The Drosophila cell adhesion molecule Klingon
is required for long-term memory formation
and is regulated by Notch
Motomi Matsunoa, Junjiro Horiuchia,b, Tim Tullyc, and Minoru Saitoea,1
aTokyo Metropolitan Institute for Neuroscience, 2-6 Musashidai, Fuchu, Tokyo 183-8526, Japan;bLaboratory of Cellular Genetics, Tokyo Metropolitan
University, 1-1 Minami-osawa, Hachioji, Tokyo, Japan, 192-0397; andcDart Neuroscience LLC, 7374 Lusk Boulevard, San Diego, CA 92121
Edited by Jeffrey C. Hall, University of Maine, Orono, ME, and approved November 19, 2008 (received for review August 5, 2008)
The ruslan (rus) mutant was previously identified in a behavioral
screen for mutants defective in long-lasting memory, which con-
ory, and protein synthesis-dependent long-term memory (LTM).
encodes a homophilic cell adhesion molecule. Klg is acutely re-
quired for LTM but not anesthesia-resistant memory formation,
and Klg expression increases upon LTM induction. LTM formation
also requires activity of the Notch cell-surface receptor. Although
defects in Notch have been implicated in memory loss because of
Alzheimer’s disease, downstream signaling linking Notch to mem-
ory have not been determined. Strikingly, we found that Notch
activity increases upon LTM induction and regulates Klg expres-
sion. Furthermore, Notch-induced enhancement of LTM is dis-
rupted by a klg mutation. We propose that Klg is a downstream
effector of Notch signaling that links Notch activity to memory.
memory consolidation ? ruslan ? Alzheimer’s disease
for protein synthesis-dependent long-term memory (LTM) for-
mation (2–4). Upon ligand binding, the Notch receptor is
proteolytically cleaved by presenilin-dependent ?-secretase (5,
6) to release a Notch intracellular domain (NICD), which
activates gene expression in the nucleus. Over-expression of a
dominant-negative Notch, which is defective for NICD function,
inhibits LTM formation (3).
Presenilin-dependent ?-secretase also cleaves the amyloid
precursor protein (APP), generating ß-amyloid peptide (Aß),
and a transcriptionally active APP intracellular domain (AID)
(7). Notably, mutations in presenilin, which cause early-onset
familial Alzheimer’s disease (AD) and selectively enhance pro-
duction of the pathogenic 42-residue Aß peptide (Aß42), also
impair NICD production (8). Furthermore, AID represses tran-
scriptional activity of the NICD by binding to cytosolic inhibitors
of Notch, Numb, and Numb-like (9). Although these observa-
tions suggest that a contributing factor to neuronal dysfunction
and memory loss in some AD patients may be a decline in Notch
signaling, downstream effectors linking Notch signaling to mem-
ory formation have not been identified.
Drosophila have 2 types of long-lasting consolidated memory,
LTM and anesthesia-resistant memory (ARM). LTM of an
aversive olfactory association is produced after spaced training,
10 training sessions with 15 min rest intervals between each
training session, but not after massed training, 10 training
sessions given successively with no rests between training ses-
sions. ARM is produced after both spaced and massed training.
LTM formation requires activity of the cyclic AMP-response
element-binding transcription factor and depends on new pro-
tein synthesis. In contrast, ARM does not require cyclic AMP-
response element-binding activity and can be produced under
he Notch signaling pathway, which plays critical roles in cell
fate specification and differentiation (1), is also important
Previously, a large-scale behavioral screen identified 60 mu-
tants with defective 1-day memory after spaced training (11).
The ruslan (rus) mutation was identified in this screen and was
proposed to be a mutation in the klingon (klg) gene, which
encodes a member of the Ig superfamily of cell adhesion
molecules (CAMs). CAMs play an important role in structural
and functional synaptic plasticity, as well as in learning and
memory. However, few CAMs have been implicated in consol-
idation to long-lasting memory (12, 13).
