FLRT3 is expressed in sensory neurons after peripheral nerve injury and regulates neurite outgrowth

www.elsevier.com/locate/ymcne Mol. Cell. Neurosci. 27 (2004) 202 – 214

FLRT3 is expressed in sensory neurons after peripheral nerve injury and regulates neurite outgrowth M. Robinson,a M.C. Parsons Perez,a L. Te´bar,a J. Palmer,b A. Patel,a D. Marks,a A. Sheasby,a C. De Felipe,c R. Coffin,b F.J. Livesey,d and S.P. Hunta,* a

Department of Anatomy and Developmental Biology, UCL, London, UK Department of Immunology and Molecular Pathology, UCL, London, UK c Instituto de Neurosciencias, Universitas Miguel Herna´ndez, Alicante 03550, Spain d Wellcome/CRC Institute of Cancer and Developmental Biology, University of Cambridge, Cambridge, UK b

Received 19 February 2004; revised 10 June 2004; accepted 15 June 2004 Available online 17 August 2004 We used a molecular screen to identify genes upregulated in regenerating adult rat dorsal root ganglion cells. FLRT3 mRNA and protein characterized by a fibronectin type III domain and a leucinerich repeat motif was upregulated in damaged sensory neurons. The protein was then transported into their peripheral and central processes where the FLRT3 protein was localized to presynaptic axon terminals. In vitro, the FLRT3 protein was expressed at the cell surface, regulated neurite outgrowth in sensory neurons, but did not exhibit homophilic binding. FLRT3 was widely expressed in the developing embryo, particularly in the central nervous system and somites. However, in the adult, we found no evidence for accumulation or reexpression of the FLRT3 protein in damaged axons of the central nervous system. We conclude that FLRT3 codes for a putative cell surface receptor implicated in both the development of the nervous system and in the regeneration of the peripheral nervous system (PNS). D 2004 Elsevier Inc. All rights reserved.

Introduction The failure of neurons contained entirely within the adult central nervous system (CNS) to regenerate after damage remains a major cause of suffering and has largely resisted therapeutic intervention. The problem is complex, requiring maintained cell survival, axonal regeneration across the site of injury, and the appropriate reconnection with targets to regain function (Fawcett and Keynes, 1990). Numerous repair strategies have been proposed that generally target either the inhibitory central environment through which neurons have to regenerate or the identification of genes that might increase the regenerative capacity of neurons of

* Corresponding author. Department of Anatomy and Developmental Biology, University College of London, Medawar Building, Gower Street, Malet Place, London, WC1E 6BT, UK. Fax: +44 020 7383 0929. E-mail address: [email protected] (S.P. Hunt). Available online on ScienceDirect (www.sciencedirect.com.) 1044-7431/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.mcn.2004.06.008

the adult CNS. The ability of neurons of the adult peripheral nervous system (PNS) to regenerate after injury compared with CNS neurons has often been exploited to identify genes that are pro-regenerative and potentially useful for increasing the regenerative capacity of damaged axons within the CNS (Livesey and Hunt, 1998; Tanabe et al., 2003). For example, it has previously been shown that a prior dconditioningT lesion of the peripheral branch of the sciatic nerve will enhance regeneration of the corresponding central branch of the sensory nerve following either crush of the dorsal root (Richardson and Verge, 1987) or when large-diameter sensory fibers have been cut within the spinal cord (Doubell et al., 1997). Direct activation of the sensory neurons by application of second messengers (Neumann et al., 2002) or inflammation (Lu and Richardson, 1991) has also proved successful. This is thought to be due to the activation of potentially pro-regenerative genes such as c-Jun (Herdegen and Waetzig, 2001; Jenkins and Hunt, 1991), ATF3 (Nakagomi et al., 2003; Tsujino et al., 2000), GAP-43 (Woolf et al., 1990), and Reg-2 (Averill et al., 2002) in sensory neurons that follow damage to their peripheral but not central processes. Furthermore, it has been postulated that successful regeneration will require activation or recapitulation of developmental processes driven by genes that initially establish the nervous system. Thus, candidate genes identified as potentially pro-regenerative in adult peripheral neurons but not present in the adult CNS would be expected to have made an appearance in central neurons at some point during development even though many may adopt different roles at different stages of development [for example, c-Jun, which can regulate apoptosis or neurite outgrowth and survival, depending on developmental stage and neuronal type (Dragunow et al., 2000; Ham et al., 2000)]. Previously, we identified the Reg-2 gene that fulfilled many of these criteria. Following peripheral nerve damage, Reg-2 protein production was upregulated by some adult sensory neurons and all alpha motor neurons, and regeneration of the peripheral nerve was attenuated by immunoneutralizing Reg-2 protein. Reg-2 was expressed by many of the same neurons during

M. Robinson et al. / Mol. Cell. Neurosci. 27 (2004) 202–214

development and in vitro, and was shown to be essential for cell survival maintained by the IL6 family of cytokines (Livesey et al., 1997; Nishimune et al., 2000). Here we present data on a second gene FLRT3 previously identified in human muscle (Lacy et al., 1999) and recently from microarray analysis of lesioned sensory neurons (Tanabe et al., 2003). The protein demonstrates many of the characteristics described above and may convey pro-regenerative capacity on peripheral and potentially central neurons.

Results Isolation and identification of a cDNA clone encoding rat FLRT3 Differential display of mRNA was used to compare gene expression between regenerating and DRG from sham-operated rats using total RNA extracted from ipsilateral and contralateral L4–L5 DRG 3 days after sciatic nerve crush. A 150-bp cDNA fragment from an upregulated gene was isolated and cloned (Livesey and Hunt, 1998). Upregulation in DRG after peripheral nerve injury and gene orientation were confirmed by radioisotopic in situ hybridization (ISH) using 35S-labelled oligonucleotides. Northern blot analysis of DRG total RNA using the 32 P-labelled cDNA fragment as probe showed a single transcript of approximately 4.2 kb. The fragment DNA sequence gave no significant matches to any known genes after sequence database searching. A rat hippocampal cDNA library was then screened using the 32P-labelled cDNA fragment as probe in an attempt to isolate cDNA encoding the full open reading frame of the gene. This yielded a 1.5-kb clone encoding the 3V region, the sequence of which also produced no significant matches to any known genes or proteins. This larger cDNA fragment was used to screen the rat hippocampal library for a second time, yielding a 2.8-kb clone encoding a putative full open reading frame of 649 amino acids. Initial database searches using the sequence of this larger clone suggested that this gene was novel, but it was subsequently identified following the isolation of the human homologue (Lacy et al., 1999; see Fig. 1A). With the sequence data of human FLRT3 for comparison, we were able to confirm that the 2.8-kb clone contained the full open reading frame and also check the accuracy of our DNA-based structural analysis of the rat FLRT3 protein. The N-terminal extracellular domain is dominated by a 320 amino acid leucine-rich region containing 10 leucine-rich repeats, which is flanked by two cysteine-rich regions. This is followed by a fibronectin type III domain. These are features characteristic of cell adhesion molecules and receptors. A putative signal sequence is also present at the N-terminus. The transmembrane domain is 20 amino acids long. The 99 amino acid C-terminal cytoplasmic tail bears no recognizable motifs. The predicted structure of rat FLRT3 protein is shown in Fig. 1B. Western blot analysis has shown that the protein is approximately 90 kDa in size, larger than its predicted size of 73 kDa, suggesting that it may be glycosylated in a similar way to human FLRT proteins (Lacy et al., 1999). FLRT3 is a cell surface protein To demonstrate that FLRT3 protein has a cell surface component, the surface proteins of cultured E15 rat cortical neurons were labeled using a biotinylation reagent incapable of

