Alzheimer’s Disease, Neuropeptides,
Neuropeptidase, and Amyloid- Peptide
Metabolism
Takashi Saito, Yoshie Takaki, Nobuhisa Iwata, John Trojanowski*, Takaomi C. Saido

Amyloid  peptide (A), the pathogenic agent of Alzheimer's disease (AD), is aphysiological metabolite in the brain. We have focused our attention and effort on
elucidation of the unresolved aspect of A metabolism, proteolytic degradation.
Among a number of A-degrading enzyme candidates, we used a novel in vivo
paradigm to identify a member of the neutral endopeptidase family, neprilysin, as the
major A catabolic enzyme. Neprilysin deficiency results in defects in themetabolism of endogenous A40 and 42 in a gene dose-dependent manner. Our
observations suggest that even partial down-regulation of neprilysin activity, which
could be caused by aging, can contribute to AD development by promoting A
accumulation. Moreover, we discuss the fact that an aging-dependent decline of
neprilysin activity, which leads to elevation of A concentrations in the brain, is anatural process that precedes AD pathology. In this Perspective, we hypothesizethat neprilysin down-regulation has a role in sporadic AD (SAD) pathogenesis andpropose that this knowledge be used for developing novel preventive andtherapeutic strategies through utilization of a G protein-coupled receptor (GPCR).
Keywords. mammalian aging, mutations, aggregates, cell senescence, cell loss, life extension,
Alzheimer's disease
f humans lived to be 120 to 140 years ofage or more, there is a serious possibility
that all would develop Alzheimer's
disease (AD) (see "Detangling Alzheimer's
Disease" http://sageke.sciencemag.org/cgi/
content/full/sageke;2001/1/oa2) in the later
years of the human life-span. This prediction
stems from the fact that the incidence of AD
increases exponentially after the 7th decadeof life, although it is not certain that thistrend continues in persons beyond age 100.
Thus, epidemiological studies indicate that
one out of two people who reach the age of
85 and nine out of 10 people who attain theage of 100 will be affected by AD or AD typebrain pathology (1) (see "Honig Case
Study" http://sageke.sciencemag.org/cgi/
content/full/sageke;2001/1/dn2). What is thesignificance of this fact? The answers are
obviously not entirely clear, as so few
individuals have been studied who haveattained these ages. But one interpretation isthat AD or AD type pathology may be an
ultimate form of brain aging. Note that here
we mean to imply that "brain aging" differsfrom "general aging" in that mitotic quality
control mechanisms play relatively minor
roles simply because differentiated neurons
in the adult brains are primarily post-mitotic
cells.

I

Laboratory for Proteolytic Neuroscience,
RIKEN Brain Science Institute, 2-1 Hirosawa,Wako-shi, Saitama 351-0198, JAPAN. *Center
for Neurodegenerative Disease Research,Institute on Aging and Department of
Pathology and Laboratory Medicine, 36th andSpruce Streets, Philadelphia, PA, 19104-4283,
USA. Email: saido@brain.riken.go.jp

aspects of what has, until now, beencon-sidered to be normal aging.
In this Perspective, we describe ourcurrent
experimental
evidence-based
hypo-thesis that might associate themetabolic homeostasis of neuropeptide(s) inthe brain with the aging-dependent
development of sporadic AD (SAD) throughalteration in the metabolism of amyloid 
peptide (A). It was established during the
1990's, a decade of phenomenal progress inunderstanding familial AD (FAD), that A[which is derived from amyloid precursor
protein(APP
http://sageke.sciencemag.org/cgi/genedata/
sagekeGdbGene;197) via the action of - and-secretase] acts as a potent pathogenic agent
in the development of AD. Such conclusions
were drawn as a consequence of theidenti-ficationof
FAD-causing
gene
mutations and analyses of their molecularand pathological phenotypes (2). We
consider the micro-tubule-associated protein
tau to be as import-ant as A, particularly inclinical terms, now that mutations in the taugene have been shown to cause tauopathy
and neurodege-neration (3), but this topic isbeyond the scope of this Perspective. We also
propose here a new approach to themetabolic control of A concentrations in the
brain
via
the
modulationofneuropeptide-receptor systems, for theprevention and treatment of AD. Note that allof the neuropeptide receptors are G
protein-coupled receptors (GPCRs), which
are the most tractable and relevant
pharma-ceutical targets in molecularpharmacological terms (4).

