Neuron, Vol. 37, 925–937, March 27, 2003, Copyright 2003 by Cell Press
APP Processing and Synaptic Function
tive disorder that is pathologically characterized by ex-
tracellular deposits of ? amyloid (A?) in senile plaques,
intraneuronal neurofibrillary tangles, depressed brain
function, and neuronal death (reviewed in Price and Si-
tion ofA?, asmall peptidewith ahigh propensity toform
aggregates, is central to the pathogenesis of disease
(Selkoe, 2000). Although the potential neurotoxic prop-
et al., 1989), it is still not known how A? participates in
a pathologic cascade resulting in progressive cognitive
decline. It is also not known if A?, which is detected in
both cerebrospinal fluid and plasma in healthy individu-
als throughout life (Seubert et al., 1992), plays a role in
The proteolytic processing pathways leading to the
formation of A? from the amyloid precursor protein
(APP), a type I membrane protein, have been well char-
acterized in a number of cell lines (Figure 1A) (Selkoe,
2000). APP is delivered to the surface membrane where
it is subject to proteolytic processing by ?-secretase.
APP molecules that fail to be cleaved by ?-secretase
can be internalized into endocytic compartments and
tase to generate A?. A fraction of A? peptides are also
generated in the Golgi apparatus and, to a lesser extent,
the endoplasmic reticulum. A? peptides generated in
the Golgi and in recycling compartments can be se-
creted into the extracellular space (Greenfield et al.,
acids in length (A?40), although the smaller fraction of
longer, 42 amino acid species (A?42) have received
to nucleate and drive production of amyloid fibrils (Jar-
rett et al., 1993).
Recent lines of experimental evidence have sug-
gested that excessive amounts of A? are deleterious to
neuronal function, in addition to, or in lieu of, its pro-
posed neurotoxic effects. First, addition of A? in various
aggregation states to neuronal preparations has been
shown to elicit electrophysiological phenotypes (Cullen
et al., 1997; Freir et al., 2001; Hartley et al., 1999; Kim
ing with the peptides, whose biological properties can
depend on aggregation states and peptide size and
composition (Fezoui et al., 2000; Walsh et al., 1999).
Furthermore, the relevant subcellular sites and (patho)-
physiological concentrations are not known and thus
difficult to mimic by exogenous application. Alternative
various naturally occurring familial AD-linked mutants
ological and behavioral consequences of excessive A?
production and accumulation (Chapman et al., 1999;
Fitzjohn et al., 2001; Hsia et al., 1999; Larson et al., 1999;
Westerman et al., 2002). However, the interpretations
of these studies are complicated by the fact that the
transgenes are expressed at high levels throughout de-
velopment and aging. Furthermore, several domains
Flavio Kamenetz,1,2Taisuke Tomita,4
Helen Hsieh,1,3Guy Seabrook,5
David Borchelt,6Takeshi Iwatsubo,4
Sangram Sisodia,7and Roberto Malinow1,2,3,*
1Cold Spring Harbor Laboratory
Cold Spring Harbor, New York 11724
2Graduate Program in Genetics
3Graduate Program in Neuroscience
State University of New York at Stony Brook
Stonybrook, New York 11794
4Department of Neuropathology and Neuroscience
Graduate School of Pharmaceutical Sciences
University of Tokyo
5Merck Research Laboratories
Merck & Company, Incorporated
770 Sumneytown Pike
West Point, Pennsylvania 19486
6Department of Pathology
Johns Hopkins University School of Medicine
Baltimore, Maryland 21287
7Center for Molecular Neurobiology
Department of Neurobiology, Pharmacology,
The University of Chicago
Chicago, Illinois 60637
A large body of evidence has implicated A? peptides
and other derivatives of the amyloid precursor protein
(APP) as central to the pathogenesis of Alzheimer’s
disease (AD). However, the functional relationship of
physiology is not known. Here, we show that neuronal
activity modulates the formation and secretion of A?
peptides in hippocampal slice neurons that overex-
synaptic transmission onto neurons that overexpress
sion depends on NMDA-R activity and can be reversed
by blockade of neuronal activity. Synaptic depression
from excessive A? could contribute to cognitive de-
cline during early AD. In addition, we propose that
duction may normally participate in a negative feed-
back that could keep neuronal hyperactivity in check.
