Naomasa Kakiya, Takashi Saito, Per Nilsson,
Yukio Matsuba, Satoshi Tsubuki, Nobuyuki
Takei, Hiroyuki Nawa and Takaomi C. Saido
J. Biol. Chem. 2012, 287:29362-29372.
doi: 10.1074/jbc.M112.340372 originally published online July 5, 2012

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Metabolism:
Cell Surface Expression of the Major
Amyloid- β Peptide (Aβ)-degrading
Enzyme, Neprilysin, Depends on
Phosphorylation by Mitogen-activatedProtein Kinase/Extracellular
Signal-regulated Kinase Kinase (MEK) and
Dephosphorylation by Protein Phosphatase
1aTHE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 35, pp. 29362–29372, August 24, 2012
© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

Author’s Choice

Cell Surface Expression of the Major Amyloid-␤ Peptide(A␤)-degrading Enzyme, Neprilysin, Depends on
Phosphorylation by Mitogen-activated Protein
Kinase/Extracellular Signal-regulated Kinase Kinase (MEK)and Dephosphorylation by Protein Phosphatase 1a*□S

Received for publication, January 6, 2012, and in revised form, June 27, 2012 Published, JBC Papers in Press, July 5, 2012, DOI 10.1074/jbc.M112.340372

Background: Neprilysin is a major A␤-degrading enzyme, the expression of which is reduced in the AD brain.
Results: The phosphorylation status of the neprilysin intracellular domain regulates localization and cell surface activity.
Conclusion: Regulation of neprilysin through phosphorylation influences A␤ levels.
Significance: Our results indicate that neprilysin phosphorylation/dephosporylation could be one druggable target in thedevelopment of an AD-modifying treatment.
Neprilysin is one of the major amyloid-␤ peptide (A␤)-degrading enzymes, the expression of which declines in the brain
during aging. The decrease in neprilysin leads to a metabolic A␤
imbalance, which can induce the amyloidosis underlying
Alzheimer disease. Pharmacological activation of neprilysin
during aging therefore represents a potential strategy to preventthe development of Alzheimer disease. However, the regulatory
mechanisms mediating neprilysin activity in the brain remain
unclear. To address this issue, we screened for pharmacologicalregulators of neprilysin activity and found that the neurotrophic
factors brain-derived neurotrophic factor, nerve growth factor,
and neurotrophins 3 and 4 reduce cell surface neprilysin activity. This decrease was mediated by MEK/ERK signaling, which
enhanced phosphorylation at serine 6 in the neprilysin intracellular domain (S6-NEP-ICD). Increased phosphorylation of
S6-NEP-ICD in primary neurons reduced the levels of cell surface neprilysin and led to a subsequent increase in extracellularA␤ levels. Furthermore, a specific inhibitor of protein phosphatase-1a, tautomycetin, induced extensive phosphorylation of
the S6-NEP-ICD, resulting in reduced cell surface neprilysin
activity. In contrast, activation of protein phosphatase-1aincreased cell surface neprilysin activity and lowered A␤ levels.
Taken together, these results indicate that the phosphorylation
status of S6-NEP-ICD influences the localization of neprilysin
and affects extracellular A␤ levels. Therefore, maintaining
S6-NEP-ICD in a dephosphorylated state, either by inhibition ofprotein kinases involved in its phosphorylation or by activation* This study was supported by research grants from RIKEN Brain Science Institute; the Ministry of Education, Culture, Sports, Science, and Technology;
and the Ministry of Health, Labor, and Welfare of Japan. This study was also
supported by the Junior Research Associate Program of RIKEN.
Author’s Choice—Final version full access.
□S
This article contains supplemental Figs. S1–S6.
1
Both authors contributed equally to this work.
2
To whom correspondence should be addressed. Tel.: 81-48-467-9715; Fax:
81-48-467-9716; E-mail: saido@brain.riken.jp.

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of phosphatases catalyzing its dephosphorylation, may represent a new approach to prevent reduction of cell surface neprilysin activity during aging and to maintain physiological levelsof A␤ in the brain.

Impaired metabolism of amyloid ␤ peptide (A␤)3 in the brain
is likely to play a central role in the pathogenesis of Alzheimer
disease (AD) (1). Genetically, causative genes in familial AD linkmutations in amyloid precursor protein (APP) and presenilins
to aberrant increased generation of A␤42 and A␤43, the 42and 43-mer forms of A␤, respectively. A␤42 and A␤43 havehigher amyloidogenecity and neural toxicity than A␤40 (1– 4)
and lead to the early onset of AD. However, A␤ amyloidosis insporadic AD, which comprises over 90% of all AD cases, may becaused by a decline in A␤ degradation, A␤ clearance, or both(5). We previously identified neprilysin as a major physiologicalA␤-degrading enzyme that regulates the steady-state levels ofA␤ species in the brain (6, 7). Consistent with the increase in A␤levels observed during aging and in AD, the expression levels of
neprilysin in the brain decrease with age and in the early stagesof AD (8 –12). Genetic ablation of neprilysin not only markedly
accelerates amyloid plaque formation but also leads toincreased impairment of synaptic plasticity and memory formation in APP transgenic mice (13, 14). In contrast, elevation of
neprilysin expression/activity promotes A␤ degradation andreduces the accumulation of both soluble and fibrillary A␤ in
APP transgenic mice (15–17). Based on these observations, wepreviously searched for factors that could increase neprilysin
activity pharmacologically, identifying somatostatin as a nepri3

The abbreviations used are: A␤, amyloid ␤ peptide; AD, Alzheimer disease;
APP, amyloid precursor protein; BDNF, brain-derived neurotrophic factor;
CTF, C-terminal fragment of APP; ICD, intracellular domain; NEP, neprilysin;
hNEP, human NEP; NT, neurotrophin; PP1 and PP2, protein phosphatase-1
and -2, respectively; SFV, Semliki Forest virus; MCA, methyl cumaryl amide.

