drosophila rsk negatively regulates bouton number at the neuromuscular junction

Drosophila RSK Negatively Regulates BoutonNumber at the Neuromuscular Junction

Matthias Fischer,1* Thomas Raabe,2 Martin Heisenberg,3 Michael Sendtner1

1 Institute for Clinical Neurobiology, University of Wurzburg, Wurzburg 97078, Germany

2 MSZ, University of Wurzburg, Wurzburg 97078, Germany

3 Institute for Genetics and Neurobiology, University of Wurzburg, Biozentrum,Wurzburg 97074, Germany

Received 2 October 2008; revised 10 December 2008; accepted 15 December 2008

ABSTRACT: Ribosomal S6 kinases (RSKs) are

growth factor-regulated serine-threonine kinases partic-

ipating in the RAS-ERK signaling pathway. RSKs have

been implicated in memory formation in mammals and

flies. To characterize the function of RSK at the synapse

level, we investigated the effect of mutations in the

rsk gene on the neuromuscular junction (NMJ) in

Drosophila larvae. Immunostaining revealed transgenic

expressed RSK in presynaptic regions. In mutants with

a full deletion or an N-terminal partial deletion of rsk,an increased bouton number was found. Restoring the

wild-type rsk function in the null mutant with a genomic

rescue construct reverted the synaptic phenotype, and

overexpression of the rsk-cDNA in motoneurons reduced

bouton numbers. Based on previous observations that

RSK interacts with the Drosophila ERK homologue

Rolled, genetic epistasis experiments were performed

with loss- and gain-of-function mutations in Rolled.

These experiments provided evidence that RSK medi-

ates its negative effect on bouton formation at the Dro-sophila NMJ by inhibition of ERK signaling. ' 2009

Wiley Periodicals, Inc. Develop Neurobiol 69: 212–220, 2009

Keywords: neuromuscular junction; Drosophila; ribo-

somal S6 kinase; RSK; ERK

INTRODUCTION

RSKs (90 kDa ribosomal S6 kinase) comprise a fam-

ily of serine-threonine kinases that are involved in

signaling processes mediated by the RAS-ERK path-

way. RSKs are activated in response to growth fac-

tors, neurotransmitters, peptide hormones, and other

stimuli (Frodin and Gammeltoft, 1999; Hauge and

Frodin, 2006). Four isoforms (RSK 1-4) are known in

mammals, in Drosophila melanogaster only one iso-

form has been identified (Wassarman et al., 1994).

RSKs act on cytosolic substrates like GSK3b and

BAD. In the nucleus, they phosphorylate histones and

transcription factors, for instance CREB, ATF4, c-

FOS, c-JUN, and NUR77 (Frodin and Gammeltoft,

1999). Besides other impairments, mutations in the

rsk2 gene lead to memory defects. The Coffin-Lowry

Syndrome (CLS) is an X-linked syndromic form of

mental retardation caused by mutations of the human

rsk2 gene. Memory defects were also observed in

two independent RSK2 knock-out mouse strains

(Dufresne et al., 2001; Poirier et al., 2007). Loss-of-

function mutations in the Drosophila rsk gene cause

*Present address: Department for Psychiatry, Psychosomaticsand Psychotherapy, University of Wurzburg, Fuchsleinstraße 15,Wurzburg 97080, Germany.

Correspondence to: M. Fischer ([email protected]).

Contract grant sponsor: IZKF (University of Wurzburg).Contract grant sponsor: Deutsche Forschungsgemeinschaft; con-

tract grant numbers: SFB581/B14, SFB581/B4.' 2009 Wiley Periodicals, Inc.Published online 21 January 2009 in Wiley InterScience (www.interscience.wiley.com).DOI 10.1002/dneu.20700

212

memory defects in operant and classical conditioning

(Putz et al., 2004) and in spatial memory during loco-

motion (Neuser et al., 2008).

Despite extensive studies of the behavioral conse-

quences of disturbed RSK2 function, little is known

about the function of RSKs at the cellular and synap-

tic level in neuronal cells. Beacuse Drosophila RSK

acts as a negative regulator of ERK-dependent differ-

entiation processes in the eye and the wing (Kim

et al., 2006), we investigated whether RSK influences

synapse formation. Therefore, we investigated the

effect of rsk gene mutations on the development of

the neuromuscular junction (NMJ) in Drosophila lar-

vae, which have been widely used in the past to study

the role of individual genes on synapse formation.

