© 2005 Nature Publishing Group http://www.nature.com/naturemedicine

LETTERS

Somatostatin regulates brain amyloid β peptide Aβ42
through modulation of proteolytic degradation
Takashi Saito1,2, Nobuhisa Iwata1,2, Satoshi Tsubuki1, Yoshie Takaki1, Jiro Takano1, Shu-Ming Huang1,
Takahiro Suemoto1, Makoto Higuchi1 & Takaomi C Saido1
Expression of somatostatin in the brain declines during
aging in various mammals including apes and humans1,2.
A prominent decrease in this neuropeptide also representsa pathological characteristic of Alzheimer disease3,4. Usingin vitro and in vivo paradigms, we show that somatostatin
regulates the metabolism of amyloid β peptide (Aβ), theprimary pathogenic agent of Alzheimer disease, in thebrain through modulating proteolytic degradation catalyzed
by neprilysin. Among various effector candidates, only
somatostatin upregulated neprilysin activity in primarycortical neurons. A genetic deficiency of somatostatinaltered hippocampal neprilysin activity and localization, andincreased the quantity of a hydrophobic 42-mer form of Aβ,Aβ42, in a manner similar to presenilin gene mutations thatcause familial Alzheimer disease. These results indicate thatthe aging-induced downregulation of somatostatin expression
may be a trigger for Aβ accumulation leading to late-onset
sporadic Alzheimer disease, and suggest that somatostatin
receptors may be pharmacological-target candidates forprevention and treatment of Alzheimer disease.
An abnormally high level of Aβ in brain seems to have a central rolein the pathogenesis of Alzheimer disease5. In particular, analyses ofseveral familial Alzheimer disease–causing mutations in genes encoding
amyloid precursor protein (APP) and presenilin have established theimportance of generation of Aβ42, a 42-mer form of the peptide thathas a higher amyloidogenic potency than Aβ40. Based on the hypothesis that Aβ amyloidosis in sporadic (nonfamilial) Alzheimer disease,which comprises more than 90% of all cases of Alzheimer disease, may
be caused by the deceleration of Aβ degradation, we previously identified
neprilysin as a physiological Aβ-degrading peptidase and showed thatneprilysin regulates the steady-state levels of both Aβ40 and Aβ42
in vivo6,7. Neprilysin expression in the brain decreases during aging andin the early stages of Alzheimer disease progression8,9, whereas elevatedneprilysin activity has been shown to reduce the accumulation of bothsoluble and fibrillar Aβ in APP transgenic mice10,11. Identification ofa pharmacological means to selectively upregulate brain neprilysin
activity would therefore provide a new therapeutic opportunity.

A rationale for this strategy is based on the fact that at least two cell type–specific ligands capable of upregulating neprilysin activity have been identified: opioid for monocytes12 and substance P for bone marrow cells13.
In our search for such a potential ‘activator’ of brain neprilysin, we first
performed a series of unbiased screens for a ligand that alters neprilysin
activity in primary cortical neurons using an activity staining method.
This method visualizes enzymatically active neprilysin on the cell surface
and was used because endogenous neprilysin in primary neurons isdifficult to detect by immunocytochemistry14, enzymatically active neprilysin requires a post-translational maturation (such as glycosylation)for its full activity14 and cell-surface neprilysin has a more importantrole in degrading the primary pathogenic form of Aβ, Aβ42, than theintracellular activity6,7,14. Positive control HEK293 cells transfected with
cDNA encoding neprilysin15, but not those transfected with a mock vector, produced fluorescence signals that were completely inhibited by aspecific inhibitor of neprilysin, thiorphan (Fig. 1a). We then examinedthe effect of nearly 50 reagents (Supplementary Table 1 online) on neprilysin activity in primary neurons. Only the neuropeptide somatostatin
reproducibly and significantly increased neuronal thiorphan-sensitive
neprilysin activity (Fig. 1b). Somatostatin did not generate fluorescencesignals in primary neurons prepared from neprilysin-deficient mice7. Allof the other reagents tested either had little effect or only slightly reduced
neprilysin activity. The effect of somatostatin on neprilysin activity was
clearly concentration-dependent, with a half-maximal effective dose
(ED50) of 50 nM (Fig. 1c), slightly higher than the Km values of somatostatin for somatostatin receptors, presumably owing to degradation ofsomatostatin during incubation. When we used primary neurons prepared from mice lacking somatostatin precursor (SSTP)16, the percentage
increase in neprilysin activity upon exposure to somatostatin was higher
as compared to the response of neurons from Sst+/+ mice (approximately
200% versus 160–180%). This suggests that endogenously generated
somatostatin is also involved in regulating neprilysin activity in the present experimental paradigm. Similar to the physiological subcellular location of neprilysin in the brain, neprilysin activity appeared as puncta on
neuronal processes rather than in or around soma (Fig. 1d).
We performed further mechanistic analyses of the somatostatin-induced
increase in neprilysin activity in primary neurons. The effect of somatostatin peaked at around 24 h and lasted up to approximately 36 h (Fig. 2a). The

