activity, which consequently up-regulated NEP mRNA and protein levels. Oxidative modification of NEP plays a crucial role in enzyme inactivation (Wang et al., 2009b). It indicates that NEP may also be up-regulated under the induction of increased Aβ during AD development. As a result of the oxidative modification of NEP, increased NEP is insufficient to clear the excessive Aβ that eventually results in the brain Aβ deposition and plaque formation.

Unlike Zhao et al. (2007) who found correlation between IDE immunoreactivity and the levels of Aβ in hippocampus and occipital cortex, we found no significant correlations between IDE or ECE-1 and Aβ. These results suggested that NEP is a key Aβ degrading enzyme in the aging and AD. The decrease in NEP expression in AD may be induced by accumulation of Aβ with pro-oxidant state favoring oxidative modification and inactivity of NEP. Thus, low NEP may promote Aβ formation and be part of a negative feed-forward process whereby increased Aβ leads to greater deficiencies in NEP activity (Farris et al. 2007).

In summary, our finding suggests that NEP is a key protease in Aβ degradation, although ECE and IDE may act as a second line defense. The strategies aimed at promoting NEP expression or enzymatic activities in the brain may help prevent progression of AD through mechanisms involving the clearance of the Aβ peptides from the brain.


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This work is supported by NIH grants AG025722 and AG029972 (to DSW), and an Alzheimer 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), and NIH grants to DWD and P30AG10161 and R01AG15819 (to D.A.B.).


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  • Ahn K., Herman S. B. and Fahnoe D. C. (1998) Soluble human endothelin-converting enzyme-1: expression, purification, and demonstration of pronounced pH sensitivity. Arch. Biochem. Biophys. 359, 258268.
  • Akiyama H., Kondo H., Ikeda K., Kato M. and McGeer P. L. (2001) Immunohistochemical localization of neprilysin in the human cerebral cortex: inverse association with vulnerability to amyloid beta-protein (Abeta) deposition. Brain Res. 902, 277281.
  • Apelt J., Ach K. and Schliebs R. (2003) Aging-related down-regulation of neprilysin, a putative beta-amyloid-degrading enzyme, in transgenic Tg2576 Alzheimer-like mouse brain is accompanied by an astroglial upregulation in the vicinity of beta-amyloid plaques. Neurosci. Lett. 339, 183186.
  • Barnes K. and Turner A. J. (1997) The endothelin system and endothelin-converting enzyme in the brain: molecular and cellular studies. Neurochem. Res. 22, 10331040.
  • Bates K. A., Verdile G., Li Q. X., Ames D., Hudson P., Masters C. L. and Martins R. N. (2009) Clearance mechanisms of Alzheimer’s amyloid-beta peptide: implications for therapeutic design and diagnostic tests. Mol. Psychiatry 14, 469486.
  • Bennett D. A., Wilson R. S., Schneider J. A., Evans D. A., Beckett L. A., Aggarwal N. T., Barnes L. L., Fox J. H. and Bach J. (2002) Natural history of mild cognitive impairment in older persons. Neurology 59, 198205.
  • Bernstein H. G., Ansorge S., Riederer P., Reiser M., Frolich L. and Bogerts B. (1999) Insulin-degrading enzyme in the Alzheimer’s disease brain: prominent localization in neurons and senile plaques. Neurosci. Lett. 263, 161164.
  • Caccamo A., Oddo S., Sugarman M. C., Akbari Y. and LaFerla F. M. (2005) Age- and region-dependent alterations in Abeta-degrading enzymes: implications for Abeta-induced disorders. Neurobiol. Aging 26, 645654.
  • Carty N. C., Nash K., Lee D., Mercer M., Gottschall P. E., Meyers C., Muzyczka N., Gordon M. N. and Morgan D. (2008) Adeno-associated viral (AAV) serotype 5 vector mediated gene delivery of endothelin-converting enzyme reduces Abeta deposits in APP + PS1 transgenic mice. Mol. Ther. 16, 15801586.