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Naomasa Kakiya‡§1, Takashi Saito‡1, Per Nilsson‡, Yukio Matsuba‡, Satoshi Tsubuki‡, Nobuyuki Takei§,
Hiroyuki Nawa§, and Takaomi C. Saido‡2
From the ‡Laboratory for Proteolytic Neuroscience, RIKEN Brain Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198,
Japan and the §Department of Molecular Neurobiology, Brain Research Institute, Niigata University, Niigata 951-8585, Japan

Phosphorylation Status of Neprilysin and A␤ Degradation
lysin up-regulator, although the underlining mechanisms
remain unclear (18).
In this study, we investigated the effect of the neurotrophic
factors brain-derived neurotrophic factor (BDNF), nerve
growth factor (NGF), and neurotrophins 3 and 4 (NT-3 and -4)
and found that they cause a reduction in cell surface neprilysin
activity through MAPK signaling. Moreover, our results revealthat cell surface neprilysin activity and localization are regulated by phosphorylation and dephosphorylation at the neprilysin intracellular domain, with a reduction in neprilysin activity leading to an increase in A␤ levels. We therefore speculatethat possible drug targets involving neprilysin could include
factors involved in the phosphorylation/dephosphorylation ofthis A␤-lowering enzyme.

AUGUST 24, 2012 • VOLUME 287 • NUMBER 35

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EXPERIMENTAL PROCEDURES
Materials—The reagents used in this study were purchased
as follows: BDNF, NGF, NT-3, NT-4, cyclosporin A, andFK-506 (Sigma); U0126 (Enzo Life Sciences); fostriecin, okadic
acid, and tautomycetin (Calbiochem); and recombinant humanneprilysin (R&D Systems). The phospho-human neprilysin
antibodies pS4-NEP, pS6-NEP, pT11-NEP, pT15-NEP, andpT25-NEP were obtained by immunizing rabbits with the following respective synthetic peptides containing phosphoserine(pS) or phosphothreonine (pT) residues: GKpSESQMC,
GKSEpSQC, QMDIpTDINTC, TDINpTPKPKC, and KQRWpTPLEC (19, 20). The specificities of the purified phosphoantibodies were investigated by dot blot analysis (21) (see supplemental Fig. S3).
Cell Culture—Human SH-SY5Y neuroblastoma cells wereobtained from the European Collection of Cell Cultures. Cells
were cultured in 5% CO2 at 37 °C, as previously described (22).
The medium comprised a 1:1 mixture of minimum essential
medium and Ham’s F-12 medium (Nacalai Tesque), supplemented with 1 ␮M non-essential amino acids, 100 units/ml penicillin, 100 mg/ml streptomycin, and 15% fetal bovine serum(Invitrogen). Primary cortical/hippocampal neurons derived
from wild-type or neprilysin-deficient mouse embryos wereisolated as described previously (18). The neurons were plated
at 5.0 ⫻ 104 cells/well in 96-well plates, 2.0 ⫻ 105 cells on glass
coverslips (Hamanami) placed in 24-well plates, or 1.0 ⫻ 106
cells/well in 6-well plates. After 14 days in vitro, primary neurons were subjected to the various assays.
Mutagenesis and Transfection—S4A-, S6A-, T11A-, T15Aand T25A-human neprilysin mutants were generated using a
KOD-Plus mutagenesis kit (Toyobo) according to the manufacturer’s protocol. To introduce the mutations into human neprilysin cDNA previously cloned into pcDNA3.1 (23), the following primer sets were designed: S4A, 5⬘-GCAGAAAGTCAGATGGATATA-3⬘ and 5⬘-CTTGCCCATCACCTAGGCTGC3⬘; S6A, 5⬘-GCTCAGATGGATATAACTGATATC-3⬘ and5⬘-TTCTGACTTGCCCATCACCTAGG-3⬘; T11A, 5⬘-GCTGATATCAACACTCCAAAGC-3⬘ and 5⬘-TATATCCATCTGACTTTCTG-3⬘; T15A, 5⬘-GCTCCAAAGCCAAAGAAGAAACAGC-3⬘ and 5⬘-GTTGATATCAGTTATATCCATCTG3⬘; and T25A, 5⬘-GGCTCCACTGGAGATCAGCCTCTCG-3⬘
and 5⬘-CATCGCTGTTTCTTCTTTGGCTTTG-3⬘ (underlined, original T, AG, A, A, and A, respectively). Constitutive