Until several centuries ago, most humanbeings lived only 50 to 60 years, because ofharsh living conditions that led to high infant
and early adult mortality. Infectious diseases
were rampant, and no effective therapies
existed; thus large numbers of children andadults succumbed to bacterial infections.
During this time in history, there waspracti-cally no "brain aging" of the sort that
we have been studying during the last few
Neuropeptides and Alzheimer's
decades. Therefore, the time period between
Diseasethe ages of 50-60 to 80-100 years for human
In 1980, Davies and colleagues (5)beings signals a very new biological time
dis-covered that concentrations of theperiod in the course of the evolution of life,
neuro-peptide
somatostatin
werewhich has taken hundreds of million years.
significantly re-duced in the brains of AD
Thus, in our view, understanding thepatients. This observation has beenmechanismoftheaging-dependent
develop-ment of AD is, at least inpart, equal to understanding the
new biological time period,
which did not even exist until
recently.
This reasoning intimates thatsome aspects of the biologicalmechanisms originally designed
to be beneficial in maintaining
metabolic homoeostasis throughnegative feedback processes
when humans lived to be 50 to
60 years of age could become
Animation 1. Schematized structure of the major
harmful after such ages haveA-degrading enzyme, neprilysin.
been exceeded. Therefore, an
Neprilysin is a type II membrane-associated peptidasewith its active site facing the lumen or extracellular side ofunder-standingoftheplasma membranes. This topology is favorable for themechan-isms of brain aging, thedegradation of extracytoplasmic peptides such as A.
ultimate form of which may well
Presynaptic
terminals
andnearbyintracellular
be re-presented by AD, should(lumen-sided) locations are likely to be the sites of A
help scientists to create novel
degrada-tion by neprilysin.
strate-gies for controlling somesageke.sciencemag.org/cgi/content/full/sageke;2003/3/pe1

22 JANUARY 2003

Page 1

confirmed repeatedly by others, but its
pathogenic significance has never been fully
resolved. Because concen-trations of otherneuropeptides,
such
as vaso-pressin,

pathological cascade of AD development.
Consistent with this alternative hypothesis is
the finding that the amounts of someneuropeptides decline upon aging before theFig. 1. Current list of A-degrading enzyme candidates studied using reverse genetic
techniques.
KO or KI miceA42
(Primarily pathogenic)

A40(Secondarily pathogenic)

Neprilysin-KO(-/-)*1
Neprilysin-KO(+/-)*1
ECE 2-KO (-/-)*2
ECE 2-KO (+/-)*2
ECE 1-KO (+/-)*2
IDE-KO (-/-)*3
tPA-KO (-/-)*4
uPA-KO (-/-)*5
ACE-KO (+/-)*6

2-fold increase
1.5-fold increase
1.2-fold increase
1.15-fold increase
1.2-fold increase
No significance*
No significance*
No significance*
No significance*

2-fold increase
1.5-fold increase
1.3-fold increase
1.2-fold increase
1.2-fold increase
1.1-1.2-fold increase
No significance*
No significance*
No significance*

FAD presenilin 1-KI*7(positive control)

typically 1.5-fold increase

No significance*

KO: (gene) knock-out; KI: (gene) knock-in; ECE: endothelin-converting enzyme; IDE:
insulin-degrading enzyme; tPA: tissue-type plasminogen activator; uPA: urokinase; ACE:
angiotensin-converting enzyme. *No significance if the difference compared to control mice is<10%. *1: see (18); *2: see (25); *3: see (58); *4: see (59); *5: see (60); *6: see (61); *7: see,for instance, (3). The results with mutant presenilin 1-KI mice represent typical pathogenic
alterations in the A levels, as a positive control, leading to accelerated A accumulation in thebrain. The quantification was performed using an identical ELISA developed by Nobuhiro
Suzuki (62). The mice were ~8 to 10 weeks of age. Gender is likely to be male in each study.
T.C.S. accepts the responsibility if any of the data are in error.

