DIgLONs inhibit initiation of neurite outgrowth from forebrain neurons via an IgLON-containing receptor complex

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DIgLONs inhibit initiation of neurite outgrowth from forebrain neurons via an IgLON-containing receptor complex Mohammed Akeel 1,2 , Christine J. McNamee 2 , Sahar Youssef 3 , Diana Moss⁎ Department of Human Anatomy and Cell Biology, Liverpool University, Liverpool, UK

A R T I C LE I N FO

AB S T R A C T

Article history:

IgLONs are a family of four GPI-anchored cell adhesion molecules that regulate neurite

Accepted 9 December 2010

outgrowth, synaptogenesis and may act as tumour suppressor genes. IgLONs are thought to

Available online 15 December 2010

function as monomers or homodimers and we have proposed that IgLONs also act as heterodimeric complexes termed Dimeric IgLONs or DIgLONs. Here we show that the

Keywords:

initiation of neurite outgrowth is inhibited from a subset of chick embryonic day (E) 7 or

GPI-anchored glycoprotein

8 forebrain neurons when they are cultured on CHO cell lines expressing DIgLON:CEPU-1-

Axon guidance

OBCAM and DIgLON:CEPU-1-LAMP but not on CHO cells that express single IgLONs CEPU-1

Cell adhesion molecule

or OBCAM. Surprisingly at the younger age of E6 forebrain neurons do not respond to

Heterodimeric complex

DIgLONs. Since there is little difference in expression of IgLONs on the surface of chick

Forebrain neuron

forebrain neurons at these two ages we suggest IgLONs alone are not the receptor on the responding forebrain neurons. A DIgLON heterodimeric recombinant protein DIgLON:CEPU1-OBCAM-Fc also blocked neurite outgrowth from E8 chick forebrain neurons. However, when IgLONs were removed from the surface of these E8 neurons they no longer responded to DIgLON:CEPU-1-OBCAM-Fc substrate, indicating that IgLONs form at least a component of the neuronal cell receptor complex involved in this inhibition of neurite outgrowth. Inhibitors pertussis toxin and Y27632 reversed the inhibition of neurite outgrowth on a DIgLON:CEPU-1-OBCAM and DIgLON:CEPU-1-LAMP substrate. This suggests the involvement of a G-protein coupled receptor and activation of Rho A. In summary we provide evidence that DIgLON:CEPU-1-OBCAM and DIgLON:CEPU-1-LAMP complexes regulate initiation of neurite outgrowth on forebrain neurons via an IgLON-containing receptor complex. © 2010 Elsevier B.V. All rights reserved.

⁎ Corresponding author. Sherrington Buildings, Ashton Street, Liverpool L69 3GE, UK. Fax: + 44 151 794 5517. E-mail address: [email protected] (D. Moss). Abbreviations: DIgLON, Dimeric IgLON; E6, E7, E8, embryonic day 6, 7, 8; FACS, fluorescent activated cell sorting; PIPLC, Phosphatidyl inositol phospholipase C; PTX, Pertussis Toxin 1 Current address: Faculty of Medicine, Jazan University, Jazan, P.O. Box 114, Saudi Arabia. 2 These authors contributed equally to the work. 3 Current address: Anatomy Department, Faculty of Medicine for Girls, AL-Azhar University, CAIRO-Nasr City, Egypt. 0006-8993/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2010.12.028

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1.