Previous reports provided evidence that RAS posi-

tively regulates bouton number at the DrosophilaNMJ (Koh et al., 2002). A hypomorphic mutant of

ras1 exhibits less boutons, while presynaptic expres-

sion of a transgenic wild-type or constitutively active

version of RAS increases bouton number. The selec-

tive activation of the RAS/ERK pathway by a RAS

variant that only activates ERK signaling causes the

same effect. Consistent with this result, a gain-of-

function allele of the Drosophila ERK homologue

Rolled (RL), rlSem (Brunner et al., 1994), also caused

an increase in bouton number. Both RAS and RL are

enriched at synaptic boutons of the NMJ. On the

other hand, Drosophila RSK was described as a nega-

tive regulator of ERK/RL-dependent differentiation

processes during eye and wing development (Kim

et al., 2006). Genetic epistasis experiments and inter-

action studies in these systems showed that RSK acts

at the level of RL, and that binding of RSK retains

RL in the cytoplasm. Conversely, loss of RSK func-

tion resulted in nuclear translocation of RL and in an

increase of RL-dependent gene transcription in the

developing eye.

We observed that rsk mutant Drosophila larvae

exhibit enhanced bouton numbers, and that restora-

tion of rsk function in the null mutant reverted this

effect. Overexpression of the rsk-cDNA in motoneur-

ons reduced bouton numbers. Genetic epistasis

experiments by crossings with loss- and gain-of-func-

tion erk mutants indicate that this effect occurs

through inhibition of the MAPK pathway. The inhibi-

tory function of RSK on bouton formation in Dro-sophila resembles its role as a negative regulator of

ERK in eye and wing formation (Kim et al., 2006).

Our data indicate that RSK has a physiological role in

controlling synapse architecture, and disturbance of

this effect could contribute to the learning defects

observed in flies, mice and humans with rskmutations.

MATERIALS AND METHODS

Drosophila Stocks

Df(1)ignD58/1 and Df(1)ignD24/3 were isolated in a screen for

learning mutants (Putz et al., 2004). UAS:RSK was kindly

provided by E. Hafen (Zurich, Switzerland; Rintelen et al.,

2001). GAL4-D42 was provided by the Bloomington Dro-sophila Stock Center. The loss-of-function mutation in Dro-sophila ERK, rl10a, and the gain-of-function mutation rlSem

are described in Brunner et al. (1994). Stocks were main-

tained and raised on standard cornmeal food (Guo et al.,

1996) in a 14–10-h light-dark cycle at 258C and 60% rela-

tive humidity.

Immunohistochemistry

Larval neuromuscular staining was performed as according

to the procedure described in Godenschwege et al. (2004).

For assessment of bouton number the SAP 47 antibody

nc46 (Reichmuth et al., 1995) was used (mouse; 1:100).

The mouse antibody nc82 is directed against active zones

(Wagh et al., 2006) and was used at a dilution of 1:100.

Further antibodies used were: rabbit anti-GFP (1:500; Mo-

lecular Probes, Invitrogen, Carlsbad, CA); mouse anti-CSP

(1:100; Zinsmaier et al., 1994); guinea pig anti-RSK

(1:200). Alexa Fluor 488 and Alexa Fluor 546 conjugated

secondary antibodies were purchased from Invitrogen and

used at dilutions of 1:300. For visualization of muscle tissue

Alexa Fluor 546 labeled Phalloidin (1:1000; Molecular

Probes, Invitrogen, Carlsbad, CA) was added to the second-

ary antibody solution. Preparations to be compared were

stained and processed identically. Fixed larvae were

observed under a Leica confocal microscope (TCS SP2;

Leica, Bensheim, Germany).

Generation of Drosophila RSK Antiserum

A cDNA fragment encoding the C-terminal 72 amino acids

of RSK (amino acids 840-911) was amplified by linker

PCR and cloned into EcoRI/XhoI cut pGEX-6P1 (GE

Healthcare, Munchen, Germany). Expression of the GST-

fusion protein in E. coli was induced with 0.1 mM IPTG for

4 h at 258C. The GST-fusion protein was purified according

to standard procedures and used for immunization of guinea

pigs by a commercial supplier (Eurogentec, Cologne,

Germany).

Generation of GFP-Tagged Genomic RSKRescue Construct

The 6.5 kb genomic rescue fragment described in Putz et al.