  • Cirrito J. R., May P. C., O’Dell M. A. et al. (2003) In vivo assessment of brain interstitial fluid with microdialysis reveals plaque-associated changes in amyloid-beta metabolism and half-life. J. Neurosci. 23, 88448853.
  • Cook D. G., Leverenz J. B., McMillan P. J., Kulstad J. J., Ericksen S., Roth R. A., Schellenberg G. D., Jin L. W., Kovacina K. S. and Craft S. (2003) Reduced hippocampal insulin-degrading enzyme in late-onset Alzheimer’s disease is associated with the apolipoprotein E-epsilon4 allele. Am. J. Pathol. 162, 313319.
  • Davenport A., Kuc R., Plumpton C., Mockridge J., Barker P. and Huskisson N. (1998) Endothelin-converting enzyme in human tissues. Histochemical J 30, 359374.
  • Dorfman V. B., Pasquini L., Riudavets M., Lopez-Costa J. J., Villegas A., Troncoso J. C., Lopera F., Castano E. M. and Morelli L. (2008) Differential cerebral deposition of IDE and NEP in sporadic and familial Alzheimer’s disease. Neurobiol. Aging (in press).
  • Duckworth W. C., Bennett R. G. and Hamel F. G. (1994) A direct inhibitory effect of insulin on a cytosolic proteolytic complex containing insulin-degrading enzyme and multicatalytic proteinase. J. Biol. Chem. 269, 2457524580.
  • Eckman E. A. and Eckman C. B. (2005) Abeta-degrading enzymes: modulators of Alzheimer’s disease pathogenesis and targets for therapeutic intervention. Biochem. Soc. Trans. 33, 11011105.
  • Eckman E. A., Reed D. K. and Eckman C. B. (2001) Degradation of the Alzheimer’s amyloid beta peptide by endothelin-converting enzyme. J. Biol. Chem. 276, 2454024548.
  • Eckman E. A., Watson M., Marlow L., Sambamurti K. and Eckman C. B. (2003) Alzheimer’s disease beta-amyloid peptide is increased in mice deficient in endothelin-converting enzyme. J. Biol. Chem. 278, 20812084.
  • Eckman E. A., Adams S. K., Troendle F. J., Stodola B. A., Kahn M. A., Fauq A. H., Xiao H. D., Bernstein K. E. and Eckman C. B. (2006) Regulation of steady-state beta-amyloid levels in the brain by neprilysin and endothelin-converting enzyme but not angiotensin-converting enzyme. J. Biol. Chem. 281, 3047130478.
  • Edbauer D., Willem M., Lammich S., Steiner H. and Haass C. (2002) Insulin-degrading enzyme rapidly removes the beta-amyloid precursor protein intracellular domain (AICD). J. Biol. Chem. 277, 1338913393.
  • El-Amouri S. S., Zhu H., Yu J., Gage F. H., Verma I. M. and Kindy M. S. (2007) Neprilysin protects neurons against Abeta peptide toxicity. Brain Res. 1152, 191200.
  • El-Amouri S. S., Zhu H., Yu J., Marr R., Verma I. M. and Kindy M. S. (2008) Neprilysin: an enzyme candidate to slow the progression of Alzheimer’s disease. Am. J. Pathol. 172, 13421354.
  • Fahnoe D. C., Knapp J., Johnson G. D. and Ahn K. (2000) Inhibitor potencies and substrate preference for endothelin-converting enzyme-1 are dramatically affected by pH. J. Cardiovasc. Pharmacol. 36, S22S25.
  • Farris W., Schutz S. G., Cirrito J. R. et al. (2007) Loss of neprilysin function promotes amyloid plaque formation and causes cerebral amyloid angiopathy. Am. J. Pathol. 171, 241251.
  • Fukami S., Watanabe K., Iwata N. et al. (2002) Abeta-degrading endopeptidase, neprilysin, in mouse brain: synaptic and axonal localization inversely correlating with Abeta pathology. Neurosci. Res. 43, 3956.
  • Fukumoto H., Rosene D. L., Moss M. B., Raju S., Hyman B. T. and Irizarry M. C. (2004) Beta-secretase activity increases with aging in human, monkey, and mouse brain. Am. J. Pathol. 164, 719725.