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Introduction

There are four members within the IgLON Ig superfamily of cell adhesion molecules, namely LAMP (Brummendorf et al., 1997; Hancox et al., 1997; Wilson et al., 1996; Zhukareva and Levitt, 1995), OBCAM (Schofield et al., 1989), Neurotrimin/CEPU-1 (Struyk et al., 1995) and Kilon/ Neurotractin (Funatsu et al., 1999; Marg et al., 1999; Schafer et al., 2005). Typically they are highly glycosylated proteins, consisting of three Ig domains followed by a GPI anchor that locates them to specific lipid raft regions within the plasma membrane (Simons and Toomre, 2000). IgLONs are expressed predominately in the nervous system, and both Neurotractin/Kilon and OBCAM are upregulated in astrocytes following lesion (Schafer et al., 2005; Sugimoto et al., 2010). Expression has also been observed in a number of non nervous tissues where they may have a role as tumour suppressor genes. For example in humans a loss of the OPCML gene resulting in loss of OBCAM expression is associated with epithelial ovarian cancer (Sellar et al., 2003) and also with gliomas (Reed et al., 2007). LSAMP gene is thought to act as a tumour suppressor in familial clear cell renal carcinoma (Chen et al., 2003). During development of the nervous system IgLONs regulate axonal growth and synaptogenesis, while in the adult there is evidence that LAMP expression is correlated with anxiety and other behavioural states (Catania et al., 2008; Nelovkov et al., 2003, 2006). Most experiments have been designed to investigate the activity of each IgLON in isolation. For example, a substrate of LAMP has been shown to increase the length of axons of LAMP-expressing neurons (Mann et al., 1998) while Neurotrimin has been shown to have bifunctional effects (Gil et al., 1998). Furthermore, altering the level of expression of OBCAM or Kilon on dendrites of hippocampal neurons prompts changes in synaptogenesis (Hashimoto et al., 2008; Yamada et al., 2007). In contrast a mixture of IgLONs isolated from adult chick brain, originally termed GP55, inhibited neurite outgrowth from both DRG and forebrain neurons (Clarke and Moss, 1994; Clarke and Moss, 1997; Wilson et al., 1996). In our hands, individual IgLONs failed to inhibit neurite outgrowth from these neurons (McNamee et al., 2002). More recently we have shown that two IgLONs, CEPU-1 and OBCAM, co-expressed in the plane of the membrane, inhibit neurite outgrowth from cerebellar granule cells in a similar way as GP55 (Reed et al., 2004). We have proposed that IgLONs can form heterophilic complexes within the family in cis and that these heterodimeric IgLON complexes or DIgLONs are active during the development of the nervous system to regulate axon growth and guidance (Reed et al., 2004). Recently this suggestion has been supported by experiments where co-expression of two IgLONs, differentially regulates synaptogenesis of hippocampal neurons when compared to the activity of an individual IgLON (Hashimoto et al., 2009). Here we provide further evidence that IgLON complexes composed of DIgLON:CEPU-1-OBCAM and DIgLON:CEPU-1-LAMP regulate neurite outgrowth from chick forebrain in a stage dependant manner.

2.

Results

2.1. DIgLON:CEPU-1-OBCAM and DIgLON:CEPU-1-LAMP inhibit initiation of neurite outgrowth from E7/8 forebrain neurons Previously we demonstrated DIgLON:CEPU-1-OBCAM inhibited initiation of neurite outgrowth from chick cerebellar granule cells and earlier work showed that an undefined mixture of IgLONs (GP55) also inhibited outgrowth from forebrain and DRG neurons (Clarke and Moss, 1994; Reed et al., 2004; Wilson et al., 1996). Here we have tested whether DIgLON complexes also regulate neurite outgrowth from forebrain neurons. Dissociated E 7/8 chick forebrain neurons were cultured on CHO cell lines expressing DIgLON:CEPU1-OBCAM and DIgLON:CEPU-1-LAMP (Fig. 1A). In both cases approximately 40–50% of the forebrain neurons were inhibited from extending neurites while outgrowth on CHO cell lines expressing single IgLONs CEPU-1, OBCAM and LAMP was indistinguishable from that on wild type CHO cells. Individual lengths of neurites were measured using Metamorph™ Imaging software to establish if the lengths of neurites extended from forebrain neurons on the DIgLON:CEPU-1OBCAM CHO cells varied compared to those on the wild type and CHO cells expressing single IgLONs. Lengths were found to vary between 20 and 140 μm on all the CHO cell lines and are presented as a graph showing the percentage of neurites at each length (Fig. 1B). There was no significant difference in the number of neurons at each length on DIgLON:CEPU-1-OBCAM CHO compared to CEPU-1, OBCAM or wild type CHO cell substrate. This suggested the overall length of neurite outgrowth from the neurons per se was unaffected by the DIgLON:CEPU-1-OBCAM cell substrate, but there was a subpopulation of forebrain neurons that were prevented from initiating neurite extensions. This result is similar to that observed when neurite growth from cerebellar granule cells was analysed in the same way (Reed et al., 2004).

