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Interactions between ICAM-5 and b1 integrins regulateneuronal synapse formation

Lin Ning1, Li Tian2, Sergei Smirnov2, Helena Vihinen3, Olaya Llano2, Kyle Vick4, Ronald L. Davis4,Claudio Rivera2 and Carl G. Gahmberg1,*1Division of Biochemistry and Biotechnology, Faculty of Biological and Environmental Sciences, University of Helsinki, Finland2Neuroscience Center, University of Helsinki, Finland3Institute of Biotechnology, University of Helsinki, Finland4Department of Neuroscience, Scripps Research Institute Florida, Jupiter, FL 33410, USA

*Author for correspondence (carl.gahmberg@helsinki.fi)

Accepted 15 August 2012Journal of Cell Science 126, 77–89� 2013. Published by The Company of Biologists Ltddoi: 10.1242/jcs.106674

SummaryIntercellular adhesion molecule-5 (ICAM-5) is a dendrite-specific adhesion molecule, which functions in both the immune and nervous systems.ICAM-5 is the only negative regulator that has been identified for maturation of dendritic spines so far. Shedding of the ICAM-5 ectodomain

promotes spine maturation and enhances synaptic activity. However, the mechanism by which ICAM-5 regulates spine development remainspoorly understood. In this study, we found that ablation of ICAM5 expression resulted in a significant increase in the formation of synapticcontacts and the frequency of miniature excitatory post-synaptic currents, an indicator of pre-synaptic release probability. Antibodies againstICAM-5 and b1 integrins altered spine maturation. Furthermore, we found that b1 integrins serve as binding partners for ICAM-5. b1 integrins

were immunoprecipitated with ICAM-5 from mouse brain and the binding region in ICAM-5 was localized to the two first Ig domains. b1integrins were juxtaposed to filopodia tips at the early stage of synaptic formation, but as synapses matured, b1 integrins covered the mushroomspines. Loss of b1 integrins from the pre-synaptic sites affected the morphology of the post-synaptic structures. ICAM-5 ectodomain cleavage

decreased or increased when the interaction between ICAM-5 and b1 integrins was potentiated or weakened, respectively, using antibodies. Theseresults suggest that the interaction between ICAM-5 and b1 integrins is important in formation of functional synapses.

Key words: ICAM-5, Integrin, Adhesion, Neuron, Spine

IntroductionIn the central nervous system (CNS), synapse formation is often

referred to as a process in which initial contacts between axonal

terminals and dendritic filopodia undergo changes in morphology

and molecular content resulting in mature synapses. Besides

neurotransmitter receptors and ion channels, a growing body of

evidence shows that a multitude of synaptic cell adhesion

molecules (CAMs) play important roles in regulating synapse

formation. They stabilize the initial synaptic contacts, recruit

synaptic structural proteins and trigger intracellular signaling to

the actin cytoskeleton that induces synapse formation (Nguyen and

Sudhof, 1997; Torres et al., 1998; Biederer et al., 2002; Dalva et al.,

2007; Ko et al., 2009; Mah et al., 2010; Takahashi et al., 2011).

The intercellular adhesion molecule-5 (ICAM-5, telencephalin)

belongs to the immunoglobulin (Ig) superfamily containing nine Ig-

domains. It is expressed in the soma, dendritic shafts and dendritic

filopodia/spines of excitatory neurons in the telencephalon (Oka

et al., 1990; Benson et al., 1998; Gahmberg et al., 2008; Mitsui

et al., 2005). Previous studies have revealed both immune and

neurodevelopmental functions for this molecule in the CNS (Tian

et al., 2009). The interaction of ICAM-5 with the b2 integrin

lymphocyte function-associated antigen 1 (LFA-1) has been

extensively studied (Mizuno et al., 1997; Tian et al., 1997; Tian

et al., 2000a; Tian et al., 2008; Zhang et al., 2008). It mediates the

binding of leukocytes to hippocampal neurons and induces spreading

of microglia (Mizuno et al., 1999). Furthermore, ICAM-5 induces

dendritic outgrowth by homophilic binding (Tian et al., 2000b).

ICAM-5-deficient mice exhibit decreased density of filopodia and

acceleration of spine maturation (Matsuno et al., 2006), enhanced

long-term potentiation (LTP) and altered learning performance

(Nakamura et al., 2001). In addition, loss of ICAM-5 in brain

accelerates spine maturation on thalamo-recipient cortical neurons

(Barkat et al., 2011). N-methyl-D-aspartic acid (NMDA)-induced

matrix metalloproteinase-2 and -9 (MMP-2 and -9) activation results

in ICAM-5 ectodomain cleavage, which promotes dendritic spine

development and affects LTP (Tian et al., 2007; Conant et al., 2010).

Integrins are heterodimeric trans-membrane cell adhesion

molecules formed by an a- and a b-chain (Hynes, 2002; Chan

and Davis, 2008; Gahmberg et al., 2009). In the CNS, integrins

participate in several neuronal events essential for the development

and remodeling of the brain, such as neural migration, neurite

outgrowth, synapse formation and plasticity, and formation of

working memory (Pinkstaff et al., 1998; Cohen et al., 2000; Shi and

Ethell, 2006; Gardiner et al., 2007; Webb et al., 2007; Cingolani

et al., 2008; Bahr et al., 1997; Chan et al., 2003; Chan et al., 2007;

Chan et al., 2010). However, the localization of b1 integrins in

neuronal subcellular structures is ambiguous, and no clear

correlation with their functional activity has been demonstrated.

In the present study, we found that b1 integrins are expressed on

both pre- and post-synaptic structures. We further identified b1

integrins as counter receptors for ICAM-5 and characterized the

interaction in detail. The binding involves the two first Ig-domains of

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ICAM-5. It occurs at the initial stage of synapse formation, which

may mediate loose, dynamic contacts between pre- and post-synaptic

sites. Blocking or activating of this interaction by antibodies or

knockdown b1 integrins in axons leads to altered spine morphology.

ICAM-5 ectodomain cleavage is prevented by this interaction. These

data expand our knowledge of the physical localization and

physiological function of b1 integrins in brain, and provide a

possible mechanism by which ICAM-5 regulates the maturation of

functional synapses.

ResultsICAM-5 deficiency leads to increased formation of

functional synapses

Previous studies showed that in ICAM-52/2 neurons, there

was an increase in the size and number of spines and the

overlap between synaptophysin and spine heads (Matsuno

et al., 2006), indicating that ICAM-5 not only affects spine

morphology, but also alters the formation of synaptic contacts.

To confirm this, synapsin I, a synaptic vesicle-associated

protein present in axon terminals and post-synaptic density

(PSD)-95 were used as markers for pre- and post-synapses

respectively, and the colocalization of the two markers was

used to evaluate the level of the pre- and post-synaptic

contacts. At 15 day in vitro (DIV), in ICAM-52/2 neurons,

synapsin I puncta accumulated on the spine heads while in WT

neurons, they scatter along the dendritic shafts (Fig. 1A). The

overlap between synapsin I and PSD-95 was significantly

increased in ICAM-52/2 (78%63%) neurons compared with

WT (51%65%) and the colocalization mainly increased in

spine heads (Fig. 1A,B).

Fig. 1. ICAM-5 inhibits the formation of synaptic contacts and functional synapses. (A) Representitive images of dendrites of cultured hippocampal neurons at 15 DIV

from WT (a–c) and ICAM-52/2 (d–f) mice. Colocalization of the PSD-95 (red) and synapsin I (blue) is shown (b and e). (c and f) show the merged images of EGFP-labeled

dendrites (green) and synapsin I. Arrows: synapsin I puncta overlapping with PSD-95. Arrowheads: synapsin I puncta closely associated but not overlapping with PSD-95.