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cell penetration (Mammen et al., 1997). The cells were solubilized and streptavidin beads were used to separate biotinylated surface proteins from cytosolic proteins. Western blot analysis detected the FLRT3 protein in biotinylated protein samples, confirming its surface localization (Fig. 1C). GAPDH protein was not detected in these samples, indicating that there was no contamination with cytosolic proteins. FLRT3 does not exhibit homophilic binding The cell surface localization and leucine-rich structure of FLRT3 are likely indicators of its interaction with one or more molecules. Its ligand(s) remains unknown. It was, however, important to establish whether the FLRT3 protein molecules bind to each other. A cell aggregation assay was employed to examine such homophilic binding. Suspension cultures of FLRT3-expressing CHO cells and control CHO cells (untransfected and those transfected with vector only) were agitated in a rotary incubator and the number of cell aggregates counted at hourly intervals using a hemocytometer. A decrease in the number would indicate a tendency for cells to aggregate. Western blotting and immunocytochemistry revealed at least a four-fold increase in expression of FLRT3 protein and localization to the cell membrane. There was no difference between the number of cell aggregates for FLRT3expressing and control cell cultures, in agreement with Tsuji et al. (2004), suggesting that homophilic binding had not taken place (data not shown). FLRT3 mRNA expression is upregulated after peripheral nerve injury but not by inflammation Radioisotopic in situ hybridization showed that, 1–7 days after sciatic nerve lesion, FLRT3 mRNA expression was upregulated in the majority of neurons of the adult rat L4–L5 ipsilateral DRG compared to contralateral DRG (Fig. 1D). Both large and small diameter neurons were labeled. To determine whether peripheral inflammation would also result in upregulation of the mRNA, Northern blot analysis was carried out on total RNA extracted from adult rat L4–L5 DRG after a time course of exposure to complete Freund’s adjuvant (CFA) injected into the hind paw. Total RNA extracted from the L4–L5 DRG of untreated animals and after sciatic nerve cut was also analyzed. Throughout the time course of inflammation studied (1 h to 7 days), FLRT3 mRNA expression in the DRG was detected at a similar level to that of the untreated animal (Fig. 1E). As a comparison, upregulation of the message in DRG remained high for at least 7 days after sciatic nerve cut (Fig. 1E). Six weeks after nerve crush and subsequent regeneration, levels of FLRT3 mRNA had returned to control levels (data not shown). FLRT3 protein is synthesized in the DRG after sciatic nerve lesion FLRT3 protein expression was studied after peripheral nerve injury by immunohistochemistry. At 3–7 days post sciatic nerve lesion, increased FLRT3 immunoreactivity was seen not only in the ipsilateral DRG and dorsal horn, particularly in laminae I and II, but also in deeper laminae and around a subset of dorsally located motor neurons and toward the midline around lamina X (Fig. 2A). No evidence of FLRT3 immunoreactivity was detected in control spinal cord tissue or on the contralateral side of the dorsal horn. Labeling of the dorsal horn was maximal by 7 days.

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All types of sensory fiber were labeled in the denervated zone with positive immunoreactivity concentrated in laminae I–II but extending ventrally through deeper laminae and with labeled fibers surrounding motor neurons in the dorsal sector of lamina IX (Figs. 2A and C). Superficially, the reaction product was largely punctate and occasional evidence of labeled axons was observed. Electron microscopy of the superficial dorsal horn confirmed that the reaction product was largely restricted to fine diameter preterminal axons and axon terminals making synaptic contacts with unlabelled dendrites (Fig. 2B). Immunoreactivity was associated with presynaptic specialization and a subset of small diameter synaptic vesicles (Fig. 2B). We also examined the response of motor neurons damaged by sciatic nerve cut. By in situ hybridization, cell bodies in the ventral spinal cord expressed low levels of FLRT3 mRNA 3–7 days following nerve section and required long periods of exposure (up to 3 months) to be detectable (data not shown). However, we were unable to detect increased FLRT3 immunoreactivity in motor neurons (Fig. 2C). FLRT3 immunoreactivity was also seen in the ipsilateral gracile nucleus (Fig. 2D). No FLRT3 mRNA was detected in dorsal horn in control or peripheral nerve lesioned tissue. Dorsal rhizotomy had no effect on FLRT3 protein expression in the dorsal horn (Fig. 2E). We also examined the effect of a dorsolateral quadrant lesion of the spinal cord and found no significant pile-up of FLRT3 protein in lesioned white matter (data not shown). Neurons of all diameters within the ipsilateral L4 and L5 DRG expressed FLRT3 protein. Immunoreactivity was generally found in association with the neuronal cell membrane but high levels of cytoplasmic FLRT3 staining were seen in small diameter neurons (Figs. 3A–B). Sensory neurons in the contralateral DRG were either unlabelled or more rarely (b10 cells per 40 Am DRG section) expressed above background levels of immunoreactivity. To determine whether the protein was anterogradely transported down the sciatic nerve after injury, the nerve was ligated and transected 5–8 mm distal to the ligation site. Nerves were harvested 1–3 days after transection and FLRT3 protein detected by immunohistochemistry. The intensity of FLRT3 staining was greatest proximal to the ligation site (Fig. 3C), suggesting that the protein had been transported down the nerve resulting in pileup at the ligation site. Ligation of the dorsal root alone did not result in pile-up of the protein at the site of ligation (Fig. 3D). FLRT3 immunoreactivity or mRNA was not detected in control sciatic nerve or dorsal roots. No mRNA was detected in nerve proximal to the ligation. Taken together with the mRNA expression data, the results indicate that sciatic nerve injury upregulates expression of FLRT3 message and protein in the majority of damaged sensory neurons regardless of subtype or biochemical composition. Protein is