2.2. E6 and E8 forebrain neurons respond differently to DIgLON:CEPU-1-OBCAM and DIgLON:CEPU-1-LAMP Forebrain neurons cultured from E7/8 chicks responded to DIgLON substrates so it was surprising that dissociated E6 forebrain neurons showed no similar response. The number of E6 forebrain neurons extending neurites remained the same on DIgLON:CEPU-1-OBCAM and DIgLON:CEPU-1-LAMP CHO cells as on wild type CHO controls (Fig. 2). These results prompted us to investigate the expression of IgLONs on E6 and E8 forebrain neurons. Dissociated E6 and E8 neurons were incubated with each IgLON antiserum followed by a fluorescent secondary antibody and the number of cells stained was measured by FACS. The specificity of the individual IgLON antisera was confirmed using the IgLON-expressing CHO cell lines (McNamee et al. manuscript submitted). In addition CEPU-1, OBCAM and LAMP each gave a distinct staining pattern on E18 retina (Lodge et al., 2000). FACS analysis of E6 forebrain neurons revealed that 70–80% of neurons stained with each IgLON antiserum. The results at E8 were similar with the exception of a small reduction in the number of LAMPexpressing neurons (Fig. 3A). Overlaying the E6 and E8 FACS

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%E6 forebrain neurons extending neurite outgrowth

100

75

50

25

0

WT

CO

CL

CHO cell line Fig. 2 – E6 forebrain neurons extended neurite outgrowth on DIgLON:CEPU-1-OBCAM and DIgLON:CEPU-1-LAMP CHO cell lines. Histograms represent the % of dissociated E6 forebrain neurons able to extend neurite outgrowth when cultured on a monolayer of wild type (WT n = 4) DIgLON:CEPU-1-OBCAM (CO n = 2) and DIgLON:CEPU-1-LAMP (CL n = 2) CHO cells. Expression of DIgLON:CEPU-1-OBCAM or DIgLON:CEPU-1-LAMP (lighter histogram bars) on the surface of the CHO cells did not significantly differ from the number of neurons extending neurites on wild type CHO cells (darker histogram bars, p values> 0.05, error bars represent SEM).

Fig. 1 – DIgLON:CEPU-1-OBCAM and DIgLON:CEPU-1-LAMP CHO cell lines reduced the initiation of neurite outgrowth from E7/E8 forebrain neurons. A. Dissociated E7/8 forebrain neurons were cultured on confluent mono-layers of CHO cells and the percentage of neurons able to initiate the extension of neurites at a length of at least twice the diameter of the cell body was calculated. When cultured on Wild-type (WT) CEPU-1 (C) OBCAM (O) and LAMP (L) single IgLON CHO cell lines there was no significant difference in outgrowth (lighter histogram bars, p value > 0.5, error bars represent SEM). The number of forebrain neurons extending neurites was reduced by 47% on DIgLON:CEPU-1-OBCAM (CO) and DIgLON-CEPU-1: LAMP-CHO (CL) cell mono-layers (darker histogram bars, p value < 0.01, * <0.001**) n = 3. B. The length of individual neurites was measured using Metamorph™ Imaging software and presented as a graph showing the % of neurites at lengths of 20–140 μm. There was no significance difference in the number of E7/8 forebrain neurons able to extend neurites at each length when cultured on OBCAM (O) CEPU-1 (□) wild type (X) and DIgLON:CEPU-1-OBCAM(Δ) CHO cell lines. This suggested that whilst DIgLON:CEPU-1-OBCAM may inhibit initiation of neurite outgrowth from a sub-population of forebrain neurons it does not have an effect on the overall length of neurite outgrowth.

histograms did show some changes in the distribution of staining intensity (Fig. 3B). Most striking was CEPU-1 where there were two populations at E6 with the weaker staining population decreasing by E8. A similar result was obtained for Neurotractin, whereas for OBCAM and LAMP there appeared to be a single population of

cells. FACS analysis immediately following trypsinisation and after 24 h in culture showed IgLONs to be insensitive to trypsin used to dissociate the forebrain neurons (data not shown). In conclusion there were small changes in IgLON expression on forebrain neurons between E6 and E8 however these changes are unlikely to be sufficient to explain their difference in response to DIgLON:CEPU-1-OBCAM and DIgLON:CEPU-1-LAMP.