Scale bar: 5 mm. (B) Diagram showing the quantitative analysis of synapsin I/PSD-95 colocalization. 366 (WT) and 477 (ICAM-52/2) synapsin I puncta from 3 independent

experiments were quantitatively analyzed. Mean6s.d. is shown. **P,0.01. (C) Functional synapses were analyzed by measuring mEPSCs. WT and ICAM-5-deficient

mouse hippocampal neurons were cultured until 15–18 DIV and recordings were performed on neurons of the same age (DIV) for both genotypes in each experiment. Shown

are representative recordings of spontaneous miniature glutamatergic post-synaptic currents of the neurons. (D) The cumulative probability plots from the recordings

presented in C show an increased appearance of shorter inter-event intervals in ICAM-52/2 neurons but no obvious changes in the event amplitudes. (E) Diagram showing

the overall effect of ICAM-5-deficiency on inter-event interval and event amplitude. Median inter-event intervals or amplitude of each manipulated cell have been divided by

the WT values of the same experimental day (DIV 15–18). The mean time between events was half as long in ICAM-52/2 cells as in WT cells. The amplitude of currents did

not change with any manipulation. n55 experiments, 60 cells. Mean6s.d. is shown. **P,0.01.

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To study whether the increased synaptic contacts in ICAM-

52/2 neurons are functionally active, we recorded the inter-event

intervals and amplitude of miniature excitatory post-synaptic

currents (mEPSCs) in cultured WT and ICAM-5-deficient

neurons on 15–18 DIV (60 neurons randomly selected from 5

independent experiments were patched) (Fig. 1C). Compared

with WT, ICAM-52/2 neurons exhibited a significant increase in

the frequency (decrease in the inter-event intervals) of mEPSCs,

however there was no significant effect on the amplitude

(Fig. 1C,D). The mean relative inter-event interval was

53611% shorter in ICAM-52/2 than in WT neurons implying

a significant increase in the frequency of mEPSC (Fig. 1E). The

corresponding value for event amplitudes in ICAM-52/2 was

10065% compared to WT (Fig. 1E).

In conclusion, ICAM-5 deficiency increased the probability of

successful synaptic events and the lack of change in the amplitude

of mEPSCs indicates that the efficacy of the post-synaptic

receptors is not changed. These results are not consistent with a

post-synaptic mediated mechanism in ICAM52/2 neurons.

Antibodies against ICAM-5 or b1 integrins affect spine

morphology

Conant and co-workers reported an interaction between ICAM-5

and b1 integrins (Conant et al., 2011). They did not, however,

study the localization of b1 integrins nor did they address the

physiological relevance of the interaction. In this study, we used

antibodies, which bind to the ectodomains of ICAM-5 or b1

integrins, to mimic the effect of loss-of- or gain-of-function.

Mouse hippocampal neurons were treated at 11 DIV with

antibodies against ICAM-5 or b1 integrins. The properties of the

antibodies used in the study are summarized in Table 1. The cells

were fixed at 15 DIV and immunostained for F-actin. As shown

in Fig. 2, untreated neurons exhibited 57%69% immature spines

(including thin spines and stubby spines), 28%69% filopodia and

14%67% mature spines. When cells were treated with the

ICAM-5 antibodies 179D and 179K, the ratio of immature spines

decreased and that of mature spines significantly increased. Ha2/

5, an adhesion-blocking antibody against b1 integrins, showed a

similar effect on spine maturation as ICAM-5 antibodies.

Interestingly, TS2/16 (Fig. 2A,B, TS2/16), an activating

antibody against b1 integrins, which increased the ratio of

filopodia and decreased that of the immature spines, showed an

opposite effect to the adhesion-blocking antibodies. TS2/16 is

usually described as an antibody reacting with human b1

integrins, but we saw effects on mouse neurons. To confirm

that this antibody also cross-reacts with mouse b1 integrins, we

treated mouse N2A neuroblastoma cells with TS2/16. In the

presence of 1 mM MnCl2, the reactivity of 9EG7, an antibody

recognizing activated mouse b1 integrins (Bazzoni et al., 1995),

was significantly increased in N2A cells (supplementary material

Fig. S2). Therefore, TS2/16 also activates mouse b1 integrins.

The similar function of ICAM-5 and b1 integrins suggest that

the two molecules regulate the morphological change of spines

by an interaction between them. Therefore it was important to

characterize this interaction in detail.

ICAM-5 binds to b1 integrins directly through the

ectodomain

To study ICAM-5 interactions, ICAM-5 was immunoprecipitated

from mouse forebrain homogenates. The immunoprecipitation

was confirmed by western blotting with an ICAM-5 antibody (not

shown). The integrin b1 subunit (Fig. 3A, left panel) and the a5

Table 1. Antibodies used in functional studies

Antibodies Properties Epitopes

TS2/16 Activating Human integrin b1 chain 207-218a

9EG7 Activating Mouse integrin b1 chain 495-602a

Ha2/5 Blocking Mouse integrin b1 chain ectodomainM2253z Blocking Human integrin b1 chain ectodomain179 D Blocking Mouse and human ICAM-5 1st Ig-like domain179 K Blocking Mouse and human ICAM-5 2nd Ig-like domain179 H Blocking Mouse and human ICAM-5 D2-3 Ig-like domain246 H Blocking Mouse and human ICAM-5 2nd Ig-like domain

Properties refer to the capability of these antibodies in increasing or decreasing the ligand binding function of their immunogens.aExpressed as the sequences in the primary structure.

Fig. 2. Altered ICAM-5/b1 integrin interaction results

in abnormal spine formation. (A) Hippocampal neurons

were left untreated (ctrl) or treated with antibodies from 11

to 15 DIV and then stained for F-actin. Compared with the

untreated condition, the balance of dendritic protrusions

was shifted towards more mature spines by blocking Ha2/5,

179D and 179K, yet towards filopodia by activating TS2/

16. Scale bar: 5 mm. (B) Quantitative analysis of the ratio of

different types of protrusions. The number of analyzed

protrusions is listed under the columns. Mean 6 s.d. of 3

independent experiments is shown. *P,0.01; **P,0.005;

***P,0.001.

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subunit (Fig. 3A, right panel) were detected in ICAM-5

precipitates. Reciprocally, ICAM-5 was also detected from the

precipitates with the b1 (Fig. 3B, left panel) and a5 subunits

(Fig. 3B, right panel), but not from the control precipitates. The a3

and a8 integrin subunits were also examined, but they showed very

low binding to ICAM-5 (not shown). To determine whether the

ectodomain or the cytoplasmic tail of ICAM-5 is more important in

the interaction with b1 integrins, Paju cells were transfected with

full length ICAM-5 (Paju-ICAM-5), or ICAM-5 lacking the

cytoplasmic tail (Paju-ICAM-5 Dcp). The expression level of b1

integrins was similar in the two transfectants (not shown). The b1

integrin subunit was immunoprecipitated from the cell lysates of

both Paju transfectants. ICAM-5 was detected in both precipitates

by an antibody recognizing the ectodomain of ICAM-5 without

any significant change in band intensity (Fig. 3C). Although a role

for the transmembrane segment cannot be excluded from these

experiments, the results indicate that most probably the ICAM-5

ectodomain interacts with b1 integrins.

A direct interaction between ICAM-5 and b1 integrins was

confirmed by using purified ICAM-5 D1–9-Fc and a5b1 integrin

(Fig. 3D).

Binding of ICAM-5 to b1 integrins occurs in trans

To address whether the interaction between ICAM-5 and b1 integrins

occurs in a trans manner, we performed cell adhesion assays.

We used Paju-Neo cells, which do not express ICAM-5,

making it a tool of choice to avoid homophilic binding between

ICAM-5 and study its interaction with b1 integrins. b1 integrin

expression in Paju-Neo cells was examined by flow cytometry (not

shown). Cells were seeded onto the immobilized ICAM-5-Fc

proteins D1–2 and D1–9 for 30 min and the unbound cells were

washed away. As shown in Fig. 4A, compared with negative

controls (human IgG and ICAM-2-Fc), both ICAM-5-Fc proteins

increased cell adhesion. D1–2 exhibited strong binding with 66%

bound cells. ICAM-5 D1–9 showed less binding, with 47% bound

cells. A previous study has shown that ICAM-5 D1–2 is a more

efficient binder to LFA-1 than ICAM-5 D1–9 (Tian et al., 2000a).