(2004) was cloned into the EcoRV site of the pBS vector

(Stratagene, Cedar Creek, TX) to generate pBS-genRSK. A

1.2 kb XhoI fragment derived from pBS-genRSK was

subcloned into pBS and used for in vitro mutagenesis to

change the first two codons of RSK (ATG CCG) into ATG

Drosophila RSK Regulates Bouton Number 213

Developmental Neurobiology

CAT, which corresponds to a NsiI restriction site. This site

was used to introduce the GFP coding sequence amplified

from pEGFP-C1 (BD Biosciences, Franklin Lakes, NJ).

The resulting construct was cut out with XhoI and cloned

back into XhoI cut pBS-genRSK. The complete genomic

fragment (gen-GFP-RSK) was then cloned with Asp718/

XbaI into the pW8 transformation vector (Klemenz et al.,

1987). Several independent transgenic lines were generated

by injecting Qiagen-purified plasmid DNA into w1118

embryos.

Statistical Analysis

Results are given as mean 6 SEM. Statistical significance

was assessed by unpaired t-test when comparing two

groups. For analysis of statistical differences between more

than two groups one-way ANOVA followed by Newman-

Keuls posthoc comparison tests were used. For all statistical

analyses Graph-Pad Prism software was used (GraphPad

Software, San Diego, CA).

RESULTS

Bouton Number at the NMJ is Increasedin Full and Partial Deletion Mutants

Boutons were counted at the NMJ at segment A3 in

muscle 6/7 of third instar larvae with a full deletion

of the rsk gene, Df(1)ignD58/1 (58/1) [Fig. 1(A)] and

with a N-terminal deletion of 1322 bp, removing part

of the first exon, Df(1)ignD24/3 (24/3). To minimize

the influence of the genetic background, both fly lines

were cantonized for at least eight generations to wild-

type CantonS (CS). Bouton numbers at this NMJ

were significantly higher in both the full deletion mu-

tant and the N-terminal deletion mutant compared to

the CS control [Fig. 1(B)]. We also determined bou-

ton numbers per muscle area, and the difference was

even more prominent [Fig. 1(C)]. This difference is

not because of a selective increase in the number of

one of the two types of boutons (type 1s and 1b)

Figure 1 Bouton number is increased in rsk mutants. (A) Representative images of NMJs from

wildtype CantonS (CS) and Df(1)ignD58/1 (58/1) animals stained with nc46 (SAP 47). bar ¼ 50 lm.

(B) Absolute number of boutons in CantonS (CS), Df(1)ignD58/1 (58/1) and Df(1)ignD24/3 (24/3)

(58/1: 80.71 6 4.57 boutons, N ¼ 21; 24/3: 83.14 6 6.79 boutons, N ¼ 14; WT-CS: 56.90 6 4.6

boutons, N ¼ 20; p < 0.05). (C) Bouton number per area of muscle 6/7 in segment A3 (58/1: 113.9

6 5.41 boutons/0.1 mm2, N ¼ 21; 24/3: 110.1 6 6.3 boutons/0.1 mm2, N ¼ 14; CS: 79.35 6 5.71

boutons/0.1 mm2, N ¼ 20; p < 0.001). (D) Quantification of type 1s boutons (58/1: 35.81 6 3.85

boutons, N ¼ 21; 24/3: 40.29 6 3.62 boutons, N ¼ 14; WT-CS: 24.95 6 2.38 boutons, N ¼ 20; p< 0.05). (E) Quantification of type 1b boutons (58/1: 45.00 6 3.07 boutons, N ¼ 21; 24/3: 42.86 6

4.0, N ¼ 14; WT-CS: 31.95 6 2.8 boutons, N ¼ 20; 58/1 different from WT-CS with p < 0.05; 24/3 different from WT-CS with p < 0.01).

214 Fischer et al.


within this muscle segment since both types of bou-

tons show higher absolute numbers [Fig. 1(D,E)] and

higher numbers per muscle area (data not shown) in

the mutants.

Rescue of Bouton Phenotype

To prove that deletion of the rsk gene is responsible

for the bouton number phenotype, we performed res-

cue experiments with a transgenic line carrying a 6.5

kb genomic fragment of the rsk gene on the second

chromosome (gen-RSK (T1), Putz et al., 2004). Previ-

ous Western blot analysis of whole fly lysates showed

that transgenic and endogenous RSK proteins are

expressed at comparable levels (Putz et al., 2004).