2.3. DIgLON:CEPU-1-OBCAM-Fc inhibits outgrowth from E8 forebrain neurons There is considerable evidence to suggest that IgLONs act as CAMs by binding homophilically or heterophilically within the family (Reed et al., 2004). However the mechanism by which IgLONs initiate signal transduction is not understood and the results above raised the possibility that DIgLON:CEPU-1-OBCAM might be interacting with a non IgLON surface receptor. In order to investigate the DIgLON:CEPU-1-OBCAM receptor we constructed a heterodimeric recombinant chimeric protein DIgLON: CEPU-1-OBCAM-Fc. Plasmids containing sequences of OBCAMFc with a myc tag and CEPU-1-Fc with aV5 tag were transfected into CHO cells to prepare a cell line synthesising DIgLON:CEPU1-OBCAM-Fc heterodimeric recombinant protein tagged with both myc and V5. Both CEPU-1-Fc V5 and OBCAM-Fc myc homodimeric proteins are also synthesised. Two rounds of subcloning produced a cell line that expressed DIgLON:CEPU1-OBCAM-Fc plus CEPU-1:CEPU-1-Fc and OBCAM:OBCAM-Fc and ELISA assays measured the concentration of the recombinant proteins to be approximately in a 2:1:1 ratio (Fig. 4A). We then confirmed that a substrate of DIgLON:CEPU-1-OBCAM-Fc inhibited the outgrowth of neurites from E8 forebrain neurons in the same way as DIgLON:CEPU-1-OBCAM CHO cells, whereas

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OBCAM as separate molecules which did not act in synergy to have any effect on the neurons. This confirmed that a complex between CEPU-1 and OBCAM is required to enable IgLONs to inhibit neurite outgrowth.

2.4. Forebrain neurons require IgLONs on their cell surface to respond to DIgLON:CEPU-1-OBCAM-Fc

Fig. 3 – IgLON expression on E6 and E8 forebrain neurons. A. Freshly dissociated E6 and E8 forebrain neurons were stained with specific IgLON antisera and analysed by FACS (CEPU-1 and OBCAM n = 6, LAMP and Neurotractin n = 8). The threshold for counting was set to include 1% of cells stained with secondary antibody alone. There was no significant difference in the percentage cells staining with each antiserum at E6 (lighter histogram bars). Staining at E8 (darker histogram bars) was similar although there was a significant drop in the number of LAMP-staining forebrain neurons (76.5% vs 62.5% p < 0.05*). B. FACS traces for cells stained at E6 (grey peaks) and E8 (black outlines) were overlaid and revealed a difference in distribution for some antisera. CEPU-1 overlay shows there were two populations of cells and the more weakly stained populations is still positive. By E8 the weakly stained population had decreased. A similar but less pronounced result was seen with Neurotractin. For LAMP and OBCAM there was a single population of stained cells.

CEPU-1-Fc, OBCAM-Fc and a mixture of CEPU-1-Fc plus OBCAMFc recombinant proteins had no effect (Fig. 4B). The mixture of the homodimeric recombinant proteins presented CEPU-1 and

The aim of the next experiment was to test whether IgLONs on the surface of forebrain neurons are an essential part of a receptor complex that responds to DIgLONs. IgLONs were removed from the cell surface of forebrain neurons using phosphatidyl inositol phospholipase C (PIPLC) and cultured on a substrate of DIgLON:CEPU-1-OBCAM-Fc. Following PIPLC treatment neurons no longer stained with OBCAM antiserum indicating that OBCAM had successfully been removed from the surface of forebrain neurons (Fig. 5A). CEPU-1 and LAMP antisera produced similar results (data not shown). PIPLCtreated neurons were once again able to extend neurites on DIgLON:CEPU-1-OBCAM-Fc substrate (Fig. 5B) strongly suggesting that IgLONs are a key part of the receptor complex. The identity of the DIgLON:CEPU-1-OBCAM receptor complex on the surface of the neurons and the mechanism by which signals are transduced are still unknown. Pertussis Toxin (PTX) had previously reversed GP55 inhibition of neurite outgrowth from forebrain neurons, suggesting the involvement of Go/Gi via a G protein coupled receptor in IgLON signalling (Clarke and Moss, 1994; Clarke and Moss, 1997). These results were confirmed by adding 10 ng/ml of PTX to the medium of E 7/8 forebrain neurons cultured on DIgLON:CEPU1-OBCAM and DIgLON-CEPU-1:LAMP-CHO cells lines. The forebrain neurons plus PTX in the culture medium were able to initiate the extension of neurites on DigLON substrates to a similar level as found on wild type CHO cell control suggesting that a G protein coupled receptor is involved in DIgLON signalling (Fig. 6A). Many axon guidance molecules regulate neurite outgrowth via the RHO A signalling pathway (Huber et al., 2003). 10 nm Y27632, an inhibitor to Rho-kinase (ROCK) was added to the culture medium and also reversed DIgLON: CEPU-1-OBCAM-CHO inhibition of neurite outgrowth, suggesting the potential involvement of the RHO signalling pathway in transmission of the DIgLON signal (Fig. 6B).