These results suggest that ICAM-5 promoted cell adhesion

through interactions with counter-receptor(s) on the surface of

Paju-Neo cells. To further prove that b1 integrins are responsible

for the interaction, we employed antibodies against b1 integrins

or ICAM-5 (the properties of the antibodies used in the study are

summarized in Table 1), and an arginine-glycine-aspartate

(RGD) peptide, which blocks interactions between integrin b1

subunit and several of its binding partners (Fig. 4B). Compared

with the binding to D1–2 without antibody treatment (Fig. 4B,

first bar), the b1 integrin adhesion blocking antibody 2253

inhibited the binding significantly. The ICAM-5 antibodies

179D, 179K and 246 H recognizing the first two Ig-like

domains in ICAM-5 were also effective blockers of the

binding. In contrast, 179H, which recognizes domains D2–3,

showed a minimal blocking effect. Both the GRGDS and the

SDGRG peptides slightly decreased the binding, but the decrease

was not statistically significant, which is in accordance with the

fact that ICAM-5 lacks of the RGD sequence. Importantly, TS2/

16, a b1 integrin activating antibody, increased the adhesion

Fig. 3. ICAM-5 binds to b1 integrins. (A,B) Adult mouse brain homogenates were used for immunoprecipitation with antibodies against ICAM-5 (A, left and

right), the integrin b1 chain (B, left) and a5 chain (B, right) respectively. Integrin b1 (A, left), a5 chains (A, right) and ICAM-5 (B, left and right) were detected by

western blotting from the precipitates as well as the lysates (indicated by arrowheads). In the control precipitates, no antibody was used. The apparently higher

molecular weight of the a5 subunit in lysate (A) is likely due to sample overloading. (C) Lysates from transfected Paju-ICAM-5 and Paju-ICAM-5 Dcp cells were

precipitated with an antibody recognizing the integrin b1 chain. Full length ICAM-5 and cytoplasmic tail truncated ICAM-5 (ICAM-5 Dcp) were detected by

western blotting with an antibody against the ectodomain of ICAM-5. (D) Binding of purified ICAM-5 D1–9-Fc to immobilized a5b1 integrin. Fibronectin

was used as a positive control in binding to a5b1 integrin. Mean6s.d. of 3 independent experiments is shown. **P,0.01. The right panel shows SDS-PAGE of

the integrin purification.

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significantly. We conclude that b1 integrins take part in ICAM-5-

mediated cell adhesion via binding to ICAM-5 in trans.

Moreover, the interaction between ICAM-5 and b1 integrins in

cultured neurons was further confirmed by a bead recruitment

assay. We used ICAM-5-Fc-coated beads to mimic the

endogenous ICAM-5 and studied their binding to b1 integrins.

ICAM-5 D1–9-Fc or human IgG coated beads were incubated

with cultured hippocampal neurons for 24 h. Cells were fixed and

immunostained for ICAM-5 and b1 integrins. ICAM-5 coated

beads efficiently recruited b1 integrins and the two proteins

colocalized on the surface of the beads (Fig. 4C,D).

b1 integrins are expressed in both pre- and post-synapses

The discovery of the trans interaction between ICAM-5 and b1

integrins, and the fact that ICAM-5 is a post-synaptic protein in

neurons (Benson et al., 1998; Yoshihara and Mori, 1994) led us

to study whether b1 integrins are located at the pre-synaptic sites.

Immunofluorescent staining of cultured hippocampal neurons

was performed with a rabbit monoclonal antibody (mAb) against

b1 integrins. Synapsin I and PSD-95 were used as pre- and

post-synaptic markers, respectively. The majority of synapsin I

immunoreactive puncta (72%66%) colocalized with b1 integrins

(Fig. 5Ab; Fig. 5C). PSD-95 positive puncta partially but less

abundantly colocalized with b1 integrins (32%67%) (Fig. 5Ac;

Fig. 5C). In comparison, the colocalization of ICAM-5/synapsin I

and ICAM-5/PSD-95 is also shown. ICAM-5 colocalized with

PSD-95 along the dendritic shaft and spine heads (Fig. 5Bc) and

synapsin I puncta apposed to ICAM-5-labeled dendrites and

spine heads (Fig. 5Bb). 16%63% synapsin I vs 71%63% PSD-

95 puncta colocalized with ICAM-5 (Fig. 5D). Notably, some b1

integrin puncta neither colocalized with synapsin I nor PSD-95

(Fig. 5Ab), which indicates the existence of non-synaptic b1

integrins.

Pre- and post-synaptic fractionation is an often used method to

study the distribution of synaptic proteins. Vesicular glutamate

transporter 1(VGLUT1), NMDA receptor subunit 1 (NR1) and

PSD-95 were used as the markers for the fractionation.

Synaptosomes were hence fractionated into an external

junction, a pre-synaptic and a post-synaptic fraction. The

external junction fraction constitutes plasma and vesicle

Fig. 4. In trans binding between ICAM-

5 and b1 integrins. (A) Purified ICAM-5-

Fc proteins were pre-coated on 96-well

microtiter plates. Human IgG and ICAM-2-

Fc fusion proteins were used as negative

controls. The ICAM-5-Fc proteins

increased the binding of Paju-Neo cells,

with D1–2-Fc being most efficient.

(B) Effect of antibodies and peptides on

Paju cell adhesion to ICAM-5 D1–2-Fc.

The ICAM-5 abs 179D, 179K and 246 H

and the b1 integrin adhesion blocking

antibody 2253 were effective in inhibiting

cell adhesion, while the b1 integrin

activating antibody TS2/16 increased cell

adhesion. (C) ICAM-5-Fc coated beads

recruit b1 integrins in cultured neurons. 13

DIV neurons incubated for 24 h with

ICAM-5 or human IgG-coated beads were

fixed and stained for ICAM-5 (green) and

b1 integrins (red). The DIC images were

presented for each corresponding

fluorescent image. Arrows indicate the

location of beads. Inset: higher

magnification images of the selected area.

ICAM-5 coated beads efficiently recruited

b1 integrins. Scale bar: 10 mm. The mean

fluorescent intensity within the beads area

was quantitated (D). .200 beads from 3

independent experiments were analyzed for

each treatment. Mean 6 s.d. is shown.

*P,0.05, **P,0.01, ***P,0.001.

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membranes, the pre-synaptic fraction mainly contains the pre-

synaptic active zones, and the post-synaptic fraction is composed

of PSDs. As expected, VGLUT1 was enriched in the pre-synaptic

fraction while NR1 and PSD-95 were enriched in the post-

synaptic fraction. b1 integrins were found in both pre- and post-

synaptic fractions but were enriched in the pre-synaptic fraction.

Absence of b1 integrins in the external junction fraction is

possibly due to their cytoplasmic interactions with cytoskeletal

proteins, which form a tight anchorage to the active zones and

PSDs (Fig. 5E).

The specificity of b1 integrin antibodies for the studies above was

tested using excitatory neuron-specific b1 integrin knockout mouse

brain homogenates by western blotting. AB1952 recognized a single

band at 88 kDa in WT but the intensity of a corresponding band in

b1 integrin2/2 brain was significantly lower (Fig. 5F). The same

band was also recognized by other b1 integrin antibodies (sc-6622,

Santa Cruz and Ab 52971, Abcam) (not shown). The band may

represent a proteolytic fragment of the b1 chain.

These results clearly demonstrate that b1 integrins are

expressed at both pre- and post-synaptic sites.

Development-related expression of b1 integrins atexcitatory synapses

Dendritic protrusions are classified into filopodia, thin and long

protrusions arising from the dendritic shafts, and spines, which

are relatively shorter and usually carry an enlarged head on their

tips (Ethell and Pasquale, 2005). Filopodia are usually considered

to be precursors of spines, and nascent synapses are initially

formed between axonal terminals and the dendritic filopodia. The

filopodia-to-spine transition accompanies synapse maturation and

stabilization (Ziv and Smith, 1996).