  • Funalot B., Ouimet T., Claperon A. et al. (2004) Endothelin-converting enzyme-1 is expressed in human cerebral cortex and protects against Alzheimer’s disease. Mol. Psychiatry 9, 11221128, 1059.
  • Funato H., Yoshimura M., Kusui K., Tamaoka A., Ishikawa K., Ohkoshi N., Namekata K., Okeda R. and Ihara Y. (1998) Quantitation of amyloid beta-protein (A beta) in the cortex during aging and in Alzheimer’s disease. Am. J. Pathol. 152, 16331640.
  • Goldfine I. D., Williams J. A., Bailey A. C., Wong K. Y., Iwamoto Y., Yokono K., Baba S. and Roth R. A. (1984) Degradation of insulin by isolated mouse pancreatic acini. Evidence for cell surface protease activity. Diabetes 33, 6472.
  • Guan H., Liu Y., Daily A., Police S., Kim M. H., Oddo S., LaFerla F. M., Pauly J. R., Murphy M. P. and Hersh L. B. (2009) Peripherally expressed neprilysin reduces brain amyloid burden: a novel approach for treating Alzheimer’s disease. J. Neurosci. Res. 87, 14621473.
  • Hellstrom-Lindahl E., Ravid R. and Nordberg A. (2008) Age-dependent decline of neprilysin in Alzheimer’s disease and normal brain: inverse correlation with A beta levels. Neurobiol. Aging 29, 210221.
  • Hemming M. L., Patterson M., Reske-Nielsen C., Lin L., Isacson O. and Selkoe D. J. (2007) Reducing amyloid plaque burden via ex vivo gene delivery of an Abeta-degrading protease: a novel therapeutic approach to Alzheimer disease. PLoS Med. 4, e262.
  • Hersh L. B. and Rodgers D. W. (2008) Neprilysin and amyloid beta peptide degradation. Curr. Alzheimer Res. 5, 225231.
  • Higuchi M., Iwata N. and Saido T. C. (2005) Understanding molecular mechanisms of proteolysis in Alzheimer’s disease: progress toward therapeutic interventions. Biochim. Biophys. Acta 1751, 6067.
  • Iwata N., Tsubuki S., Takaki Y., Shirotani K., Lu B., Gerard N. P., Gerard C., Hama E., Lee H. J. and Saido T. C. (2001) Metabolic regulation of brain Abeta by neprilysin. Science 292, 15501552.
  • Iwata N., Takaki Y., Fukami S., Tsubuki S. and Saido T. C. (2002) Region-specific reduction of A beta-degrading endopeptidase, neprilysin, in mouse hippocampus upon aging. J. Neurosci. Res. 70, 493500.
  • Jack Jr C. R., Lowe V. J., Weigand S. D. et al. (2009) Serial PIB and MRI in normal, mild cognitive impairment and Alzheimer’s disease: implications for sequence of pathological events in Alzheimer’s disease. Brain 132, 13551365.
  • Johnson G. D. and Ahn K. (2000) Development of an internally quenched fluorescent substrate selective for endothelin-converting enzyme-1. Anal. Biochem. 286, 112118.
  • Kim M., Hersh L. B., Leissring M. A. et al. (2007) Decreased catalytic activity of the insulin-degrading enzyme in chromosome 10-linked Alzheimer disease families. J. Biol. Chem. 282, 78257832.
  • Leal M. C., Dorfman V. B., Gamba A. F., Frangione B., Wisniewski T., Castano E. M., Sigurdsson E. M. and Morelli L. (2006) Plaque-associated overexpression of insulin-degrading enzyme in the cerebral cortex of aged transgenic tg2576 mice with Alzheimer pathology. J. Neuropathol. Exp. Neurol. 65, 976987.
  • Leissring M. A., Farris W., Chang A. Y., Walsh D. M., Wu X., Sun X., Frosch M. P. and Selkoe D. J. (2003) Enhanced proteolysis of beta-amyloid in APP transgenic mice prevents plaque formation, secondary pathology, and premature death. Neuron 40, 10871093.