3.

Discussion

This work has provided additional evidence for the DIgLON complexes first proposed by Reed et al. (2004). A mixture of CEPU1-Fc and OBCAM-Fc did not influence forebrain neurons whereas DIgLON:CEPU-1-OBCAM-Fc had a similar activity to that of the DIgLON:CEPU-1-OBCAM-CHO cells. This result provided further support for the hypothesis that CEPU-1 and OBCAM must form a complex to be active rather than acting in synergy. IgLONs are thought to be cell adhesion molecules and there are numerous reports that demonstrate their ability to bind in trans (between cells). However, there is as yet, no known mechanism by which IgLONs can transduce signals and initiation downstream signal transduction pathways. In addition the small differences in IgLON expression between

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E6 and E8 make it unlikely that IgLONs alone act as their receptor. The removal of GPI-anchored glycoproteins from the surface of neurons did block the neuronal response to DIgLON: CEPU-1-OBCAM-Fc so we propose that the DIgLON:CEPU1-OBCAM receptor is composed of one or more IgLONs complexed with an as yet unknown transmembrane receptor. There are numerous examples of GPI-anchored glycoproteins forming part of a receptor complex, for example, p75 associates with the GPIanchored NOGO receptor NgR (Wang et al., 2002). Results with PTX suggest a G protein coupled receptor may be a candidate and DIgLON:CEPU-1-OBCAM-Fc will provide a useful tool to further characterise the nature of the DigLON receptor complex on neurons. It remains to be seen whether there may be distinct receptors for each IgLON pair. Further work is required to understand the contribution IgLONs make to the growth and guidance of forebrain axons and the selection of synaptic partners. Pairs of specific IgLON antisera have been used to stain forebrain neurons and confocal microscopy revealed each of the six combinations of IgLON pairs co-localised as DIgLONs to a specific region on the forebrain neuron (McNamee et al. manuscript submitted). Further detailed analysis of the pattern of IgLON expression within the forebrain may reveal whether combinations of IgLONs are expressed in specific regions or perhaps they are expressed as a gradient. Previous work has largely examined the role of IgLONs as individual proteins and it is likely that there are circumstances where IgLONs act as monomers or homodimers. Gil et al. show that Neurotrimin/CEPU-1 can form homodimers on the surface of

A Absorbance 450nm

1.5

1.0

0.5

0.0

CO-FC

OO-FC

CC-FC

Recombinant protein

%E7/8 forebrain neurons extending neurite outgrowth

B

100

* 50

0

O-Fc

C-Fc

O-Fc

C-Fc

C+O-Fc

CO-FC

CO-Fc

31

transfected cells (Gil et al., 1998), results we have confirmed (Reed and Moss unpublished). LAMP alone modulates the length of axons and Neurotrimin alone has opposite effects on axon extension from DRG and sympathetic neurons (Gil et al., 1998; Mann et al., 1998). However both IgLONs and DIgLONs have been shown to regulate synaptogenesis between hippocampal neurons (Hashimoto et al., 2009). For example LAMP and OBCAM upregulate synapse formation when overexpressed individually, but have no effect when they are both over expressed. Over expression of Neurotrimin alone has no effect, whereas Neurotrimin and OBCAM together down regulate the number of synapses. Thus the activity of two IgLONs together is distinct from that of individual members of the family (Hashimoto et al., 2009). The combinatorial activity of four IgLONs and six DIgLONs may play an important role in specifying synapse formation. Further characterisation of the molecular interactions of the IgLON family is critical in determining the function of this family in neural differentiation, axon extension, synaptogenesis and plasticity, also as a tumour suppressor gene in various different cancers.