1Laboratory for Proteolytic Neuroscience, RIKEN Brain Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan. 2These authors contributed equally tothis work. Correspondence should be addressed to T.C.S. (saido@brain.riken.go.jp) or N.I. (iwatan@brain.riken.go.jp).

Published online 20 March 2005; doi:10.1038/nm1206

434

VOLUME 11 | NUMBER 4 | APRIL 2005 NATURE MEDICINE© 2005 Nature Publishing Group http://www.nature.com/naturemedicine

LETTERSdecline after 24 h seems to be the result of somatostatin degradation andreceptor desensitization because the quantity of exogenously administeredsomatostatin in culture media decreased to 19% in 36 h and readministration of somatostatin led to a smaller increase in neprilysin activity (<50% of
the initial increase). We then inspected the effect of a somatostatin receptor
antagonist17 (BIM23056), a Gi inhibitor (pertussis toxin) and a Gs inhibitor (cholera toxin), on the somatostatin-induced elevation of neprilysin
activity (Fig. 2b). BIM23056 and pertussis toxin, but not cholera toxin,
inhibited neprilysin activation, indicating that Gi-coupled somatostatin
receptors18 are involved in the signal transduction leading to neprilysin
activation. RT-PCR analysis using a set of primers that recognize all threeneprilysin transcript variants7 indicated that somatostatin administration did not change the quantity of mRNA 12 h after the treatment to an
extent that can be detected by this method (data not shown). We also madeefforts to detect neprilysin in primary neurons by immunocytochemistry
and western blotting, but the protein levels were below the detection limit.
The results indicate that the effect of somatostatin on the transcriptionof the gene encoding neprilysin, Mme, is limited and that somatostatin
might have exerted its effect mainly through post-translational events.
Notably, somatostatin treatment resulted in selective and significant
reduction of Aβ42, but not Aβ40, in the culture media of primary neurons(Fig. 2c). Somatostatin did not induce this selective reduction of Aβ42 when
primary neurons prepared from neprilysin-knockout mice were used, indicating that neprilysin is directly involved in the somatostatin-regulated
Aβ42 metabolism (Fig. 2d).

