12
Development of the nervous system requires differentiating neurons to grow axons over great distances following long- and short-range signals that are controlled by distinct guidance systems. In general, long-distance acting mole- cules, such as growth factors, are soluble and bind receptors on the neuronal surface. In contrast, short-range membrane-associated molecules interact with surrounding cells and extracellular matrix, allowing for growth through diverse and changing environments. However, the bound- aries between long-range diffusible ligands and short-range contact-mediated factors have blurred. Cell adhesion molecules (CAMs) have increasingly been shown to participate in signaling cascades with classical receptors, Received October 31, 2011; revised manuscript received December 17, 2011, accepted January 10, 2012. Address correspondence and reprint requests to Giampietro Schiavo, Cancer Research UK London Research Institute, 44 Lincoln’s Inn Fields, WC2A 3LY London, UK. E-mail: [email protected] Abbreviations used: ALCAM, activated leukocyte cell adhesion molecule; BSA, bovine serum albumin; CAM, cell adhesion molecule; DMEM, Dulbecco’s minimum essential medium; EDL, extensor digi- torum longus; GDNF, glial cell-derived neurotrophic factor; H C, car- boxy-terminal fragment of tetanus toxin; Ig, immunoglobulin; LAL, levator auris longus; MION, monocrystalline iron oxide nanoparticles; NGF, nerve growth factor; NMJ, neuromuscular junction; p75 NTR, p75 neurotrophin receptor; PBS, phosphate-buffered saline; pERK1/2, phosphorylation of ERK1/2; pTrkA, phosphorylation of TrkA; SDS, sodium dodecyl sulfate; Trk, tropomyosin-receptor-kinase. , , , *Molecular NeuroPathobiology Laboratory, Cancer Research UK London Research Institute, London, UK  Sobell Department of Motor Neuroscience and Movement Disorders, Institute of Neurology, University College London, Queen Square, London, UK àCEA, IRTSV, Biologie a ` Grande Echelle, Grenoble, France §INSERM, U1038, Grenoble, France Universite ´ Joseph Fourier, Grenoble 1, France Abstract Cell adhesion molecules of the immunoglobulin superfamily (IgCAMs) have been shown to modulate growth factor sig- naling and follow complex trafficking pathways in neurons. Similarly, several growth factors, including members of the neurotrophin family, undergo axonal retrograde transport that is required to elicit their full signaling potential in neurons. We sought to determine whether IgCAMs that enter the axonal retrograde transport route co-operate with neurotrophin sig- naling. We identified activated leukocyte cell adhesion mole- cule (ALCAM), a protein involved in axon pathfinding and development of the neuromuscular junction, to be associated with an axonal endocytic compartment that contains neuro- trophins and their receptors. Although ALCAM enters carriers that are transported bidirectionally in motor neuron axons, it is predominantly co-transported with the neurotrophin receptor p75 NTR toward the cell body. ALCAM was found to specifically potentiate nerve growth factor (NGF)-induced differentiation and signaling. The extracellular domain of ALCAM is both necessary and sufficient to potentiate NGF-induced neurite outgrowth, and its homodimerization is required for this novel role. Our findings indicate that ALCAM synergizes with NGF to induce neuronal differentiation, raising the possibility that it functions not only as an adhesion molecule but also in the modulation of growth factor signaling in the nervous system. Keywords: axonal transport, CD166, motor neuron, neurite outgrowth, neurotrophin receptor, signaling endosome. J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07658.x JOURNAL OF NEUROCHEMISTRY | 2012 doi: 10.1111/j.1471-4159.2012.07658.x Ó 2012 The Authors Journal of Neurochemistry Ó 2012 International Society for Neurochemistry, J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07658.x 1

Activated leukocyte cell adhesion molecule modulates neurotrophin signaling

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Page 1: Activated leukocyte cell adhesion molecule modulates neurotrophin signaling

Development of the nervous system requires differentiatingneurons to grow axons over great distances followinglong- and short-range signals that are controlled by distinctguidance systems. In general, long-distance acting mole-cules, such as growth factors, are soluble and bindreceptors on the neuronal surface. In contrast, short-rangemembrane-associated molecules interact with surroundingcells and extracellular matrix, allowing for growth throughdiverse and changing environments. However, the bound-aries between long-range diffusible ligands and short-rangecontact-mediated factors have blurred. Cell adhesionmolecules (CAMs) have increasingly been shown toparticipate in signaling cascades with classical receptors,

Received October 31, 2011; revised manuscript received December 17,2011, accepted January 10, 2012.Address correspondence and reprint requests to Giampietro Schiavo,

Cancer Research UK London Research Institute, 44 Lincoln’s InnFields, WC2A 3LY London, UK.E-mail: [email protected] used: ALCAM, activated leukocyte cell adhesion

molecule; BSA, bovine serum albumin; CAM, cell adhesion molecule;DMEM, Dulbecco’s minimum essential medium; EDL, extensor digi-torum longus; GDNF, glial cell-derived neurotrophic factor; HC, car-boxy-terminal fragment of tetanus toxin; Ig, immunoglobulin; LAL,levator auris longus; MION, monocrystalline iron oxide nanoparticles;NGF, nerve growth factor; NMJ, neuromuscular junction; p75NTR, p75neurotrophin receptor; PBS, phosphate-buffered saline; pERK1/2,phosphorylation of ERK1/2; pTrkA, phosphorylation of TrkA; SDS,sodium dodecyl sulfate; Trk, tropomyosin-receptor-kinase.

,

, ,

*Molecular NeuroPathobiology Laboratory, Cancer Research UK London Research Institute, London,

UK

�Sobell Department of Motor Neuroscience and Movement Disorders, Institute of Neurology, University

College London, Queen Square, London, UK

�CEA, IRTSV, Biologie a Grande Echelle, Grenoble, France

§INSERM, U1038, Grenoble, France

¶Universite Joseph Fourier, Grenoble 1, France

Abstract

Cell adhesion molecules of the immunoglobulin superfamily

(IgCAMs) have been shown to modulate growth factor sig-

naling and follow complex trafficking pathways in neurons.