Fig. 4 – DIgLON:CEPU-1-OBCAM-Fc is synthesised by CHO cells and inhibits neurite outgrowth. A. CHO cells were doubly transfected with constructs coding for CEPU-1-Fc-V5 and OBCAM-Fc-myc. The heterodimeric recombinant protein DIgLON:CEPU-1-OBCAM-Fc tagged with V5 and myc, along with homodimeric proteins OBCAM-Fc-myc and CEPU-Fc-V5 were secreted into the culture medium. Following two rounds of subcloning medium produced by these cell lines was assayed using a capture ELISA assay to determine the ratio of heterodimeric recombinant protein to homodimeric proteins. DIgLON:CEPU-1-OBCAM-Fc (CO-Fc) was captured with anti myc and detected with anti-V5-HRP; OBCAM-Fc-myc (OO-Fc) was captured with anti-myc and detected with anti-myc-HRP and CEPU-Fc-V5 (CC-Fc) was captured with anti-V5 then detected with anti-V5-HRP. Neat medium from the cell line was assayed in triplicate and the ratio of expression was approximately 2:1:1 DIgLON:CEPU-1-OBCAM-Fc : homodimeric OBCAM-Fc-myc: homodimeric CEPU-Fc-V5 (n = 3). B. Forebrain neurons were grown on substrates of protein A plus recombinant proteins and Poly-L-Lysine (PL). Protein A was added to capture the Fc portion of the recombinant protein and orientate the IgLON monomers. There was no significant difference in the number of E 7/8 forebrain neurons extending neurites on a CEPU-1-Fc (C-Fc) or OBCAM-Fc (O-Fc) substrate (p value < 0.05, error bars represent SEM, data normalised to 100%). A mixture of the two homodimeric recombinant proteins (C-Fc + O-Fc) had no effect on the percentage of forebrain neurons extending neurite outgrowth, n = 3 (p value < 0.05). However there was a significant reduction in the number of forebrain neurons extending neurites on DIgLON:CEPU-1-OBCAM-Fc (CO-Fc) heterodimeric recombinant protein substrate (darker histogram bar p value < 0.01*, error bars represent SEM). Cartoons of the recombinant proteins illustrate their structure.

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Fig. 5 – Removal of IgLONs from the surface of forebrain neurons reverses DIgLON:CEPU-1-OBCAM-Fc inhibition of neurite outgrowth. A. Dissociated E8 forebrain neurons were treated with PIPLC and stained with OBCAM antisera. Phase and fluorescence images of untreated forebrain neurons are clearly stained with OBCAM antisera. In contrast the neurons treated with PIPLC (second pair of images) demonstrates the lack of OBCAM staining suggesting IgLONs have been removed from the surface of these neurons. B. Forebrain neurons cultured on recombinant CEPU-1-Fc (C−), OBCAM-Fc (O−) DIgLON:CEPU-1-OBCAM-Fc (CO-Fc−) proteins without treatment with PIPLC are shown as light histograms. Only those forebrain neurons cultured on DIgLON:CEPU-1-OBCAM-Fc show a reduction in neurite outgrowth (*p value > 0.05 compared to C-Fc−, O-Fc− data normalised to 100%). The cultured forebrain neurons treated with PIPLC (dark histograms bars) show no significant reduction in neurite outgrow on DIgLON:CEPU-1-OBCAM-Fc (CO-Fc+) the inhibition in neurite outgrowth had been reversed back to a similar level as found on CEPU-1-Fc (C+) and OBCAM-Fc (O+) homodimeric recombinant proteins.

4.

Experimental procedures

4.1.

Preparation of DIgLON recombinant proteins

α2OBCAM-Fc, α2βCEPU-1-Fc and α1LAMP-Fc recombinant proteins were prepared either from stably transfected J558L mouse myeloma or CHO cell lines as described (Howard et al., 2002). A stably transfected CHO cell line secreting DIgLON: CEPU-1-OBCAM-Fc recombinant protein was prepared as follows. CEPU-1-Fc-V5-His6 was constructed by cloning

CEPU-1-Fc, obtained by PCR, into pBudCE4.1 (Invitrogen) such that V5-His6 epitope tag was in frame. OBCAM-Fc-myc-His6 was constructed by cloning OBCAM-Fc, obtained by PCR, into pcDNA6 (Invitrogen) such that the myc-His6 epitope tag was in frame. Both these constructs were co transfected into CHO cells and doubly transfected cells were selected using blasticidin 5 μg/ml and zeocin 200 μg/ml. After two rounds of subcloning, recombinant proteins produced were characterised by ELISA using anti myc or anti V5 to capture the proteins and HRP-labelled anti V5 or anti myc (Invitrogen) to detect the protein. The method was essentially as described in the

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%E7/8 forebrain neurons extending neurite outgrowth

A

pH 5 containing 0.03% sodium perborate (Sigma) incubated at RT for 30 min. The reaction was terminated with 1 M H2SO4 and the OD at 450 nm was read in triplicate for each sample to estimate the amount of proteins produced.