active protein phosphatase-1a (PP1a)-T320A (24) was generated using the KOD-Plus mutagenesis kit, using the following
primer set: 5⬘-GCCCCACCCCGCAATTCCGCCAAA-3⬘
and 5⬘-GATGGGTCGGCCTCCAGGGTTCAG-3⬘ (underlined, original A to G mutation). The mutant genes were transfected into SH-SY5Y cells using FuGENE transfection reagent(Roche Applied Science) according to the manufacturer’s
instructions. Cells were harvested with lysis buffer containing
0.1 M Tris-HCl, pH 7.5, EDTA-free complete protease inhibitor
mixture (Roche Applied Science), 1% Triton X-100, 0.15 M
NaCl, 1 mM Na3VO4, 1 mM NaF, and 1 ␮g/ml pepstatin A (Peptide Institute) 48 –72 h after transfection.
Cell Surface and Whole-cell Neprilysin Activity—Activity
staining of neprilysin using primary neurons was performed as
described previously (18), with slight modifications. Because
the endogenous neprilysin activity was too low in the primaryneurons and SH-SY5Y cells, the cells were infected with Semliki
Forest virus containing human neprilysin cDNA (SFV-hNEP),
as previously described (25). Twenty-four h postinfection at day
in vitro 14, neurotrophic factors or other reagents were added,
and the cells were incubated for 24 h. They were then fixed with
1.5% paraformaldehyde in 50 mM phosphate buffer (pH 6.8) for
5 min at room temperature. The fixed neurons were incubatedin substrate solution (0.25 mM glutaryl-Ala-Ala-Phe-methoxy2-naphthylamide in 50 mM Tris-HCl, pH 7.4) at 37 °C for 2 h.
Leucine aminopeptidase (Sigma), phosphoramidon (Peptide
Institute), and nitrosalicylaldehyde (Sigma-Aldrich) were thenadded to the substrate solution at a final concentration of 50
␮g/mg, 10 ␮M, and 0.6 mM, respectively, and incubated for 30
min at 37 °C. Quantification of the fluorescence signal arisingfrom cell surface neprilysin activity was performed as described
previously (18). Cell surface and whole-cell neprilysin activityof SH-SY5Y cells expressing mutant neprilysin were measured
as described previously (26), with slight modifications (supplemental Fig. S5). Before the addition of neurotrophic factors, thecells were starved for 48 h to eliminate the effect of serum. Aftera 24-h treatment with neurotrophic factors, cells or lysates wereincubated with substrate mixture (50 ␮M suc-Ala-Ala-PheMCA (Peptide Institute) and 10 nM benzyloxycarbonyl (Z)Leu-Leu-Leucinal in 50 mM MES, pH 6.5, with or without 10 ␮Mthiorphan (neprilysin-specific inhibitor)) at 37 °C for 30 min.
Following this, 0.1 mg/ml leucine aminopeptidase (Sigma) and
0.1 mM phosphoramidon were added, and the reaction mixture
was incubated at 37 °C for a further 30 min. 7-Amino-4-methylcoumarin fluorescence was measured at excitation and emission wavelengths of 380 and 460 nm, respectively. After measurement, cells were collected and subjected to Western blot
analysis to evaluate neprilysin levels.
Cell Surface Biotinylation—The cell membrane of cortical/
hippocampal neurons or SH-SY5Y cells was biotinylated withsulfo-NHS-SS-biotin (Pierce), according to the manufacturer’s
instructions. The samples were subsequently subjected toimmunocytochemical study or pull-down assay. Biotinylated
cell surface proteins were pulled down using Biotin-Capture
beads (Adar Biotech).
Immunocytochemical Study—To visualize and quantify
neprilysin localization in cortical/hippocampal neurons, thecells were infected with SFV-hNEP, and the cell surface wasPhosphorylation Status of Neprilysin and A␤ Degradation

labeled with biotin. The cells grown on coverslips were fixedwith 100% ice-cold MeOH for 10 min at ⫺20 °C and permeabilized in 100% ice-cold acetone for 1 min at ⫺20 °C. After blocking with blocking buffer (phosphate-buffered saline containing
5% skim milk, 5% goat serum, and 0.05% Tween 20) for 30 min
at room temperature, the samples were incubated with primaryanti-human neprilysin antibody (1:100, Novocastra) in blocking buffer for 1 h at room temperature, followed by secondaryanti-mouse Alexa 488 (1:500, Invitrogen) and Streptavidin-Alexa 546 (1:500; Molecular Probes) antibody for 30 min at room
temperature. The fluorescence signals observed by confocal
microscopy were quantified by counting signal dots, as
described previously (27).
Immunoprecipitation and Western Blot Analysis—Celllysates from primary cortical/hippocampal neurons infectedwith SFV-hNEP were immunoprecipitated with mouse monoclonal anti-human neprilysin (SN5c/L4 –1A1, Ancell). Samples
were subjected to Western blot analysis using the following
antibodies: phospho-human neprilysin antibodies (supplemen-

29364 JOURNAL OF BIOLOGICAL CHEMISTRY

tal Fig. S3), anti-human neprilysin (56C6, Novocastra), antimouse neprilysin (421126, Techne), antibodies recognizing the
N-terminal region of APP (22C11, Chemicon) or the C-terminal region of APP (A8717, Sigma), anti-PP1A (Thr(P)-320)(EP1512Y, Novus), anti-PP1␣ (Santa Cruz Biotechnology, Inc.,
Santa Cruz, CA), anti-phospho-TrkA (Tyr-490, Cell Signaling),
anti-Trk (B-3, Cell Signaling), anti-phospho-Erk1/2 (Thr-202/
Tyr-204, Cell Signaling), anti-Erk1/2 (Cell Signaling), anti-Myc(9B11, Cell Signaling), anti-G3PDH/GAPDH (Trevigen), or
anti-␤-actin (AC-15, Sigma-Aldrich).
A␤ ELISA—Conditioned medium from control cortical/hippocampal neurons or those treated with neurotrophic factors
for 24 h and from SH-SY5Y cells transiently expressing wildtype neprilysin (WT-NEP), S6A-NEP, WT-PP1a, and T320APP1a were collected, and guanidine HCl was added as described
previously (4). In order to achieve a measurable level of A␤40 in
the conditioned medium from SH-SY5Y cells transfected with
WT-NEP or S6A-NEP, 200 pM A␤40 peptide was added to thesamples. Samples were analyzed using A␤-ELISA kits (Wako)
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FIGURE 1. Neurotrophic factors reduce cell surface neprilysin activity in primary cortical/hippocampal neurons. A, primary cortical/hippocampal neurons infected with SFV-hNEP were incubated with BDNF (100 ng/ml), NGF (100 ng/ml), NT-3 (100 ng/ml), or NT-4 (100 ng/ml) for 24 h, after which they were
subjected to the neprilysin activity-staining assay. The top panels show fluorescence images representing neprilysin activity, and the bottom panels showphase-contrast images merged with the top panels. Scale bar, 100 ␮m. B, quantification of the fluorescence signal areas, indicated as average ⫾ S.D. (error bars)(n ⫽ 5). ***, p ⬍ 0.01 compared with control. C, primary neurons were incubated with BDNF (100 ng/ml), NGF (100 ng/ml), NT-3 (100 ng/ml), or NT-4 (100 ng/ml)
for 30 min and then subjected to Western blot analysis to measure the phosphorylation level of the neurotrophic factor receptor, Trk, using antibodies against
Trk receptor (bottom) and phosphorylated Trk (p-Trk; top). At least three independent experiments were repeated.