Although Klg can mediate homophilic adhesion and participates
in the development of the R7 photoreceptor neuron (14), its
function in the adult stage has not been defined. In the present
study, we demonstrate that rus is an allele of klg and Klg is a CAM
required for memory consolidation to LTM but not ARM. Fur-
thermore, we provide evidence indicating that Klg is a downstream
effector linking Notch activity to memory.
ruslan Is a Klingon Mutant and Disrupts Consolidation to LTM. ruslan
consists of an insertion of a P-GAL4 transposon into the first
exon of the klingon (klg) gene (Fig. 1A), suggesting that ruslan is
a new allele of klg [(11), J. Dubnau personal communication,
T.T. unpublished data]. Therefore, we examined Klg expression
in rus mutants and determined that Klg expression is reduced to
?50% of WT in rus mutants (Fig. 1B). We next examined 1-day
memory in a klg mutant. While homozygotes of 2 klg mutations,
dex.html] and klgE226(14), are larval lethal, the klgGS10439/klgE226
heteroallelic mutant is viable and expresses similar amounts of
Klg as rus mutants (see Fig. 1B). We observed a disruption of
1-day memory after spaced training in klgGS10439/klgE226flies
similar to rus flies (Fig. 1C). In contrast, we observed normal
1-day memory after massed training in both rus and klgGS10439/
klgE226flies (see Fig. 1C). Both mutant flies showed normal
learning (3-min memory after single-cycle training), normal
short-term memory (30-min memory after single cycle training)
(Fig. 1D) and normal sensorimotor responses to odors and foot
shocks used for training [supporting information (SI) Fig. S1].
These results indicate that rus and klg mutants are specifically
defective for LTM and this memory defect is not because of
impaired learning or sensorimotor responses.
Both rus and klgE226mutations are recessive for memory
defects. However, rus/klgE226heterozygous flies are defective for
Author contributions: M.M. and M.S. designed research; M.M., T.T., and M.S. performed
research; M.M., J.H., T.T., and M.S. analyzed data; and J.H., T.T., and M.S. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Freely available online through the PNAS open access option.
1To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2008 by The National Academy of Sciences of the USA
January 6, 2009 ?
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1-day memory after spaced training (Fig. 1E), demonstrating
that rus and klg are unable to complement each other. From
these data, we conclude that rus is a new allele of klg (klgrus) and
Although the klgGS10439mutation is also recessive for impaired
memory, a klgrus/klgGS10439line does not have LTM defects (Fig.
S2). This is likely because the klgrusmutation results from
insertion of a P-GAL4 transposon, while the klgGS10439mutation
results from insertion of a P-UAS transposon (see Fig. 1A).
GAL4-dependent induction of klg expression rescues the LTM
defects in klgrus/klgGS10439flies.
Klg Protein Increases After LTM Induction and Adult klg Function Is
Required for LTM Formation. Because LTM formation requires
new protein synthesis, we investigated whether Klg expression
protein in fly heads did not change immediately after spaced
training but increased to a statistically significant level within
24 h (Fig. 2A and Fig. S3). Importantly, the increase in Klg was
specific to LTM, because protein amounts did not change after
massed training or after exposure to spaced training controls,
including US (shock) or CS (odors) alone (see Fig. 2A). In
contrast to protein amounts, we did not observed an increase in
that the increase in Klg upon LTM induction occurs at a
To address whether Klg is physiologically required for LTM
formation, we next conditionally expressed a klg transgene (klg?)
in the adult klg mutant (Fig. 2B). The klgGS10439mutation results
from a P-UAS transposon insertion (15) 75 bp upstream of the
klg transcription start site, oriented in the same direction as klg
transcription (see Fig. 1A and 2C). Consequently, klg can be
inducibly expressed in adult mutants by crossing this line to one
containing a GAL4 driver under heat-shock promoter (hs-
GAL4) control (16).
When hs-GAL4/?;klgGS10439/klgE226(hs-GAL4/?;GS/E) flies
were transferred from 18 °C to 37 °C for 15 min, 3 h before
training, 1-day memory after spaced training improved signifi-
cantly compared to nonheat-shocked controls (see Fig. 2B).
Although the P-UAS insertion in klgGS10439flies is also upstream
of the CG6660 and CG31281 genes, heat-shock increased ex-
pression of klg but not these other neighboring genes in hs-
GAL4/?;GS/? flies (see Fig. 2C), indicating that acutely in-
duced Klg complements klg memory defects.