Similarly, several growth factors, including members of the

neurotrophin family, undergo axonal retrograde transport that

is required to elicit their full signaling potential in neurons. We

sought to determine whether IgCAMs that enter the axonal

retrograde transport route co-operate with neurotrophin sig-

naling. We identified activated leukocyte cell adhesion mole-

cule (ALCAM), a protein involved in axon pathfinding and

development of the neuromuscular junction, to be associated

with an axonal endocytic compartment that contains neuro-

trophins and their receptors. Although ALCAM enters carriers

that are transported bidirectionally in motor neuron axons, it is

predominantly co-transported with the neurotrophin receptor

p75NTR toward the cell body. ALCAM was found to specifically

potentiate nerve growth factor (NGF)-induced differentiation

and signaling. The extracellular domain of ALCAM is both

necessary and sufficient to potentiate NGF-induced neurite

outgrowth, and its homodimerization is required for this novel

role. Our findings indicate that ALCAM synergizes with NGF to

induce neuronal differentiation, raising the possibility that it

functions not only as an adhesion molecule but also in the

modulation of growth factor signaling in the nervous system.

Keywords: axonal transport, CD166, motor neuron, neurite

outgrowth, neurotrophin receptor, signaling endosome.

J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07658.x

JOURNAL OF NEUROCHEMISTRY | 2012 doi: 10.1111/j.1471-4159.2012.07658.x

� 2012 The AuthorsJournal of Neurochemistry � 2012 International Society for Neurochemistry, J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07658.x 1

Page 2: Activated leukocyte cell adhesion molecule modulates neurotrophin signaling

or as ligands and receptors themselves (Maness andSchachner 2007; Schmid and Maness 2008). For example,the best characterized immunoglobulin (Ig) CAM, neuralcell adhesion molecule, is a glial cell-derived neurotrophicfactor (GDNF) co-receptor, and is involved in neuriteoutgrowth and neuron survival in cooperation with fibro-blast growth factor signaling (Saffell et al. 1997; Paratchaet al. 2003).

Neurotrophins are released by target tissues and activatetropomyosin-receptor-kinase (Trk) and p75 neurotrophinreceptor (p75NTR) to mediate neuronal growth and survival.Activated receptor complexes undergo long-range axonaltransport, which is required to elicit their full survivalresponse (Zweifel et al. 2005; Cosker et al. 2008). In motorand sensory neurons, this axonal compartment is specificallyentered by an atoxic carboxy-terminal binding fragment ofthe tetanus toxin (HC) (Lalli and Schiavo 2002). Thisproperty has been exploited to purify these organelles usingmagnetic affinity chromatography (Fig. 1) and has allowedthe determination of their composition via proteomic analysis(Deinhardt et al. 2006b). In addition to the neurotrophinreceptors TrkB and p75NTR, and small GTPases controllingprogression along the endosomal pathway (Deinhardt et al.2006b), this strategy yielded several plasma membraneproteins, of which many were found to be CAMs (seeTable 1). Considering the growing evidence that CAMs havediverse roles in growth factor signaling, we investigatedwhether the CAMs identified in the proteome of HC-containing carriers undergo retrograde transport with neuro-trophins and their receptors, and whether or not they canmodulate their signaling.

In this study, we focused on activated leukocyte celladhesion molecule (ALCAM), known also as CD166, aclassical IgCAM with two distal variable Ig domains, threeconstant type-2 Ig domains, followed by a transmembraneregion and a short cytoplasmic tail (Swart 2002). ALCAMexpression is developmentally regulated in a wide-range oftissues and has been associated with cells undergoingdynamic growth and migration (Swart et al. 2005). In thedeveloping nervous system, ALCAM is involved in axonaloutgrowth and pathfinding (Ott et al. 1998, 2001; Avciet al. 2004). However, research has mainly focused on itsrole as a substrate permissive to neurite elongation (Ka-wauchi et al. 2003; Avci et al. 2004). Our work reveals forthe first time that ALCAM is not merely a substrate forneurite outgrowth, but participates in neurotrophin signaling.We found that ALCAM undergoes bidirectional movementin axons, and that it colocalizes with p75NTR duringretrograde transport. Over-expression and down-regulationstudies revealed a novel role for ALCAM in the potentiationof neurite outgrowth in PC12 cells, which occurs byenhancing TrkA phosphorylation in response to nervegrowth factor (NGF).

Methods

Endosome purificationAmino-derivatized monocrystalline iron oxide nanoparticles(MION) (Moore et al. 1997) were coupled to cysteine-rich taggedHC (Deinhardt et al. 2006b) or bovine serum albumin (BSA; Sigma-Aldrich, Gillingham, Dorset, UK) (Fig. 1a). Briefly, 4 mg MIONswere incubated with 1 mM EDTA and 4 mM succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (Thermo Fisher Scien-tific, Cramlington, Northumberland, UK) for 30 min at 22�Cfollowed by 2 h at 4�C on a rotor wheel. Activated MIONs werepurified on a PD10 column (GE Healthcare, Little Chalfont,Buckinghamshire, UK) and divided into two batches, which werelabeled for 48 h at 4�C with 200 lg HC or BSA previously reducedwith 1 mM Tris (2-carboxyethyl) phosphine (Pierce) for 30 minat 22�C. The reaction was blocked by addition of 10 mMb-mercaptoethanol overnight. Conjugated MIONs were purified onSephacryl S100HR (GE Healthcare). Figure 1a shows a schematicof the procedure used for the purification of HC-MIONs endosomes(Deinhardt et al. 2006b). Ventral horn motor neuron cultures (Arceet al. 1999) were incubated with conjugated MIONs at 37�C incomplete medium for 1 h. After cooling on ice, cells were scraped inHank’s balanced salt solution pH 7.4 supplemented with a proteaseinhibitor cocktail (Roche Diagnostics, Burgess Hill, West Sussex,UK), centrifuged at 170 g for 5 min at 4�C and resuspended inbreaking buffer (0.25 M sucrose, 10 mM HEPES–KOH, pH 7.2,1 mM EDTA, 1 mM magnesium acetate and protease inhibitors).Neurons were passed 15 times through a cell cracker (18 lmclearance; European Molecular Biology Laboratories, Heidelberg,Germany) and clarified at 690 g for 10 min at 4�C. AS columns(Miltenyi Biotech, Bisley, Surrey, UK) were equilibrated withbreaking buffer supplemented with 0.4% BSA, placed inside aSuperMACS II (Miltenyi Biotech), and loaded with the post-nuclearsupernatant. After washing with breaking buffer, columns wereremoved from the magnetic field and elution was carried out withbreaking buffer containing 300 mM KCl. Proteins were precipitatedand analyzed by sodium dodecyl sulfate (SDS)–polyacrylamide gelelectrophoresis using 4–12% gradient gels (Life Technologies,Paisley, UK).