Phosphorylation Status of Neprilysin and A␤ Degradation
to quantify A␤40 and A␤42, according to the manufacturer’s
instructions.
Statistical Analysis—All of the values are presented asmean ⫾ S.D. Statistical analysis was performed by one-way
analysis of variance with Scheffe’s F test.

RESULTS

AUGUST 24, 2012 • VOLUME 287 • NUMBER 35

FIGURE 2. Reduction of neprilysin activity by neurotrophic factors
involves the MEK1/2 signaling pathway. A, primary cortical/hippocampal
neurons infected with SFV-hNEP were co-incubated with BDNF (100 ng/ml),
NGF (100 ng/ml), NT-3 (100 ng/ml), or NT-4 (100 ng/ml) and the MEK1/2 inhibitor U0126 (1 ␮M), for 24 h, after which they were subjected to the neprilysin
activity-staining assay. Representative fluorescence images merged withphase-contrast images are shown. Scale bar, 100 ␮m. B, quantification of thefluorescence signal derived from neprilysin activity was performed, and theresults are presented as average ⫾ S.D. (error bars) (n ⫽ 5). ***, p ⬍ 0.01
compared with control. C, primary neurons were incubated with or without
U0126, as indicated, and neurotrophic factors for 30 min and were then subjected to Western blot analysis to measure the phosphorylation levels of
MAPK using antibodies against Erk1/2 (bottom) and phosphorylated Erk1/2(p-Erk1/2; top). At least three independent experiments were repeated.

tered 30 min after the addition of the neurotrophic factors (supplemental Fig. S1E). Rather, the effect became clearly visible
24 h poststimulation (Fig. 1, A and B). Taken together, thesefindings indicate that neurotrophic factors reduce cell surface
neprilysin activity by binding to neuronal Trk receptors andsubsequent activation of the MAPK pathway. The mechanistic
explanation behind the delayed effect is currently not known
but could include changes in, for example, gene expression of
additionally required factors.
Effect of NT-3 on A␤ Levels—A reduction in neprilysin
induced by neurotrophic factors would presumably lead toincreased A␤ levels. We therefore measured A␤ levels in conditioned medium from primary neurons infected with SFVhNEP, using the same conditions as for the activity assay. HowJOURNAL OF BIOLOGICAL CHEMISTRY

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Neurotrophic Factors Reduce Neprilysin Activity via MAPK
Signal Transduction—Membrane-bound neprilysin located on
the cell surface participates in extracellular A␤ degradation andtherefore plays a key role in A␤ metabolism. We have previously established an activity-staining method for primary neurons that visualizes neprilysin activity on the cell surface (18,25). In a search for regulators of neprilysin activity, we identified somatostatin, which was found to exert an up-regulatory
effect (18). To further unravel the mechanisms regulating
neprilysin activity, we screened the neurotrophic factors
BDNF, NGF, NT-3, and NT-4 using our activity-staining technique on primary cortical/hippocampal neurons. Representative images of such activity-staining experiments are shown in
Fig. 1. Interestingly, BDNF, NT-3, and NT-4 all significantly
reduced neprilysin activity, whereas NGF had no effect. However, given that cortical/hippocampal neurons do not express
the TrkA receptor subtype to which NGF binds (28), we also
examined the effect of NGF on primary neurons derived fromthe striatum. Indeed, exposing striatal primary neurons to NGF
induced a reduction in neprilysin activity (supplemental Fig. S1,
A and B). To confirm activation of the signal transduction pathway leading to a reduction in neprilysin, phosphorylation of
Tyr-490 in Trk was measured (29). Upon exposure of the primary neurons to BDNF, NGF, NT-3, or NT-4, phosphorylationof Trk significantly increased (Fig. 1C and supplemental Fig.
S1C), indicating that the Trk receptors were activated.
To further elucidate the Trk receptor-induced transduction
pathway, we next investigated the effects of U0126, LY294002,
and calphostin C, inhibitors of MAPK/ERK kinase 1/2 (MEK1/2), phosphatidylinositol-3 kinase, and protein kinase C, respectively, on cell surface neprilysin activity (30). First, we
co-treated neurons with BDNF, NGF, NT-3, or NT-4 together
with U0126 and found that U0126 completely inhibited the
reduction in cell surface neprilysin activity induced by the neurotrophic factors (Fig. 2), indicating the involvement of MAPK/ERK kinase in the signaling pathway. We then focused on NT-3
because it binds to all Trk receptor subtypes (i.e. TrkA, -B, and-C) (28). Simultaneous treatment of neurons with NT-3 andcalphostin C did not inhibit the reduction in neprilysin activity.
Finally, LY294002 treatment alone produced a decrease in cellsurface neprilysin activity. However, because LY294002 incombination with NT-3 did not inhibit the NT-3-induced
reduction in neprilysin activity (supplemental Fig. S1D), it ispossible that the effect of LY294002 involves a pathway independent of the Trk signaling pathway. Thus, together theseresults indicate that neurotrophic factors reduce cell surface
neprilysin activity in neurons via the MEK/ERK signaling
pathway.
Although binding of neurotrophic factors to Trk receptors is
known to activate the MAPK pathway within 30 min (31) (Fig.
1C), the levels of cell surface neprilysin activity remained unal-Phosphorylation Status of Neprilysin and A␤ Degradation