To further verify the physiological role of klg in LTM, we
acutely inhibited klg expression using a heat-shock inducible klg
RNAi construct in hs-GAL4/UAS-klgRNAi flies. We observed
that a 37 °C heat-shock for 30 min, 5 h before training caused a
significant decrease in 1-day memory after spaced training in
these flies, compared to nonheat-shocked controls (Fig. 2D). In
contrast, the same heat-shock regimen did not disrupt short-
term memory in hs-GAL4/UAS-klgRNAi flies (Fig. S5). The
above results taken together suggest that klg has an acute
physiological role for LTM.
Klg Expression Is Regulated by Notch. Similar to Klg, Notch is
required for LTM formation (2–4) and development of the R7
photoreceptor neuron (17). These phenotypes led us to study
possible interactions between Klg and Notch. To test whether
Klg expression is regulated by Notch signaling, we measured Klg
protein in transgenic flies expressing a dominant negative Notch
transgene (hs-N?cdc10rpts) or a WT Notch transgene (hs-N?)
under heat-shock promoter control (3).
of a P-GAL4 transposon in the first exon (?97 bp), oriented in the opposite direction to klg transcription [(11), J. Dubnau personal communication, T.T.
unpublished data]. The klgGS10439mutation results from an insertion of a P-UAS transposon 75 bp upstream of the proposed transcription start site [(15)
http://gsdb.biol.metro-u.ac.jp/?dclust/index.html]. The klg ORF is entirely deleted in the null klgE226mutation, although the 3? end of the deletion has not been
determined (14). (B) Western blotting shows reduced expression of Klg protein (60 kDa) in adult heads of klgrus(rus) and klgGS10439/klgE226flies (GS/E). ?-tubulin
(Tub) was used as an internal control. (C) Comparison of long-lasting memory produced by spaced or massed training. The klg mutations significantly reduce
1-day memory after spaced training. (*, P ? 0.05 vs. WT by t test). (D) Olfactory learning and short-term memory (memory retention quantified 3 min and 30
min after a single training session, respectively) were not affected in klg mutants. (E) While 1-day memory after spaced training is normal in ?/klgE226(?/E) and
rus/? flies, memory defects in rus are not complemented in rus/klgE226flies (**, P ? 0.005 vs. WT by t test).
Matsuno et al.
January 6, 2009 ?
vol. 106 ?
no. 1 ?
A previous study has demonstrated that a 37 °C heat-shock for
30 min disrupts LTM formation in transgenic hs-N?cdc10rptsflies,
while the same heat-shock enhances LTM formation in trans-
genic hs-N?flies (3). Accordingly, we investigated Klg levels in
transgenic hs-N?cdc10rptsflies and hs-N?flies 5 h after a 37 °C
heat-shock for 30 min. As seen in Fig. 3A, Klg levels in
hs-N?cdc10rptsflies were significantly reduced after heat-shock
compared to nonheat-shocked controls. Conversely, Klg expres-
sion was significantly increased in hs-N?flies after the same
heat-shock treatment compared to nonheat-shocked controls. In
contrast to Klg protein, expression of klg transcripts was not
dependent on Notch activity (Fig. S4B). Our results suggest that
Notch regulates Klg protein expression at a posttranscriptional
step, such as at protein synthesis and turnover.
The N?cdc10rptsprotein binds ligands normally but lacks NICD
activity and is defective for signaling downstream from Notch
(3). This led us to hypothesize that generation of the NICD may
be important for LTM formation and we measured NICD
protein levels after spaced training. We observed a significant
increase in NICD amounts in the heads of spaced-trained flies
3 and 6 h after training, as compared to nontrained flies (Fig.
3B). We also observed a slight increase in NICD amounts in
heads from mass trained flies, but this increase was not signif-
icantly different from untrained control flies and was signifi-
cantly lower than in spaced-trained flies.