rapidly transported from the DRG along the injured nerve and to the dorsal horn where it accumulates in presynaptic axon terminals. FLRT3 mRNA expression is present during development and in the adult brain In situ hybridization (ISH) studies have shown that FLRT3 message is regulated during embryonic development in both rat and mouse. Expression has been studied in rat and mouse using radioisotopic and non-radioisotpic ISH, and patterns of expression were found to be similar. At E12.5 in mouse and E12 in rat, expression was observed in brain, in two areas of the somite corresponding to the lateral lip of the dermomyotome and the dorsal somite, DRG, and also around the eye (Figs. 4A and F). From E16.5 to E17.5, expression was weaker in mouse but still evident in the cortical plate and DRG (Figs. 4D and E). Postnatally, FLRT3 message rapidly declines over the first postnatal week but in the adult is still highly expressed in the dentate gyrus and field CA3 of the hippocampus (Fig. 4G) and cerebellar granule cells. FLRT3 immunoreactivity was undetectable in the adult brain and no labeled fibers were seen 3 days after lesion of the cortex or hippocampus (data not shown). Antisense knockdown of FLRT3 protein reduces neurite outgrowth from DRG neurons in vitro Antisense knockdown of FLRT3 protein in cultured neuronal cells produced a decrease in neurite length of approximately 44% and a decrease in neurite number per cell of approximately 43%, relative to control cultures with sense oligonucleotide added (Figs. 5Ai–ii). These decreases were significant (P b 0.001, two-way ANOVA). Data were accumulated from three separate experiments. Western blot analysis of protein lysates from DRG cell cultures taken 24 h after oligonucleotide application confirmed antisense knockdown of FLRT3 protein (Fig. 5Aiii). HSV-mediated overexpression of FLRT3 protein promotes neurite outgrowth from DRG neurons in vitro Overexpression of FLRT3 protein in vitro produced an increase of approximately 51% in the length of the longest neurite per cell relative to control cultures overexpressing GFP. The number of neurites per cell increased by approximately 65% compared to control (Figs. 5Bi–ii). Data were from three experiments. Statistical analysis using a two-tailed Mann–Whitney test showed that the increase in neurite length was significant (P b 0.001). The increase in neurite number was also significant (P b 0.001, experiments 1 and 3; P b 0.01, experiment 2—two-tailed Mann–Whitney test). Western blot analysis of protein lysates from 27/12/M:4 cell cultures taken 48 h after virus application confirmed overexpression of FLRT3 protein by the virus (Fig. 5Biii).

Fig. 1. FLRT3 is a transmembrane protein located on the cell surface whose message levels are upregulated in the DRG after sciatic nerve injury but not peripheral inflammation. (A) Alignment of rat and human FLRT3 protein sequences. (B) The predicted structure of rat FLRT3 protein. (C) Cell surface localization of FLRT3. The cell surface proteins of E15 rat cortical cells were biotinylated in vitro. Both biotinylated (BIOT) and cytosolic (CYTO) protein samples were analyzed by Western blot. FLRT3 was clearly detectable in the biotinylated cell surface protein samples (n = 3). GAPDH was not detected in these samples. A sample DRG lysate (DRG CONT) was included as a control for immunostaining. (D) FLRT3 mRNA was upregulated in most adult rat ipsilateral L4–L5 DRG neurons after sciatic nerve injury. FLRT3 message expression was analyzed 3 days post-crush in contralateral (CON) and ipsilateral (IPSI) L4–L5 DRG using radioisotopic ISH (scale bar = 50 Am). (E) Northern blot analysis showed that FLRT3 mRNA was upregulated in adult rat L4–L5 DRG after sciatic nerve lesion but not after peripheral inflammation. L4–L5 DRGs were taken over a time course (h = hour; d = day) after peripheral inflammation (by CFA injection into the hind paw) and after sciatic nerve lesion.

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Discussion We have described the isolation and characterization of rat FLRT3 from axotomised sensory neurons confirming recent

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observations using microarray analysis (Tanabe et al., 2003; Tsuji et al., 2004). We have shown that the gene is widely expressed during development and, using antisense and viral transfer approaches, that the protein strongly influences neurite outgrowth.

A

B

FLRT3_[Homo_sapiens] FLRT3rat

MISAAWSIFLIGTKIGLFLQVAPLSVMAKSCPSVCRCDAGFIYCNDRFLT MISPAWSLFLIGTKIGLFFQVAPLSVMAKSCPSVCRCDAGFIYCNDRSLT *** *** ********** **************************** **

FLRT3_[Homo_sapiens] FLRT3rat

SIPTGIPEDATTLYLQNNQINNAGIPSDLKNLLKVERIYLYHNSLDEFPT SIPVGIPEDATTLYLQNNQINNVGIPSDLKNLLKVQRIYLYHNSLDEFPT *** ****************** ************ **************

FLRT3_[Homo_sapiens] FLRT3rat

NLPKYVKELHLQENNIRTITYDSLSKIPYLEELHLDDNSVSAVSIEEGAF NLPKYVKELHLQENNIRTITYDSLSKIPYLEELHLDDNSVSAVSIEEGAF **************************************************

FLRT3_[Homo_sapiens] FLRT3rat

RDSNYLRLLFLSRNHLSTIPWGLPRTIEELRLDDNRISTISSPSLQGLTS RDSNYLRLLFLSRNHLSTIPGGLPRTIEELRLDDNRISTISSPSLHGLTS ******************** ************************ ****

FLRT3_[Homo_sapiens] FLRT3rat

LKRLVLDGNLLNNHGLGDKVFFNLVNLTELSLVRNSLTAAPVNLPGTNLR LKRLVLDGNLLNNHGLGDKVFFNLVNLTELSLVRNSLTAAPVNLPGTSLR *********************************************** **

Predicted Structure of Rat FLRT3 Protein

N Single pass transmembrane protein 320 aa LRR

N

N-terminus

Extracellular domain 530 aa

Cysteine rich region FLRT3_[Homo_sapiens] FLRT3rat

KLYLQDNHINRVPPNAFSYLRQLYRLDMSNNNLSNLPQGIFDDLDNITQL KLYLQDNHINRVPPNAFSYLRQLYRLDMSNNNLSNLPQGIFDDLDNITQL **************************************************

Leucine rich repeat (LRR) Fibronectin type III (Fn III) domain

FLRT3_[Homo_sapiens] FLRT3rat

ILRNNPWYCGCKMKWVRDWLQSLPVKVNVRGLMCQAPEKVRGMAIKDLNA ILRNNPWYCGCKMKWVRDWLQSLPVKVNVRGLMCQAPEKVRGMAIKDLSA ************************************************ *