Previous studies have shown that ICAM-5 is enriched in

filopodia and immature spines, but becomes excluded from

mature spines (Matsuno et al., 2006; Tian et al., 2007). To further

clarify the distinctive role of b1 integrins at different synaptic

developmental stages, we studied b1 integrin expression in

different types of dendritic protrusions. To visualize the fine

structure of dendritic protrusions, neurons were transfected with

Enhanced Green Fluorescent Protein (EGFP) and immunostained

for b1 integrins on 15 and 22 DIV after fixation. The

colocalization of b1 integrins and different subtypes of

protrusions was studied. At 15 DIV, in some filopodia, b1

integrins were weakly expressed at their tips (Fig. 6Aa and

Fig. 6Ad, narrow arrows), but increased in immature (Fig. 6Aa

and Fig. 6Ad, wide arrows) and mature spines (Fig. 6Aa and

Fig. 6Ad, arrowheads). Interestingly, b1 integrins were more

often found juxtaposed to the heads of immature spines rather

than colocalized with them (Fig. 6Ba, 66%67% vs 23%65%).

However, most of mature spines (Fig. 6B, 78%68%) had b1

integrins overlapping with the enlarged spine heads. At 22 DIV,

even though the proportion of mature spines greatly increased

compared with 15 DIV, the correlation of b1 integrin reactivity

with subtypes of spines remains similar (Fig. 6Bb).

The distribution of the two proteins during synapse maturation

was also examined. Cultured hippocampal neurons were used to

study the colocalization of ICAM-5 and b1 integrins in different

dendritic protrusions. On 13 DIV (Fig. 6Ca–c), dendrites

contained abundant filopodia and ICAM-5 was enriched in

them. b1 integrins were scattered around dendritic shafts and

were apposed to some filopodia (Fig. 6Ca, arrows). A fragment

of an axon (ICAM-5 negative), weakly seen but indicated by red

arrowheads (Fig. 6Ca), made contacts with filopodia, in which

Fig. 5. Expression of b1 integrins on synapses. Mouse

hippocampal neurons were fixed at 15 DIV and triple

stained for PSD-95 (green), b1 integrins (red) and synapsin

I (blue) (A) or PSD-95 (green), ICAM-5 (red) and synapsin

I (blue) (B). (A) Colocalization of b1 integrins with

synapsin I (b), or with PSD-95 puncta (c). b1 integrins

colocalize with synapsin I at a higher level than with PSD-

95. In comparison, ICAM-5 colocalizes with PSD-95 along

the dendritic shaft and protrusions (c), but not with

synapsin I (b). Arrows indicate the area where two proteins

colocalized. Scale bar: 5 mm. Small windows: higher

magnification view of the area marked by dashed frames.

(C,D) The colocalization of b1 integrin/synapsin I, b1

integrin/PSD95, ICAM-5/synapsin I and ICAM-5/PSD-95

was quantitated. The number of puncta analyzed is shown

above the columns. Mean 6 s.d. from 3 independent

experiments is shown. ***, P,0.001. (E) Mouse forebrains

were used to isolate pre- and post-synaptic fractions. 30 mg

protein from each following fractions was applied in

duplicate to SDS-PAGE followed by western blotting:

homogenate, crude synaptosome, external junction fraction,

pre-synaptic fraction, and post-synaptic fraction. b1

integrins were found on both pre- and post-synaptic

fractions but enriched in the pre-synaptic fraction.

(F) 50 mg brain homogenates from WT and b1 integrin

conditional knockout mice were applied to SDS-PAGE

followed by western blotting. The b1 integrin antibody

recognized a single band at 88 kDa, which was present in

WT but almost absent in b1 integrin2/2 mouse brain

homogenates. This shows that the antibody is specific.

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two ICAM-5-stained filopodia are associated with b1 integrins. A

small population of mushroom spines were also observed at this

stage, in which ICAM-5 did not colocalize with b1 integrins

(Fig. 6Ca, arrowheads). On 22 DIV (Fig. 6Cd–f), dendrites

contained more spines than filopodia (Fig. 6Ce, F-actin).

ICAM-5 was well colocalized with F-actin in filopodia and

immature spines, but was partially excluded from the mushroom

spines. In filopodia and immature spines, b1 integrins were

opposite to ICAM-5 at the tip of filopodia or immature spines

(Fig. 6Cd and Fig. 6Cf, arrows). In mature mushroom spines, b1

integrins covered most of the spine heads. However, ICAM-5

was found on one side of the spine, and minimally overlapped

with b1 integrins (Fig. 6Cd and Fig. 6Cf, arrowheads).

These results indicate that at the early stage of synapse

formation, b1 integrins are more often expressed in the pre-

synaptic structures, making contacts with the tips of filopodia and

immature spines from the counter neuron. It is difficult to

determine the localization of b1 integrins after mature spines

formed. The enhanced overlap of b1 integrins with mature spine

heads may result from increased expression of post-synaptic b1

integrins or a shortened distance between pre- and post-synaptic

membranes at the late stage of synapse formation.

ICAM-5 binding to b1 integrins affects its ectodomain

shedding

The ectodomains of ICAM-5 seem important in delaying synapse

maturation as MMP-dependent cleavage of ICAM-5 promotes

spines maturation and synaptic activity (Tian et al., 2007; Conant

et al., 2010; Conant et al., 2011). Moreover, MMP-9 enzymatic

activity has been shown to increase the surface trafficking of

NR1 through a b1 integrin-dependent pathway (Michaluk et al.,

2009; Wang et al., 2008). We found that the interaction of ICAM-

5 with b1 integrins affects spine structures. Therefore it was

tempting to study the putative role of ICAM-5/b1 integrin

interaction in ICAM-5 ectodomain cleavage. Mouse hippocampal

neurons were pre-treated with antibodies against the b1 integrins

(TS2/16, 9EG7 and Ha2/5) and ICAM-5 (179D and 179K) and

purified ICAM-5 D1–2-Fc protein for 3 days, and changed into

HBSS/Ca++ medium. After 16 h incubation, cell lysates and

culture media were collected. The levels of sICAM-5 released

Fig. 6. b1 integrins localize opposite to early spines and colocalize with mature spines. (A) Rat hippocampal neurons were transfected with EGFP 24 h before

fixation (15 DIV or 22 DIV) and immunostained for b1 integrins. The colocalization of the b1 integrins with EGFP-labeled dendritic protrusions was studied. b1

integrins were weakly expressed at the tip of filopodia (narrow arrows), juxtaposed to immature spines (wide arrows), and colocalized with the head of mushroom

spines (arrowheads). Scale bar: 10 mm. (B) Quantitative analysis of the correlation of the b1 integrin expression with different spines was performed using 355

immature spines and 304 mature spines from 3 independent experiments. Data from 15 (a) and 22 (b) DIV cultures are shown, respectively. Colocalized: .50%

areas of spine heads overlapping with b1 integrin staining; juxtaposed: integrin b1 expression associated with the tip of filopodia or the spines from the counter

cell and ,50% areas of spine heads overlapping with b1 integrin staining; no expression: b1 integrin staining not seen in the protrusions. Mean6s.d. is shown.

**P,0.01, ***P,0.001. (C) Representative images of rat hippocampal neurons at 13 and 22 DIV immunostained for b1 integrins (green), ICAM-5 (red) and

F-actin (blue). Arrows indicate filopodia or immature spines, in which b1 integrins were opposite to ICAM-5 on the tips of the protrusions. Arrowheads indicate

mature spines, which were mostly covered by b1 integrins but with minimal overlap with ICAM-5. Note that in panel a, red arrowheads show a fragment

of an axon, which was making contacts with filopodia. Scale bar: 5 mm.

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into the culture media (Fig. 7A, left panel) and full-length

ICAM-5 present in cell lysates (Fig. 7A, right panel) were

measured by western blotting. Two sICAM-5 fragments of 85

and 110 kDa were detected in the culture media as previously

reported (Tian et al., 2007). Our cell adhesion assays showed that

TS2/16 enhanced the interaction between ICAM-5 and b1

integrins, while 179D and 179K inhibited this interaction

(Fig. 3B,C). Interestingly, we found that these antibodies also

affected ICAM-5 cleavage. The adhesion blocking antibodies

Ha2/5, 179D and 179K increased the level of soluble sICAM-5.

In contrast, the b1 integrin-activating antibodies TS2/16 and

9EG7 effectively reduced the level of sICAM-5. Notably, ICAM-

5 D1–2-Fc treatment also led to increased sICAM-5 levels

released into the culture media (Fig. 7).