neuropeptideY,substanceP,andcorticotropin-releasing factor (CRF), havealso been reported to be reduced in AD
brains (6-12), the general interpretation has
been that the reduction is likely to be, at leastin part, a onsequence of the degeneration ofneuropeptide-generating neurons in AD
brains. This assumption does not, however,exclude the possibility that reduction of a
specific neuropeptide(s) may be causal in theonset of AD (13,14).
Meanwhile, our laboratory has been
en-gaged in an effort to identify thephysiologi-cally
relevant
peptidase
responsible for the in vivo degradation of A
in the brain (15-18); as described in the next
section, the prime suspect, neprilysin, (seeAnimation1
http://
sageke.sciencemag.org/cgi/content/full/sagek
e;2003/3/pe1/DC1) is not only involved in

the metabolism of neuropeptide(s), but also
is likely to be regulated by neuropeptide(s) as
part of the homeostatic mechanisms present
in the brain.
Accordingly, what Davies and others
discovered in the 1980's (5) and what we
identified during the past few years as the
major in vivo A-degrading enzyme (18)now may be combined to form-ulate aplausible strategy to eluci-date the mostfun-damental question in AD research: Whatcauses the aging-dependent accumu-lation ofA
leads to the development of
SAD? Moreover, thoughtful integration ofthese seemingly disparate findings may leadto the development of a new therapy for AD.
Such a therapy would not be expected tohave the types of side effects that are
anticipated for inhibitors of - and-secretase, which may also inhibit thebiologically indispensable processing of APP
http://sageke.sciencemag.org/cgi/genedata/sagekeGdbGene;197 and other important
substrates (19-21).
Metabolism of A: The Current Status
of Research on the A-degrading
Enzymes
Because A is constantly anabolized andcatabolized in the brain, the steady-state
concentrations of A are determined by the
dynamic balance between anabolic andcatabolic activities (22,23). In contrast to
most cases of dominantly inherited FAD andmouse models of A amyloidosis, elevationof A anabolism in the brain prior to theoccurrence of the A pathology is rarely
observed in normal aging. One logicalpresumption, then, is that A deposition in alarge number of SAD cases might be caused
primarily by its reduced catabolism [see (23)forfurther
details].
Duringthepast few years,
severalgroupshave proposed avarietyofcandidates for the"real,"orauthentic
A-degrading
enzymes[see(22,24,25)
forreviews]. Fig. 1
describes
thecurrent list ofseemingly
relevant

Fig. 2. Cleavage sites of several neuropeptides and A in
pro-teolysis catalyzed by neprilysin and endothelin-converting
enzyme-1 (ECE-1). Neprilysin and ECE-1, which both belong to theM13 clan of peptidases, share similar cleavage site specificity withrespect to their substrates. See (15, 25, 26, 29, 31).

Page 2sageke.sciencemag.org/cgi/content/full/sageke;2003/3/pe1

22 JANUARY 2003

Animation 2. Regulation of neprilysin activity/expression byligand-receptor mechanisms. Morphine and substance P havebeen shown to increase neprilysin activity/expression in neutrophils
and fibroblasts, respectively. All of the receptors for such peptide
ligands identified thus far are GPCRs.

can-didates studied using reverse genetics
tech-niques. Among these candidates,
nepri-lysin
andendothelin-converting
enzymes (ECEs), which both belong to theM13 clan of metallopeptidases, appear toaccount for 60 to 80% of the totalA-degrading activity in brain tissue [see(24) for the mathematical basis of thisassumption]. Because of the differences intheir enzymatic and cellular properties,
neprilysin and ECEs are likely to playcomplementary roles in distinct subcell-ular
compartments; the former degrades A
inside secretory vesicles and on theextra-cellular surface, while the latter does soin acidic compartments represented by thetrans-Golgi network (23-25). Incidentally,
these peptidases share some neuropeptides ascommon substrates (15,25,26) and thus could
be grouped on this basis into a particularfamily of neuropeptidases (see Fig. 2).
In any case, at the present time,
nepri-lysin (see Animation 1 http://sageke.
sciencemag.org/cgi/content/full/sageke;2003/3/pe1/
DC1) is the most potent A-degrading
enzyme identified in vivo. It is noteworthy
that even a heterozygous deficiency ofnepri-lysin leads to an approximately
1.5-fold increase in A concentrations inbrain tissues, a phenotype comparable tothose of the FAD-causing gene mutations.
Assuming that the parallel with FAD isrelevant [see Figure 1.8 of (24)], this finding
suggests that if neprilysin activity is reducedin human brains to ~50% at the age of 50, theaffected individuals will develop AD in 30 to
60 years after this time. Consistent with thishypo-thesis, the McGeer group has
demonstrated that, compared to control
mRNAs, neprilysin mRNA concentrations in
SAD patients, at relatively early stages of thedisease process (Braak Stage II), aresignificantly and select-ively reduced in the
hippocampus (a part of the limbic system
located in the medial tem-poral lobe andimportant in memory forma-tion), themidtemporal gyrus (a convolution or ridgeon the surface of the cerebrum), and, to alesser extent, the cerebellum (the part of thebrain that lies just below the posterior part ofthe cerebrum and plays an essential role incoordinating
voluntary
movement,
controlling muscle tone, and maintainingbalance). The fact that the amounts of control
neuronal mRNAs that encode cyclophilin
and microtubule-associated protein 2(MAP2) were not changed in tissues from
SAD patients indicates that down-regulationof neprilysin expression is not simply acon-sequence of neurodegeneration (27, 28).
Neprilysin, Neuropeptides, and Aging
Enkephalinis a5-aminoacid
neuropep-tide that is primarily involved inanalgesia (pain regulation). Neprilysin was
originally identified biochemically as an