FLRT3_[Homo_sapiens] FLRT3rat

ELFDCKDSGIVSTIQITTAIPNTVYPAQGQWPAPVTKQPD---------ELFDCKDSGIVSTVQITTAIPNTAYPAQGQWPAPVTKQPDGQWPAPVTKQ ************* ********* ****************

FLRT3_[Homo_sapiens] FLRT3rat

--IKNPKLTKDHQTTGSPSRKTITITVKSVTSDTIHISWKLALPMTALRL PDIKNPKLTKDQRTTGSPSRKTILITVKSVTPDTIHISWRLALPMTALRL ********* ********** ******* ******* **********

FLRT3_[Homo_sapiens] FLRT3rat

SWLKLGHSPAFGSITETIVTGERSEYLVTALEPDSPYKVCMVPMETSNLY SWLKLGHSPAFGSITETIVTGELSEYLVTALEPESPYRVCMVPMETSNLY ********************** ********** *** ************

FLRT3_[Homo_sapiens] FLRT3rat

LFDETPVCIETETAPLRMYNPTTTLNREQEKEPYKNPNLPLAAIIGGAVA LFDETPVCIETQTAPLRMYNPTTTLNREQEKEPYKNPNLPLAAIIGGAVA *********** **************************************

FLRT3_[Homo_sapiens] FLRT3rat

LVTIALLALVCWYVHRNGSLFSRNCAYSKGRRRKDDYAEAGTKKDNSILE LVSIALLALVCWYVHRNGSLFSRNCAYSKGRRRKDDYAEAGTKKDNSILE ** ***********************************************

FLRT3_[Homo_sapiens] FLRT3rat

IRETSFQMLPISNEPISKEEFVIHTIFPPNGMNLYKNNHSESSSNRSYRD IRETSFQMLPISNEPISKEEFVIHTIFPPNGMNLYKNNLSESSSNRSYRD ************************************** ***********

FLRT3_[Homo_sapiens] FLRT3rat

SGIPDSDHSHS SGIPDLDHSHS ***** *****

Transmembrane domain (20 aa) Intracellular domain (99 aa)

C

MEMBRANE

C

C Biotinylation of Cell Surface FLRT3

* - single, fully conserved residue - no consensus _ - predicted N-glycosylation site

D

Radioactive In Situ Hybridisation

C-terminus

BIOT

CYTO

DRG CONT

92kDa

FLRT3 GAPDH

E CON

Northern Blot Analysis

FLRT3

4.2kb

Cyclophilin N

1h 5h

24h 48h 7d

CFA IPSI

24h 48h 7d

Sciatic Cut

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A

IPSI

B

CONT

C

Lamina VI

D

IPSI

CONT

E

IPSI

CONT

Fig. 2. FLRT3 protein appears strongly in the dorsal horn and gracile nucleus by 7 days post lesion but is unaffected by dorsal rhizotomy. (A) FLRT3 protein was upregulated in adult rat lumbar dorsal horn by 7 days post-sciatic nerve lesion (IPSI = ipsilateral; CONT = contralateral; scale bar = 500 Am). The protein was detected immunohistochemically. (B) Electron micrograph showing FLRT3 immunoreactivity (indicated by arrows) at dorsal horn presynaptic axon terminals 7 days post-sciatic nerve lesion (scale bar = 250 nm). (C) Higher magnification micrograph of ipsilateral adult rat lumbar spinal cord 7 days postsciatic nerve lesion. Arrows indicate FLRT3-labeled fibers (scale bar = 100 Am). (D) FLRT3 immunoreactivity was detected in the ipsilateral gracile nucleus (see arrow) by 7 days post-sciatic nerve lesion (scale bar = 100 Am). (E) Immunohistochemistry showed that FLRT3 protein expression in adult rat lumbar spinal cord remained unaffected by dorsal rhizotomy 7 days post-surgery (IPSI = ipsilateral; CONT = contralateral; scale bar = 200 Am).

The protein accumulates in damaged sensory neuron cell bodies and is exported into their peripheral and central processes. Within the spinal cord, the FLRT3 protein is localized to axon terminals and there is no change in FLRT3 message levels in the dorsal horn, implying that all synthesis occurs within sensory neurons. Protein structure analysis predicts that FLRT3 is a single pass transmembrane protein and we confirmed a cell surface location by biotinylation of cell surface proteins in vitro and by immunolocalization to the cell surface of dorsal root ganglion cells. Electron microscopy implied that in dorsal horn the protein has a presynaptic location close to the presynaptic density. The FLRT3 protein has been postulated to be a receptor, signal transducer, or participant in cell–cell contact (Lacy et al., 1999; Tsuji et al., 2004). Cell aggregation assay showed that FLRT3 does not exhibit homophilic binding, in agreement with Tsuji et al. (2004). FLRT3 has homology with proteins such as FLRT1 that contain a laminin-binding motif (Lacy et al., 1999). However, FLRT3, unlike FLRT1, does not have an obvious laminin-binding motif and other extracellular proteins may be required as ligands. A recent study has shown an approximately 10% increase in neurite outgrowth from neonatal cerebellar granule cells grown on CHO

cells expressing FLRT3 (Fig. 6) (Tsuji et al., 2004). Recent data (Bottcher et al., 2004) have implicated FLRT3 in the positive modulation of fibroblast growth factor (FGF)-MAP kinase signaling during Xenopus development and FLRT3 may play a similar role in rodents. FGFs and their receptors (FGFRs) constitute an elaborate signaling system in vertebrates that participate in many developmental, plasticity, and repair processes, including areas of neurogenesis, cell survival, axonal growth, neuroprotection, lesion repair, and learning and memory (Reuss and Bohlen und, 2003). FGF and FGFRs are found constitutively expressed by subsets of sensory neurons and expression is upregulated in most DRG neurons following axotomy as well as in Schwann cells, satellite cells, and invading macrophages (Grothe et al., 2001). FLRT3potentiated FGF signaling at the site of axon regeneration could result in an increased production of FGF by neurons (through autoinduction) and increased Schwann cell mitogenesis and suppression of myelin related gene expression (Davis and Stroobant, 1990; Grothe and Nikkhah, 2001; Morgan et al., 1994)—a series of events remarkably similar in outcome to that seen by manipulation of Reg-2 signaling. Reg-2 protein is produced in adult motor neurons and subsets of DRG neurons following peripheral nerve