a

b

c

d

NATURE MEDICINE VOLUME 11 | NUMBER 4 | APRIL 2005

Because the above observations might be in vitro artifacts, we next
investigated whether somatostatin contributes to the regulation of
neprilysin activity and, consequently, of Aβ levels in vivo using SSTP
knockout mice. These mice were developmentally and morphologicallynormal16. Immunofluorescence indicated that the level of hippocampal
somatostatin, which is absent in the Sst−/− mouse brain, did not significantly differ between Sst+/+ and Sst+/− mice (Fig. 3a), suggesting that therelationship between the somatostatin level and SSTP gene dosage is
not linear. This presumably reflects the fact that transcription, mRNAstability, translation or post-translational processing of SSTP have arate-limiting role in the generation of somatostatin to some extent.
Neprilysin activity in the hippocampus of Sst−/− mice was significantly
lower than in Sst+/+ mice in agreement with the above in vitro observations, whereas the difference between Sst+/+ mice and Sst+/− mice was
not significant (Fig. 3b). Nevertheless, there was a tendency for the
enzyme activity in Sst+/− mice to be consistently weaker.
We then quantified Aβ40 and Aβ42 levels in the hippocampus from Sst+/+,Sst+/− and Sst−/− mice. Although somatostatin deficiency seemed to resultin the elevation of both Aβ species, the increase in Aβ42 in Sst−/− mice
was statistically significant (P = 0.0011), but the increase in Aβ40 was not(P = 0.2221). Thus, the effect of somatostatin deficiency was more pronounced for Aβ42 in this brain region. The difference in the Aβ42/Aβ40
ratio was not statistically significant. (Therefore, the effect of somatostatin deficiency is similar, but not identical, to that of familial Alzheimer
disease–causing mutations in presenilin genes, which selectively increase
the level of Aβ42 relative to Aβ40.) This observation is consistent with thein vitro effect of somatostatin on the Aβ levels in culture media (Fig. 2c,d).
The presence of cortistatin, a somatostatin homolog capable of activating
somatostatin receptors, in the brain19 might have contributed to the basal
neprilysin activity in Sst−/− mice, although the nature of this relatively newneuropeptide family member has not yet been thoroughly explored.
In contrast, we observed no difference in neprilysin activity in the
cerebellum between Sst+/+ and Sst−/− mice (Fig. 3c). There was also no
difference in the quantities of Aβ40 or Aβ42 in the cerebellum between
Sst+/+ and Sst−/− mice. These observations are in accordance withthe human pathology of Aβ amyloidosis initially taking place in the
hippocampus and the temporal cortex and only modestly affecting
the cerebellum9. We also examined whether somatostatin deficiency
affected APP metabolism in the hippocampus by western blot analysis
of full-length APP, soluble extracellular APP fragment generated by
α-secretase (APPsα), soluble extracellular APP fragment generated by
β-secretase (APPsβ), C-terminal fragment (CTF)-β, CTF-α and CTF-γ(Fig. 3d,e). There was no difference in the quantity of APP or any of the
APP fragments, whereas neprilysin expression was significantly reducedin Sst−/− mice, excluding the possibility that somatostatin might haveaffected Aβ levels through alteration of APP metabolism.

Figure 1 In vitro screening for a neprilysin activating ligand using primaryneurons. (a) HEK293 cells transfected with mock vector or Mme cDNA15
were subjected to activity staining in the presence and absence of 1
µM thiorphan as indicated. The upper panels are fluorescence images
representing neprilysin activity and the lower panels are phase-contrast
images. Bar, 20 µm. (b) Primary cortical neurons prepared from Mme+/+and Mme−/− mice treated with or without 1 µM somatostatin (SST; 14-mer)were analyzed by activity staining in the presence or absence of thiorphan as
indicated. The fluorescence signals in the upper panels are superimposedon the phase-contrast images in the lower panels. Relative intensities of thefluorescence signals are indicated below each panel as an average ± s.d.
(n = 4). n.d., not detectable. *P < 0.05 as compared to controls. Bar, 120µm. (c) Effect of somatostatin concentration on neprilysin activity. The bars
indicate 1 s.d. (n = 4). (d) Higher power images of activity-stained primaryneurons treated with somatostatin as above. Bar, 40 µm.

435

LETTERS© 2005 Nature Publishing Group http://www.nature.com/naturemedicine

a

b

c

d

Figure 2 Mechanism of somatostatin action and effect on Aβ clearance in vitro. (a) Time course of neprilysin activity changes in primary neurons treated with(closed circles) or without (open circles) somatostatin as in Figure 1b. The bars indicate 1 s.d. (n = 6). (b) Effect of somatostatin receptor agonist BIM23056(BIM, 100 µM), pertussis toxin (100 ng/ml) and cholera toxin (100 ng/ml) on the somatostatin (SST;1µm)-induced neprilysin activity. The bars indicate averages± 1 s.d. (n = 8). *, P < 0.05 as compared to controls; #, P < 0.05 as compared to somatostatin-treated neurons by Student-Newman-Keuls multiple range test.
(c) Effect of somatostatin treatment on the concentrations of Aβ40 and Aβ42 in the culture media of primary neurons prepared from wild-type mice. Culture
media were collected 24 h after somatostatin treatment, and soluble Aβ levels were quantified as previously described14. The experiments were performed
7 d after neurons had been cultured at a concentration of 6 × 105 per well in 12-well plates. The bars indicate the averages ± 1 s.d. (n = 12). *, P < 0.05.
(d) Effect of somatostatin treatment on the concentrations of Aβ40 and Aβ42 in the culture media of primary neurons prepared from neprilysin knockout mice.
The experiments were performed in an identical manner for those in c (n = 12).