Mass spectrometry and protein identificationAfter SDS–polyacrylamide gel electrophoresis, bands were excisedfrom the Coomassie blue-stained gel and in-gel digestion wasperformed as previously described (Brun et al. 2007). Gel pieceswere then sequentially extracted with 5% (vol/vol) formic acidsolution, 50% acetonitrile, 5% (vol/vol) formic acid, and acetoni-trile. After drying, the tryptic peptides were resuspended in 0.5%aqueous trifluoroacetic acid. Samples were injected into a CapLCnanoLC system (Waters, Elstree, Hartfordshire, UK) and first pre-concentrated on a 300 lm · 5 mm pre-column (PepMap C18;Dionex, Camberley, Surrey, UK). The peptides were then elutedonto a C18 column (75 lm · 150 mm; Dionex). Chromatographicseparation was performed using a gradient transition from solutionA (2% acetonitrile, 98% water and 0.1% formic acid) to solution B(80% acetonitrile, 20% water and 0.08% formic acid) over 60 min ata flow rate of 200 nL/min. The LC system was directly coupled to amass spectrometer (QTOF Ultima; Waters). MS and MS/MS data

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2 | A. Wade et al.

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were acquired and processed automatically using MassLynx 4.0software (Waters). Database searching was performed usingMASCOT 2.1 software (Matrix Science). Two databases wereused: an in-house list of well-known contaminants (keratins andtrypsin) and an updated compilation of the SwissProt and Trembldatabases.

Axonal transport assaysMotor neurons were exposed to an antibody against the ALCAMectodomain labeled using the Monoclonal Antibody Labeling Kit(Life Technologies) alone or in combination with an AlexaFluor-conjugated p75NTR antibody (Deinhardt et al. 2007), HC555 (Lalliand Schiavo 2002) or LysoTracker (Life Technologies) at 37�C for30 min. Neurons were washed with imaging medium (Dulbecco’sminimum essential medium without phenol red, DMEM-), ribofla-vin, folic acid and penicillin/streptomycin, supplemented with30 mM HEPES–NaOH pH 7.4), placed in a 37�C humidifiedchamber and an image taken every 5 s with a Zeiss LSM 510confocal laser scanning microscope using a 63· 1.4 NA Plan-Apochromat oil-immersion objective (Lalli and Schiavo 2002).Tracking was performed on time lapse sequences using AQMsoftware as previously described (Bohnert and Schiavo 2005).

ImmunofluorescencePC12 cells were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS), quenched with 50 mM NH4Cl pH 7.5,washed with PBS and mounted directly, or surface ALCAM waslabelled with a goat antibody directed to ALCAM ectodomain and acorresponding fluorescent secondary antibody. Alternatively, neu-rons were incubated with a fluorescent antibody against theextracellular domain of the protein of interest, for example, ALCAM(R&D Systems, Abingdon, Oxfordshire, UK) or p75NTR (Deinhardtet al. 2007) at 37�C for 1–2 h, acid washed (Deinhardt et al. 2006a)and then processed for immunofluorescence.

Neurite outgrowth quantificationPC12 cells were maintained in DMEM (Life Technologies)supplemented with 7.5% horse serum, 7.5% fetal bovine serum,4 mM glutamine, 1% penicillin/streptomycin and transfected withLipofectamine 2000 (Life Technologies) according to manufac-turer’s instructions. The transfection medium was changed after4 h to differentiation medium (DMEM supplemented with 0.1%horse serum, 0.1% fetal bovine serum, 4 mM glutamine) with orwithout the appropriate differentiation stimulus [NGF (LifeTechnologies), 100 ng/mL, dibutyryl-cAMP (Sigma-Aldrich),0.1 mM or 0.5 mM, GDNF (Life Technologies), 100 ng/mL inthe presence of 10 lg/mL GDNF receptor alpha chimera (GFRa-Fc; R&D Systems)]. Human Fc (Hu-Fc), CD6-Fc or ALCAM-Fcchimeras (R&D Systems) were used at 1–10 lg/mL. For knock-down experiments, PC12 cells were treated with 1 lg of shRNAvector (CSHCTR001-LvmH1) or shRNA ALCAM clone 1(RSH051336-1-LvmH1; OS377393, Genecopoeia, Source Bio-Sciences, Nottingham, Nottinghamshire, UK) pre-mixed with1.5 ll of Lipofectamine 2000 in Opti-MEM I Reduced SerumMedium, GlutaMAX (Life Technologies). After 5 h, cells werereturned to normal medium for 48 h and then differentiated for24 h in DMEM supplemented with 4 mM glutamine, 1%penicillin/streptomycin and 100 ng/mL NGF.

Cells were fixed in 4% paraformaldehyde in PBS at 1, 3, 5 or7 days after transfection as indicated. Cells were processed forimmunofluorescence and nuclei were stained with 1 : 2000 DRAQ5(BioStatus, Shepshed, Leicestershire, UK). At least 10 random fieldswere selected per coverslip and an average of 48 cells were countedper condition. Cells were regarded as exhibiting neurite outgrowth ifthe longest neurite was at least twice the widest diameter of thesoma. In the knockdown experiments optimum cell viability wasobtained after 1 day NGF treatment, therefore we classified cells asexhibiting neurite outgrowth whose longest neurite was at least aswide as the soma.

Quantitative RT-PCRPC12 cells were seeded in 6-well plates to 70% confluence inDMEM supplemented with 7.5% horse serum, 7.5% fetal bovineserum, 4 mM glutamine and transfected with shRNA vector andALCAM shRNA as above. Cells were left in normal medium for72 h or after 48 h recovery transferred to serum-free DMEMsupplemented with 100 ng/mL NGF for 24 h. The medium was thenremoved from the cells and total RNA extracted as per Qiageninstructions (Crawley, West Sussex, UK). First-strand cDNA wasgenerated using the Vilo kit (Life Technologies). The cDNA wasthen used in qPCR reaction with the following primers: rat ALCAMforward TGAGGAGTTCATGTTTTACTTACCA and reverseCGTCTGTCAGTGTGTAAGTGTTTG; rat tubulin forward CAGAGCCATTCTGGTGGAC and reverse GCCAGCACCACTCTGACC mixed with Fast SYBR� Green (Life Technologies) asinstructed by manufacturer and then run on an Applied Biosystems7900HT PCR.