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Fig. 3. FLRT3 protein is synthesized in the DRG after sciatic nerve lesion, localized to the cell membrane, and transported along the injured nerve. (A) FLRT3 immunoreactivity was increased in injured (IPSI) adult rat L4–L5 DRG compared to uninjured (CONT) DRG 7 days after sciatic nerve lesion (scale bar = 100 Am). (B) High magnification image shows distinct membrane as well as cytoplasmic localization of the FLRT3 protein (N = nucleus; S = satellite cell; scale bar = 50 Am). (C) Immunohistochemistry showed that, after adult rat sciatic nerve ligation and lesion, the FLRT3 protein was anterogradely transported from the DRG down the injured nerve and, by 3 days, had accumulated proximal to the ligation site, indicated by the arrow (P = proximal; D = distal; scale bar = 250 Am). (D) Transport of FLRT3 protein along adult rat dorsal root was not induced 3 days after ligation of the root (scale bar = 100 Am).

damage (Averill et al., 2002; Livesey et al., 1997), is secreted into the extracellular environment at the site of damage, and stimulates Schwann cell division. Blocking the action of Reg-2 with antibodies reduces the rate of axon regeneration in part by reducing the activation of Schwann cells. It seems likely therefore that the potentially pro-regenerative effect of FLRT3 in the PNS may be mediated by potentiation of FGF signaling and the subsequent stimulation of Schwann cell mitogenesis (Davis and Stroobant, 1990). However, in contrast to the results of Tsuji et al. (2004), who isolated FLRT3 mRNA from nerve distal to a peripheral nerve cut, we were only able to locate protein proximally to a cut. This suggests that in some areas of the nervous system (see below) mRNA was not translated to protein. FLRT3 may respond to its own extracellular ligand or act autonomously on adjacent proteins within the axonal membrane. In Xenopus, the transmembrane domain of FLRT3 and extracellular face of the molecule were sufficient to bind FGFRs, but the smaller cytoplasmic C-terminal portion together with the transmembrane domain was sufficient for signal transduction through the MAPkinase pathway, possibly by recruiting cofactors to the membrane. FLRT3 is widely expressed during development but not in the adult brain, and any interaction with FGFRs would be restricted to the damaged peripheral nerve and its site of termination within the dorsal horn, although the possibility that FLRT3 has its own extracellular ligand cannot be excluded. Nevertheless, there are widespread actions of FGF in the adult brain (Reuss and Bohlen und, 2003), which clearly do not require FLRT3 expression. The interaction between FLRT3 and FGFRs may be restricted to areas of the nervous system where particular signaling pathways need to be recruited. For example, Bottcher et al. (2004) reported that the response to FLRT3/FGF activation was through the MAP-kinase pathway but not through the PI3kinase signaling cascade.

Exogenously applied FGF also promotes axon outgrowth and, within the dorsal horn, FGF encourages regeneration of damaged (but not undamaged) sensory axons (Romero et al., 2001). The localization of the protein to the presynaptic membrane implies a role in the events that follow peripheral axotomy. At the time point studied, there is electrophysiological and anatomical evidence that, following peripheral axotomy, considerable neuronal reorganization has occurred within the dorsal horn. Some atrophic change in synaptic terminals has been described, but neurophysiologically the majority of neurons within the substantia gelatinosa now respond not only to high but also to low threshold inputs from the periphery albeit via polysynaptic circuits (Kohno et al., 2003). There is also some evidence for the uncovering of dsilentT synapses perhaps through the recruitment of glutamate receptor subunits into the postsynaptic membrane and sprouting of a few small and large myelinated sensory fibers within the dorsal horn following peripheral axotomy (Baba et al., 2000; Woolf et al., 1992). Again, many of these events may be, in part, the result of upregulation of the FGF signaling pathway. Peripheral nerve ligation results in increased FGF-2 expression by astrocytes in the dorsal horn (Madiai et al., 2003) whereas FGF has been reported to induce growth of damaged sensory fibers and produce an increase in numbers of microglia (Goddard et al., 2002). Activated microglia have recently been shown to contribute to the hyperalgesia and allodynia found after peripheral nerve damage (Tsuda et al., 2003). Also, levels of NMDA glutamate receptors are reduced in the hippocampus following exogenous FGF treatment (Mattson et al., 1993), and a decrease in NMDA receptor binding is seen in the spinal cord following sciatic nerve lesion (Croul et al., 1998) and may result from increased efficacy of the FGF signaling pathway following upregulation of FLRT3 in sensory afferents.

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Fig. 4. FLRT3 message is regulated during nervous system development. (A–E) FLRT3 expression was studied in E12.5–E17.5 mouse using radioisotopic ISH. Eii shows an E17.5 control slide to check for nonspecific binding of the oligonucleotide probe. Scale bar = 20 mm. (Fi) Whole mount non-radioisotopic ISH showing FLRT3 expression in E12 rat. D, diencephalon; E, eye; LD, lateral lip of the dermomyotome; S, somites; FL, forelimb; B, branchial arch. Scale bar = 1.25 mm. Fii and Fiii show, in dark field, sections through the embryo in Fi [(Fii) FLRT3 mRNA expression in the somite S and lateral lip of the dermomyotome LD (SC, spinal cord; scale bar = 150 Am) and (Fiii) in the telencephalic vesicle tv—scale bar = 100 Am]. (G) Radioisotopic ISH showing FLRT3 mRNA expression in adult rat brain; expression is restricted to the hippocampal area (DG = dentate gyrus; scale bar = 200 Am). n = 3 embryos per developmental stage.

FLRT3 is highly expressed at early stages of cortical development. We first observed FLRT3 expression at around E11.5 in rat within a small region of the telencephalic vesicle. By E17, this expression had spread to include other areas of the cortex and subcortical areas but was also very evident in the somites. A downregulation in mRNA expression was observed postnatally, but the message was still detectable in the adult hippocampus. The widespread and early expression of FLRT3 during development implies a role not only related to axon outgrowth but also to many other areas including developmental patterning and neurogenesis. FGF is crucial for many of these processes, and differential expression of FLRT3 may act as a local signal to potentiate FGF signaling. This may be particularly true in the developing cerebral cortex where evidence implies that FGFs confer anterior–posterior positional information to the cortical primordium (Grove and Fukuchi-Shimogori, 2003). FLRT3 may also be involved in cell patterning in the somites. It is expressed by cells of the lateral lip of the dermomyotome that migrate to form the limb and hypaxial

musculature (Dietrich, 1999) while FGF-4 also aids the formation of these migratory muscle precursors (Alvares et al., 2003). The failure of FLRT3 protein to express at high levels in motor neurons following peripheral nerve section or in the brain following injury (and the absence of FLRT3 protein in damaged central axons) suggests that [unlike Reg-2 protein (Livesey et al., 1997)] the regenerative potential of FLRT3 may not be available to central neurons. However, the ability of transplanted adult sensory neurons to survive and regenerate within central white matter of the adult brain (Davies et al., 1997a) could imply that FLRT3 has an important pro-regenerative role within the CNS and that the environmental cues for signaling with FLRT3 may remain available in the adult brain. In the adult nervous system, FLRT3 is highly expressed in neurons of the hippocampus, an area where neurons demonstrate long-term potentiation (LTP) and that is closely associated with learning and memory. The plasticity of the hippocampus and the establishment of both early and late phases of LTP can be reduced by disrupting adhesion molecule function