Female Df(1)ignD58/1 flies were crossed with gen-RSK (T1) male flies, and bouton numbers were

counted in the male progeny (Df(1)ignD58/1/Y; gen-RSK (T1)/+ in comparison to Df(1)ignD58/1/Y; +/+).

Bouton number was reduced in the presence of the

gen-RSK (T1) construct [Fig. 2(A)]. The same was

true for bouton number per muscle area [Fig. 2(B)],

thus demonstrating that the effect could be rescued

by transgenic rsk expression in the null mutant. The

experiments with the genomic rescue construct do not

allow distinguishing whether RSK function is

required at the pre- or postsynaptic level. Therefore,

we used the GAL4/UAS system (Brand and Perri-

mon, 1993) to express RSK in larval motoneurons in

Df(1)ignD58/1 flies. In third instar larvae, the driver

strain Gal4-D42 expresses Gal4 in the CNS and, at

high levels, in motoneurons, including nerve termi-

nals and synaptic boutons (Yeh et al., 1995). We

observed a significant rescue but bouton number was

still at a higher level than in the control larvae, so we

cannot fully exclude a postsynaptic component of

RSK function [Fig. 2(C,D)]. We also expressed RSK

in WT flies with the Gal4-D42 line. By this we

induced the opposite effect than observed with the

rsk deletion mutants. Bouton number was reduced

compared to controls carrying only the Gal4-D42 or

the UAS:RSK transgene [Fig. 2(E,F)]. This is a further

hint that a function of RSK at the presynapse is re-

sponsible for the phenotype at the NMJ.

Average Number of Synapses isUnchanged in rsk Mutant

A change in the number of boutons may be accompa-

nied by a compensatory change in the number of syn-

apses, as previously shown for mutations of the

cAMP pathway. In larvae over-expressing the dncphosphodiesterase or in the rut1 mutant, both of

which have a reduced numbers of boutons, the

amount of dense bodies per synapse and the area of

synapses in individual boutons were increased

(Shayan and Atwood, 2000). To see if RSK has an

impact on the number or size of individual synapses

we measured the mean fluorescence intensity of nc82

staining which should be proportional to the number

and size of active zones. The antibody nc82 recog-

nizes the active zone protein Bruchpilot [Wagh et al.,

2006; Fig. 3(A)]. We quantified the signal at muscle

6/7 in segment A3 of third instar larvae either as

mean fluorescence intensity (intensity per active zone

area as identified by nc82 staining) or as the 5000

brightest pixel to reduce the influence of background.

Moreover, we calculated the average area of a single

active zone and the number of active zones per bou-

ton area. There was no difference in mean fluores-

cence intensity [Fig. 3(B)] and 5000 brightest pixel

[Fig. 3(C)]. There was a tendency towards a reduced

fluorescence intensity in the Df(1)ignD58/1 null mu-

tant, which could be interpreted as a compensatory

reduction in synapse number but this effect was not

significant. The number of active zones per bouton

area and the average active zone area was unchanged

as well [Fig. 3(D,E)]. We conclude that RSK does not

regulate bouton number independently from synapse

number as shown for cAMP.

Localization of RSK at the NMJ

To determine where the RSK protein localizes at the

NMJ, we raised an antiserum against the C-terminal

region of RSK. In Western blots of whole fly lysates

from wild-type flies, the antiserum recognizes a band

that corresponds in size to the RSK protein, and this

band is absent in lysates from Df(1)ignD58/1 animals

[Fig. 4(A)]. Unfortunately, this antiserum was not

sensitive enough to recognize endogenous RSK at the

NMJ of wild-type CS third instar larvae. Only a faint

signal could be detected, which was probably unspe-

cific because a similar signal was observed in the

Df(1)ignD58/1 mutant (data not shown). However,

when the RSK protein was over-expressed in moto-

neurons by crossing UAS:RSK transgenic flies with

the Gal4-D42 driver line, a strong signal became de-

tectable in boutons that colocalized with the Cystein

String Protein (CSP), a marker of synaptic vesicles

[Mastrogiacomo et al., 1994; Fig. 4(B)]. Double-

staining with the antibody nc82, which recognizes the

Bruchpilot protein at presynaptic active zones (Wagh

et al., 2006), revealed only a partial overlap [Fig.

4(C)]. Thus, the RSK protein localizes to the presy-

napse but is not specifically enriched at active zones.