Knockdown of b1 integrins in axons affects synapse

formation

Interfering with the ICAM-5/b1 integrin interaction by antibody

treatments resulted in altered spine morphology (Fig. 2).

However, because b1 integrins are present in both pre- and

post-synaptic structures, the effects may also come from the cis

interaction of ICAM-5/b1 integrins. To eliminate this possibility,

we studied the loss-of-function of pre-synaptic b1 integrins in

synapse formation. Ten DIV hippocampal neuron cultures were

transfected with small hairpin RNA (shRNA) for b1 integrins or

control plasmids and the expression levels of b1 integrins were

examined by immunofluorescent staining. A prominent decrease

of b1 integrin expression was found in the soma (Fig. 8A,

indicated by arrowheads) and neurites (Fig. 8A, indicated by

dash lines) of shRNA transfected neurons. The mean fluorescent

intensity of soma was quantitated and b1 integrin expression was

found to be downregulated by ,70% by shRNA in transfected

cells (Fig. 8B). To study the interaction of pre- and post-synaptic

structures, neurons were stained with axon marker Tau (Fig. 8C,

blue) and F-actin (Fig. 8C, red), which visualizes the morphology

of spines. The neurites labeled with GFP and Tau are axons,

which originate from b1 integrin knocked-down neurons

(Fig. 8C, cyan). Since shRNA downregulated b1 integrins in

axons as well as dendrites, in this study, we only focused on those

dendrites which were not transfected. At 12 DIV, in the control

cultures, dendrites exhibit 63% of immature (including filopodia,

thin spines and stubby spines) and 35% of mature (mushroom)

spines among all the protrusions and there is no significant

difference between the dendrites with or without contact with

GFP labeled axons (Fig. 8C–E). In b1 integrin shRNA

transfected cultures, dendrites, when in contact with transfected

axons, carry an increased number of mature spines (61%) and a

decreased number of immature spines (37%) (Fig. 8C,D); these

dendrites, which failed to make contact with shRNA transfected

axons, have a similar proportion of filopodia and spines with the

control neurons (Fig. 8E).

DiscussionICAM-5 was first described as a telencephalon-specific molecule

(telencephalin) with high homology to the previously described

ICAM-molecules (Gahmberg, 1997; Yoshihara and Mori, 1994).

In the immune system, the receptor of ICAM-5 is the b2 integrin

LFA-1 expressed on peripheral blood leukocytes and microglia

(Tian et al., 1997; Mizuno et al., 1999; Tian et al., 2000a; Zhang

et al., 2008; Ransohoff and Cardona, 2010). In the CNS, the roles

of ICAM-5 in stimulating dendrite outgrowth, delaying spine

maturation and increasing LTP have been extensively studied

(Tian et al., 2000b; Nyman-Huttunen et al., 2006; Matsuno et al.,

2006; Nakamura et al., 2001). Upon stimulation of NMDA

receptors and MMP activation, ICAM-5 ectodomain cleavage is

promoted, which induces spine maturation (Tian et al., 2007).

Furthermore, the addictive drug methamphetamine stimulates

ICAM-5 cleavage, and this cleavage was blocked with MMP

inhibitors (Conant et al., 2010). These findings highlight

important roles of ICAM-5 in the CNS. Finding and

Fig. 7. Antibody treatments against ICAM-5

and b1 integrins affect ICAM-5 ectodomain

cleavage. (A) Hippocampal neurons were left

untreated or treated with 20 mg/ml antibodies

between days 11 to 14. After changing the cell

culture medium to HBSS with 1.8 mM CaCl2 for

16 h, cell lysates and the conditioned media was

collected separately, and ICAM-5 was detected by

an antibody against the ICAM-5 cytoplasmic tail

(ICAM-5cp, cell lysate) or ectodomains (1000J,

culture medium). Soluble ICAM-5 85- and 110-

kDa fragments were released to the culture media.

Antibodies Ha2/5, 179D and 179K caused an

increased level of sICAM-5. Purified ICAM-5 D1–

2-Fc protein and the b1 integrin-activating

antibodies TS2/16 and 9EG7 inhibited the release

of sICAM-5. Actin in cell lysates was used to

quantitate the amount of cellular material from

which the culture media were collected.

(B) Intensity of the released (85-kDa fragment)

and membrane-bound ICAM-5 was quantified by

ImageJ. Mean 6 s.d. of 3 independent experiments

is shown. *P,0.05; **P,0.01; ***P,0.001.

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characterizing the counter receptor for ICAM-5 in the brain

would significantly advance our understanding of the neuronalfunctions of ICAM-5.

In this study, we have characterized the interaction between

ICAM-5 and b1 integrins in detail. Importantly, we found that byregulating the ectodomain cleavage of ICAM-5, b1 integrinsmodulate spine morphology and synapse maturation.

The interaction was first observed by coimmunoprecipitation.

According to previous studies, a3, a5 and a8 integrins are presentin synaptic regions and play distinctive roles in synapse formationand plasticity. We found that the a5b1 integrin is the predominant

binding partner of ICAM-5 in brain, whereas binding of a3b1 anda8b1 integrins was not clearly observed (not shown). The bindingbetween ICAM-5 and b1 integrins is direct and occurs through the

extracellular domains of the two proteins.

The cell adhesion results show that ICAM-5 binds to b1integrins through its ectodomain and the ICAM-5-D1–2 domainsform the most important binding region. The lower binding

capability of the longer polypeptide D1–9 probably results fromintra-molecular interactions that partially mask the binding site ofICAM-5 to b1 integrins. A similar finding was observed for its

binding to LFA-1 (Tian et al., 2000a). Several extracellular matrix(ECM) proteins are well-known ligands for b1 integrins (Hynes,2002) and many studies have shown a role of the interaction

between ECM proteins and b1 integrins in synapse differentiation.Although RGD-containing peptides block the interactions betweenb1 integrins and some proteins, such as fibronectin (FN) (Ruoslahti

and Pierschbacher, 1986), the peptides did not inhibit the bindingof b1 integrin-mediated cell adhesion to ICAM-5-Fc proteins. Infact, ICAM-5 does not contain an RGD-sequence. b1 integrins do

not contain an I-domain found in the LFA-1 integrin known to bindICAM-5 (Zhang et al., 2008). Therefore, the binding site in b1integrins must differ from that of LFA-1.

As synaptic adhesion molecules, b1 integrins are oftenconsidered to be located at the post-synaptic site, even though

Hellwig and co-workers earlier showed a pre-synapticlocalization of b1 integrins by electron microscopy (EM)(Hellwig et al., 2011). Our studies using fluorescentimmunostaining and synaptosome fractionation show that,

besides being present on dendrites, b1 integrins are also foundin pre-synaptic structures. A similar expression pattern of b1integrins was also shown by immune-EM (supplementary

material Fig. S3). It is possible that the pre- and post-synapticb1 integrins play different roles during development. In nascentsynapses, b1 integrins appear to be more predominant on the pre-

synaptic sites, suggesting that they may serve as counter-receptors for ICAM-5. Taking into account the expressionpattern of ICAM-5, we speculate that the in trans interaction ofICAM-5 and b1 integrins is more important during early stages

of synapse formation. In fact, soluble ICAM-5-coated beadsfailed to induce pre-synaptic protein clustering (supplementarymaterial Fig. S1) even though they recruited b1 integrins

efficiently, suggesting that the ICAM-5/b1 integrin interaction isirrelevant for synaptic protein clustering. They may form a looseand dynamic contact between pre- and post-synaptic membranes

and the interaction is likely to be released upon to furthersignaling.

ICAM-5 is known as a negative regulator of spine maturation.When we used antibodies recognizing the first and second Ig-likedomain of ICAM-5, the neurons exhibited an increased number

Fig. 8. Pre-synaptic b1 integrin knockdown alters synapse formation. 10 DIV cultured hippocampal neurons were transfected with GIPZ b1 integrin-shRNA

or control plasmids and cultured for another 48 h before fixation. (A) Cells were stained for b1 integrins (red). Arrowheads indicate the soma of transfected cells.