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Fig. 5. FLRT3 promotes neurite outgrowth in DRG neuronal cells in vitro. (Ai) DRG neuronal cells in vitro 24 h after application of sense or antisense oligonucleotides. The cells have been fixed and immunostained for the pan-neuronal marker PGP9.5. Scale bar = 250 Am. (Aii) Antisense knockdown of FLRT3 protein in DRG neuronal cells in culture resulted in a significant reduction in both maximum neurite length and number (mean values F SEM are shown). ***P b 0.001, two-way ANOVA. (Aiii) Western blot analysis of cultured DRG neuronal cell lysates 24 h after application of sense (S) or antisense (aS) oligonucleotides confirmed antisense knockdown of FLRT3 protein. FLRT3 protein levels are shown relative to GAPDH (mean values F SEM are shown in the graph; Sense n = 5, Antisense n = 3). An adult rat L4–L5 DRG lysate sample was included on the gel as a control (DRG Con) for immunostaining. (Bi) DRG neuronal cells in vitro 24 h after application of GFP- or FLRT3-overexpressing HSV-1 virus. The cells have been fixed and immunostained for the panneuronal marker PGP9.5. Scale bar = 250 Am. (Bii) Overexpression of FLRT3 by HSV-1 in DRG neuronal cells in vitro resulted in a significant increase in both maximum neurite length and number (mean values F SEM are shown). ***P b 0.001, **P b 0.01—two-tailed Mann–Whitney test. (Biii) Western blot analysis of cultured 27/12/M:4 cell lysates 48 h after application of a control virus overexpressing LacZ (LZ) or the virus overexpressing FLRT3 (F3) confirmed viral overexpression of FLRT3. A sample lysate of uninfected (U/I) cultured cells was also included on the gel. The blot was reprobed for GAPDH to show protein loading. n = 6.

without, in most cases, altering presynaptic release of transmitter (Luthi et al., 1994; Muller et al., 1996; Sakurai et al., 1998; Tang et al., 1998; Yamagata et al., 1999). A role for FLRT3 in mediating LTP by modulating synaptic architecture is clearly suggested. However, lesions of the hippocampus and cortex did not upregulate

FLRT3 protein expression, implying that neurons of the CNS only express FLRT3 protein during development and that this is not recapitulated following axon damage in the adult. This may also explain the presence of mRNA for FLRT3 in the distal segment of a sectioned peripheral nerve (Tsuji et al., 2004), but no protein.

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A

Dorsal rhizotomy, dorsal root ligation, and spinal cord lesion Adult Sprague–Dawley rats (200–300 g) were anesthetized by halothane inhalation and a lumbar laminectomy was performed. Three to five dorsal roots (n = 3) were sectioned or ligated (n = 3) under a dissecting microscope, taking care not to damage the spinal cord, or a unilateral lesion of the dorsolateral quadrant of the dorsal horn (n = 4) was made using fine dissecting scissors. Animals were allowed to survive for 3 or 7 days then perfused and the tissue processed for immunohistochemistry.

B

Lesions of cortex and hippocampus

U/T

V

FLRT3

Fig. 6. Immunostaining of FLRT3-transfected CHO cells and analysis of FLRT3 protein expression by Western blotting. (A) Shows a confocal image of cell surface FLRT3 labelling. Immunocytochemistry was carried out using the N-terminal FLRT3 antibody (which recognises the extracellular domain) under non-permeabilising conditions. (B) Western blot analysis of FLRT3 protein expression in untransfected (U/T) CHO cells, CHO cells transfected with pCMV-Tag5 A vector (V) only, and CHO cells transfected with pCMV-Tag5 A/FLRT3 (FLRT3).

In summary, we present data support the hypothesis that FLRT3 is a cell surface receptor critical for neurite outgrowth and possibly involved in the remodeling of connections between neurons or other cell types and the extracellular matrix. Therapeutically, we suggest that reintroduction of the gene into the adult CNS may encourage neuronal repair and regeneration possibly through potentiation of the FGF-signaling pathway.

Adult Sprague–Dawley rats (200–300 g; n = 3) were anesthetized by halothane inhalation and a lesion of the cortex and underlying hippocampus was made. A small scalpel blade was lowered through the cortex and hippocampus to a depth of 3.5 mm from the cortical surface and moved 1 mm rostrally and caudally, cutting connections between regions CA3 and CA1 and damaging the frontal parietal cortex. Animals were allowed to survive for 3 days then perfused and the tissue processed for immunohistochemistry. Inflammation Inflammation was induced in adult male Sprague–Dawley rats (200–300 g) (n = 24) by subcutaneous injection of 100 Al of complete Freund’s adjuvant (CFA) (Sigma, UK) into the plantar surface of the left hind paw under halothane anesthesia. The CFA injection produced a localized area of erythema and edema. CFAtreated animals were terminally anaesthetized with sodium pentabarbitone at time points ranging from 1 h to 7 days posttreatment. DRGs were dissected and total RNA extracted. Total RNA extraction and differential display of mRNA

Experimental methods Animals and surgical procedures All surgery was carried out according to UK Home Office guidelines for the ethical treatment of animals. Sciatic nerve surgery Adult Sprague–Dawley rats (200–300 g) were anesthetized by halothane inhalation. The left sciatic nerve was exposed in the upper thigh and crushed twice with watchmaker’s forceps (No. 5) close to the sciatic notch (n = 10) or ligated and transected 5–8 mm distal to the ligation site (n = 48). Treatment time courses ranged from 24 h to 28 days. Animals were sacrificed by CO2 inhalation for Northern blot analysis, in situ hybridization, and Western blot analysis. Tissue removed was frozen immediately on dry ice and stored at 808C. For immunohistochemistry, animals were terminally anaesthetized with sodium pentabarbitone and perfused transcardially with ice-cold 2% PLP fix (2% paraformaldehyde in 0.056 M phosphate buffer plus 74.5 mM l-lysine monohydrochloride and 1 mM sodium metaperiodate, pH 7.4). Dissected tissue was post-fixed in 2% PLP at 48C and stored at 48C in 30% sucrose/0.1 M phosphate buffer/0.02% sodium azide. Tissue dissected included L4–L5 DRG, sciatic nerve, brain, and spinal cord.