Figure 2 (A) Expression of a genomic rsk construct in the full deletion mutant reduced absolute

bouton number to WT levels (Df(1)ignD58/1/Y; +/+: 78.40 6 3.29 boutons, N ¼ 43; Df(1)ignD58/1/Y;gen-RSK (T1)/ +: 65.00 6 2.82 boutons, N ¼ 45; p < 0.01). (B) Also bouton number per muscle

area was rescued (Df(1)ignD58/1/Y; +/+: 118.5 6 5.29 boutons/0.1 mm2, N ¼ 43; Df(1)ignD58/1/Y;gen-RSK (T1)/ +: 102.2 6 4.39 boutons/0.1 mm2, N ¼ 45; p < 0.05). (C) Presynaptic expression of

RSK in the full deletion mutant significantly reduced absolute bouton number (Df(1)ignD58/1/Y;UAS:RSK: 83.34 6 3.6 boutons, N ¼ 41; Df(1)ignD58/1/Y; UAS:RSK; GAL4-D42: 70.58 6 3.99

boutons, N ¼ 33; GAL4-D42: 57.38 6 6.253 boutons, N ¼ 8; p < 0.05). (D) Also bouton number

per muscle area was rescued (Df(1)ignD58/1/Y; UAS:RSK: 136.8 6 7.31 boutons/0.1 mm2, N ¼ 41;

Df(1)ignD58/1/Y; UAS:RSK; GAL4-D42: 101.1 6 7.05 boutons/0.1 mm2, N ¼ 33; GAL4-D42: 85.38

6 13.47 boutons/0.1 mm2, N ¼ 8; p < 0.01) (E) Absolute bouton number is reduced in larvae over-

expressing RSK in motoneurons (GAL4-D42/UAS:RSK) compared to larvae carrying the GAL4

driver (GAL4-D42) or the UAS-construct alone (UAS:RSK) (UAS:RSK: 81.41 6 6.14 boutons, N ¼22; GAL4-D42/UAS:RSK: 64.27 6 4.75 boutons, N ¼ 22; GAL4-D42: 82.56 6 4.71 boutons, N ¼27; p < 0.05). (F) Same data as in (E) but normalized to the area of muscle 6/7 in segment A3

(UAS:RSK: 111.1 6 7.24 boutons/0.1 mm2, N ¼ 22; GAL4-D42/UAS:RSK: 84.45 6 6.29 boutons/

0.1 mm2, N ¼ 22; GAL4-D42: 119.4 6 6.04 boutons/0.1 mm2, N ¼ 27; p < 0.001).

To verify whether the endogenous RSK protein

localizes at the NMJ, we modified the genomic rskconstruct (gen-RSK) used for the rescue experiments

described before. The GFP encoding sequence was

inserted 50 to the rsk open reading frame to allow

expression of a GFP-RSK fusion protein from the

native rsk promoter (gen-GFP-RSK). Western blot

analysis of the corresponding transgenic flies verified

expression of the GFP-RSK protein with the expected

molecular weight and with an expression level that

corresponds to the endogenous RSK protein [Fig.

4(D)]. Like the original gen-RSK construct [Fig.

2(A)], the gen-GFP-RSK construct is also able to res-

cue the bouton number defect of Df(1)ignD58/1 ani-

mals [Df(1)ignD58/1/Y; +/+: 93.43 6 7.84 boutons, N¼ 7; Df(1)ignD58/1/Y; gen-GFP-RSK (transgenic line

T2)/+: 60.71 6 5.2 boutons, N ¼ 7; p < 0.01] thus

verifying functionality of the GFP-RSK protein.

Staining of transgenic larvae with an anti-GFP anti-

body confirmed expression of the GFP-RSK protein

at the NMJ [Fig. 4(E)]. Again, the GFP-RSK protein

colocalized with the CSP protein at the presynapse

but was also found in a narrow ring-like structure sur-

rounding the CSP expression domain. This probably

reflects accumulation of the GFP-RSK protein also at

the postsynaptic site because at the NMJ the presyn-

aptic zone faces the postsynaptic region from the

inside. Together with our mutational analysis we con-

clude that RSK is expressed at the NMJ, where it reg-

ulates synaptic bouton number.