Somatostatin deficiency reduced Mme transcription only slightly(by 15% as analyzed by northern blotting, although this decrease
was statistically significant; Fig. 3e), so the effect of somatostatin
on neprilysin is probably mediated mostly by post-translational
processes such as metabolism and cellular localization of neprilysin, as implied by the in vitro experiments. The difference in theactivity and quantity of neprilysin between Sst+/+ and Sst−/− mice
was already demonstrated (Fig. 3b,d,e), but we also investigateda possible alteration of neprilysin localization in hippocampus bya triple immunofluorescence technique using antibodies against
neprilysin (red), vesicular GABA transporter (VGAT; green) andtau (blue) (Fig. 4a). Quantification of the results by image analysis
indicated that somatostatin deficiency significantly reduced neprilysin expression in the outer molecular layer of dentate gyrus to 78%(Fig. 4b). Colocalization of neprilysin with a presynapse marker, VGAT,
was reduced even further in both lacunosum moleculare and outer molceluar layer, to 58% and to 68%, respectively (Fig. 4c), whereas colocalization with an axonal marker, tau, remained unchanged (Fig. 4d). Theseobservations, indicating that somatostatin also regulates presynaptic
localization of neprilysin, were confirmed by biochemical fractionationof cortical tissues showing that synaptic membrane-associated neprilysin was reduced and that ER-Golgi-microsome-associated neprilysin
was increased in Sst−/− mice while synaptic vesicle-associated neprilysin
remained unchanged (Supplementary Fig. 1 online).
The relatively high expression of somatostatin-induced neprilysin
activity on the cell surface both in vitro (Fig. 1) and in vivo (Fig. 4) mightperhaps account for the more pronounced effect of somatostatin on Aβ42
levels (Fig. 2c and Fig. 3c). Whereas neprilysin deficiency resulted inincreases of both Aβ40 and Aβ42 in an identical manner7, infusion of thebrain with the cell-impermeable neprilysin inhibitor, thiorphan, elevatedAβ42 but not Aβ406. This suggests that Aβ40 is largely degraded intracellularly, whereas Aβ42 degradation takes place on or near the cell surface
in vivo. We recently confirmed such differential cellular degradation ofAβ40 and Aβ42 by expressing chimeric neprilysin constructs targeted todifferent cellular compartments in primary cortical neurons14. The ‘cell
surface’ probably corresponds to the outer surface of presynaptic processes in vivo. A possible explanation for the differential intra- and extracellular degradation of Aβ40 and Aβ42 may be the acidic conditions insidesecretory vesicles, in which the pH is around 5 (ref. 20). We examineddegradation of synthetic Aβ40 and Aβ42 by recombinant human neprily-436

sin at pH 5.0 and pH 7.2 and found that, whereas Aβ40 was degraded tothe same extent under these different pH conditions, Aβ42 degradation
was reduced to approximately one-tenth at pH 5.0 as compared to that at
pH 7.2 (Supplementary Fig. 2 online). Although this result is consistent
with the notion that Aβ42 degradation occurs largely on or near the cellsurface rather than inside secretory vesicles, there remains a possibility
that another somatostatin-induced Aβ-degrading mechanism may exist
in neurons. The mechanism of the somatostatin-induced neprilysintranslocation remains unclear but might be associated with soluble
N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE)
proteins involved in intracellular membrane fusion because the quantityof syntaxin, a SNARE family member, in the synaptic vesicle fraction
was apparently increased in Sst−/− mice (Supplementary Fig. 1 online).
There also remains a possibility that unknown neprilysin-interacting or
modifying factors might be involved.
The finding that the amount of Aβ42 increased around 1.5-fold in
the brain of somatostatin-deficient mice is interesting given that anelevation of Aβ42 is the common phenotype of virtually all of the familial
AD-causing gene mutations5. Notably, it has been shown that SSTP
mRNA is one of the very few transcripts (0.5% of 11,000 examined) consistently reduced in human brains upon aging2. These observations lead
us to draw the following hypothetical scenario for development of lateonset sporadic Alzheimer disease. Downregulation of somatostatin and,possibly, related neuropeptide(s)19 in human brain in the early stage of
aging may initiate gradual elevation of Aβ levels by lowering neprilysin
activity and, in a decade or two, lead to Aβ amyloidosis that eventuallytriggers a cascade of Alzheimer disease–associated pathological events.
Our findings also indicate that increasing somatostatin concentrationor agonizing somatostatin receptors in the brain will contribute to theclearance of this primary pathogenic form of Aβ when somatostatinlevels in the brain drop to as low as 10–20% of control levels during
aging or Alzheimer disease development1,3. Also notable is the fact thatsomatostatin is likely to be directly involved in memory formation apartfrom its involvement in Aβ regulation19. This suggests that agonizing somatostatin receptors in brain may generate dual independent
benefits for the improvement of neuronal function. Thus far, there
have been five somatostatin receptor subtypes identified, all of which
are G-protein-coupled receptors19. Among the five receptor subtypes,
types two and four may be the primary candidate targets because they
are relatively potently expressed in neocortex and hippocampus19,21.