Western blottingHEK293 cells were seeded in a 6-well plate to 70% confluence inDMEM supplemented with 10% fetal bovine serum. Cells weretransfected the following day as for PC12 cells and left to recoverfor 72 h. Cells were harvested in cold PBS, washed twice and thenlysed in 10 mM Tris–HCl pH 8.0, 150 mM NaCl, 1 mM EDTA 1%NP40 supplemented with HALT� Protease and PhosphataseInhibitor cocktail (Thermo Fisher Scientific), vortex mixed at 4�Cfor 30 min followed by 15 min of centrifugation at 16 200 g. Thesupernatant was removed, protein concentration determined and10 lg of lysate subjected to western blotting. The polyvinylidenedifluoride membrane was probed with a goat anti-ALCAM antibody(R&D Systems) at 1 : 300 or anti-actin monoclonal antibody(Sigma AC-15) at 1 : 5000 dilution for loading control overnight,washed three times in TBST and incubated for a further 1 h in theappropriate horseradish peroxidase-conjugated secondary antibody(Dako, Ely, Cambridgeshire, UK). Blots were developed with ECL(GE Healthcare).

Signaling experimentsCells starved of serum and neurotrophins for 5 h were stimulatedwith 100 ng/mL NGF for the indicated times, washed once withPBS prior to addition of 100 lL of high SDS-containing lysisbuffer (2% SDS, 0.05 Tris–HCl pH 6.8) at 100�C, sonicated threetimes for 10 s and then analyzed by western blot. Quantificationof phosphorylated proteins was carried out using ImageJ andnormalized according to the total protein in the sample. Thechange in phosphorylation observed by western blot is likely to

� 2012 The AuthorsJournal of Neurochemistry � 2012 International Society for Neurochemistry, J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07658.x

Regulation of neurotrophin signaling by ALCAM | 3

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be under-estimated because of the limited transfection efficiency(�40%).

Statistical analysisNeurite outgrowth was analyzed using one-way ANOVA followed bya post hoc Bonferroni’s test on selected groups or using an unpairedt-test. For the signaling experiments, predicted intensity values weregenerated using a model that removed experiment and time-trendeffects allowing data to be pooled across all experiments and timepoints (Fig. 5b). ANOVA was then used to determine whether thedifference between Dendra2 and ALCAM-D predicted phosphory-lation values were significant.

Results

ALCAM undergoes retrograde transport in HC-containingaxonal carriersTo confirm the association of ALCAM with HC-containingaxonal carriers, motor neurons were incubated with HC- orBSA-conjugated MIONs at 37�C and the endosomalcompartment containing internalized MIONs was purifiedby magnetic affinity chromatography (Fig. 1a). The pro-teins present on organelles eluted from the magneticcolumn were then analyzed by western blot. As shown inFig. 1b, ALCAM was found associated with the HC beads,but not with the BSA control pellet. Similarly, p50/dynactin, a component of the dynein motor complex(Fig. 1b), the neurotrophin receptors TrkB and p75NTR

(Deinhardt et al. 2006b) and the small GTPases Rab5 andRab7 (Deinhardt et al. 2006b) were also found specificallyin the HC-MION pellet. This result confirmed the associ-ation between ALCAM and HC-containing endosomes,which are known to undergo axonal transport (Lalli andSchiavo 2002).

A fluorescent antibody directed towards the ALCAMextracellular domain (aALCAM488) was used to visualizethe endocytosis and axonal transport of ALCAM (Fig. 1c andFigure S1). In spinal cord motor neurons, ALCAM undergoesbidirectional transport and a proportion of ALCAM positivepuncta moving in the retrograde direction colocalize with HC

(% overlap ± SD; 59.7% ± 11.4) (Fig. 1c; n = 3 indepen-dent experiments). Importantly, the direct conjugation of thisantibody with fluorophores does not alter its ability torecognize ALCAM (Figure S1), and this staining is specific,because no signal is detected in motor neurons isolated fromALCAM knockout mice (Figure S2) (McKinnon et al. 2000;Weiner et al. 2004). As shown in Fig. 1d, the speeddistribution of ALCAM is similar to that of HC-containingcarriers, although ALCAM transport endosomes have anincreased tendency to pause when compared with HC

carriers. This is shown as a distinct peak at 0 lm/s in theALCAM speed distribution profile (Fig. 1d; green line) andwas confirmed by quantifying the proportion of carriers thatpause during transport (Fig. 1e).

Further analysis of the properties of axonal transport inspinal cord motor neurons revealed that more than half of theALCAM-positive carriers (59.8% ± 9.3) also contain theneurotrophin receptor p75NTR, as shown by a fluorescentlylabeled anti-p75NTR antibody (ap75NTR555) (Fig. 2a; n = 3independent experiments). This high level of colocalizationwas also found in the cell body at later time points (Fig. 2b).

In contrast, very limited overlap was observed betweenALCAM or p75NTR, and LAMP2, a lysosome marker,suggesting that transported ALCAM is not targeted fordegradation (Fig. 2b). Co-transport experiments with aAL-CAM488 and LysoTracker supported the conclusion thatALCAM does not enter a degradative route, because only aminor fraction of transported ALCAM colocalized withacidic compartments in axons (11.7% ± 12.6; Fig. 2c) andno colocalization with Lysotracker was observed in the cellbody (Fig. 2d). These results demonstrate that ALCAMundergoes axonal transport in a non-acidic compartment,which contains neurotrophin receptors.

ALCAM and other CAMs share the same axonal transportcompartmentFurther characterization of ALCAM-containing endosomesrevealed that ALCAM was transported together with canineadenovirus 2 (Fig. 3a). The uptake and axonal transport ofthis neurotrophic virus are mediated by the CAM familymember coxsackievirus and adenovirus receptor (Salinaset al. 2010), which has been previously shown to associatewith this transport compartment in neurons (Salinas et al.2009). Partial overlap with neural cell adhesion molecule,another member of the CAM family found in the HC

proteome (Table 1), was observed in the cell body (Fig. 3b).However, ALCAM does not directly contribute to retro-

grade transport per se, because the speed distribution of fastaxonal retrograde carriers containing HC (Fig. 3c), and byinference TrkB (Deinhardt et al. 2006b), p75NTR (Deinhardtet al. 2007), brain-derived neurotrophic factor (Deinhardtet al. 2006b) and Rab7 (Deinhardt et al. 2006b), wasunaltered in motor neurons lacking ALCAM. As shown inFig. 3c, the speed distribution profiles of retrograde transportcarriers quantified in motor neurons isolated from wild type,heterozygous (ALCAM+/)) or homozygous (ALCAM)/))ALCAM knockout mice (McKinnon et al. 2000; Weineret al. 2004) were almost completely overlapping.