Dash lines indicate fragments of GFP labeled neurites. a and e, Scale bar: 20 mm (a,e) 5 mm (c,g). (B) Mean fluorescent intensity in somas was quantitated. Mean

6 s. d. of 3 independent experiments is shown. **P,0.01. (C) Transfected axons were labeled with GFP and Tau (blue) and fine structures of dendrites

were visualized by F-actin (red). Dendrites, which make contacts with b1 integrin shRNA transfected axons, exhibit an increased number of spines. Arrows

indicate filopodia and immature spines. Arrowheads indicate mature spines. (D) Quantification from dendrites in contact with GFP labeled axons.

(E) Quantification from dendrites not in contact with GFP labeled axons. ,300 mm of dendritic fragments randomly picked from 10 untransfected neurons were

analyzed for each group. The number of filopodia and spines was quantitated. Mean6s. d. from 3 independent experiments is shown. **P,0.005.

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of spines and a higher ratio of mature/immature spines, which

indicates enhanced spine maturation. Interestingly, a blockingantibody against b1 integrins showed a similar effect on spinemorphology. These phenomena resemble the phenotype of

ICAM-5 knockout neurons. In contrast, neurons treated with anactivating antibody against b1 integrins showed little effect onmature spines but exhibited a significant increase in the filopodia/immature spines ratio. This indicates a block in the filopodia-to-

immature spine transition, but less effect on mature spines.This observation is similar to the phenotype of ICAM-5-overexpressing neurons. Therefore, an important function of the

interaction of ICAM-5/b1 integrins is to promote filopodiaelongation or to maintain the morphology of filopodia-like spinesbefore they develop into mature spines. Although we cannot

exclude the possibility that the antibodies interfere with thebinding of b1 integrins to other ligands, the effects are at leastpartially due to the ICAM-5/b1 integrin interaction.

Downregulating b1 integrin expression by shRNA in axons

resulted in a decreased number of filopodia and an increasednumber of spines in dendrites making contact. This indicates thatthe pre-synaptic b1 integrins are important in the filopodia-to-

spine transition of the post-synaptic structures. Thus the effects ofantibodies on spine morphology mainly come from themodulation of the trans interaction. These and other findings in

the present manuscript suggest that the functional phenotype inICAM-52/2 neurons that show higher frequency but no change inamplitude, is not derived from a post-synaptic but rather pre-synaptic mechanism.

The regulation of ICAM-5/b1 integrins on spine morphologylikely occurs through fine-tuning of ICAM-5 ectodomaincleavage. At the early stage of synapse formation, b1 integrins

likely start to interact with ICAM-5 when the initial contactforms between the axonal terminal and the filopodia tip. Possibly,upon binding to b1 integrins, the ICAM-5 ectodomain is partiallycovered, which makes the proteolytic sites less accessible to

MMPs. Thus, the interaction of ICAM-5 and b1 integrins seemsto provide a protecting mechanism affecting ICAM-5 ectodomaincleavage early during spine maturation.

ICAM-5 ectodomain cleavage was efficiently inhibited byactivating b1 integrins, whereas the cleavage was enhanced byblocking antibodies. Importantly, ICAM-5 mAbs also showedsimilar effects. Interestingly, sICAM-5 treatment also inhibited

ICAM-5 ectodomain cleavage. In fact, when treated with ICAM-5-Fc proteins, cultured hippocampal neurons exhibited anincreased number and length of filopodia but an unchanged

number of spines (Tian et al., 2007), which resembles thephenotype of b1 integrin-activating antibody-treated neurons.Added sICAM-5 may compete out the binding of b1 integrins to

the endogenous ICAM-5. It has earlier been shown that solubleICAM-1 and ICAM-2 promote b2 integrin-dependent Tlymphocyte adhesion (Kotovuori et al., 1999). It is possible

that sICAM-5 binding also activates b1 integrins and increasestheir binding affinity to the post-synaptic ICAM-5. ICAM-5 alsoshows homophilic binding (Tian et al., 2000b), and this bindingmay protect the ectodomain of ICAM-5 from being cleaved. The

exact mechanism how sICAM-5 works remains to be elucidated.

In conclusion, we show that b1 integrins are also pre-synapticand that they regulate synapse formation through the interaction

with ICAM-5. This interaction, which inhibits the filopodia-to-spine transition, is most important at the early stage of synapseformation.

Materials and MethodsReagents and antibodiesHuman IgG, poly-L-lysine, GRGDS and SDGRG peptides were obtained fromSigma-Aldrich (St. Louis, MO). The PEF-BOS-ICAM-5 construct was made asdescribed (Tian et al., 1997).

The polyclonal antibody (pAb) anti-ICAM-5cp against the mouse ICAM-5cytoplasmic tail was a gift from Dr Y. Yoshihara (Brain Science Institute/Institute ofPhysical and Chemical Research, Wako City, Japan). The pAb 1000J and mAbs127E, 179D, 179K, 246H, 246A and 179H against ICAM-5 ectodomains were giftsfrom P. Kilgannon (ICOS Corporation, Seattle, WA). The b1 integrin activatingantibody TS2/16 was a gift from Dr T. A. Springer (Harvard Medical School, MA).The rabbit pAb against human FN was a gift from Dr Antti Vaheri (University ofHelsinki). The rabbit anti-a5 integrin pAb (sc-10729), the goat anti-b1 integrin pAb(sc-6622), and the rabbit anti-b1 integrin pAb (sc-8978) were purchased from SantaCruz Biotechnology (Santa Cruz, CA). The rabbit anti-b1 integrin pAb (AB1952),the mouse anti-human b1 integrin adhesion blocking mAb 2253, the rabbit anti-a5integrin pAb and the guinea pig anti-VGLUT1 pAb were purchased from Millipore(Billerica, MA). The hamster b1 integrin adhesion blocking mAb Ha2/5, whichcross-reacts with mouse and rat b1 integrins (Michaluk et al., 2009), and the mouseanti-NR1 mAb were purchased from BD Biosciences (San Jose, CA). The rabbitanti-b1 integrin mAb (9EG7) and the PSD-95 mAb were obtained from Abcam(Cambridge, UK). Horseradish peroxidase (HRP)-conjugated anti-mouse, rabbit,goat and human IgGs were obtained from GE Healthcare Life Sciences (Uppsala,Sweden). Alexa488-, Cy3-, and Cy5-conjungated mouse, and rabbit IgGs, TRITC-conjugated phalloidin and Zenon labeling kit for rabbit IgG were all obtained fromInvitrogen (Carlsbad, CA). Cy5-conjugated synapsin I mAb was obtained fromSynaptic Systems (Gottingen, Germany).

AnimalsICAM-5 knockout mice were generated by gene targeting (Nakamura et al., 2001).Excitatory neuron-specific b1-integrin knockout mice were generated by crossingfloxed b1-integrin and a-calcium/calmodulin-dependent protein kinase II-cAMPresponse element Cre (CaMKII-Cre) mice (Chan et al., 2006). The geneticbackground of all animals was normalized by backcrossing at least six generationswith C57Bl/6 and maintained as homozygous lines. All experiments wereapproved by and performed according to the guidelines of the ethic committees foranimal research at the University of Helsinki and Scripps Florida.

Cell linesThe stable neuronal cell lines, Paju-Neo, Paju-ICAM-5 and Paju-ICAM-5Dcp, weremade as described earlier (Nyman-Huttunen et al., 2006). Cells were cultured inDulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum(FBS) (Invitrogen), 1% penicillin-streptomycin, and 1% L-glutamine (Lonza GroupLtd, Switzerland), in the presence of 0.5 mg/ml G418 (Sigma-Aldrich). COS-1 cellswere from ATCC and maintained as described above without G418.