VOLUME 11 | NUMBER 4 | APRIL 2005 NATURE MEDICINELETTERS© 2005 Nature Publishing Group http://www.nature.com/naturemedicine

a

c

d

b

Figure 3 Neprilysin activities, Aβ levels and APP
metabolism in somatostatin-deficient mousebrains. (a) Hippocampal sections from Sst+/+, Sst+/−and Sst−/− mice (10–12 weeks old) as indicatedwere immunostained with anti-somatostatin
antibody. Bar, 200 µm. (b) Hippocampal neprilysin
activities and Aβ levels in Sst+/+, Sst+/− and Sst−/−mice. The bars indicate averages ± s.e.m.
(n = 14 for Sst+/+, n = 21 for Sst+/− and 10 for
Sst−/−). (c) Cerebellar neprilysin activities and Aβlevels in Sst+/+, Sst+/− and Sst−/− mice.
(d) Western blot analysis of neprilysin, full-length
APP (fl-APP), APPsα, APPsβ, CTF-β, CTF-α andCTF-γ in the hippocampus of Sst+/+ and Sst−/−mice. A monoclonal antibody, 22C11, was used
to detect fl-APP and APPs; a polyclonal antibody,
A8717, against the C-terminal region of APP
was used to detect CTF-β, CTF-α and CTF-γ.
(e) Quantification of fl-APP immunoreactivity,
neprilysin immunoreactivity normalized against
fl-APP (n = 8) and neprilysin mRNA normalizedagainst actin mRNA (n = 4) in the hippocampus of
Sst+/+ and Sst−/− mice. Mme and Actb mRNA levels
were quantified by northern blotting. Reduction of
Mme mRNA in Sst−/− mice was also confirmed byquantitative RT-PCR for type 1 transcript.
* P < 0.05; ** P < 0.002.

eSynthesis of blood brain barrier–permeable agonists that can distinguish between different somatostatin receptor subtypes, or such useof an ‘antidementia’ compound as FK960 that elevates hippocampal
somatostatin levels22 in the brain may be potential approaches to reducing Aβ levels. One potential benefit of harnessing neprilysin activity byagonizing somatostatin receptors, among other Aβ-reducing strategies,
is that, if used modestly, it is unlikely to accompany major adverse side
effects because a deficiency of neprilysin does not seem to alter thelevels of ‘neprilysin substrate’ neuropeptides such as enkephalin, cholecystokinin, neuropeptide Y, substance P and somatostatin in the brain(N.I. & T.C.S., unpublished data), indicating the presence of redundantbuffering mechanisms to metabolize these peptides. This new approach
might be combined with other approaches targeting at β-secretase23
and γ-secretase24 as a cocktail therapy strategy to achieve a selective
effect on Aβ levels in the brain with minimum adverse side effects.
METHODS
Activity staining of primary neurons. Cortical neurons were isolated fromembryonic C57BL/6J mice at the development stage of 16–18 d as previously
described25. Cells (1 × 105 cells) were cultured on poly-L-lysine (PLL)-coatedglass cover slips (12 mm/0.12–0.17 mm thickness; Matsunami) in 24-well plate(Iwaki). AraC (final concentration, 1 µM) was added after the first 3 d of culture,and the culture medium was changed once per week thereafter and 4 h before
treatment. After 28 d in vitro, cultured neurons were treated with one of thereagents listed in Supplementary Table 1 online for 24 h at 37 °C, and thisprocess repeated on different cultures for each of the reagents. Cells were thenwashed twice with Tris-buffered saline (pH 7.4), fixed with 1.5% paraformaldehyde in 50 mM phosphate buffer (pH 6.8) for 12 min at 20 °C, and washed