Because Klg protein levels are regulated by Notch, we next
hypothesized that the increase in Klg protein after induction of
LTM may also depend on Notch signaling activity. Thus, we
investigated the amount of Klg in hs-N?cdc10rptsflies after spaced
training (Fig. 3C). In the absence of heat shock, hs-N?cdc10rpts
flies displayed a significant increase in Klg protein 24 h after
spaced training, similar to WT flies. However, this increase was
abolished when a 37 °C heat-shock was given for 30 min 5 h
before training, suggesting that the LTM-associated increase in
Klg requires NICD activity.
Klg Is Required for Notch-Dependent LTM Formation. We next asked
whether klg is required for Notch’s effects on LTM formation.
Acute over-expression of Notch in transgenic hs-N?flies, in-
duced by a 37 °C heat-shock for 30 min, has previously been
shown to enhance 1-day memory after a single-cycle training
session (3). Although aversive olfactory memory formed by
single-cycle training is normally consolidated into ARM and not
LTM (10), the enhanced 1-day memory in hs-N?flies is blocked
by the protein synthesis inhibitor, cycloheximide, indicating that
Notch over-expression specifically enhances LTM (3). We there-
fore examined the effect of a klg mutation on the enhancement
of LTM formation by Notch.
Significantly, memory enhancement upon Notch over-
expression was prevented in klg mutants. While heat-shocked
hs-N?flies showed a greater than 4-fold increase in 1-day
memory after single-cycle training, neither hs-N?;klgrusnor
hs-N?;klgGS10439/klgE226flies showed this increase (Fig. 4), dem-
onstrating that regulation of Klg by Notch is an essential step for
Notch-dependent LTM formation.
In the present study, we demonstrate that the memory mutant,
rus, is a previously uncharacterized allele of klg. klg was originally
identified as a gene which encodes a homophilic CAM involved
24 h after spaced training (***, P ? 0.001), but not after massed training or after spaced application of the unconditioned stimulus (US) or conditioned stimuli
after spaced training in hs-GAL4/?;klgGS10439/klgE226(hs-GAL4/?;GS/E) flies (*, P ? 0.04), indicating that the klg memory defect can be complemented by
conditional expression of klg?. (C) Comparison of the expression of P-UAS downstream gene transcripts in hs-GAL4/?;klgGS10439/? flies 3 h after the heat-shock
(?hs). While heat-shock enhanced the expression of klg and GAL4 transcripts, it did not alter the expression of two other downstream genes, CG6660 and
CG31281. RNA was isolated from Drosophila whole bodies and transcripts were quantified by semiquantitative RT-PCR. (D) RNAi-mediated silencing of klg
disrupts LTM. A 37C°C heat-shock (?hs) for 30 min significantly disrupted 1-day memory after spaced training in hs-GAL4/UAS-klgRNAi (hs-GAL4/klgRNAi) flies
(**, P ? 0.005).
Klg increases upon LTM induction and LTM depends on Klg expression. (A) Klg protein amounts, measured from fly head extracts, increase significantly
www.pnas.org?cgi?doi?10.1073?pnas.0807665106Matsuno et al.
using 2 independent genetic interventions: induced over-
expression of klg?in klg mutants (see Fig. 2B) and acute
silencing of klg in WT flies (see Fig. 2D).
Functional roles of CAMs in learning and memory have been
previously studied. For example, Drosophila Fas II, a homologue
of vertebrate neural CAM, is a homophilic CAM that regulates
synaptic stabilization and growth in an activity-dependent man-
in the adult stage (19). Drosophila ?-integrin, a molecule that
mediates cell adhesion and signal transduction, is required for
early-phase memory (20) and also regulates structural and
functional synaptic plasticity (21). Likewise, neural CAM and
integrin-mutant mice display impaired learning and memory as
well as reduced long-term potentiation (22, 23). Besides roles in
learning and early-phase memory, CAMs have also been impli-
cated in memory consolidation. N-cadherin is synthesized upon
induction of late-phase long-term potentiation, a putative cel-
lular basis for LTM, and recruited to newly formed synapses
(12). Functional modification of neural CAM by polysialic acid
Our results demonstrate that Klg is another CAM critical for
memory consolidation. Interestingly, immunohistochemical ex-
periments demonstrate extensive localization of Klg protein at
the junctures between the neuropil and neuropil glia, including
the junctures between the lobes and calyces of the mushroom
bodies and the surrounding glial cells (data not shown). The
?-lobes of the mushroom bodies have been implicated in LTM
formation (24) and inhibition of Notch activity in the mushroom
bodies impairs LTM formation (4). These results suggest that
Klg may be involved in acute neuron-glia interactions required
for LTM formation.