Mouse brain homogenization and coimmunoprecipitationTwo adult mouse forebrains were homogenized in 10 volumes of ice-coldhomogenization buffer (1% Triton X-100 in phosphate buffered saline (PBS),16protease inhibitor cocktail (Roche Diagnostics GmbH, Germany), 16phosphataseinhibitor (Roche)), and centrifuged at 100,000 g for 1 h. The supernatant wasprecleared with 1 ml 50% Protein G Sepharose (Invitrogen) at 4 C for 1 h, anddivided into 1 ml aliquots, which were left untreated or incubated with 2 mg/mlantibodies against ICAM-5 (246A, ICOS, WA), b1 (sc-6622, Santa Cruz) and a5(sc-10729, Santa Cruz) subunits respectively, overnight at 4 C. 30 ml Protein GSepharose was added and the samples were incubated with rotation for an additionalhour at 4 C. The precipitates were washed with ice-cold homogenization buffer threetimes and resuspended in 25 ml 26SDS sample loading buffer.

Recombinant protein purificationICAM-5 ectodomain cDNAs coding for ICAM-5 Ig domains 1–2 (D1–2) and 1–9(D1–9) were prepared as described earlier (Tian et al., 1997). COS-1 cells weretransiently transfected with recombinant ICAM-5 cDNAs by using Fugene 6transfection reagent (Roche), and recombinant ICAM-5 ectodomains were purifiedfrom the cell culture media by Protein A sepharose CL 4B (Invitrogen). Purifiedproteins were examined by SDS-PAGE and their concentrations determined usingthe BCA assay (Pierce, Rockford, IL).

Purification of a5b1 integrinThe a5b1 integrin was purified from human placenta by affinity chromatographyusing a wheat germ agglutinin agarose column and an affinity matrix of Sepharosecoupled with the 110 kDa thermolysin fragment of FN (Argraves and Tran, 1994;Forsberg et al., 1994).

Enzyme-linked immunoassay (ELISA)One mg of purified a5b1 integrin was attached to flat-bottom 96-well microtiterplates (Nunc, Denmark) in 25 mM Tris, pH 8.0, 150 mM NaCl, 1 mM CaCl2 and

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1 mM MgCl2 by incubation overnight at 4 C. The wells were blocked with 1%milk powder for 1–2 h at room temperature (RT). Ten mg ICAM-5 D1–9-Fc or FNwere then incubated on the plates at 1 mg/100 ml/well for 1 h at RT. The boundproteins were detected by HRP-conjugated anti-human IgG (for ICAM-5 D1–9-Fc)or FN rabbit pAb followed by HRP-conjugated anti-rabbit IgG (for FN). Afterwashing, the plates were incubated in 100 ml/well 0.1 M phosphate-citrate, pH 5.0containing 0.8 mg/ml OPD tablet (Dako, Finland), at 37 C for 30 min. Thereaction was stopped by adding 100 ml 1 M H2SO4 and the absorbance at 492 nmwas measured by spectrometry.

Cell adhesion assays

100 ml of purified recombinant ICAM-5-Fc, ICAM-2-Fc and human IgG (8.8 mM)were pre-coated on 96-well microtiter plates (Nunc) overnight at 4 C. Afterwashing with PBS, the wells were blocked with 1% Bovine Serum Albumin(BSA)/PBS for 2 h at RT. Paju cells were detached, washed, and resuspended inDMEM containing 1% BSA, 2 mM MnCl2, 20 mM HEPES pH 7.4, at 106 cells/ml. 66104 cells were added per well and incubated for 30 min at RT. For blockingexperiments, ICAM-5-Fc protein-coated wells were treated with 100 mg/mlantibodies against ICAM-5: 179D, 179K, 246H and 246K. Meanwhile, the cellswere treated with 100 mg/ml RGD-containing peptides or b1 integrin antibodiesfor 30 min at RT before being added to 96-well microtiter plates. Non-adherentcells were removed by gentle washing with PBS. After washing, the bound cellswere lysed in 100 ml/well phosphatase substrate-containing lysis buffer (1% TritonX-100, 50 mM sodium acetate, pH 5.0, 3 mg/ml P-nitrophenyl phosphate), andincubated at 37 C for 30 min. The reaction was stopped by adding 50 ml/well 1MNaOH. The absorbance at 405 nm was measured by spectrometry. The percentageof bound cells was calculated as:

% bound cells~A405 bound cell=A405 total amount of cells|100%:

Western blotting

Samples were separated using 4–12% SDS-PAGE (Invitrogen) and transferredto nitrocellulose membranes (Whatman GmbH, Germany). After blocking,membranes were incubated with primary antibodies followed by incubation withHRP-conjugated secondary antibodies (Invitrogen). After washings with TBS and0.05% Tween 20, labeled bands were visualized with an ECL kit (Pierce). Bandintensity was quantified by ImageJ (NIH).

Isolation of pre- and post-synaptic fractions

The method was adapted from Bouvier et al., 2008 with modification. The wholeprocedure was carried out at 0–4 C unless otherwise stated. Forebrains from fourmale adult C57Bl/6 mice were homogenized in ice-cold sucrose/HEPES buffer(0.32 M sucrose, 10 mM HEPES, pH 7.4) containing protease inhibitors andcentrifuged at 1000 g for 10 min. The supernatant was centrifuged at 17,500 g for30 min. The pellet (P1, crude synaptosome) was resuspended in sucrose/HEPESbuffer and layered on top of a discontinuous sucrose gradient (2.6 M, 1.2 M,0.8 M). After ultra-centrifugation at 110,000 g for 2 h, the fraction between the0.8 M/1.2 M sucrose interfaces, which contains the purified synaptosomal fractionwas collected, and treated with 50 mM CaCl2, 1% Triton X-100, 20 mM Tris,pH 6.0 for 30 min. After centrifugation at 40,000 g for 30 min, the supernatant(S2, external junction), which mainly contains plasma and vesicle constituents,was saved. The pellet, constituting the pre-synaptic active zone and PSD proteins,was resuspended in 1% Triton X-100, 20 mM Tris, pH 8.0 and incubated for30 min. The increase of pH from 6 to 8 released pre-synaptic active zones (S3)from PSDs (P3). Proteins in S2 and S3 were precipitated with acetone at 220 Covernight and all pellets were solubilized in 5% SDS.

The protein concentrations of the fractions were determined by the BCA assay.30 mg protein/sample was applied in duplicates to SDS-PAGE followed by westernblotting.

Primary hippocampal neuron cultures

Primary cultures of hippocampal neurons were prepared from C57Bl/6 mousefetuses at gestational day 18 as previously described (Nyman-Huttunen et al.,2006). Dissociated neurons were counted and seeded on poly-L-lysine coated cellculture surfaces at the concentrations of 66104/well for 24-well plates and 106/wellfor 6-well plates. All hippocampal neurons were cultured in glial cell-conditionedNeurobasal medium (Invitrogen) containing 2% B27 and 1% L-glutamine. Onethird of the culture media were refreshed every 3–4 days.

Immunofluorescence microscopy

Hippocampal neurons were transfected with the pEGFP-N1 plasmid byLipofectamine 2000 (Invitrogen) 24 h before fixation and cultured until 15–22DIV. Neurons were fixed with 4% paraformaldehyde (PFA) in PBS andpermeabilized with 0.2% saponin. After blocking with 5% BSA/saponin/PBS,the cells were incubated with primary antibodies for 2 h at RT or overnight at 4 C,followed by incubation with the secondary antibodies for 1 h at RT. For ICAM-5/

b1 integrin co-staining (Fig. 6C), ICAM-5-cp pAb was labeled with Alexa488-conjugated rabbit IgG by Zenon antibody labeling kit (Invitrogen) before beingmixed with b1 integrin pAb. Images were obtained with a confocal laser-scanningmicroscope under 636magnification (TSC SP2 AOBS, HCX OIL APO636objective/1.4-0.6; Leica) using a charge-coupled device camera (Leica) andthe LCSLite software.

Cell stimulation

Primary hippocampal neurons were cultured in 6-well plates at 106 cells/well. Theb1 integrin adhesion blocking antibody Ha2/5 and activating antibody TS2/16, andICAM-5 antibodies 179D and 179K, were added to the culture mediumrespectively at 20 mg/ml on 11 DIV. On 14 DIV, cell culture medium wasreplaced with 1 ml Hank’s Buffered Salt Solution (HBSS) containing 1.8 mMCaCl2. After 16 h, 1-ml aliquots of the conditioned medium were concentrated 20-fold by vivaspin centrifugal concentrators (Sartorius Ltd), and the cells werestripped with Laemmli sample buffer. 10% (v/v) lysates and the culture mediawere applied to SDS-PAGE.