Total RNA extraction was performed by the guanidinium-acid phenol method (Chomczynski and Sacchi, 1987). Differential display RT-PCR was carried out on total RNA extracted from control and regenerating DRG as previously described (Livesey and Hunt, 1998). The three anchored oligo-dT primers were as described in Liang et al. (1994), while the arbitrary primers were designed using the recommendations of Bauer et al. (1993). The arbitrary primers (5V–3V) used were as follows: DD1 GCTTAC A A C G A G G ; D D 2 G C T T G C AT T G G T C ; D D 3 G CTCTTTCTACCC; DD 4 GCTTTTTGGCTCC; DD5 GCTGGAACCAATC; DD6 GCTAAACTCCGTC; DD7 GCTTCGATACAGG; DD8 GCTTGGTAAAGGG; DD9 GCTTCGGTCATAG; DD10 GCTGGTACTAAGG; DD11 GCTTACCTAAGCG; DD12 GCTCTGCTT GATG. The sequences of cloned cDNAs were compared to the GenBank non-redundant and EST databases using the BLAST algorithm (Altschul et al., 1990). cDNA library screening A rat hippocampal cDNA library (Stratagene Ltd., UK) was screened for FLRT3. Briefly, for the primary screen, the library was plated out at a density of 5  106 pfu over two 220  220 mm plates. Plaque lifts were taken on Hybond-N+ nylon membrane (Amersham, UK) and these processed as described (Sambrook et

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al., 1989). Two sets of library screens were carried out. The first used the FLRT3 fragment obtained from differential display. This yielded a 1.5-kb FLRT3 fragment, which was used as probe in the second screen. Oswel Research Products Ltd. (UK) carried out the DNA sequencing. The cDNA clone sequences were compared to the GenBank databases as before. Protein structural analysis and sequence alignments were carried out using the ExPASy website. Northern blot analysis The procedures for formaldehyde gel electrophoresis of total RNA (10 Ag per sample) and RNA transfer to nylon membrane (Hybond-N, Amersham) were as described (Sambrook et al., 1989). After prehybridization, the membrane was hybridized with a 1.5-kb fragment of rat FLRT3 (labeled with a32PdATP by random priming) and washed free of unbound probe as described in Sambrook et al. (1989). Signal was visualized by exposing X-ray film to the membrane at 808C. The membrane was reprobed for cyclophilin as a comparison of total RNA levels. In situ hybridization A 45-base oligonucleotide complementary to the sequence of rat FLRT3 [5V-GCTGTGCTTATTCTACAAGGATCTCAAGTGAACGCAGTCCTGATT-3V, (desalted, from Sigma-Genosys Ltd., UK)] was 3V end labeled with a35SdATP and hybridized to 15 Am cryostat tissue sections. Tissue preparation and in situ protocol are as described in Wisden et al. (1991). Non-radioisotopic in situ hybridization was carried out for FLRT3 on whole mount rat embryos as described in Wilkinson and Nieto (1993). The DIG-labeled riboprobe template used was prepared as previously described (Sambrook et al., 1989). Generation of rat FLRT3 antibodies Two polyclonal rat FLRT3 antibodies were raised in rabbit: the first recognizes the following peptide sequence from the Cterminal intracellular tail-H2N-NRS YRD SGI PDL DHS HCONH2 (amino acids 632–647); the second was raised to protein comprising amino acids 30–551 of the N terminus of rat FLRT3. Immunoreactivity was completely adsorbed by 10 Ag/ml of native peptide added to diluted antiserum. FLRT3 immunohistochemistry Immunohistochemistry was performed on 20-Am slide-mounted cryosections or 40-Am free-floating freezing microtome sections. All tissue was perfused and post-fixed with 2% PLP fix and stored in 30% sucrose in 0.1 M phosphate buffer/0.02% sodium azide at 48C. The following steps were performed with gentle agitation for free-floating sections. The sections were blocked for 1 h in TTBS (0.05 M Tris saline, pH7.4/0.3% Triton X-100) plus 5% normal goat serum (NGS) at room temperature. They were then incubated 24–72 h at 48C with FLRT3 C-terminal primary antibody (diluted 1:1000 in TTBS plus 3% NGS). Three 10-min washes in 0.1 M phosphate buffer were followed by a 2-h incubation at room temperature with biotinylated goat anti-rabbit secondary antibody (from Vector Laboratories, UK; diluted 1:500 in TTBS plus 3% NGS). A further three 10-min washes in 0.1 M phosphate buffer were followed by a 45-min incubation at room temperature with Cy3-conjugated Streptavidin (Jackson ImmunoResearch Labora-

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tories, inc., West Grove, PA; diluted 1:4000 in TTBS plus 3% NGS). A tyramide enhancement method was also used for free-floating sections. The primary and secondary antibodies were diluted 1:50,000 (in block plus 3% NGS) and 1:400 (in TTBS), respectively. Sections were incubated with primary and secondary antibodies as described previously and then incubated for 30 min with an avidin–biotin complex with horseradish peroxidase [dAT and dBT solutions from Vectastain ABC Elite kit (Vector Laboratories), diluted in TTBS]. After three 10-min washes in 0.1 M phosphate buffer, sections were incubated for 7 min in biotinylated tyramide solution from the TSA Biotin System kit (Perkin-Elmer, Boston, MA), prepared according to the manufacturer’s instructions. Sections were washed and incubated for 2 h with FITC avidin (diluted 1:600 in TTBS). Alternatively, we used the avidin–biotin complex with horseradish peroxidase to produce a colored reaction product. After a 2h incubation with goat anti-rabbit biotinylated secondary antibody at room temperature, sections were washed three times for 10 min in 0.1 M phosphate buffer and incubated for 1 h in dA + BT solution from the Vectastain ABC Elite kit (Vector Laboratories). Staining was visualized by peroxidase reaction using the DAB Brown Substrate kit (Vector Laboratories). After three final 10-min washes in 0.1 M phosphate buffer (where applicable, sections were then mounted onto slides), slides were cover-slipped using aqueous mounting medium for fluorescent stain or DPX (BDH, UK) for DAB stain and images taken using image analysis software from Leica Microsystems (UK) Ltd. Immuno-electron microscopy Tissue was perfused as described above with 2% or 4% PLP, postfixed for 2 h, and cryoprotected in 7% sucrose in 0.1 M phosphate buffer. Tissue sections were taken on a freezing microtome as previously described (Hunt et al., 1980) and incubated in 0.1% Triton-X 100 for 10–20 min before incubation in antibodies as described above except that detergent was omitted at all subsequent stages. Following development of the colored peroxidase reaction product, areas of interest were processed as described in Davies et al. (1997b). Briefly, tissue was osmicated, stained in uranyl acetate in sodium buffer, dehydrated in ethanols, cleared in polypropylene oxide, and embedded in Araldite between two sheets of Melenex (ICI, UK). Semithin (1 Am) sections were cut with glass knives adjacent to thin sections cut with a diamond knife. The sections were collected on mesh grids coated with a thin formavar film, counterstained with lead citrate, and viewed in a Jeol 1010 electron microscope. Western blot analysis Tissue or cells were homogenized or lysed in standard PAGE gel loading buffer (Sambrook et al., 1989) and denatured at 1008C for 5 min. Protein extracts were separated by electrophoresis on precast 10% polyacrylamide gels (Bio-Rad Laboratories Ltd., UK), transferred to polyvinylidene difluoride membrane, and blocked at room temperature for at least 1 h in 6% dried skimmed milk in PBS/0.1% Tween 20. Blots were probed (48C, overnight) with Nor C-terminal rat FLRT3 primary antibody (1:5000 or 1:2000, respectively in block) and detected by peroxidase-linked secondary antibody enhanced chemiluminescence. For normalization purposes, blots were then stripped and re-probed with primary antibody