The importance of Notch signaling in memory formation has
been demonstrated both in Drosophila and mice (2–4). Increas-
ing Notch activity facilitates LTM formation, while reducing
activity severely impairs LTM (3, 4). Because presenilin-
dependent ?-secretase activity generates the transcriptionally
active NICD, and familial AD-associated mutations in presenilin
disrupt this step, a decline in Notch signaling activity has been
proposed to be a causal factor in memory impairment in AD
(25). In the present study, we provide evidence indicating that
Klg is a downstream effector molecule linking Notch activity to
memory formation. Furthermore, we demonstrate an increase in
NICD amounts upon induction of LTM (spaced training).
Although we also observed a slight increase in NICD amounts
after massed training, this increase is significantly lower than
after spaced training and does not cause an increase in Klg
protein. These results are consistent with the observation that
massed training is insufficient to generate LTM. Because the
NICD is generated by a presenilin-dependent cleavage, our
results further suggest that presenilin-dependent ?-secretase
activity may be required for LTM formation. In support of this
in the mouse forebrain results in severe impairment of spatial
and contextual memory and impaired synaptic plasticity (26).
Although transcriptional activity of Notch regulates Klg pro-
tein levels, Notch is unlikely to directly regulate transcription of
the klg gene, because the increase in Klg protein upon LTM
induction is not accompanied by an increase in klg transcripts.
These results suggest that in response to induction of LTM,
Notch signaling is activated to regulate either synthesis or
turnover of the Klg protein. Supporting this idea, we observed
a delay in the increase of Klg protein compared to the increase
of the NICD. While Klg protein gradually increases over 24 h
after spaced training, the increase in the NICD reaches a plateau
within 6 h after training. In addition to transcriptional regula-
tion, recent studies have demonstrated the importance of strict
regulation of protein synthesis and degradation in LTM forma-
tion (27–29). We suspect that the NICD activates transcription
of a second factor, which in turn stabilizes or increases Klg
Materials and Methods
humidity. w(CS10), derived from outcrossing w1118to Canton-S for 10 gener-
ations, was used as the WT control as previously described (30). klgGS10439and
null klgE226lines were gifts from T. Aigaki (Tokyo Metropolitan University,
Japan) and Y. Hiromi (National Institute of Genetics, Japan), respectively;
w;hs-GAL4/CyO (31) was a gift from Y. Hiromi and transgenic hs-N?and
UAS-klgRNAi line (klg36162) was obtained from the Vienna Drosophila RNAi
require Notch activity. (A) Heat-shock (?hs, 37 °C for 30 min) induction of
a dominant-negative N?cdc10rptstransgene significantly decreases Klg pro-
tein amounts in head extracts from hs-N?cdc10rpts(hs-N?cdc) flies. Conversely,
Klg amounts are up-regulated by induction of a WT Notch transgene in
hs-N?flies. Flies were heat-shocked 5 h before fly head collection. The
Klg/Tub ratio in nonheat-shocked WT flies was defined as 1.0. *P ? 0.05. (B)
Generation of the NICD upon LTM induction. The amounts of NICD in head
extracts of naive flies (n) and flies harvested 3 and 6 h after massed (m) or
spaced (s) training were measured. The amounts of NICD (120 kDa) and full
length of Notch (NFL, 350 kDa) were determined using an anti-NICD mono-
clonal antibody. NICD levels increased significantly in spaced-trained flies
compared to naive and massed-trained flies. The NICD/Tub ratio in naive
WT flies was defined as 1.0.*, P ? 0.03 and**, P ? 0.005. (C) Induction of
the dominant negative N?cdc10rptstransgene in hs-N?cdcflies suppresses the
LTM-dependent increase in Klg protein in fly heads. The N?cdc10rptstrans-
gene was induced by heat-shocking flies for 30 min at 37 °C, 5 h before
collection. Relative Klg amounts in naive flies and in spaced-trained flies at
0 and 24 h after training are indicated.*, P ? 0.05.