ever, due to the high viral expression of neprilysin, the A␤ levels
were too low to be detected. Therefore, we instead measured
the conditioned medium from non-infected primary cortical/
hippocampal neurons. NT-3 treatment significantly increased
the steady-state levels of A␤40 and A␤42, an effect that was
abolished by the addition of U0126 (Fig. 3, A and B). Importantly, treating primary neurons derived from neprilysin-deficient mice with NT-3 did not affect A␤ levels, showing that theincreased A␤ levels generated from wild-type neurons are due
specifically to a decrease in neprilysin activity (Fig. 3, C and D).
Furthermore, full-length APP levels and the levels of the C-terminal fragments of APP, CTF␣, CTF␤, and APP intracellular

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domain, which are produced by ␣-, ␤- and ␥-secretase, respectively, and used as an index of secretase activity (32) were notaltered by NT-3 treatment (Fig. 3, E and F). These results
together suggest that the increased steady-state levels of A␤
upon NT-3 treatment are due specifically to a decrease in cellsurface neprilysin activity rather than increased A␤ generationin the neurons. Finally, to ascertain that the increased A␤ levels
observed upon NT-3 treatment were specifically due todecreased levels of cell surface neprilysin, the levels of theA␤-degrading enzymes endothelin-converting enzyme-1 (33)and insulin-degrading enzyme (34) were investigated. Neither
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FIGURE 3. NT-3 induces increased A␤ levels via the MEK/ERK pathway. A–D, the culture medium of primary neurons derived from wild-type (A and B) or
NEP-deficient mouse embryos (C and D) was collected 24 h after NT-3 (100 ng/ml) treatment with or without U0126 (1 ␮M) and then subjected to A␤ ELISA.
A␤40 (A and C) and A␤42 (B and D) levels were measured using an A␤-ELISA kit. Each column with error bar represents the mean ⫾ S.D. (n ⫽ 4). *, p ⬍ 0.05
compared with control and co-treatment. E and F, the effect of neurotrophic factors on A␤ generation was investigated by measuring the levels of full-length
APP (fl-APP), CTF␣, CTF␤, APP intracellular domain (AICD), and ␤-actin in the primary neurons by Western blot. Intensities of each band were quantified bydensitometric analysis, and data represent the mean ⫾ S.D. (n ⫽ 5).

Phosphorylation Status of Neprilysin and A␤ Degradation

degrading enzyme nor the activity of insulin-degrading enzyme
were affected by NT-3 treatment (supplemental Fig. S2, A and
B). These data, together with the lack of changes in A␤ levels
secreted from primary neurons prepared from neprilysin
knock-out mice and stimulated with neurotrophic factors, lead
us to conclude that decreased levels in cell surface neprilysin
expression are responsible for the increased A␤ levels.
NT-3 Alters Neprilysin Localization in Neurons—To address
whether the NT-3-induced reduction in neprilysin was due tochanges in neprilysin expression, we quantified neprilysin levels in whole-cell lysates before and after NT-3 treatment. Western blot data clearly showed that neprilysin expression levels
were not altered after NT-3 treatment (Fig. 4, A and B). Therefore, we next performed immunocytochemical experiments tovisualize neprilysin in the neurons. Proteins located on the cellsurface were biotinylated, after which the cells were immunocytochemically stained with avidin and anti-neprilysin antibody and analyzed as described under “Experimental Procedures.” Quantitative image analysis revealed that the level ofneprilysin located on the cell surface was significantly reduced
upon NT-3 treatment (Fig. 4, C and D). Importantly, the addition of U0126 restored this level to normal. Taken together,
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FIGURE 4. NT-3 regulates neprilysin localization via the MEK/ERK pathway. A and B, the effect of NT-3 on expression levels of neprilysin in primaryneurons was analyzed by Western blot analysis. The experiments wererepeated four times, and the results are presented as mean values ⫾ S.D.
(error bars). C and D, double staining for neprilysin and biotinylated proteinslocated on the cell surface. Primary cortical neurons infected with SFV-hNEP
were treated with NT-3 (100 ng/ml) for 24 h. The cell surface proteins were
subsequently cross-linked with biotin, after which the cells were doublestained with neprilysin antibody (56C6; green) and Alexa 546-conjugatedstreptavidin (red). The images of the green and the red signals were merged,
yellow representing cell surface neprilysin. Scale bar, 50 ␮m. The ratio of cellsurface neprilysin levels was quantified by image analysis. Data represent themean ⫾ S.D. (n ⫽ 15). *, p ⬍ 0.05 compared with control and co-treatment.