enkephalin-degrading enzyme in a test(see Fig. 1).
tube-based ex-perimental paradigm and was
It is of interest that some substrates areonce termed "enkephalinase" (29-31).
known to regulate the activity/expression of
However, because enkphalin concentrations
neprilysin. Morphine, a mimic of opioid
remain essentially unchanged in the brains of
pep-tides, increases neprilysin enzyme
neprilysin-knock-out (KO) mice (32),
activity in neutrophils in vivo (36), while
neprilysin does not appear to be the major
substance P induces neprilysin mRNA
rate-limiting enkepha-lin-degrading enzyme
expression in fibroblasts in vitro (37) (seein the brain as a whole. These observations
Animation
2
illuminate the danger of depending too much
http://sageke.sciencemag.org/cgi/content/full/on in vitro assays to predict the in vivo
sageke;2003/3/pe3/DC1). In both cases, thefunctions of a given mole-cule, an important
receptors involved are likely to be GPCRs
caveat in AD research. On the basis of a(4). Such mechanisms are beneficial incollection of in vivo and in vitro experiments, main-taining
neuropeptide
homeostasis
we predict that relatively small peptides thatthrough the formation of negative feedback
can be degraded by exo-peptidases--such assystems; ex-cessively large amounts ofaminopeptidases, dipep-tidyl peptidases,
substrate ligands would be reduced bytripeptidyl peptidases, carboxy peptidases,
increased neprilysin activity, while very
andpeptidyl
dipepti-dases—probably do not
require neprilysin activity for
their cata-bolism as long as they
are acce-ssible to these peptidases.
Becau-se neprilysin is incapableof de-grading peptides larger than
~5 kDa (~50 amino acids)(29-31), peptides composed of~10 to 40 amino acid residues arelikely to be relevant substrates.
However, the secondary andtertiary
struc-turesofthesubstrates also need to be taken
into account, because the 3-D
structure of neprilysin indicatesthat only substrates that fit into Animation 3. Metabolic regulation of A levels
the active site of the en-zyme can through modulation of a peptide ligand-receptor
be proteolyzed effici-ently (33). mechanism in the brain. We hypothesize that a peptide
Indeed, an opioid pep-tide 5 ligand-receptor mechanism regulates both the ligand andamino acids in length was shown A levels in the brain. Bases for this hypothesis include
the presence of a similar mechanism in other tissues
to be elevated in a limited region (see Animation 2).
of the hippocampal forma-tion of
neprilysin-KO mice (34), contrary
to the results of the bulk
biochemical quantification (32).
This finding indicates that therelative function of neprilysin
de-pendsonwhetherit
colocalizes with its substrate andon whether other redundant
enzymes exist in the area (34).
Other candidate substrateneuropeptides for neprilysin
in-clude
somatostatin
andsubstance P, both of which are
decreased in AD brains (5, 8). Animation 4. Decline with aging of neprilysin
Indeed, these peptides are good expression in the dentate gyrus. We havedemonstrated that neprilysin expression is prominently
substrates for neprilysin-catalyzed reduced in the outer molecular layer, the inner molecular
proteolysis
inatest-tube layer, and the polymorphic cell layer of the dentate gyrus
paradigm, and sub-stance P has upon aging. See (24, 39, 40) for more details.
been shown to be metabolically Abbreviations: po, polymorphic cell layer; gr, granule cell
regulated by nepri-lysin in the layer; iml, inner molecular layer; mml, middle molecular
colon
under
inflama-tory layer; oml, outer molecular layer.
conditions
in
vivo
(35).
Incidentally, insulin-degrading enzyme (IDE), small amounts of ligands would have longer
lifespans due to the reduced catabolic activity.
which has been most strongly implicated by
We thus hypo-thesize that there may also
some groups (22), has now been shown toexist a similar mechanism in the brain (seeplay a relatively minor role in A catabolism