  • Llovera R. E., De Tullio M., Alonso L. G., Leissring M. A., Kaufman S. B., Roher A. E., De Prat Gay G., Morelli L. and Castano E. M. (2008) The catalytic domain of insulin-degrading enzyme forms a denaturant-resistant complex with amyloid beta peptide: implications for Alzheimer disease pathogenesis. J. Biol. Chem. 283, 1703917048.
  • Marr R. A., Rockenstein E., Mukherjee A., Kindy M. S., Hersh L. B., Gage F. H., Verma I. M. and Masliah E. (2003) Neprilysin gene transfer reduces human amyloid pathology in transgenic mice. J. Neurosci. 23, 19921996.
  • Marr R. A., Guan H., Rockenstein E., Kindy M., Gage F. H., Verma I., Masliah E. and Hersh L. B. (2004) Neprilysin regulates amyloid Beta peptide levels. J. Mol. Neurosci. 22, 511.
  • Masliah E., Mallory M., Hansen L., DeTeresa R., Alford M. and Terry R. (1994) Synaptic and neuritic alterations during the progression of Alzheimer’s disease. Neurosci. Lett. 174, 6772.
  • Meilandt W. J., Cisse M., Ho K., Wu T., Esposito L. A., Scearce-Levie K., Cheng I. H., Yu G. Q. and Mucke L. (2009) Neprilysin overexpression inhibits plaque formation but fails to reduce pathogenic Abeta oligomers and associated cognitive deficits in human amyloid precursor protein transgenic mice. J. Neurosci. 29, 19771986.
  • Miller B. C., Eckman E. A., Sambamurti K., Dobbs N., Chow K. M., Eckman C. B., Hersh L. B. and Thiele D. L. (2003) Amyloid-beta peptide levels in brain are inversely correlated with insulysin activity levels in vivo. Proc. Natl Acad. Sci. USA 100, 62216226.
  • Miners J. S., Van Helmond Z., Chalmers K., Wilcock G., Love S. and Kehoe P. G. (2006) Decreased expression and activity of neprilysin in Alzheimer disease are associated with cerebral amyloid angiopathy. J. Neuropathol. Exp. Neurol. 65, 10121021.
  • Miners J. S., Kehoe P. G. and Love S. (2008a) Immunocapture-based fluorometric assay for the measurement of insulin-degrading enzyme activity in brain tissue homogenates. J. Neurosci. Methods 169, 177181.
  • Miners J. S., Verbeek M. M., Rikkert M. O., Kehoe P. G. and Love S. (2008b) Immunocapture-based fluorometric assay for the measurement of neprilysin-specific enzyme activity in brain tissue homogenates and cerebrospinal fluid. J. Neurosci. Methods 167, 229236.
  • Miners J. S., Baig S., Palmer J., Palmer L. E., Kehoe P. G. and Love S. (2008c) Abeta-degrading enzymes in Alzheimer’s disease. Brain Pathol. 18, 240252.
  • Miners J. S., Baig S., Tayler H., Kehoe P. G. and Love S. (2009) Neprilysin and insulin-degrading enzyme levels are increased in Alzheimer disease in relation to disease severity. J. Neuropathol. Exp. Neurol. 68, 902914.
  • Morishima-Kawashima M., Oshima N., Ogata H., Yamaguchi H., Yoshimura M., Sugihara S. and Ihara Y. (2000) Effect of apolipoprotein E allele epsilon4 on the initial phase of amyloid beta-protein accumulation in the human brain. Am. J. Pathol. 157, 20932099.
  • Perez A., Morelli L., Cresto J. C. and Castano E. M. (2000) 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. 25, 247255.
  • Qiu W. Q. and Folstein M. F. (2006) Insulin, insulin-degrading enzyme and amyloid-beta peptide in Alzheimer’s disease: review and hypothesis. Neurobiol. Aging 27, 190198.