these results indicate that neurotrophic factors modulate
neprilysin localization through MEK/ERK signaling, without
affecting neprilysin expression levels, leading to a decrease incell surface neprilysin activity and ultimately resulting inincreased extracellular A␤ levels.
MEK/ERK Signaling Modulates the Phosphorylation State of
the Neprilysin Intracellular Domain—The localization ofplasma membrane-associated proteins is often regulated byphosphorylation/dephosphorylation at the intracellular domain (ICD) of the protein (35). Because neprilysin is a type IImembrane protein, its N-terminal side is located in the cytoplasmic lumen. As shown in Fig. 5A, there are five potential
amino acid residues in human neprilysin and four in mouseneprilysin ICD (NEP-ICD) that could possibly be phosphorylated. In order to accurately determine the phosphorylation
state of the NEP-ICD, we first generated the phosphoserineand phosphothreonine-specific antibodies pS4-NEP, pS6-NEP,
pT11-NEP, pT15-NEP, and pT25-NEP, which specifically recognize individual phosphorylated threonine and serine residues in the NEP-ICD, respectively. The specificities of these
antibodies are summarized in supplemental Fig. S3. We thenprobed cell extracts of primary neurons using this battery ofphospho-antibodies. Although the endogenous expression
level of neprilysin is low, we were able to detect phosphorylation of Ser-4, Ser-6, and Thr-11 in neprilysin (Fig. 5). We were
not able to detect pT25-NEP due to limited affinity of the antibody. Interestingly, upon exposing primary neurons to NT-3,
phosphorylation of Ser-6 increases significantly. Simultaneously treating the cells with NT-3 and U0126 inhibited theincrease in phosphorylation of Ser-6 induced by NT-3, implying that the MAPK pathway is involved in the regulation of
NEP-ICD phosphorylation (Fig. 5, B and C).
Because the endogenous neprilysin level in primary neurons
is low and our aim was to study the regulatory mechanism of
human neprilysin, we continued our studies using SFV-hNEPinfected primary neurons. After infection, we investigated thephosphorylation state of NEP-ICD. Consistent with phosphorylation of Ser-6 of endogenous neprilysin, we detected thatoverexpressed human neprilysin also was phosphorylated at
Ser-6 upon treatment with NT-3. Further, the effect was
inhibited by the addition of U0126. In addition, a trend
toward increased Ser-4 phosphorylation was detected
together with increased phosphorylation of Thr-15 (Fig. 5, B
and C). Subsequently, we examined whether neprilysin
localized to the cell surface exhibits different phosphorylation statuses compared with intracellularly located neprilysin. Biotinylated proteins located on the cell surface of SFVhNEP-infected primary neurons were first pulled down fromcell lysates using Biotin-Capture beads, after which intracellular neprilysin was collected using a neprilysin-specificantibody. Although all potential phosphorylation sites in the
NEP-ICD of intracellularly located neprilysin were phosphorylated, no phosphorylation of Ser-6 or Thr-25 could be
detected in neprilysin present in the cell surface fraction(Fig. 5D). These results together indicated that treating thecells with NT-3 for 24 h resulted in extensive phosphorylation of Ser-6 and Thr-15 of cell surface-located neprilysin.
Although activation of Trk receptors was detected 30 min

Phosphorylation Status of Neprilysin and A␤ Degradation
after the addition of NT-3 to the cells, there was no difference in the phosphorylation state of NEP-ICD after a 30-min
treatment by NT-3 (data not shown). The reasons behind thedelayed effect are currently not known. Furthermore, theincrease in phosphorylation of Ser-4, Ser-6, and Thr-15
induced by NT-3 was inhibited by the addition of U0126,

indicating the involvement of MAPK in the signaling pathway (Fig. 5, B and C). Together, the data indicate that phosphorylation of Ser-6 is involved in the regulation of cell surface neprilysin.
To further clarify the role of human NEP-ICD phosphorylation in neprilysin activity, we generated five mutants in which

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Phosphorylation Status of Neprilysin and A␤ Degradation
treatment with the PP1a inhibitor tautomycetin resulted in significantly increased phosphorylation of the S6-NEP-ICD (Fig.
6, A and B) and decreased cell surface neprilysin activity (Fig.
6C). In addition to S6-NEP-ICD, phosphorylation of S4- andT11-NEP-ICDs was also increased by tautomycetin. However,phosphorylation of Ser-4 and Thr-11 did not influence cell surface neprilysin activity (Fig. 5 and supplemental Fig. S6).
The increased phosphorylation of Ser-6 upon PP1a inhibition suggests an association of neprilysin and PP1a. Previously,
PTEN (phosphatase and tensin homolog deleted from chromosome 10), which is a tumor suppressor and acts as a tyrosine
phosphatase, has been shown to be associated with the NEPICD (36). Interestingly, an amino acid sequence found in the
NEP-ICD, KKKQRW, is similar to a PP1a-interacting sequence(37, 38). This finding prompted us to investigate a possible
interaction between neprilysin and PP1a. We therefore performed immunoprecipitation experiments using SH-SY5Y celllysates from mock and WT-NEP transfectants. The results
revealed that neprilysin co-immunoprecipitates not only withPTEN but also with PP1a, indicating that the proteins are
directly or indirectly associated (Fig. 6D).
To confirm the role of PP1a in neprilysin dephosphorylationand localization, and because PP1a is activated by dephosphorylation at the threonine 320 residue, we prepared a constitutive
active mutant PP1a, which harbors a T320A mutation (Fig. 6E)(24). Expression of both WT-PP1a and T320A-PP1a in SFVhNEP-infected SH-SY5Y cells induced an increase in cell surface neprilysin activity compared with mock transfectants.
Moreover, constitutive active T320A-PP1a transfectants
exhibited significantly higher cell surface neprilysin activity
compared with WT-PP1a transfectants (Fig. 6F).
In addition, extracellular A␤ levels were significantly reduced byboth WT-PP1a and T320A-PP1a expression (Fig. 6G). Taken
together, these results suggest that activation of PP1a increases cellsurface neprilysin activity/localization is due, at least partly, todephosphorylation of Ser-6 in the NEP-ICD.