sageke.sciencemag.org/cgi/content/full/sageke;2003/3/pe1

22 JANUARY 2003

Page 3

Animation3
http://sageke.sciencemag.org/cgi/content/full/sageke;2003/3/pe1/DC1), although we do not
yet know the exact identity of theligand-receptor system. There are several
other rea-sons to predict the presence of sucha mechanism in the brain besides theobser-vations outlined above. These can besum-marized as follows: (i) neprilysin
expression in the hippocampus and neocortex(a portion of the cerebral cortex that has
distinct territories involved in sensory, motor,
and association functions) is closely
associated with interneurons (inhibitory
neurons), which are the major sources ofneuropeptides (38); (ii) interneurons possess
auto- and hetero-receptors (38) for various
ligands by which they regulate thehomeostatic levels of the ligands via a
modulation of the associated signaltransduction
mechanisms
(38);
(iii)
neprilysin is present inside secretory vesicles
and at presynaptic terminals (39, 40) andcould thus modulate the intra- andinter-cellular signals generated by these
peptidic ligands, which are produced insynaptic vesi-cles and released frompresynapses; and (iv) the mechanism
proposed above can account for the
aging-dependent
declineofcertainneuropeptides and of neprilysin (see next
paragraph).
We have reproducibly observed anaging-dependent, selective reduction of
neprilysin
activity/expression
inthehippocampus and neocortex (24, 39, 40). For
these experiments, neprilysin-KO mice wereused as a negative control. In the
hippocampus (see Fig. 3), a prominent local
neprilysin reduction was de-tected in thepolymorphic cell layer, the outer molecularlayer, and the inner molecular layer of thedentate gyrus, a region of the hippocampal
formation involved in the relay of
information from the entorhinal cortex toother areas of the hippocampus primarily
re-presented by the CA3 sector. Local
nepri-lysin reduction was also detected in the
stria-tum lucidem--a subcortical mass thathas an important excitatory or inhibitory rolein pro-cessing cortical signals--of the CA3
sector. The CA3 sector processes
information coming in through the perforantpath (see Fig. 3) and out to hippocampal CA
sectors 1-3 of the ipsilateral and contralateralsides of the brain (40) (see Animation 4
http://sageke.
sciencemag.org/cgi/content/full/sageke;2003/
3/pe1/DC1). These results indicate that aging
causes a local elevation of A concentrations
at the presynapses in these hippocampal
areas. Anatomically speaking, these areas
correspond to the presynaptic terminal zonesof the perforant path and mossy fibers
origi-nally projecting from the entorhinal
cortex (see Fig. 3), where the initialneurodegenera-tion takes place in the early
Page 4stages of the onset/progression of AD (41).
The observed decrease in neprilysin
expression is not due to a loss of neurons orof presynapses, be-cause presynaptic markers
remain unchanged upon aging.
It is not, however, so easy to prove or
disprove the mechanism shown in
Animation 3 (http://sageke.sciencemag.org/
cgi/content/full/sageke;2003/3/pe1/DC1)
using in vitro paradigms, for the following
reasons. Neprilysin is expressed mainly at
the presynaptic membranes of interneurons
in the brain (39). Although the number ofinterneurons is smaller than that of excitatoryneurons in the hippocampus and neocortex,
the number of presynapses formed byinter-neurons is 100 to 1000 times greaterthan the number of presynapses formed byexcitatory neurons (38, 42). Unless it
becomes possible to culture interneurons in amanner that would allow formation of as
many pre-synapses as occurs in vivo, it will
be difficult to analyze the hypotheticalligand-regulated
neprilysin