In summary, these results suggest that ALCAM undergoesaxonal transport in an endosomal compartment containingCAMs and neurotrophin receptors, but is not required for therecruitment of molecular motors, nor directly modulates thetransport properties of this axonal retrograde transportcompartment.

ALCAM potentiates NGF-induced neurite outgrowthBased on the presence of neurotrophin receptors andALCAM in the same transport compartment, we went on

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Page 5: Activated leukocyte cell adhesion molecule modulates neurotrophin signaling

Fe

H2N NH2

NH2

SMCC HcFe

* *Fe

Hc Hc

FeHc

Hc

Hca) Cell homogenisationb) Magnetic separationc) Analysis (e.g. MS/MS)

a) Bindingb) Internalisation

CouplingEndosome purification(a)

(b)

HcBSA*

BeadsFTInput

BSA BSA BSAHc Hc Hc

MW(kDa)

10075

(c)

(d) (e)

αALCAM488Hc

0

0.02

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0.1

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–0.6 –0.2 0.2 0.6 1 1.4 1.8 2.2 2.6 3 3.4 3.8

Rel

ativ

e ca

rrie

r fre

quen

cy

Speed (μm/s)0

0.4

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Car

riers

/min

αALCAM488 Hc

Hc555 αALCAM488

*

*

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*

*

*

0 s

7.5 s

15.0 s

22.5 s

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FeHc

Hc

Hc

αALCAM

p50/dynamitin50

*

*

*

*

*

*

*

*

*

*

*

*

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*

Merge

Fig. 1 ALCAM undergoes axonal retrograde transport in HC-contain-

ing endosomal carriers. (a) MIONs were activated with heterobifunc-

tional cross-linkers, allowing the immobilization of HC or BSA via

sulfhydryl groups (Deinhardt et al. 2006b). Modified MIONs underwent

binding and internalization in motor neurons. Axonal carriers contain-

ing MIONs were purified from post-nuclear supernatants and their

proteome analyzed by mass spectrometry (Table 1) (Deinhardt et al.

2006b) or western blot (b). A portion of ALCAM and p50/dynamitin

present in the cell lysate (input) were associated with HC-MION beads,

but not with BSA-coated beads (FT, flow through). (c) Consecutive

frames from a movie of HC555 and aALCAM488 (scale bar, 10 lm).

Arrowheads indicate a retrograde transport vesicle containing HC and

ALCAM moving towards the cell body (black arrow). Asterisks indicate

HC transport carriers that do not contain ALCAM. Squares mark

stationary organelles that contain both HC and ALCAM (closed) or

ALCAM alone (open). (d) The speed distribution profile of ALCAM

carriers overlaps with that of HC (n = 183 ALCAM and n = 231 HC

carriers, recorded in 38 axons from eight cultures). (e) The box and

whisker plot shows that a higher proportion of aALCAM488-positive

carriers pause when compared with HC-containing vesicles.

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Regulation of neurotrophin signaling by ALCAM | 5

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to investigate whether the latter has a role in neurotrophinsignaling. PC12 cells were transfected with ALCAM taggedwith the fluorescent protein Dendra2 (ALCAM-D) (Gurs-kaya et al. 2006) or Dendra2 alone, and treated or not with100 ng/mL NGF. ALCAM-D is expressed on the cell surfacewhere it was recognized by an antibody directed toward theALCAM extracellular domain, as well as in internalorganelles, which are not labeled by the antibody undernon-permeabilizing conditions (Fig. 4a). Interestingly, upontransfection with ALCAM-D, the percentage of PC12 cellsthat displayed neurites was significantly increased whencompared with cells transfected with Dendra2 alone (Fig. 4b;p = 0.0059 at 3 days). Although this trend was alreadyvisible after 1 day of NGF treatment, it did not reachstatistical significance (Fig. 4b; p = 0.0996). In contrast, noeffect of ALCAM on neuritogenesis was found in absence ofNGF (Fig. 4c), suggesting that ALCAM over-expressionper se does not directly initiate neurite outgrowth.

To further investigate this process, we transfected cellswith ALCAM-D or Dendra2 and compared their neuritegrowth response to a variety of differentiation stimuli(Fig. 4c). Strikingly, ALCAM over-expression had no effecton neurite outgrowth observed in response to dibutyryl-cAMP, a cell-permeable cAMP analog. This suggests thatALCAM does not act on a signaling cascade controlled bycAMP (Fig. 4c). However, in addition to robustly increasing

NGF-induced neurite elongation, ALCAM over-expressionalso potentiated GDNF-induced differentiation at 7 dayspost-transfection (Fig. 4c; p = 0.0433), suggesting thatALCAM acts as a modifier of specific growth factor-dependent differentiation pathways.

The specificity of these gain-of-function experiments isconfirmed by a loss-of-function approach in which ALCAMhas been down-regulated in PC12 cells using an shRNAstrategy (Fig. 4d–f). Cells were transfected for two days withan shRNA vector, targeting rat ALCAM, and then differen-tiated for one day in the presence of 100 ng/mL NGF.These conditions, which have been selected to maximizecell viability, yielded a robust, yet partial, knockdownof ALCAM both at mRNA (Fig. 4d) and protein (Fig. 4e) lev-els, as shown by quantitative real-time PCR and western blot,respectively. Crucially, under these conditions, NGF-depen-dent neurite outgrowth was significantly impaired in PC12cells in which ALCAM was down-regulated when comparedwith vector-treated controls (Fig. 4f).

Both cis- and trans- interactions of ALCAM contributeto NGF-dependent neurite outgrowthWe generated truncation mutants in order to determinewhich domains of ALCAM were required for the potenti-ation of NGF-dependent neurite outgrowth (Fig. 5a). Dele-tion of the cytoplasmic domain of ALCAM, a short 34

(a)αp75 555 αALCAM4880 s

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Merge αp75 555NTR

LAMP2(d)

Lysotracker Merge884MACLAα

(c)

0 s

7.5 s

15.0 s

22.5 s

Fig. 2 ALCAM is co-transported with p75NTR and CAMs in non-

acidic organelles. (a) Consecutive frames showing co-transport of

ALCAM and p75NTR (arrowheads). (b) ALCAM (pseudocolored in

white in the lower left panel) also colocalizes with p75NTR in vesicles

in the cell body (white arrowheads). There is little or no colocal-

ization of ALCAM with LAMP2, although rare LAMP2-positive en-

dosomes that also contain p75NTR are visible (empty arrowhead).