Bead recruitment assay

4.84 mm diameter polystyrene beads (Bangs Laboratories) were washed with PBSand mixed with ligand proteins at 40 mg/ml overnight at 4 C. The beads were thenblocked with 0.1% BSA for 1 h at RT, washed three times with PBS andresuspended in Neurobasal medium. 1–1.56105 beads/coverslip were added toneuron cultures on 12–13 DIV and incubated for 24 h before fixation.

Patch clamp recordings, data acquisition and analysis

mEPSCs were recorded from cultured mouse hippocampal neurons (15–18 DIV) ina whole-cell voltage-clamp configuration at RT. The composition of theextracellular solution was: 124 mM NaCl, 3 mM KCl, 2 mM CaCl2, 1.1 mMNaH2PO4, 1.3 mM MgSO4, 20 mM HEPES, and 10 mM D-glucose, pH 7.4.Neurons were in the extracellular solution maximally for 2 h. To eliminatesynchronized action potential-induced spontaneous post-synaptic currents, allexperiments were performed in the presence of 1 mM tetrodotoxin (TTX).mEPSCs of glutamatergic origin were isolated by extracellular application of10 mM bicuculline methiodide. Miniature spontaneous events were completely andreversibly blocked by NBQX and AP5 (data not shown). Patch pipettes werefabricated from borosilicate glass (GC-150F; Harvard Apparatus); and theirresistance, when filled with intracellular solution containing 18 mM KCl, 111 mMpotassium gluconate, 0.5 mM CaCl2, 5 mM BAPTA, 2 mM Mg-ATP, 10 mMHEPES, 10 mM glucose, and 2 mM NaOH, at pH 7.3, ranged from 6 to 8 MV.Membrane potential was clamped at 260 mV. Only cells with access resistancethat did not exceed 20 MV were accepted for analysis. Access- and cell membraneresistance were regularly monitored by 10-mV hyperpolarizing voltage pulses100 ms long, and recordings were terminated if any of these resistances changedby 10% or more.

A patch-clamp amplifier (EPC 9; HEKA) was used for voltage clamp and dataacquisition. Data were acquired at sampling rate of 2 kHz and then band-passfiltered (1 Hz–1 kHz). Synaptic events were detected and analyzed using MiniAnalysis software (version 6; Synaptosoft Inc.). After automatic screening(amplitude threshold set to 5 pA), events were approved manually according totheir onset and decay. Because glutamatergic synaptic activity strongly depends onthe age of neurons, the effect of ICAM-5 deficiency was assessed from recordingsobtained on the same DIV. Only recordings that contained at least 200 synapticevents were accepted for analysis. The inter-event interval and event amplitude foreach individual cell were characterized by their median values. The mean ofmedian inter-event intervals and event amplitudes for each experimental paradigmwas normalized to corresponding mean control values obtained on the sameexperimental day.

Lentivirus-based b1 integrin knockdown

Mouse GIPZ lentiviral shRNAmir plasmids for b1 integrin subunit (ClonesV2LMM_39157, V2LMM_188403 and V3LMM_429934) and a control GIPZempty plasmid were purchased from Open Biosystems/ThermoFisher. The level ofb1 integrin knockdown was accessed by transfecting N2A cells and the mosteffective shRNA plasmids were selected for transfecting neurons (supplementarymaterial Fig. S4). Ten DIV cultured hippocampal neurons were transfected with b1integrin-shRNA or control plasmids and cultured for an additional 48 h beforefixation.

b1 integrin activation by TS2/16 in N2A cells

N2A cells were grown in DMEM with 10% FBS, 1% penicillin-streptomycin and1% L-glutamine for 24 h and changed into HBSS without or with 1 mM MnCl2.10 mg/ml mIgG or b1 integrin-activating antibody TS2/16 were added into cellsand incubated for 30 min at 37 C. Cells were then fixed with 4% PFA andimmunostained with b1 integrin monoclonal antibody 9EG7. This antibody hasbeen previously found to recognize the ligand-bound conformation of b1 integrins(Bazzoni et al., 1995).

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Electron microscopy

12–16 weeks old male C57 Bl/6 mice were anaesthetized and perfused with PBSand 0.1 M phosphate buffer, pH 7.4, followed by 4% PFA. Hippocampi weredissected into 1 mm3 cubes and further fixed in 4% PFA at 4 C for 4 days. Toimprove the plasticity of tissue blocks, tissue blocks were infused with 1.8 Msucrose and 20% (w/v) polyvinylpyrrolidone and frozen in liquid nitrogen(Tokuyasu, 1989). Frozen blocks were trimmed and cut into 60-nm-thick sectionsat 2120 C. After being blocked with 1% fish skin gelatin (FSG), 1% FBS in50 mM NH4Cl/PBS, pH 7.4, sections were incubated with anti b1 integrin pAb at5 mg/ml (AB1952, Millipore), followed by protein A-conjugated 10 nm goldparticles (University of Utrecht, The Netherlands). Excessive washing wasperformed after each antibody incubation. To increase the contrast, sectionswere incubated with 2% neutral uranyl acetate for 10 min at RT, followed byincubation with 1.8% methyl cellulose/0.4% uranyl acetate for 15 min on ice.Images were taken with an electron microscope (Tecnai 12, FEI Company,Holland) running at 80 kV under 11,0006magnification. The labeling of b1integrins in active zones was quantified from ,150 synapses which were randomlyimaged from four grids. The active zone was divided horizontally into four equalsections along the pre-synaptic membrane. The two sections in the middlecomposed the ‘central’ sub-region, while the two sections on both sides of theactive zone were named as the ‘peripheral’ sub-region. The number of goldparticles was quantified and presented as the percentage of b1 integrin labeling oneach sub-region: % b1 integrin labeling on the sub-region5number of goldparticles (sub-region)/number of gold particles (whole active zone)6100%.

Quantitative analysis for immunofluorescence

Quantification was performed on more than 10 neurons, which were randomlyselected from three independent experiments. Segments of dendrites less than100 mm apart from the somas were used for quantification. For colocalizationanalysis, region of interest (ROI) was selected manually in a random manner. Onlythose puncta which have $50% area fallen in the ROIs were counted as‘colocalized’. Analysis was performed with the same criteria in all experimentsand the genotypes or treatments were unknown to the analyzer.

For spine analysis, dendritic protrusions were categorized by the followingcriteria: mushroom spine: length ,3 mm and with an enlarged head; thin spine:length .3 mm and with an enlarged head; filopodium: length53–10 mm, withoutan enlarged head. To quantify the correlation of b1 integrins with the EGFP-labeled spines, .50% areas of spine heads overlapping with b1 integrin stainingwas defined as ‘colocalized’, while ,50% was ‘juxtaposed’. Images wereprocessed with Photoshop and ImageJ.

Statistical analysis

Mann–Whitney U tests were used to measure the significance of inter-groupdifferences between datasets.

AcknowledgementsWe thank Dr Yoshihiro Yoshihara for providing the anti-ICAM-5cppAb; Dr Patrick Kilgannon for rat ICAM-5 mAbs and the pAb1000J; Dr Timothy A. Springer for the b1 integrin activatingantibody TS2/16; Dr Antti Vaheri for the rabbit anti-fibronectin pAb;Juha Kuja-Panula for help with Lentiviral shRNA; LeenaKuoppasalmi, Seija Lehto, Outi Nikkila, Erja Huttu and MariaAatonen for technical assistance; and Yvonne Heinila and LeeaSokura for secretarial help.

FundingThis study was supported by the Academy of Finland, the SigridJuselius Foundation, the Magnus Ehrnrooth Foundation, the FinnishMedical Association, Wilhelm och Else Stockmanns stiftelse and theLiv och Halsa Foundation. Additional support was from the NationalInstitutes of Health [grant number MH060420 to R.L.D.]. Depositedin PMC for release after 12 months.

Supplementary material available online at

http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.106674/-/DC1

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