NATURE MEDICINE VOLUME 11 | NUMBER 4 | APRIL 2005

twice with the Tris-buffered saline for subsequent
staining for neprilysin activity. The fixed neurons
were incubated in 0.2 ml of substrate solution (0.5mM glutaryl-Ala-Ala-Phe-methoxy-2-naphthylamide26 (Sigma) in 62.5 mM Tris-HCl (pH 7.4)) at 4 °C
for 48 h. Leucine aminopeptidase, phosphoramidon
and nitrosalicylaldehyde were then added to the substrate solution at final concentrations of 50 µg/ml,
10 µM and 0.6 mM, respectively, and incubated for
0.5 h at 37 °C. The cells were rinsed twice with ice-cold 50 mM Tris-HCl (pH7.4) and subjected to immunofluorescence analysis with a filter for rhodamine
visualization using argon laser excitation. The activity staining is used to visualize neprilysin activity on the cell surface because leucine-aminopeptidase does
not enter the cells. We also made efforts to perform intracellular activity staining
by permeabilizing cells with detergents but did not succeed because detergent
treatment resulted in destruction of cell structures in the present experimental
paradigm. Quantification of fluorescence images was performed as previously
described8,11. All animal experiments were performed in compliance with theinstitutional guidelines.
RT-PCR and northern blot analysis. RT-PCR to detect Mme mRNA was carried out using total RNA as previously described27 with the following pairs ofprimers: 5′-GCTGTAGTCAATGCATT-3′ and 5′-GAAAGGCATCTGCAAAC3′ for all three types of transcripts; 5′-CTCAGTCGCCTCCAACTTCTCC-3′
and 5′-GCATAGAGAGCGATCATTGTCACC-3′ for type 1 transcript7. The
following pair of primers for gyceraldehyde-3-phospate dehydrogenase
was used for calibration: 5′-TGACCACAGTCCATGCCATCAC-3′ and 5′TCCACCACCCTGTTGCTG-3′. PCR products were electrophoresed on 5%
polyacrylamide gels. Gels were stained with ethidium bromide (1 µg/ml), andthen DNA fragments were visualized by UV illumination and photographed.
For northern blotting, polyadenylated RNA was isolated from hippocampus
using Micro-Fast Track 2.0 kit (Invitrogen). Samples were electrophoresed in 1%
formaldehyde agarose gels and transferred to nylon membranes. The membranes
were probed with a [α-32P]dCTP-labeled 520-base-pair mouse Mme cDNA fragment generated by PCR using a pair of primers, 5′-GCTGTAGTCAATGCATT3′ and 5′-GAAAGGCATCTGCAAAC-3′. The gels were stained with ethidium
bromide and the membranes were reprobed with an actin cDNA (Sigma) to
confirm the quantity of loaded DNAs.

437

LETTERS
Figure 4 Localization of neprilysin immunoreactivity in Sst+/+ andSst+/– mice. (a) Hippocampal sections from Sst+/+ mice (left panels) andSst−/− mice (right panels) were subjected to triple-immunofluorescenceexamination using antibodies to neprilysin (red), VGAT (green), andtau (blue) as indicated. Superimposed images of neprilysin-VGAT andneprilysin-tau are shown in the lower two pairs of panels. Areas indicated bysquares in the top panels were quantitatively analyzed at higher resolution.
Bars, 200 µm in the top panels and 40 µm in the others. (b) Neprilysin
immunoreactivity in stratum lacunosum-moleculare (LM), middle molecularlayer (MMo) and outer molecular layer (OMo) was quantified by image
analysis. The bars represent averages ± s.e.m. (n = 4). (c) Colocalization of
neprilysin and VGAT, as an indicator of presynapse-associated neprilysin, wasquantified as in b. (d) Colocalization of neprilysin and tau, as an indicator ofaxon-associated neprilysin, was quantified as in b. *P < 0.05; **P < 0.01.