(c) LysoTracker and aALCAM488 are transported along axons to-

ward the cell body (empty and white arrows, respectively) in dif-

ferent compartments. (d) aALCAM488 and LysoTracker are not

found in the same endosomal organelles in the cell body. Scale

bars, 10 lm.

Journal of Neurochemistry � 2012 International Society for Neurochemistry, J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07658.x� 2012 The Authors

6 | A. Wade et al.

Page 7: Activated leukocyte cell adhesion molecule modulates neurotrophin signaling

(a)

(c)

αALCAM488

Merge αp75 555NTR

NCAMic

(b)

Wild typeALCAMALCAM

Rel

ativ

e ca

rrie

r fre

quen

cy

Speed (μm/s)

+/–

–/–

00.020.040.060.08

0.10.120.140.160.18

0.2

–0.6 –0.2 0.2 0.6 1 1.4 1.8 2.2 2.6 3 3.4 3.8

Hc555

CAV-2

αALCAM488

Merge0 s

6.9 s

13.8 s

20.8 s

Fig. 3 ALCAM and other CAMs share the same axonal transport

compartment. Panel (a) shows consecutive frames from a transport

experiment after incubation with aALCAM488, HC555 and CAV-2-Cy3.

CAV-2 and HC-positive endosomes, moving towards the cell body

contain or lack ALCAM (white or empty arrowheads respectively). (b)

Internalized aALCAM488 and p75NTR colocalize with NCAM in motor

neuron soma as revealed by an antibody directed to its intracellular

domain (NCAMIc). ALCAM and p75NTR are found in organelles with

(empty arrowheads) or without NCAM (white arrowheads). Scale bars,

10 lm. (c) ALCAM does not directly contribute to retrograde transport.

Speed analysis performed in wild type, ALCAM+/) and ALCAM)/)

motor neurons revealed no significant difference in the speed distri-

bution profile of HC transport in the three genotypes (wild type: n = 92

carriers, 22 axons, 4 independent cultures; ALCAM+/): n = 100 carri-

ers, 17 axons, 3 independent cultures; ALCAM)/): n = 101 carriers, 22

axons and 4 independent cultures).

Table 1 Plasma membrane proteins associated with the axonal retrograde transport compartment. Components of integrin, selectin and cadherin

binding complexes are coloured in orange, green and yellow respectively. IgCAMs are coloured in blue and ALCAM, which has been investigated

in this work, is in bold. Other cell membrane proteins identified are also shown. NCAM, CAR and Junction plakoglobin had already been confirmed

by other techniques to associate with the HC-endosome

RP, TeNT HC-associated lipid microdomain proteome; LC, live-cell imaging; IF, immunofluorescence; WB, western blot.aK. Deinhardt and G. Schiavo, unpublished results.bSalinas et al. 2009;.cThis study.

� 2012 The AuthorsJournal of Neurochemistry � 2012 International Society for Neurochemistry, J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07658.x

Regulation of neurotrophin signaling by ALCAM | 7

Page 8: Activated leukocyte cell adhesion molecule modulates neurotrophin signaling

amino acid region with no known interaction motifs, had noeffect on this process (Fig. 5a). Surprisingly, truncations ofthe most distal extracellular domains in DD1-ALCAM-Dand DD1D2-ALCAM-D, which have been previouslyshown to abolish ALCAM interactions in trans (vanKempen et al. 2001), had only a limited impact ondifferentiation (Fig. 5a). Indeed, over-expression of thesemutants still potentiated neurite outgrowth in response to

NGF, although to a lesser extent than the full-length protein(Fig. 5a). However, the extracellular domain of ALCAM isessential for this effect, because a CD8-ALCAM chimeracontaining its transmembrane and cytoplasmic domains wasnot capable of potentiating NGF-dependent neurite out-growth (Fig. 5a). The differential effects of the ALCAM-Dconstructs on neurite outgrowth were not attributable totheir level of expression (Figure S3).

(a)

(c)(b)

0

1.0

1.5

2.0

2.5

5DPT-

5DPTGDNF

5DPT0.1 mMdbcAMP

5DPT0.5 mMdbcAMP

Dendra2ALCAM-D

3DPTNGF

7DPTGDNF1 DPT 3 DPT

0

1

2

3

4

Dendra2 Dendra2ALCAM-D ALCAM-D

*

Neu

rite

outg

row

th (n

orm

alis

edto

con

trol)

Dendra2 ALCAM-D αALCAM

***

*

0.5

Neu

rite

outg

row

th (n

orm

alis

edto

Den

dra2

NG

F)

ALCAM

Actin

Contro

l vec

tor

ALCAM sh

RNA

100

0

150

50

Controlvector

ALCAMshRNA

Neu

rite

outg

row

th (n

orm

alis

edto

con

trol v

ecto

r)(f)(e)(d)

100

0

150

50

mR

NA

leve

ls (

norm

alis

edto

con

trol v

ecto

r)

Controlvector

ALCAMshRNA

*

Fig. 4 ALCAM enhances NGF-induced neurite outgrowth. (a) PC12

cells were transfected with Dendra2 or ALCAM-D and differentiated in

the presence of 100 ng/mL NGF for 3 days (scale bar, 10 lm). Fluo-

rescence is cytosolic in Dendra2-transfected cells whilst it is pre-

dominantly at the plasma membrane in ALCAM-D cells where the

fusion protein is recognized by an anti-ALCAM antibody (aALCAM).

ALCAM-D organelles, which are not labeled by this antibody in non-

permeabilized conditions, are also visible (arrowheads). (b) ALCAM-D

significantly potentiated the percentage of cells that displayed neurite

outgrowth at 3 days post-transfection (DPT) (p = 0.0059; n = 3; error

bars = SEM). (c) Quantification of neurite outgrowth in the presence of

specific differentiation stimuli. ALCAM-D did not induce neuronal dif-

ferentiation in the absence of differentiation stimuli ()) or in response

to dibutyryl-cAMP (dbcAMP; 0.1 or 0.5 mM) 5 days after transfection

(5DPT), but significantly potentiated it in response NGF at 3DPT and

increased outgrowth following treatment with GDNF at 7DPT. Data

were analyzed using one-way ANOVA with Bonferroni’s multiple com-

parison test (*p < 0.05, ***p < 0.001; n = 4; error bars = SEM). (d–f)

ALCAM knockdown was followed both at the level of mRNA by

quantitative real-time PCR (d) and protein level by western blot (e). (f)

Neurite outgrowth upon NGF treatment was significantly impaired in

PC12 cells down-regulated for ALCAM when compared with vector-

treated control cells. Data were analyzed using an unpaired t-test

(*p < 0.05, error bars = SEM).