activity/expression mechanism in an in vitro
paradigm. The most relevant as well as
realistic approach would be to quanti-fy
neprilysin activity and A levels in the brainsof genetically manipulated mice de-ficient ina candidate neuropeptide precursor, although
this would take more time than the
shorter-term in vitro approaches.
Hypothesis: The Mechanism ofAging-dependentA
Deposition
Associatedwith
Neuropeptide
Metabolism in the Brain
On the basis of the presumption that themechanism schematized in Animation 3(http://sageke.sciencemag.org/cgi/content/ful
l/sageke;2003/3/pe1/DC1) also exists in thebrain, as it does in some other tissues, wesuggestamechanism
fortheaging-depend-ent accumulation of A inhuman brains. If we suppose that aging
causes a decrease in the synthesis of aneuropeptide (13, 43) that happens to be aligand involved in the regu-lation ofFig. 3. Diagram of thephysiological localization
andaging-associatedre-duction of neprilysininthehippocampal
forma-tion and entorhinal
cor-tex.
Previously
described
quantitative
immunofluo-rescenceobservations (24, 39, 40)
are summarized in the
figure. See also Anima-tion4.
Abbreviations:Or,
stratum oriens; Py, stratum
pyramidale; Ra, stratum
ra-diata;
Lm,stratumlacuno-sum-moleculare;
Mo, mole-cular layer; Gr,
granule cell layer; Po,
l
hi
ll l
L

Animation 5. Hypothesis: aging, neuropeptide, neprilysin, and A metabolism. Suppose
aging causes a decrease in the synthesis of a neuropeptide, which happens to be a ligand
involved in the regulation of neprilysin activity/expression as well as it being a practicallyselective substrate for neprilysin in vivo, then the activity/expression of neprilysin will decreasein a negative feedback manner in order to maintain the homeostasis of the neuropeptide levels.
Such a mechanism is probably beneficial to the brain in the relatively short terms bycompensating for the reduced synthesis of the ligand peptide. An apparent adverse side effectof this negative feedback mechanism is an increase in A levels particularly at presynapses.
This could cause presynaptic toxicity on a long-term basis, generating a positive feed-backvicious cycle that would further lower the neuropeptide levels and elevate the A levels. Further
sageke.sciencemag.org/cgi/content/full/sageke;2003/3/pe1
22 JANUARY 2003
to this, the presence of two other positive feedback/vicious cycles, likely to contribute to A
accumulation, has been implicated. One involves an increased synthesis of APP. Another one
involves an increase in the expression of -secretase, or BACE 1.

neprilysin activity/expression, as well as aselective substrate for neprilysin in vivo, thenthe activity/expression of nepri-lysin will
decrease in a negative feedback manner inorder to maintain the homeostasis of theneuropeptide concentrations (see Animation
5
http://sageke.sciencemag.org/
cgi/content/full/sageke;2003/3/pe1/DC1).
Such a mechanism would probably bebene-ficial to the brain in the short term, as it
would compensate for the reduced synthesisof the ligand peptide. This would have beentrue particularly in the times when mosthuman beings lived only 50 to 60 years.

accumulation have been described (seeAnimation 5 http://sageke.sciencemag.org/
cgi/content/full/sageke;2003/3/pe1/DC1).
One involves an increased synthesis of APP
http://sageke.sciencemag.org/cgi/genedata/
sagekeGdbGene;197, as observed by someinvestigators in AD brains (28). This increaseis presumably associated with theneuro-protective functions of the soluble
form of APP (28) in response to thepathological conditions present. A second
process involv-es an increase in theexpression of -secretase (also known as-site APP cleaving enzyme 1, or BACE 1)(as observed in thefrontal cortex of AD
patients)(46),

Animation 6. Generation of A from APP. APP first undergoes
limited proteolysis catalyzed by -secretase, or BACE 1, and thencleaved by -secretase, in which presenilin is an essential
component, generating A. A pathway alternative to -secretasecleavage of APP is proteolysis conducted by -secretase(s), thecandidates for which include ADAMs 9, 10, and 17. The former
pathway is considered to be amyloidogenic while the latter
non-amyloidogenic.