DISCUSSION
Pharmacological maintenance or enhancement of the majorA␤-degrading enzyme neprilysin during aging could offer apossible treatment for the prevention of AD. Accumulating evi-

FIGURE 5. Dephosphorylation of the neprilysin intracellular domain localizes neprilysin to the cell surface. A, scheme of human and mouse neprilysin
domain structure and the N-terminal amino acid sequences corresponding to the neprilysin intracellular domain. ICD and TM, intracellular domain andtransmembrane domain of neprilysin, respectively. Potential phosphorylation sites are indicated in boldface type. Below, the substituted serines/threonines toalanines are indicated in the five phosphoneprilysin mutants, S4A-, S6A-, T11A-, T15A-, and T25A-NEP. B, effects of NT-3 on the phosphorylation state of theintracellular domain of neprilysin. Primary neurons (left) were untreated or treated with NT-3 (100 ng/ml) or NT-3 together with U0126 for 24 h, as indicated,
after which neprilysin was immunoprecipitated from 1 mg of cell lysate with neprilysin antibody, and the immunoprecipitates were analyzed by immunoblotting with phosphoneprilysin antibodies. Primary neurons expressing human neprilysin by infection with SFV-hNEP (right) were treated the same way as the
non-infected primary neurons, as indicated. 150 ␮g of cell lysate was immunoprecipitated and immunoblotted with phosphoneprilysin antibodies, as indicated to the right. N/A, not applicable; N.D, not determined. C, densitometric quantification of Western blot data in B. Data represent the mean ⫾ S.D. (error bars)(n ⫽ 3). *, p ⬍ 0.05, treated sample compared with non-treated or co-treated. D, cell surface-located proteins were biotinylated and pulled down using
streptavidin beads, followed by immunoprecipitation (IP) of non-biotinylated neprilysin using a neprilysin antibody. Immunoprecipitates were subjected toWestern blot analysis using the phosphoneprilysin antibodies pS4-NEP, pS6-NEP, pT11-NEP, pT15-NEP, pT25-NEP, and NEP (56C6). Western blot data ofsamples from two independent experiments are shown. At least three independent experiments were performed, and a similar band pattern was repeatedly
verified. E, cell surface neprilysin activity of SH-SY5Y cells transiently expressing WT-, S4A-, S6A-, T11A-, T15A-, or T25A-NEP were evaluated using suc-Ala-AlaPhe-MCA substrate. The neprilysin activities were normalized to neprilysin expression levels quantified by Western blot. Data represent the mean ⫾ S.D. (n ⫽3). **, p ⬍ 0.01 compared with WT-NEP. F, SH-SY5Y cells transiently expressing WT-NEP or S6A-NEP were biotinylated, and the amount of biotinylated neprilysin
was evaluated by a pull-down assay. Data represent the mean ⫾ S.D. (n ⫽ 3). ***, p ⬍ 0.01 compared with WT-NEP. G, A␤ levels in the conditioned medium from
SH-SY5Y cells transiently expressing WT-NEP or S6A-NEP. Data represent the mean ⫾ S.D. (n ⫽ 4). **, p ⬍ 0.01 compared with WT-NEP. H, neprilysin activity inwhole-cell lysates from WT-NEP and S6A-NEP transfectants was measured. Data represent the mean ⫾ S.D. (n ⫽ 3). I, the effect of NT-3 on cell surface S6A-NEP
activity. SH-SY5Y cells transiently expressing S6A-NEP were treated with NT-3 for 24 h. Cell surface neprilysin activity was measured using suc-Ala-Ala-Phe-MCA
substrate. Data represent the mean ⫾ S.D. (n ⫽ 3).

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the putative serines/threonines in the NEP-ICD were substituted with alanines (i.e. S4A-, S6A-, T11A-, T15A-, and T25ANEP, respectively) (Fig. 5A). The selection of a cell line for the
mutational analysis was based on two criteria: 1) it should be ahuman neuronal cell line, and 2) it should express endogenous
neprilysin to assure that the regulatory mechanisms of neprilysin are preserved. The endogenous expression of neprilysin in
SH-SY5Y was confirmed with Western blot (Fig. S5A). Thus,
the mutants were introduced into SH-SY5Y cells, and cell surface neprilysin activity was measured and normalized toexpression levels. Interestingly, S6A-NEP transfectants showedsignificantly higher cell surface activity than S4A-, T11A-,
T15A-, and WT-NEP transfectants (Fig. 5E and supplemental
Fig. S4). The effect of T25A-NEP on neprilysin activity could
not be evaluated due to its low expression (supplemental Fig.
S5). We also subsequently measured the amount of cell surfacelocated S6A-NEP and found it to be significantly higher thanthat of WT-NEP (Fig. 5F). In accordance with the increased
cell surface activity of S6A-NEP, the extracellular A␤40 levelin conditioned medium from S6A-NEP transfectants was
significantly lower than that for WT-NEP transfectants (Fig.
5G). Furthermore, the total neprilysin activity, normalized
to expression levels, in whole-cell lysates from WT-NEP andS6A-NEP transfectants were similar (Fig. 5H). This indicatesthat the increase in cell surface neprilysin activity observedin the case of S6A-NEP was not due to an allosteric effectinduced by the mutation. Last, the cell surface neprilysin
activity of S6A-NEP transfectants was not affected by NT-3
treatment (Fig. 5I). Taking all of the data together, we conclude that phosphorylation of Ser-6 of the NEP-ICD regulates neprilysin localization and thereby neprilysin cell surface activity via MEK/ERK signaling.
PP1a Facilitates Cell Surface Neprilysin Activity—Given that
phosphorylation of Ser-6 in the NEP-ICD influences neprilysin
cell surface localization, we next evaluated the effects of different phosphatase activities on neprilysin localization and activity. Primary cortical/hippocampal neurons infected with SFVhNEP were treated with the following specific inhibitors forserine/threonine phosphatases: tautomycetin (PP1a inhibitor),
fostriecin and okadaic acid (PP2A inhibitors), and cyclosporin
A and FK-506 (PP2B/calcineurin inhibitors). Interestingly,