  • Sato M., Ikeda K., Haga S., Allsop D. and Ishii T. (1991) A monoclonal antibody to common acute lymphoblastic leukemia antigen (neutral endopeptidase) immunostains senile plaques in the brains of patients with Alzheimer’s disease. Neurosci. Lett. 121, 271273.
  • Takaki Y., Iwata N., Tsubuki S. et al. (2000) Biochemical identification of the neutral endopeptidase family member responsible for the catabolism of amyloid beta peptide in the brain. J. Biochem. 128, 897902.
  • Vardy E. R., Catto A. J. and Hooper N. M. (2005) Proteolytic mechanisms in amyloid-beta metabolism: therapeutic implications for Alzheimer’s disease. Trends Mol. Med. 11, 464472.
  • Vepsalainen S., Hiltunen M., Helisalmi S., Wang J., Van Groen T., Tanila H. and Soininen H. (2008) Increased expression of Abeta degrading enzyme IDE in the cortex of transgenic mice with Alzheimer’s disease-like neuropathology. Neurosci. Lett. 438, 216220.
  • Wang D. S., Iwata N., Hama E., Saido T. C. and Dickson D. W. (2003) Oxidized neprilysin in aging and Alzheimer’s disease brains. Biochem. Biophys. Res. Commun. 310, 236241.
  • Wang D. S., Lipton R. B., Katz M. J., Davies P., Buschke H., Kuslansky G., Verghese J., Younkin S. G., Eckman C. and Dickson D. W. (2005) Decreased neprilysin immunoreactivity in Alzheimer disease, but not in pathological aging. J. Neuropathol. Exp. Neurol. 64, 378385.
  • Wang D. S., Dickson D. W. and Malter J. S. (2006) beta-Amyloid Degradation and Alzheimer’s Disease. J. Biomed. Biotechnol. 2006, 58406.
  • Wang S., Simon B. P., Bennett D. A., Schneider J. A., Malter J. S. and Wang D. S. (2007) The significance of Pin1 in the development of Alzheimer’s disease. J. Alzheimers Dis. 11, 1323.
  • Wang R., Wang S., Malter J. S. and Wang D. S. (2009a) Effects of 4-hydroxy-nonenal and Amyloid-beta on expression and activity of endothelin converting enzyme and insulin degrading enzyme in SH-SY5Y cells. J. Alzheimers Dis. 17, 489501.
  • Wang R., Wang S., Malter J. S. and Wang D. S. (2009b) Effects of HNE-modification induced by Abeta on neprilysin expression and activity in SH-SY5Y cells. J. Neurochem. 108, 10721082.
  • Weeraratna A. T., Kalehua A., Deleon I. et al. (2007) Alterations in immunological and neurological gene expression patterns in Alzheimer’s disease tissues. Exp. Cell Res. 313, 450461.
  • Wilson R. S., Beckett L. A., Barnes L. L., Schneider J. A., Bach J., Evans D. A. and Bennett D. A. (2002) Individual differences in rates of change in cognitive abilities of older persons. Psychol. Aging 17, 179193.
  • Yasojima K., McGeer E. G. and McGeer P. L. (2001a) Relationship between beta amyloid peptide generating molecules and neprilysin in Alzheimer disease and normal brain. Brain Res. 919, 115121.
  • Yasojima K., Akiyama H., McGeer E. G. and McGeer P. L. (2001b) Reduced neprilysin in high plaque areas of Alzheimer brain: a possible relationship to deficient degradation of beta-amyloid peptide. Neurosci. Lett. 297, 97100.
  • Yfanti C., Mengele K., Gkazepis A., Weirich G., Giersig C., Kuo W. L., Tang W. J., Rosner M. and Schmitt M. (2008) Expression of metalloprotease insulin-degrading enzyme insulysin in normal and malignant human tissues. Int. J. Mol. Med. 22, 421431.
  • Zhao Z., Xiang Z., Haroutunian V., Buxbaum J. D., Stetka B. and Pasinetti G. M. (2007) Insulin degrading enzyme activity selectively decreases in the hippocampal formation of cases at high risk to develop Alzheimer’s disease. Neurobiol. Aging 28, 824830.