An apparent adverse side effect of thisnegative feedback mechanism is an increase
in A concentrations particularly at
pre-synapses, as demonstrated by reverse
gene-tics experiments using neprilysin-KO
mice
(18,
39).
If
A
n
remaineleva-ted long enough to cause presynaptic
dys-functionordegeneration,asdemonstrated by a number of studies using
human tissues and mouse models thatoverexpress APP (44, 45), this side effectwould positively feed back on itself in amanner that would further lower theneuropeptide levels, due to a reduction in theabilityofneuronstosynthesize
neuro-peptides. Such a process would resultin a further elevation of the concentration ofA. The presumed presence of such a
positive feedback-based, vicious cycle ismathematic-ally consistent with the fact thatthe accumulation of A in the brain takes
place very slowly in the initialpresymptomatic stages of AD, but thenincreases gradually, and finally escalates
catastrophically in what might evencorrespond to an essentially exponential
process (23).
Further, two other positive feedback/vicious cycles likely to contribute to A

the accumulation of A with advancing age
leading to the development of SAD, adisorder in which aging is by far thestrongest risk factor.
A New Strategy to Control Brain A
Concentrations
Using
G
Protein-coupled Receptor (GPCR)
Ligand(s)Although we have not yet identified theligand-receptor system involved in the
main-tenanceof
neprilysin
expression/activity
schematizedinAnimation3,
http://sageke.
sciencemag.org/cgi/content/full/sageke;2003/
3/pe1/DC1 somatostatin is a strong candidate
ligand for the following reasons: (i)Animation 7. Proposal of a new strategy to control brain Aconcentrations through modulating GPCR. Although we have notyet identified the ligand-receptor system involved in the maintenanceof neprilysin expression/activity, somatostatin is a strong candidate
ligand for various reasons (see the main text). Because somatostatin
receptor subtype 4 is exclusively expressed in the hippocampal
formation and neocortex, if somatostatin is one of the major ligands inthe neprilysin maintenance system, then a non-peptidic agonist
specific for this receptor would selectively and specifically mobilize theligand-receptor system only in these brain regions and thus should
yield minimal systemic side effects. This strategy has three majoradvantages: (i) increased A degradation, (ii) enhanced memory, and(iii) possibly the activation of -secretase(s) represented by some of
the ADAMs family members.

indicating that theincrease in BACE 1
occurs at a relatively
late stage in the
cascadeofAD
development,
according to the different pathologi-cal
stages of AD development established by
Braak and Braak (47). See Animation 6
http://sageke.sciencemag.org/cgi/content/full/sageke;2003/3/pe1/DC1 for the generation ofA by BACE 1 (-secretase) and thepresenilin complex (-secretase).
While none of these cyclic processes arelikely to have had any apparent pathogenic
effects during past epochs of the human race
when most people died prior to the age of 60,
taken together with an increasing life-span,they could synergistically result in the
pro-gressive accumulation of A in the brain
and thereby contribute (in conjugation withother genetic and epigenetic risk factors) toan in-creased propensity to develop AD. Thisis essentially true of countries wherein theaver-age life-span has approached or isapproa-ching 80 years. While provocative, it
is note-worthy that no published studies on
AD or AD-related topics support orcontradict this hypothesis. However, it
provides a logical, relevant explanation forsageke.sciencemag.org/cgi/content/full/sageke;2003/3/pe1

somato-statin precursor is highly expressedin the hippo-campus and neocortex (43); (ii)soma-tostatin has been shown, in a very
reproduc-ible manner, to be decreased in thebrains of AD patients; (iii) the somatostatin
receptor subtype 4 is exclusively expressedin the hippocampus and neocortex (see nextpara-graph); (iv) somatostatin is anextremely
good
substratefor
neprilysin-catalyzed pro-teolysis (48); (v)even in primary cultured neurons, anddespite the difficulty pointed out in the fourth
section of this Perspective, somatostatinseems to up-regulate neprilysin activity tosome extent (49); (vi) somato-statin has beenshown to improve long-term potentiation(LTP), a principal measure for memory, inhippocampal slices (50); and (vii) unlike inneutrophils, treatment of mice with morphine
does not have any effect on neprilysin
activity or A levels in the brain (51), thus
excluding the opioid peptides as candidate
ligands.
As indicated above, the type 4