© 2005 Nature Publishing Group http://www.nature.com/naturemedicineaImmunochemical procedures. Somatostatin immunostaining of brain sections was performed as described28 using a rabbit anti-somatostatin antiserum, T-4103 (Peninsula). The assay for brain tissue neprilysin was described
previously in detail8,11. Quantification of brain Aβ40 and Aβ42 was performed
using sandwich ELISA with antibodies BNT77/BA27 and BNT77/BC05 as previously described7,11. Western blot analyses of APP and APP fragments were
performed using a monoclonal antibody to the N-terminal region of APP(22C11; Chemicon) and a rabbit polyclonal antibody to the C-terminal regionof APP (A8717; Sigma). Western blot analysis of neprilysin was performed as
described11,14 except that a polyclonal antibody (421126; Techne) was used.
Immunoreactive signals were detected with ECL Advance Western Blotting
Detection kit (Amersham) and quantified with a LuminoImager, LAS-3000 and
Science Lab 2001 Image Gauge software (Fuji Photo Film). For triple-immunofluorescence analysis of the hippocampal formation, representative 4-µm
thick paraffin sections were stained with antibodies against neprilysin (mousemonoclonal 56C6, 1:400; Novocastra), VGAT (rabbit polyclonal 117G4, 1:1,500;
Synaptic Systems) and tau (T49, mouse monoclonal, 1:500; gift of J. Trojanowski
and V. M.-Y. Lee, University of Pennsylvania School of Medicine) based on
the previously described method29 with a TSA-Direct kit (NEN Life Science
Products). Specificity of neprilysin staining was confirmed using neprilysin-deficient mice as a negative control8. Quantification of immunofluorescence images
was performed using MetaMorph (version 6.1) imaging software (Universal
Imaging Corporation) as previously described8,11. The statistical significancewas examined by Student’s t-test after confirming the equality between the groupvariances unless otherwise stated.
Other reagents. BIM23056 was synthesized in the Research Resource Center,
RIKEN BSI. Pertussis toxin and cholera toxin were purchased from CalBiochem
and Sigma, respectively.
Note: Supplementary information is available on the Nature Medicine website.

bc

ACKNOWLEDGMENTS
We thank E. Hosoki, M. Sekiguchi, Y. Matsuba, N. Yamazaki and K. Watanabe fortechnical assistance and A. Miyawaki for valuable discussions. We express our
gratitude to U. Hochgeschwender, Oklahoma Medical Research Foundation forproviding SSTP knockout mice and helpful comments. We also thank Takeda
Chemical Industries Ltd. and C. Gerard, Harvard Medical School, for providing themonoclonal antibodies to Aβ for ELISA and neprilysin knockout mice, respectively.
The research was supported by research grants from RIKEN Brain Science Institute,
the Ministry of Education, Culture, Sports, Science and Technology of Japan, andLife Science Foundation (Tokyo).
COMPETING INTERESTS STATEMENT
The authors declare that they have no competing financial interests.

dReceived 25 August 2004; accepted 3 January 2005
Published online at http://www.nature.com/naturemedicine/
1. Hayashi, M., Yamashita, A. & Shimizu, K. Somatostatin and brain-derived neurotrophic
factor mRNA expression in the primate brain: decreased levels of mRNA during aging.
Brain Res. 749, 283–289 (1997).
2. Lu, T. et al. Gene regulation and DNA damage in the ageing human brain. Nature 429,
883–891 (2004).
3. Davies, P., Katzman, R. & Terry, R.D. Reduced somatostatin-like immunoreactivity in

438

VOLUME 11 | NUMBER 4 | APRIL 2005 NATURE MEDICINE© 2005 Nature Publishing Group http://www.nature.com/naturemedicine

LETTERS
cerebral cortex from cases of Alzheimer’s disease and Alzheimer senile dementia.
Nature 288, 279–280 (1980).
4. van de Nes, J.A.P., Sandmann-Keil, D. & Braak, H. Interstitial cells subjacent to theentorhinal region expressing somatostatin-28 immunoreactivity are susceptible todevelopment of Alzheimer’s disease–related cytoskeletal changes. Acta Neuropathol.
104, 351–356 (2002).
5. Hardy, J. & Selkoe, D.J. The amyloid hypothesis of Alzheimer’s disease: progress andproblems on the road to therapeutics. Science 297, 353–356 (2002).
6. Iwata, N. et al. (2000). Identification of the major Aβ1–42-degrading catabolic pathway
in brain parenchyma: Suppression leads to biochemical and pathological deposition.
Nat. Med. 6, 143–151 (2000).
7. Iwata, N. et al. Metabolic regulation of brain Aβ by neprilysin. Science 292, 1550–
1552 (2001).
8. Iwata, N., Takaki, Y., Fukami, S., Tsubuki, S. & Saido, T.C. Region-specific reductionof Aβ-degrading endopeptidase, neprilysin, in mouse hippocampus upon aging. J.
Neurosci. Res. 70, 493–500 (2002).
9. Yasojima, K., Akiyama, H., McGeer, E.G. & McGeer, P.L. Reduced neprilysin in highplaque areas of Alzheimer brain: a possible relationship to deficient degradation of
β-amyloid peptide. Neurosci. Lett. 297, 97–100 (2001).
10. Leissring, M.A. et al. Enhanced proteolysis of β-amyloid in APP transgenic mice
prevents plaque formation, secondary pathology, and premature death. Neuron 40,
1087–1093 (2003).
11. Iwata, N. et al. Presynaptic localization of neprilysin contributes to efficient clearanceof amyloid-β peptide in mouse brain. J. Neurosci. 24, 991–998 (2004).
12. Wang, T.-L., Chang, H., Hung, C.-R. & Tseng, Y.-Z. Morphine preconditioning attenuates neutrophil activation in rat models of myocardial infarction. Cardiovasc. Res. 40,
557–563 (1998).
13. Joshi, D.D. et al. Negative feedback on the effect of stem cell factor on hematopoiesis
is partly mediated through neutral endopeptidase activity on substance P: a combinedfunctional and proteomic study. Blood 98, 2697–2706 (2001).
14. Hama, E., Shirotani, K., Iwata, N. & Saido, T.C. Effects of neprilysin chimeric proteinstargeted to subcellular compartments on amyloid β peptide clearance in primaryneurons. J. Biol. Chem. 279, 30259–30269 (2004).
15. Shirotani, K. et al. Neprilysin degrades both amyloid β peptides 1–40 and 1–42 most
rapidly and efficiently among thiorphan- and phosphoramidon-sensitive endopepti-