RSK Reduces Bouton Numberby Inhibition of ERK

Because Drosophila RSK was described as a negative

regulator of ERK/RL-dependent differentiation proc-

esses during eye and wing development (Kim et al.,

2006) we tested whether RSK controls bouton

Figure 3 The average number/bouton and size of synapses is not affected by the deletion of the

rsk gene. (A) Representative image of a WT NMJ stained with anti-Brp; bar ¼ 10 lm. Mean fluo-

rescence intensity (B; 58/1: 116.5 6 3.3, N ¼ 22; CS: 125.3 6 4.8, N ¼ 24; p > 0.05) and 5000

brightest pixels of anti-Brp staining (C; 58/1: 156.6 6 10.2, N ¼ 22, CS: 175.1 6 13.0, N ¼ 24; p >

0.05) were not significantly different between 58/1 and wild-type CS. (D) The number of synapses

per bouton area was not significantly different between 58/1 and CS (58/1: 22.84 6 1.6, N ¼ 22;

CS: 23.09 6 0.8, N ¼ 24; p > 0.05). (E) The average size of a single synapse was not significantly

different between 58/1 and CS (58/1: 0.52 6 0.037 lm2, N ¼ 22; WT: 0.48 6 0.03 lm2, N ¼ 24;

p > 0.05).



number by regulation of RL activity. The rlSem muta-

tion, which results in the substitution of aspartate res-

idue 334 into an asparagine residue (Brunner et al.,

1994), abolishes binding of the RL protein to RSK

and thus should relieve the inhibitory influence of

RSK on RL-dependent processes. So, the effect of the

rlSem mutation on bouton number should not be sig-

nificantly enhanced in the absence of RSK function

because the RLSem activity is independent of the pres-

ence or absence of RSK. Conversely, down-regula-

tion of RL signaling by removal of one copy of the rlgene should suppress the RSK null phenotype. First,

we confirmed the findings by Koh et al. (2002) that

the loss-of-function allele rl10a does not significantly

affect bouton number in heterozygous rl10a/+ male

larvae, while rlSem/+ larvae have a higher number of

boutons (see Fig. 5). Male progeny from a cross

between homozygous Df(1)ignD58/1 females and rl10a/+ males which were selected for the genotype

Df(1)ignD58/1/Y; rl10a/+ had significantly less boutons

per muscle area than male progeny from a control

cross between Df(1)ignD58/1 females and CS males.

Only the relative bouton number compared to muscle

area was significantly lower but not the absolute bou-

ton number, which could be explained by the fact that

these animals still carry one functional copy of the rlgene (see Fig. 5). From these genetic epistasis experi-

ments we concluded that RSK not only affects differ-

entiation processes during wing and eye development

(Kim et al., 2006) but also bouton formation by inhi-

bition of ERK/RL activity. If this effect is achieved

by binding of RSK to RL, the Df(1)ignD58/1/Y; rlSem/

+ double mutant should have the same number of

boutons as rlSem. This is indeed the case. The double

mutant did not have a higher number of boutons than

each single mutant (see Fig. 5). Because the single

effects on bouton number are nonadditive in the dou-

ble mutant we conclude that the function of RSK in

synaptic growth is mainly mediated by negative regu-

lation of RL activity.

Figure 4 Localization of RSK at the NMJ. (A) Western blot analysis of whole fly lysates from

wild-type (wt) and Df(1)ignD58/1 flies with an antiserum directed against the C-terminal region of

RSK. (B,C) Representative images of single boutons of GAL4-D42/UAS:RSK animals stained with

anti-RSK and anti-CSP (B) and anti-RSK and anti-Brp antibodies (C). The overlay is shown in the

third column. (D) Western blot of wild-type and transgenic flies expressing the GFP-RSK protein

with the RSK antiserum. (E) Larvae expressing the GFP-RSK protein were costained with anti-

GFP and anti-CSP antibodies. Bar ¼ 5 lm.

218 Fischer et al.


DISCUSSION

We have investigated the effect of rsk loss of function

mutations in Drosophila, and found higher numbers

of synaptic boutons in these mutants. The effect could

be rescued by transgenic rsk expression. Vice versaoverexpression of RSK reduced bouton numbers. Fur-

thermore, removal of one allele of the Drosophilaerk/rl gene normalized the effect of rsk loss of func-

tion on bouton formation, indicating that RSK medi-

ates its effect through ERK/RL. Indeed, RSK and

ERK/RL proteins interact directly with each other,

and this interaction is abolished in the rlSem mutant.

Furthermore, rlSem mutants show enhanced bouton

numbers, similarly as rsk mutants, indicating that

RSK negatively regulates ERK/RL activity at the

NMJ and thus modulates bouton formation.