Klg is regulated by Notch and LTM-dependent increases in Klg
Matsuno et al.
January 6, 2009 ?
vol. 106 ?
no. 1 ?
Center. Other lines used in this study were obtained from the Bloomington
Stock Center of Indiana University. For all experiments, 3- to 7-day-old flies
were used. All fly lines, except klgE226, were out-crossed to w(CS10) flies for at
least 6 generations.
Fly Behavior. All memory tests were performed in an environmental room
maintained at 25 °C and 60% humidity.
Single-Cycle Training. Standard single-cycle olfactory conditioning was per-
formed as previously described (30, 32). Two aversive odors (OCT and MCH)
were used for CS, and 1.5-s pulses of 60V DC electric shocks were used as the
(the odor that was paired to the US during training) and the CS? (unpaired
with the US). A performance index was calculated so that a 50:50 distribution
(no memory) yielded a performance index of zero and a 0:100 distribution
away from the CS? yielded a performance index of 100 (10).
Spaced and Massed Training. Spaced and massed training sessions were per-
training sessions, with a 15-min rest interval between successive training
cycles. Massed training consists of 10 cycles of training, where one session
immediately follows the previous one. Memory was measured 1 day after
spaced or massed training to evaluate LTM and ARM.
Sensorimotor Responses. Peripheral control experiments, including odor acu-
to verify that sensitivity to the odors and shock were unaffected in our
mutants. About 100 naive flies were tapped into the choice point of a T-maze
in which they had to choose between an odor (OCT or MCH) and mineral oil
and a tube where they were not shocked (shock reactivity). A performance
index was calculated as previously described (10).
Western Blotting. Rabbit anti-Klg polyclonal antibodies were generated
against a 26-mer peptide sequence, CKGSGNPVPSIYWTKKSGANKSTARI, from
the second IgG domain of the Klg protein in New Zealand White rabbits.
Affinity-purified serum obtained from peptide cross-linked to a Hi-Trap af-
finity column (Amersham) recognized the same size Klg band as described
previously (14). An affinity purified rabbit anti-Klg antibody (1:25) and a
monoclonal antibody against NICD (1:100, C17.9C6 from Hybridoma bank)
were used for quantification of Klg and NICD, respectively. A 1:1,000 dilution
of mouse anti-?-tubulin antibody (#DM1a, Seikagaku Kogyo) was used for
normalization. Head extracts were made in homogenization buffer [25 mM
Hepes, 100 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 0.2% Trion X-100, 0.2%
Nonidet P-40 and protease inhibitors (Roche)]. Immunoreactive bands were
were quantified using ImageJ (National Institutes of Health, http://
Quantification of Transcripts by RT-PCR. Total RNA from Drosophila heads or
whole bodies was extracted with TRIzol reagent (Invitrogen) and cDNA was
synthesized using a High Capacity cDNA Archive Kit (Applied Biosystems) as
25 cycles and for qPCR, an Applied Biosystems model 7500 machine was used.
GPDH1 and rp49 transcripts were used for normalization. Primer sequences
are shown in Table S1.
Heat-Shock Treatment. Collected flies were maintained in an 18 °C incubator
for at least 1 day before heat shock treatment to minimize leaky expression.
For heat-shock, flies were transferred to preheated vials and submerged in a
returned to food vials during the recovery period (3 h or 5 h).
Statistical Analyses. All data are expressed as mean ? SEM. Sample sizes are
indicated in each bar in all graphs. Graph Pad Prism version 4.01 was used for
statistical analyses. Unless noted otherwise, data were analyzed by ANOVA
followed by Bonferroni/Dunn posthoc comparisons.
ACKNOWLEDGMENTS. We thank Drs. Y. Hiromi, T. Aigaki, and Y. Zhong for
fly stocks. We also thank H. Ueda for assistance with histology and Dr. T.
was funded by Grant-in-Aid for Scientific Research (B) 14380374 and a grant
from The Uehara Memorial Foundation (to M.S.).
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