NATURE MEDICINE VOLUME 11 | NUMBER 4 | APRIL 2005

dases. J. Biol. Chem. 276, 21895–21901 (2001).
16. Zeyda, T., Diehl, N., Paylor, R., Brennan, M.B. & Hochgeschwender, U. Impairment
in motor learning of somatostatin null mutant mice. Brain Res. 906, 107–114(2001).
17. Shimon, I. et al. Somatostatin receptor subtype specificity in human fetal pituitarycultures. J. Clin. Invest. 99, 789–798 (1997).
18. Garcia-Jimenez, A., Fastbom, J., Winbland, B. & Cowburn, R.F. G-protein regulationof signal transduction in Alzheimer’s disease. Brain Aging 2, 7–15 (2002).
19. Moller, L.N., Stidsen, C.E., Hartmann, B. & Holst, J.J. Somatostatin receptors.
Biochim. Biophys. Acta 1616, 1–84 (2003).
20. Miesenbock, G., De Angelis, D.A. & Rothman, J.E. Visualizing secretion and synaptictransmission with pH-sensitive green fluorescent proteins. Nature 394, 192–195(1998).
21. Bruno, J.F., Xu, Y., Song, J. & Berelowitz, M. Molecular cloning and functional
expression of a brain-specific somatostatin receptor. Proc. Natl. Acad. Sci. USA 89,
11151–11155 (1992).
22. Doggrell, S.A. The potential of activation of somatostatinergic neurotransmission with
FK960 in Alzheimer’s disease. Expert Opin. Investig. Drugs 13, 69–72 (2004).
23. Citron, M. β-Secretase: progress and open questions. in Aβ Metabolism and Alzheimer’s
Disease. (ed. Saido, T.C.) 17–25 (Landes Bioscience, Georgetown, 2003).
24. Wolfe, M.S. γ-Secretase and presenilin. in Aβ Metabolism and Alzheimer’s Disease.
(ed. Saido, T.C.) 33–47 (Landes Bioscience, Georgetown, 2003).
25. Hama, E. et al. Clearance of extracellular and cell-associated amyloid β peptide by viral
expression of neprilysin in primary culture. J. Biochem. 130, 721–726 (2001).
26. Back, S.A. & Gorestein, C. Histochemical visualization of neutral endopeptidase24.11 (enkephalinase) activity in rat brain: cellular localization and codistribution
with enkephalins in the globus pallidus. J. Neurosci. 9, 4439–4455 (1989).
27. Takano, J., Watanabe, M., Hitomi, K. & Maki, M. Four types of calpastatin isoforms
with distinct amino-terminal sequences are specified by alternative first exons anddifferentially expressed in mouse tissues. J. Biochem. 128, 83–92 (2000).
28. Jinno, S. & Kosaka, T. Patterns of expression of neuropeptides in GABAergic nonprincipal neurons in the mouse hippocampus: Quantitative analysis with optical disector.
J. Comp. Neurol. 461, 333–349 (2003).
29. Wang, G. et al. Tyramide signal amplification method in multiple-label immunofluorescence confocal microscopy. Methods 18, 459–464 (1999).

439