A role of vertebrate RSK2 in inhibition of the

RAS/ERK pathway has been proposed in several

studies, but different underlying mechanisms have

been suggested. In isolated mouse motoneurons,

RSK2 is a negative regulator of axon growth by in-

hibiting ERK phosphorylation (M.F. and M.S.,

unpublished data). In skeletal muscles of RSK2

knock-out mice, increased ERK activation has been

observed (Dufresne et al., 2001). This could be

explained by lack of inhibition of the ERK pathway

via RAS guanine exchange factor SOS (Douville and

Downward, 1997). In Drosophila, this inhibition

seems not to occur through SOS (Kim et al., 2006).

Knockdown of RSK2 leads to increased ERK phos-

phorylation in PC12 cells and cortical neurons (Clark

et al., 2007). Moreover basal and 5HT2A receptor-

mediated ERK 1/2 phosphorylation is increased in

RSK2 knock-out fibroblasts (Sheffler et al., 2006).

These data are consistent with our results showing

that RSK interacts with ERK/RL and that this interac-

tion leads to inhibition of ERK/RL activity in bouton

formation at the NMJ.

Previous studies on RSK and RL in the developing

eye and wing imaginal disc provided evidence that

RSK inhibits translocation of ERK/RL from the cyto-

plasm to the nucleus and thereby controls RL depend-

ent gene transcription (Kim et al., 2006). However,

the NMJ constitutes a separate part of the cell, and it

is also conceivable that the effects of RSK and RL

are mediated locally and do not involve nuclear trans-

location of these proteins. RSK seems to be present

in the presynapse, but its distribution is diffuse and

not restricted to active zones. This corresponds to the

known distribution of RL in axon terminals (Koh

et al., 2002). Thus, it is possible that RSK determines

the localization of RL within synaptic boutons. Inter-

estingly, an antibody that only recognizes active,

phosphorylated RL showed a restricted localization

to spots most likely corresponding to active zones

(Koh et al., 2002). Thus one could speculate that

RSK binds ERK/RL in axon terminals, thus inhibiting

its activation, and only ERK/RL that is unbound can

be activated by phosphorylation and move to active

zones.

In conclusion, our data indicate that RSK nega-

tively regulates bouton formation at the NMJ, and

that negative regulation of RL signaling is involved

in this effect. Thus, Drosophila RSK seems to have a

Figure 5 Genetic interaction of rsk and rl mutations. (A)

The gain-of-function mutant rlSem has more boutons than

CS (CS: 68.20 6 6.23 boutons, N ¼ 15; rlSem/+: 96.64 6

6.49 boutons, N ¼ 14). The number of boutons is not fur-

ther increased by additional removal of the rsk gene

(Df(1)ignD58/1/Y; rlSem/+: 98.42 6 6.44 boutons, N ¼ 12).

The rl10a mutation does not influence bouton number in het-

erozygous animals (rl10a/+: 66.69 6 4.93 boutons, N ¼ 16).

It suppresses the bouton defect of Df(1)ignD58/1, but this

effect is not significant (Df(1)ignD58/1/Y: 90.53 6 7.99 bou-

tons, N ¼ 17; Df(1)ignD58/1/Y; rl10a/+: 74.22 6 5.67 bou-

tons, N ¼ 18); (p < 0.05 for all significant differences). (B)

Same data as in (A) but normalized to the area of muscle 6/

7 in segment A3; the rl10a mutation significantly suppresses

the bouton defect of Df(1)ignD58/1 (CS: 98.00 6 10.78 bou-

tons, N ¼ 15; rlSem/+: 141.3 6 10.1, N ¼ 14; Df(1)ignD58/1/Y; rlSem/+: 160.6 6 13.1, N ¼ 12; Df(1)ignD58/1/Y: 161.5 6

15.6, N ¼ 17; rl10a/+: 103.9 6 5.6, N ¼ 16; Df(1)ignD58/1/Y; rl10a/+: 113.3 6 8.8, N ¼ 18; p < 0.01).



similar function as the RSK2 isoform in vertebrates.

Therefore, the memory defects observed in flies,

mice, and human CLS patients with mutations in rskcould be caused by dysregulated synapse architecture,

as observed in the Drosophila model.

The authors thank Heike Wecklein for excellent techni-

cal assistance and Ernst Hafen for providing fly stocks.

They also thank Stephan Sigrist and Vanessa Nieratschker

for helpful comments.

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