100

75

4.2.

50

*

** 25

0

WT-

WT+

CO-

CO+

CL-

CL+

+/-PTX

%E7/8 forebrain neurons extending neurite outgrowth

B

33

100

75

50

Transfected cell lines

CHO cell lines stably expressing cell surface α2OBCAM, α2βCEPU-1 and α1LAMP IgLON sequences (GenBank accession numbers: Q98892, Q90773, and Y08171 respectively) were prepared as described previously (Lodge et al., 2001; McNamee et al., 2002) and cultured in DMEM/F12 plus 0.584 mg/ml glutamine (Sigma) with 10% foetal calf serum (FCS). DIgLON: CEPU-1-OBCAM and DIgLON:CEPU-1-LAMP expressing CHO cell lines were prepared using the pBudCE4.1 (Invitrogen) plasmid vector containing the α2CEPU-1 sequence cloned downstream of the EF-1α promoter and the α2OBCAM or α1LAMP sequence cloned downstream of the CMV promoter as described previously (Reed et al., 2004).

**

4.3.

CO-

IgLONs were removed from the surface of forebrain neurons by incubating the dissociated neurons in 1 unit/ml phosphatidylinositol-specific phospholipase C (PIPLC) at 37 °C for 1 h prior to culture. The neurons were then cultured for 24 h in medium containing 0.1 unit/ml PIPLC (Invitrogen).

PIPLC removal of IgLONs

25

0

WT-

WT+

CO+

+/-Y27632 Fig. 6 – Inhibitory effect of DIgLON:CEPU-1-OBCAM and DIgLON: CEPU-1-LAMP CHO cells on neurite outgrowth from E7/8 forebrain neurons is reversed by the specific inhibitors PTX and Y27632. A. When E7/8 forebrain neurons were cultured on a monolayer of CHO cells expressing DIgLON:CEPU-1-OBCAM (CO−) and DIgLON:CEPU-1-LAMP (CL−) without the addition of PTX to the medium, initiation of neurite outgrowth from forebrain neurons was significantly reduced (dark histogram bar) compared to wild type (WT− first light histogram bar, p values<0.001**, <0.01* respectively). The addition of PTX to the medium of the cultured neurons on DIgLON:CEPU-1-OBCAM (CO +) and DIgLON-CEPU-1:LAMP-CHO (CL+) cell lines reversed this inhibition to a similar level to that found on wild type CHO (WT+, lighter histogram bars, n=4, error bars represent SEM). B. In a second experiment Y27632 was added to the medium of forebrain neurons. Neurons cultured without Y27632 were inhibited by DIgLON:CEPU-1-OBCAM-CHO cells (CO− dark histogram), but the addition of Y27632 reversed this inhibition (CO+, light histogram bar, p value>0.5, n=3, error bars represent SEM) back to a similar level on wild type CHO cell substrate (WT ± light histogram bars).

product details except the ELISA plates were coated with unlabelled V5 and myc antibodies in Na2CO3 buffer pH 9.5. DIgLON:CEPU-1-OBCAM-Fc recombinant protein was assayed by capturing the protein with anti myc and detecting it with anti V5-HRP. Any CEPU-1:CEPU-1-Fc homodimeric recombinant protein was captured with anti V5 and detected with anti V5-HRP, OBCAM:OBCAM-Fc was similarly detected by anti myc and anti myc-HRP. HRP was visualised with freshly prepare 0.1 mg/ml 3,3′,5,5′-titramethylbenzidine dihydrochloride (TMB) substrate prepared in 0.05 M phosphate-citrate buffer

4.4.