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FIGURE 6. PP1a regulates neprilysin activity through dephosphorylation. A, primary neurons infected with SFV-hNEP were treated with specific inhibitorsof serine/threonine phosphatases, tautomycetin (1 ␮M), fostriecin (100 nM), okadaic acid (10 nM), cyclosporin A (1 ␮M), and FK-506 (1 ␮M), and the phosphorylation state of neprilysin was measured by immunoprecipitation using a neprilysin antibody and immunoblot with phosphospecific antibodies. B, densitometric quantification of phosphorylation levels after tautomycetin treatment for 24 h as analyzed by Western blot in A. Data represent the mean ⫾ S.D. (errorbars) (n ⫽ 3). *, p ⬍ 0.05; **, p ⬍ 0.01 compared with non-treated. C, the effect of tautomycetin on cell surface neprilysin activity in primary neurons infected with
SFV-hNEP. Data represent the mean ⫾ S.D. (n ⫽ 3). **, p ⬍ 0.01 compared with control. D, immunoprecipitation (IP) of SH-SY5Y cell lysates with PTEN and PP1aantibodies. SH-SY5Y cells were transfected with a mock or WT-NEP expression vector. Lysates were prepared and immunoprecipitated using PTEN and PP1aantibodies as indicated. The immunoprecipitates were subjected to Western blot analysis, using anti-neprilysin antibody for detection. E, verification of WTand T320A-PP1a expression in SH-SY5Y transfectants by Western blot and using antibodies as indicated to the right. F, the effect of PP1a activation on cellsurface neprilysin activity. SH-SY5Y cells infected with SFV-hNEP were transfected with WT- or T320A-PP1a followed by measurement of cell surface neprilysin
activity. Data represent the mean ⫾ S.D. (n ⫽ 4). *, p ⬍ 0.05; **, p ⬍ 0.01 compared with control. G, effect of PP1a activation on A␤ levels. The A␤ levels in
conditioned medium from WT- and T320A-PP1a-transfected SH-SY5Y cells, infected with SFV-hNEP, were quantified by ELISA. Data represent the mean ⫾ S.D.
(n ⫽ 4). **, p ⬍ 0.01 compared with control.

dence indicates the feasibility of a neprilysin-based approach.
For example, overexpression of neprilysin leads to a decrease inA␤ load and improved memory in AD model mice. Anotherfactor that argues in favor of a neprilysin-based treatment is thelimited number of possible side effects. For example, the levels

29370 JOURNAL OF BIOLOGICAL CHEMISTRY

of neuropeptides in neprilysin knock-out mice are unaltered,indicating that proteases other than neprilysin are involved intheir metabolism. Furthermore, neprilysin transgenic mice do
not display other behavioral abnormalities (15–17, 39). However, in order to develop a neprilysin-based AD treatment, it is
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AUGUST 24, 2012 • VOLUME 287 • NUMBER 35

of the NEP-ICD amino acid sequence revealed a peptide
sequence, KKKQRW, similar to the PP1a binding motif, suggesting that the NEP-ICD might be a target of PP1a. Indeed,
PP1a was found to be associated with neprilysin in SH-SY5Y
cell extracts, and overexpression of PP1a stimulated cell surface
neprilysin activity, leading to reduced A␤ levels (Fig. 6, D–G).
Therefore, activation of PP1a (e.g. pharmacologically) would bea possible strategy to increase cell surface neprilysin activity.
Interestingly, it has been reported that PP1a activity was regulated by DARPP32, an endogenous regulator of PP1a (45), andthat DARPP32 levels are reduced in the brains of somatostatin
receptor-1 (SSTR1) and SSTR5 double knock-out mice (46).
These observations, considered together with our previous data
showing that presynaptic localization of neprilysin is significantly decreased in the hippocampus of somatostatin-deficient
mice (18), indicate that somatostatin possibly regulates neprilysin activity through PP1 activity. Thus, SSTR agonists might
be successful drug candidates for the treatment of AD.
With the development of new gene therapy technologies,
application of a neprilysin gene transfer strategy could be apotential way to decrease A␤ levels and delay the onset of amyloidosis. Considering the increased activity of S6-NEP, an alternative would be to introduce the S6A-NEP mutant, possibly
leading to even more efficient A␤ degradation compared withthat of WT-NEP.
In conclusion, our results show that the phosphorylation
state of the intracellular domain regulates cell surface neprilysin activity and extracellular A␤ levels due to the modulation of
neprilysin localization. We propose that enhancing cell surface
neprilysin activity, for example by maintaining S6 of the NEPICD in a dephosphorylated state, either through activation ofphosphatases or through inhibition of the kinase responsible
for phosphorylation at the S6-NEP-ICD, would be beneficial forthe prevention of A␤ amyloidosis.
Acknowledgment—We thank Dr. C. Gerard for providing neprilysindeficient mice.

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essential to have a more detailed understanding of the mechanism of neprilysin activation and to identify a regulator of
neprilysin activity.
In this report, we show that neurotrophic factors reduce cellsurface neprilysin activity (Fig. 1 and supplemental Fig. S1)
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and that the reduction of neprilysin activity leads to increasedA␤ levels (Fig. 3). On the other hand, previous extensive studies
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neprilysin located at the cell surface (Fig. 5D). Alternatively, theintroduced mutation could destabilize the protein and induce
aggregation, although the S6A/T25A double mutant was normally expressed in the transfectants (data not shown).
The addition of the PP1a inhibitor tautomycetin to primarycortical/hippocampal neurons extensively increased the phosphorylation state of the NEP-ICD (Fig. 6, A and B) and reduced
cell surface neprilysin activity (Fig. 6C). A closer examination

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