Journal of Neurochemistry � 2012 International Society for Neurochemistry, J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07658.x� 2012 The Authors

8 | A. Wade et al.

Page 9: Activated leukocyte cell adhesion molecule modulates neurotrophin signaling

To our knowledge, this is the first molecular characteriza-tion of ALCAM function in neurite outgrowth and suggeststhat the protein itself does not engage in downstreamsignaling via its cytoplasmic tail; rather, ALCAM appearsto act as a modifier of differentiation pathways involvingdifferent classes of trophic factors, such as neurotrophins andGDNF. In support of this possibility, incubation of PC12with the soluble ALCAM extracellular domain is sufficient topotentiate NGF-dependent neurite outgrowth (Fig. 5b). Incontrast, the glycoprotein CD6, a well-known heterophilicALCAM binding partner (Bowen et al. 1995), was incapableof potentiating this process (Fig. 5c). These results illustratethat distinct ALCAM ligands differentially affect theALCAM-mediated potentiation of neurite outgrowth inducedby NGF.

ALCAM over-expression affects TrkA signalingWe then assessed the effects of ALCAM-D or Dendra2 inNGF-dependent signaling by treating starved PC12 cells with100 ng/mL of NGF and analyzing the phosphorylation ofTrkA and ERK1/2 (pTrkA and pERK1/2) (Klesse and Parada1999). As shown in Fig. 6a, ALCAM-D over-expressingPC12 cells displayed increased pTrkA levels at 3, 15 and

30 min after NGF activation when compared with Dendra2-expressing cells (Fig. 6a). These differences are highlysignificant for pTrkA (Fig. 6b; upper panel; p = 0.00038),but not for pERK1/2 (Fig. 6b; lower panel; p = 0.19) (seeDiscussion section).

Discussion

In this study, we have shown that ALCAM undergoes axonaltransport in an endosomal compartment associated withneurotrophin signaling and provided evidence that ALCAMhas a cell-autonomous role in promoting differentiation bypotentiating TrkA phosphorylation and NGF signaling.Our findings contribute to the growing body of evidencesuggesting that the interplay between classical IgCAMs andneurotrophic signaling is a widespread function of thisprotein family, rather than the exception. Many CAMs havesimilar properties to ALCAM that may be important forgrowth factor signaling. These properties include site-specificcleavage to release soluble ectodomains that alter adhesivefunctions, but could also modulate growth factor responses,as shown for the ALCAM ectodomain in NGF signaling andneurite outgrowth.

0

0.5

1.5

2.0

2.5

Neu

rite

outg

row

th(n

orm

alis

ed to

Den

dra2

)

Dendra

2

ΔD1-A

LCAM-D

ALCAM-D

ΔC-A

LCAM-D

ΔD1D

2-ALC

AM-D

CD8-ALC

AM-D

*(b)(a)

(c)

Hu-Fc ALCAM-Fc

*

Dendra2ALCAM-D

0.5

1.0

1.5

2.0

Hu-Fc CD6-Fc Hu-Fc CD6-Fc0

****** ***

0

0.5

1.0

1.0 Neu

rite

outg

row

th(n

orm

alis

ed to

Hu-

Fc)

1.5

2.0

Neu

rite

outg

row

th(n

orm

alis

ed to

Den

dra2

Hu-

Fc)

Fig. 5 The extracellular domain of ALCAM is required for potentiation

of NGF-dependent neurite outgrowth. (a) Neurite outgrowth in PC12

cells transfected with ALCAM mutants (lower panel) and differentiated

with 100 ng/mL NGF for 3 days (upper panel). Truncations in the

extracellular domain (DD1-ALCAM-D and DD1D2-ALCAM-D) reduced

the potentiating effect of ALCAM. In contrast, removal of the entire

intracellular domain (DC-ALCAM-D) did not. Consistently, CD8-AL-

CAM-D, which lacks the ALCAM extracellular domain did not poten-

tiate neurite outgrowth. Data analyzed using one-way ANOVA

with Bonferroni’s multiple comparison test (*p < 0.05, ***p < 0.001;

n = 3–8; error bars = SEM). (b) Quantification of Dendra2-transfected

cells with extended neurites 3 days after transfection in response to

100 ng/mL NGF in the presence of 1 lg soluble ALCAM-Fc or human-

Fc (Hu-Fc). A significant difference was seen between samples trea-

ted with ALCAM-Fc and Hu-Fc (p = 0.0285). Data were analyzed

using an unpaired t-test (*p < 0.05; n = 4; error bars = SEM). (c) No

significant potentiation of neurite outgrowth was observed in the

presence of soluble CD6-Fc (1 lg/mL) in either Dendra2 or ALCAM-D

transfected cells treated with NGF for 3 days. Data were analyzed

using unpaired t-test analysis (n = 3; error bars = SEM).

� 2012 The AuthorsJournal of Neurochemistry � 2012 International Society for Neurochemistry, J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07658.x

Regulation of neurotrophin signaling by ALCAM | 9

Page 10: Activated leukocyte cell adhesion molecule modulates neurotrophin signaling

We were unable to determine the exact mechanism bywhich ALCAM potentiates neurotrophin signaling, althoughwe did find an increase in TrkA phosphorylation. No directbinding between TrkA and ALCAM was detected byimmunoprecipitation (data not shown). However, ALCAMcould increase TrkA activation in several ways that wouldnot require direct interaction, including promoting the localconcentration of growth factors at the cell surface, or alteringthe distribution of neurotrophin receptors on the membrane.

We have shown that ALCAM undergoes long-range axonaltransport in a non-degradative compartment associated withneurotrophin receptors, as well as other CAMs. Moreover, wedid not find evidence to suggest that the transported protein isdegraded in the cell body. As the ALCAM extracellulardomain is required for potentiating NGF-signaling, andALCAM does not synergize with the secondary messengercAMP, it is possible that ALCAM’s role in potentiating TrkAsignaling occurs at the plasma membrane or within signalingendosomes, as suggested previously for neurotrophin recep-tors (Moises et al. 2007; Cosker et al. 2008).