Neurite outgrowth assays

Neurite outgrowth assays were carried out on substrates of transfected IgLON CHO cell lines and IgLON-Fc recombinant proteins (McNamee et al., 2002). To prepare neuronal cultures the chick forebrain was dissected, torn into pieces and dissociated in 0.05% trypsin in Ca2+ Mg2+ free Hanks buffered saline solution (HBSS−) containing 100 μg/ml DNAse (Sigma) for 30 min at 37 °C, washed in HBSS and 1 mg/ml soyabean trypsin inhibitor (Sigma). Neurons were resuspended in DMEM/F12 plus 0.584 mg/ml glutamine medium (Sigma) containing 1.5% glucose, 100 μg/ml transferrin (Sigma), 10 μg/ml insulin/0.67 μg/ml selenium (Invitrogen) plus 100 units of penicillin and 100 μg/ml of streptomycin (Invitrogen) (Aizenman et al., 1986; Clarke and Moss, 1994). To prepare the CHO cell substrate wild type or stably transfected single IgLON-CHO cell lines, plus DIgLON:CEPU-1-OBCAM and DIgLON:CEPU-1LAMP cell lines were seeded at a density of 2 × 105 cells/well onto glass coverslips and cultured overnight to confluence in DMEM/F12/glutamine medium containing 10% FCS plus 100 units of penicillin and 100 μg/ml of streptomycin (Invitrogen). Forebrain neurons were seeded at a density of 2 × 105 cells/well onto the CHO cell monolayers and cultured in forebrain growth medium for 28–31 h at 37 °C in a 5% CO2 atmosphere, fixed with ice cold methanol and stained with anti-chicken GAP-43 antiserum diluted 1:500 in 0.12 M phosphate buffer containing 1% Bovine Serum Albumin, (Allsopp and Moss, 1989) followed by 1:200 dilution of Texas Redconjugated anti-Rabbit IgG (Jackson ImmunoResearch). Alternatively neurons were cultured on coverslips coated sequentially with 30 μl nitrocellulose (Schleicher & Schuell) prepared by dissolved a 2 cm square of nitrocellulose in 2 ml of

34

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methanol; followed by 10 μg/ml Protein A (Sigma) plus recombinant proteins and 10 μg/ml of poly L lysine, and finally blocked with 1% BSA. In all cases the total IgLON-Fc concentration was 100 μg/ml. However, since the recombinant DIgLON:CEPU-1-OBCAM-Fc contains a mixture of DIgLON: CEPU-1-OBCAM-Fc, homodimeric CEPU-1-Fc and OBCAM-Fc in a ratio of approximately 2:1:1, DIgLON:CEPU-1-OBCAM-Fc concentration was estimated to be approximately 50 μg/ml. After 24 h in culture coverslips were fixed with 2% gluteraldehyde in 0.12 M phosphate buffer pH 7.4, stained at RT for 5 min with 1% weight: volume Cresyl violet crystals (Sigma) dissolved in PBS, containing 1% acetic acid to visualise neurites. Counts of forebrain neurons extending neurites were made without knowing the conditions to avoid prejudice. In each experiment 100–200 individual neurons per cover slip, from triplicate cover slips, per experiment were counted and the percentage of neurons showing extension of neurites twice that of the diameter of the cell body calculated. The neurons counted were carefully selected as single neuronal cell bodies, avoiding clumps which may have influence overall neurite outgrowth. Neurite lengths from >100 neurons on randomly chosen microscope fields were measured using Metamorph™ Imaging software and the % of neurites at each length between 20 and 140 μm was calculated. Statistics were compiled with Excel, analysed with SPSS (SPSS, Inc.) using One-Way ANOVA.

4.5.

Fluorescent activated cell sorting analysis

Forebrains from E6 and E 8 chick embryos were dissociated as above and cell aggregates were allowed to settle for 10–15 min. 80–90% of the cell suspension was then removed and the cells counted. 1 × 106 cells were centrifuged at 1700 × g for 5 min and resuspended in 25 μl of 0.12 M phosphate pH 7.4, 2 mM EDTA, 0.1% BSA and 0.1% NaN3 (buffer A) containing IgLON antibodies. Following incubation for 30 min at 4 °C the cells were diluted to 0.5 ml in buffer A and centrifuged. Cells were then resuspended in 25 μl of buffer A containing 1:100 dilutions of either anti rat Alexa 488 (Invitrogen) or anti rabbit Phycoethryin (PE, Stratech) secondary antibodies. Antibodies were removed and cells were resuspended in 1.0 ml of buffer A. Cells were analysed on a BD FACS calibur.

Acknowledgments This work was partially supported by a studentship to Mohammed Akeel from Jazan University, Saudi Arabia and by an Egyptian Government studentship to Sahar Youssef.

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