22 JANUARY 2003

Page 5somato-statin receptor subtype is exclusively
ex-pressed in the hippocampus and neocortex(52). Therefore, if somatostatin is one of the
major ligands in the neprilysin maintenancesystem, then a non-peptidic agonist specificfor the type 4 somatostatin receptor would
selectively and specifically mobilize theligand-receptor system only in these brain
regions and thus should yield minimal
systemic side effects (see Animation 7
http://sageke.sciencemag.org/cgi/content/full/sageke;2003/3/pe1/DC1). This strategy has
three major advantages: (i) increased A
de-gradation, (ii) enhanced memory (throughsupplementing a factor that is beneficial tomemory formation and, at the same time,lacking in AD brains), and (iii) possible
activation of -secretase(s), including the
ADAM proteins (which are disintegrins andmetalloproteases)(see
Animation6
http://sageke.sciencemag.org/cgi/content/full/sageke;2003/3/pe1/DC1), as has been
de-monstrated in cardiac tissues undernormal condi-tions (53). Activation of
-secretase(s) in the brain would lead to a
reduction
inthesynthesisofA
reducing the relative in-volvementof -secretase in APP processing(see Animation 6 http://sageke.sciencemag.org/cgi/content/full/sageke;2003/3/pe1/
DC1).
The advantages of utilizing neprilysin
activity to modulate A concentrations in thebrain include the following: (i) neprilysin
does not influence the processing of APP andother secretase substrates; (ii) neprilysin has
similar Km values for all of its substrates andthus preferentially proteolyzes a substrate inlarge abundance, for example, A in thebrains of patients with AD and with mild
cognitive impairment (MCI), a precursor toAD (16); (iii) neprilysin degrades both
extra-cellular and cell-associated A;(iv) neprilysin is a constitutively active
enzyme and thus does not require activationof an inactive precursor; (v) unlike matrix
metallo-proteinases and plasmin, neprilysin
does not degrade matrix proteins and thus
would not be destructive to structuralproteins (29-31); and (vi) neprilysin degrades
inflammation-associated peptides, such as
substance P, and neurokinins 1 and 2 (29-31)
and thus may attenuate neuroinflammation(see
McGeerReview
http://sageke.sciencemag.org/cgi/
content/full/sageke;2002/29/re3).
These
properties make the use of neprilysin activity
for the prevention and therapy of AD
complementary to the present mainstream
approaches represented by - and -secretaseinhibitors and A vaccination (19-21, 56).
Future Perspectives
We predict that some of the present andfuture anti-A approaches to treat AD, manyof which are based on different strategies,
will be optimally combined in a mannerPage 6similar to that of the "cocktail therapy"
em-ployed for the treatment of Acquired
Immunodeficiency Syndrome (AIDS) (57).
In this latter treatment protocol usedworld-wide, a cocktail of three different
drugs suppresses disease development,
whereas the use of one or two of the three
agents generally fails to be effective.
Moreover, combining anti-A strategies withother stra-tegies, such as those that target
inflammation, oxidative stress, gonadal
steroid
deficiencies,
cholesterolmis-matabolism, tauopathies, etc., will makefuture prevention and therapy options for AD
even more promising.
We are optimistic that the efforts of the
AD research community will eventually
make it possible for A concentrations inhuman brains to be controlled. If this can beaccomplished in the early stages of AD
development,
beforemassiveneurodegenera-tion takes place, it will serve
as a post-symptomatic form of therapy. If it
becomes possible to pre-diagnose the MCI
prodromal
phase
of
AD
then
pre-symptomatic inter-vention can beinitiated. The conversion of what has beeninterpreted as "normal aging" to AD via MCI
appears to be a continuous process caused by
the gradually accelerating accumulation ofseveral brain pathologies, among which
lesions formed by A are prominent. Thus, it
may even be possible to partially control
certain aspects of brain aging by maintaining
low A concentrations throughout our lives.
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63. Acknowledgements: Our research was
supported by grants from RIKEN BSI,
Ministry of Education, Culture, Sports, Scienceand Technology, and Ministry of Health, Labor,and Welfare of Japan, as well as from the
National Institutes of Health (National Instituteon Aging) and the Alzheimer’s Association,and by a personal donation from Shigeru
Sawada, whose mother died of Alzheimer’s
disease.

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