Our results also have implications for the study of the role ofALCAM in other tissues and in pathological conditions, suchas cancer. ALCAM is over-expressed in a diverse range ofcancers where it has been associated with tumor growth andmetastasis (Swart et al. 2005; Ofori-Acquah and King 2008).Soluble ALCAM and ALCAM truncation mutants disrupt

ALCAM adhesion and alter metalloprotease activity andcancer cell migration (van Kilsdonk et al. 2008). Moreover,we showed that ALCAMpotentiates growth factor signaling, acondition found in many types of cancers and linked withtumor progression (Spencer-Cisek 2002). Importantly, certainforms of cancer in which ALCAM is over-expressed, such asepithelial ovarian cancer (Rosso et al. 2007), also show highlevels of expression of NGF and TrkA (Campos et al. 2007).Our results predict that ALCAMover-expression in these cellswould potentiate TrkA signaling, possibly contributing to cellsurvival and enhanced tumor progression.

ALCAMcould function as a site of integration for signaling,adhesion and migration. Its clustering at the plasma mem-brane at points of cell-cell contact could provide additionalspatio-temporal control of growth factor receptor signaling atthese specific sites. These signaling hotspots may provide apositive feedback mechanism so that growth and survivalsignals are potentiated at specific points of cell–cell interac-tion, which in turn could help in the stabilization of neuronalnetworks during development.

Further work is still needed to determine the role ofendogenous ALCAM in established neurotrophin-dependentprocesses using cells from ALCAM)/) mice as well asknockdown experiments. Based on the lack of overtdevelopmental effects in ALCAM)/) mice (McKinnon et al.2000; Weiner et al. 2004), a common occurrence in single

pTrkA

panTrk

pERK1/2

ERK1/2

0 03 315 1530 30

Dendra ALCAM-D (+NGF)

min

Trk

Aphosphory

lation

(au)

0

Dendra ALCAM-D

ER

K1/2

phosphory

lation

(au)

1.0

2.0

1.5

0.5

0

1

2

3

4

5

***

0.5

1.5

1.0

2.0

Trk

Aphosphory

lation

(xfo

ldin

cre

ase)

0

0 3 15 30Time

+NGF(min)

DendraALCAM-D

Dendra ALCAM-D

(a) (b)

Fig. 6 ALCAM enhances TrkA phosphorylation in response to NGF. (a)

pTrkA and pERK1/2 are shown at 0, 3, 5 and 30 min after addition of

100 ng/mL NGF to PC12 cells starved for 5 h. ALCAM-D expressing

cells show higher TrkA phosphorylation than control cells in response to

NGF. Four independent experiments are assessed in the lower panel

using one-way ANOVA (p = <0.0004). However, Bonferroni’s multiple

comparison test failed to detect a significant effect of transfection. (b) An

alternative statistical approach that removes variability attributed to

different experiments and timepoints after NGF stimulation is shown in

the box and whisker plot for TrkA (upper panel) and ERK1/2 phos-

phorylation (lower panel) (au, arbitrary units). A significant increase in

TrkA phosphorylation in ALCAM-D transfected cells was found in

comparison to Dendra2 controls (***p < 0.001). In contrast, no signifi-

cant difference in ERK1/2 phosphorylation was detected.

Journal of Neurochemistry � 2012 International Society for Neurochemistry, J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07658.x� 2012 The Authors

10 | A. Wade et al.

Page 11: Activated leukocyte cell adhesion molecule modulates neurotrophin signaling

CAM knockouts caused by the compensatory effect of otherCAMs (Cremer et al. 1994; Cohen et al. 1998), and thefinding that ALCAM is up-regulated in response to injury(Fournier-Thibault et al. 1999); we tested whether itsabsence influences sprouting at the neuromuscular junctionin response to botulinum neurotoxin A (Angaut-Petit et al.1990). No significant difference in sprouting was seen inmice lacking ALCAM (Figure S4), suggesting that ALCAMdoes not play a role in this process. However, botulinumneurotoxin A-induced nerve terminal sprouting may not relyon the neurotrophin pathway potentiated by ALCAM, oralternatively, other proteins may compensate for its loss inthis context.

A better experimental model to validate our studies andextend our conclusions to other neurotrophins could be totest the effects of ALCAM on the plasticity of the dendriticnetwork in response to TrkB receptor signaling (Horch2004). It would be also very interesting to check the effectsof ALCAM deletion in conjunction with other neurotrophinmodifiers, such the scaffolding protein Kidins220/ARMS(Iglesias et al. 2000; Kong et al. 2001).

In conclusion, the results of this study provide strongevidence that CAMs possess functions beyond their adhesiveproperties and undergo complex and long-range endocyticpathways in neurons.

Acknowledgements

We thank Anna Moore, Department of Radiology, MassachusettsGeneral Hospital, for providing MIONs; Anne Gonzalez de Peredo,CEA, Grenoble, for the MS analysis; Katrin Deinhardt, New YorkUniversity School of Medicine, for the purification of axonalendosomes; Joshua Weiner, University of Iowa, for providing theALCAM)/) mice and Gavin Kelly, Cancer Research UK LondonResearch Institute, for help with statistical analysis. AW was inreceipt of a PhD Studentship from the Wellcome Trust. LG is theGraham Watts Senior Research Fellow supported by the BrainResearch Trust. MT was supported by the Marie Curie RTN‘ENDOCYTE’’ from the European Union FP6 Program and GS byCancer Research UK. The authors have no actual or potentialconflict of interest to declare.

Supporting information

Additional supporting information may be found in the onlineversion of this article:

Appendix S1. Supplemental methods.Figure S1. Direct conjugation of the anti-ALCAM antibody

(aALCAM) to fluorophores does not alter the ability of thisantibody to recognize native ALCAM.

Figure S2. aALCAM does not bind to motor neurons isolatedfrom ALCAM knockout mice.

Figure S3. Expression of ALCAM-D constructs does not reflectthe level of neurite outgrowth in response to NGF.

Figure S4. ALCAM does not modulate botulinum neurotoxinA-induced nerve terminal sprouting in vivo.

As a service to our authors and readers, this journal providessupporting information supplied by the authors. Such materials arepeer-reviewed and may be re-organized for online delivery, but arenot copy-edited or typeset. Technical support issues arising fromsupporting information (other than missing files) should beaddressed to the authors.

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