Disruption of this feedback system could contribute
to disease progression in AD.
Alzheimer’s disease (AD), the most common form of
Figure 1. Neural Activity Controls Formation
of APP Cleavage Derivatives
(A) Biochemical pathways leading to the for-
mation of A? from APP. ? and ? cleavage of
APP result in the production of a large, solu-
ble ectodomain (APPs) and a membrane-
associated carboxy-terminal fragment (CTF).
Cleavage of APP by ?-secretase precludes
production of A?. ?-secretase cleavage of
CTFs produce small peptides (A? and p3)
which can be secreted and a truncated CTF
from mice expressing human APPSwe were
trodoxin [TTX], 1 ?M; 10 mM MgCl2; picro-
toxin [PTX], 100 ?M; flunitrazepam [Flu], 1
?M). Media aliquots were collected and ana-
lyzed for A?40 and A?42. Values expressed
as percentage of secretion seen in sister
slices maintained in control media. All values
different from control, p ? 0.05.
(C) Rat organotypic slices were infected (APP
WT, APP WT ? TTX, APP WT ? Mg) or not
dia (Uninf, APP WT), in culture media with 1
uM TTX (APP WT ? TTX), or in culture media
with 10 mM MgCl (APP WT ? Mg) for 24 hr
(n ? 3 for each). Superfusate media was col-
lected and analyzed for A?40 or A?42 as
(D) Western blots of extracts from organo-
typic APPSWEslices in the presence of ?-secre-
therefore aiding fragment detection) treated
with (n ? 8) or without (n ? 8) picrotoxin (100
?M) and 8 mM CaCl2for 36 hr. Blots were
probed with an antibody specific for ?1
BACE cleavage site (3D6) and subsequently
reprobed using CT-15 for detection of full-
length APP. Bands corresponding to the
BACE APP cleavage product and full-length
APP were quantified by scanning.
(E) Western blots of extracts form organotypic APPSWEslices in the presence of L-685,458 and treated with (n ? 3) or without (n ? 3) TTX for
36 hr as in (D). Note that levels of full-length APP are not significantly changed by either blocking or enhancing neuronal activity (control:
1.0 ? 0.13, TTX: 1.0 ? 0.04, p ? 0.9; control: 1.0 ? 0.11, PTX/Ca2?: 1.2 ? 0.15, p ? 0.3).
(F) IP-Western blot of culture media from organotypic APPSWEslices treated for 36 hr with 1 ?m TTX (n ? 8), picrotoxin (100 mm) and 8 mM
CaCl2(n ? 8), or untreated (n ? 8). APPSWEs?was precipitated from culture media using antibody 54, which specifically recognizes the C
terminus of APPSWEs?, and subsequently blotted with the antibody 22C11, which recognizes the N terminus domain of APP. 1 ?m L-685,458
was present in all samples to parallel (D) and (E) above.
within APP have been described that may have impor-
tant physiological functions (Cao and Sudhof, 2001;
Kamal et al., 2001), and thus, the consequence of over-
expressing APP may have unanticipated effects on
physiological and behavioral measures of learning and
memory. We thus sought to develop a system where
the physiologic effects of acute APP overexpression
could be ascertained and in which the relevant molecu-
lar determinants could be dissected. We employed the
Sindbis expression system (Hayashi et al., 2000; Schle-
singer and Dubensky, 1999; Shi et al., 1999; Zhu et al.,
in organotypic hippocampal slices over a period of be-
tween 12 and 72 hr (Stoppini et al., 1991). In this experi-
mental setting, we could determine the effects of APP
overexpression on synaptic transmission, and the ef-
fects of synaptic transmission on APP processing.
The regulatory mechanisms that control A? biosyn-
thesis have received much interest, as these are attrac-
tive targets for therapeutic intervention. While a number
of proteins have been identified whose expression ap-
et al., 1998; Yu et al., 2000), there is little information
about the neuronal mechanisms that modulate A? pro-
duction. Here, we show that neuronal activity regulates
the production and secretion of A? by controlling APP
we report that A? modulates synaptic strength. Taken
together, these two observations suggest a negative
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