Neprilysin-2 is an important β-amyloid degrading enzyme - PubMed (original) (raw)

Neprilysin-2 is an important β-amyloid degrading enzyme

Daniel Hafez et al. Am J Pathol. 2011 Jan.

Abstract

Proteases that degrade the amyloid-β peptide (Aβ) are important in protecting against Alzheimer's disease (AD), and understanding these proteases is critical to understanding AD pathology. Endopeptidases sensitive to inhibition by thiorphan and phosphoramidon are especially important, because these inhibitors induce dramatic Aβ accumulation (∼30- to 50-fold) and pathological deposition in rodents. The Aβ-degrading enzyme neprilysin (NEP) is the best known target of these inhibitors. However, genetic ablation of NEP results in only modest increases (∼1.5- to 2-fold) in Aβ, indicating that other thiorphan/phosphoramidon-sensitive endopeptidases are at work. Of particular interest is the NEP homolog neprilysin 2 (NEP2), which is thiorphan/phosphoramidon-sensitive and degrades Aβ. We investigated the role of NEP2 in Aβ degradation in vivo through the use of gene knockout and transgenic mice. Mice deficient for the NEP2 gene showed significant elevations in total Aβ species in the hippocampus and brainstem/diencephalon (∼1.5-fold). Increases in Aβ accumulation were more dramatic in NEP2 knockout mice crossbred with APP transgenic mice. In NEP/NEP2 double-knockout mice, Aβ levels were marginally increased (∼1.5- to 2-fold), compared with NEP(-/-)/NEP2(+/+) controls. Treatment of these double-knockout mice with phosphoramidon resulted in elevations of Aβ, suggesting that yet other NEP-like Aβ-degrading endopeptidases are contributing to Aβ catabolism.

Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

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Figures

Figure S1

Figure S1

Examples of genotyping assays for the NEP2/NEP knockout and APPtg mice. UV images of ethidium bromide-stained agarose electrophoresis gels showing DNA PCR products using specific primers (see Methods section) to amplify genomic template DNA extracted from mouse ear clippings.

Figure S2

Figure S2

Aβ levels in the cerebral cortex of APPtg mice in the presence and absence of the NEP2 gene. Relative levels of Aβ42 (A) and Aβ40 (B) measured by specific ELISA. Data are presented as the fold change compared with the average value from APPtg/NEP2+/+ control mice. Values are means ± SEM.

Figure S3

Figure S3

Analysis of intracellular Aβ accumulation in the presence and absence of the NEP2 gene. Representative fluorescent images of immunohistochemistry staining of Aβ from sagittal brain sections from APPtg/NEP2+/+ (A, B) and APPtg/NEP2−/− (C, D) mice from the hippocampus (A, C) and cerebral cortex (B, D). Quantitation of the % area staining positive for Aβ in the hippocampus (E) and cerebral cortex (F) from APPtg/NEP2+/+ (n = 4) and APPtg/NEP2−/− (n = 5) mice. Values are averages ± SEM.

Figure S4

Figure S4

Real-time PCR analysis of NEP2 expression on APP transgenic and non-transgenic mice. Coding DNA samples from mouse brain tissues (2 months old) were analyzed for NEP2 gene expression using the ΔΔCt-method (compared with cyclophilin expression) of quantitative real-time PCR. Data are presented as the mean of arbitrary expression units (resultant) ± SEM.

Figure 1

Figure 1

Amyloid β (Aβ) levels are increased in NEP2 knockout (KO) mice. Total Aβ42 (A) and Aβ40 (B) levels in the hippocampus of 10-month-old NEP2 wild-type (+/+) (n = 9) and NEP2 KO (−/−) (n = 7) mice. Relative levels of Aβ42 (C) and Aβ40 (D) are reported as a percentage of wild-type mice from hippocampus (Hipp), cerebral cortex (Ccx), brainstem/diencephalon (BsD), and cerebellum (Cb). Values are averages ± SEM. *P < 0.05, **P < 0.01.

Figure 2

Figure 2

Aβ40 levels are increased in the absence of NEP2 in APPtg mice. Total Aβ42 (A) and Aβ40 (B) levels in 10-month-old APPtg mice in the presence (+/+) (n = 4) and absence (−/−) (n = 5) of the NEP2 gene. Relative levels of Aβ42 (C) and Aβ40 (D) are reported as a percentage of APPtg/NEP2+/+ control mice from the hippocampus (Hipp), brainstem/diencephalon (BsD), and cerebellum (Cb). Values are averages ± SEM. **P < 0.01.

Figure 3

Figure 3

Aβ plaque deposition is increased in the absence of NEP2 in APPtg mice. Representative fluorescent images of immunohistochemistry staining of deposited Aβ (green) from sagittal brain sections from APPtg/NEP2+/+ mice (A) and APPtg/NEP2−/− mice (40×) (B). Arrowheads indicate examples of plaques. Cx, cerebral cortex; Hp, hippocampus. Quantitation of the % area staining positive for plaque deposition in the hippocampus (C) and cerebral cortex (D) from APPtg/NEP2+/+ mice (n = 4) and APPtg/NEP2−/− mice (n = 5). Values are averages ± SEM. *P < 0.05.

Figure 4

Figure 4

Murine NEP2α degrades Aβ42. Quantitative analysis of remaining Aβ42 in conditioned cell culture medium containing 50 pmol/L Aβ42 after incubation (5 hours) with HEK293T cells transfected with endopeptidase expressing plasmids (mNEP2α, hNEP) or control plasmid (green fluorescent protein, GFP) in the presence (+T) or absence of thiorphan (100 μmol/L) (n = 4). Values are averages ± SEM. **P < 0.01 compared with GFP.

Figure 5

Figure 5

Aβ levels are increased in the absence of NEP2 in NEP KO mice. Total Aβ42 (A) and Aβ40 (B) in the hippocampus of 10-month-old NEP KO mice homozygous (n = 8), heterozygous (n = 5), and deficient (n = 10) for the NEP2 gene. Relative levels of Aβ42 (C, E) and Aβ40 (D, F) from 10-month-old (C, D) and 14-month-old (E, F) NEP/NEP2 DKO mice reported as a percentage of control (NEP−/−/NEP2+/+) mice from hippocampus (Hipp), cerebral cortex (Ccx), brainstem/diencephalon (BsD), and cerebellum (Cb). For the 14-month group, n = 9 for NEP2+/+ and n = 7 for NEP2−/−. Values are averages ± SEM. *P < 0.05, **P < 0.01.

Figure 6

Figure 6

Increased Aβ levels after phosphoramidon administration in NEP/NEP2 DKO mice. Relative levels of Aβ42 (A) and Aβ40 (B) in DKO mice (10 months old, n = 3) infused with phosphoramidon (i.c.v., 10 mmol/L, 0.11 μL/hr). Data are presented as the percentage of control (saline) Aβ levels from the hippocampus (Hipp), cerebral cortex (Ccx), brainstem/diencephalon (BsD), and cerebellum (Cb). Values are means ± SEM. *P < 0.05, **P < 0.01.

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