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to GAPDH (Chemicon Europe, Ltd., UK; 1:750 in block containing 4% dried skimmed milk). Films were analyzed by quantitative densitometry. Preparation of herpes simplex viral vectors Genomic herpes simplex viral vectors were derived from herpes simplex virus (HSV)-1 strain 17syn + (Brown et al., 1973). The viruses used in this study (1764) have a mutation in the gene encoding the transactivator VP16 (Ace et al., 1989) and are deleted for the gene encoding ICP34.5 (Coffin et al., 1996). The viruses were also deleted for ICP4 (Lilley et al., 2001; Palmer et al., 2000). The pR19 promoter cassettes have previously been described (Palmer et al., 2000). The pR19GFP and pR19FLRT3 plasmid constructs consist of a CMV-GFP or CMV-FLRT3 cassette in LAT flanking regions that allow homologous recombination into the LAT region of the HSV genome (Palmer et al., 2000). Plasmids were recombined into viral DNA by standard calcium phosphate methods and recombinant viruses were identified by reporter gene expression and plaque purified. Genome structures of pure viruses were confirmed by Southern blotting and transgene expression was confirmed by visualization of GFP by fluorescence microscopy (1764 4-pR19GFP virus) or Western blotting (1764 4-pR19FLRT3 virus). All viruses used in this study were produced and grown on BHK-derived complementing cells (27/12/M:4) as described previously (Thomas et al., 1999) in Dulbecco’s modified Eagle media (DMEM, from Invitrogen Ltd., UK) supplemented with 10% fetal calf serum, 5% tryptose phosphate broth, and 3 mM hexamethylenecbisacetamide (HMBA). High titer viral stocks were produced by filtration of 500 ml of supernatant of virally infected cells through a 0.45-Am filter and centrifugation at 12,000 rpm for 2 h at 48C. The viral pellet was resuspended in 500 Al of serumfree DMEM and tittered on 27/12/M:4 cells. Dissociated DRG culture Lumbar DRGs from two 250- to 300-g adult male rats (sacrificed by CO2 inhalation) were dissected out into prechilled modified Eagles medium (MEM) with HEPES (Invitrogen Ltd.) and cultures prepared as described (Lindsay, 1988). The cell suspension (100 Al) in 2 ml of Neurobasal medium (with lglutamine, penicillin–streptomycin, and B27 serum supplement, from Invitrogen Ltd.) was plated in the center of 22-mm diameter glass coverslips (in 35 mm tissue culture dishes) coated with a polydl-ornithine-laminin substratum. The cells were left to adhere to the coverslips for 1 h at 378C in 5% CO2, after which time a further 1 ml of Neurobasal medium was added. The cells were then incubated for 24 h at 378C in 5% CO2. The Neurobasal medium was removed and the following FITC-conjugated oligonucleotides (desalted, from Sigma-Genosys Ltd.) sense 5V-CTGCTGACCATGATCAGCCCA-3V; antisense 5V-TGGGCTGATCATGGTCAGCAG-3; scrambled 5V-AGTCGCGATCATTGCGGTGAG-3V were applied to the cells at a concentration of 0.5 AM in pluronic gel (from Sigma, UK, 30% in PBS (Becker et al., 1999). Neurobasal medium (2 ml) was then reapplied. The cells were incubated for a further 24 h, fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH7.4), and immunostained for the pan-neuronal marker PGP9.5 using the Vectastain ABC kit (Vector Laboratories) and visualization by peroxidase reaction. Neurite outgrowth measurements (length of longest branch point of each neurite and number of

neurites per cell) were carried out using Q Win software from Leica Microsystems (UK) Ltd., UK. Neuronal cells (200, 150, and 250) were measured for each treatment in experiments 1, 2, and 3, respectively. Measurements were carried out blind. Random fields of view were selected and all neuronal cells within them measured. For overexpression of rat FLRT3 in cultures of dissociated adult rat DRG using a herpes simplex virus delivery system, the cultures were prepared as for the antisense study using lumbar DRG from one adult male rat. Neuronal cells were left in culture for 24 h before the application of 1764 4-pR19FLRT3 virus or control 1764 4-pR19GFP virus. DRG cultures were infected by incubation with virus at a multiplicity of infection of 10 for 1 h in serum-free F14 medium. After 1 h, the virus was removed and the cultures overlayed with full growth medium. The cells were then incubated for a further 24 h, fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH7.4), and immunostained for PGP9.5. Neurite outgrowth was measured as described for the antisense study, determining the length of the longest neurite and the number of neurites per cell. Neuronal cells (50 and 100) were measured for each treatment in experiments 1 and 2, respectively. For experiment 3, 75 and 73 neuronal cells were measured for cultures treated with 1764 4-pR19FLRT3 virus and control 1764 4-pR19GFP virus, respectively. CHO cell culture and transfection CHO cells were cultured at 378C in CO2-buffered MEM-Alpha medium (Invitrogen Ltd.) supplemented with 2 mM l-glutamine, penicillin–streptomycin (100 stock solution from Invitrogen Ltd.), and 10% fetal bovine serum. The expression vector pCMV-Tag5A/rat FLRT3 was prepared using standard protocols (Sambrook et al., 1989) by inserting rat FLRT3 cDNA encoding the full open reading frame into the pCMV-Tag5A vector (Promega, UK). The pCMV-Tag5A/rat FLRT3 and pCMV-Tag5A plasmids were transfected into CHO cells using Lipofectamine Reagent (Invitrogen Ltd.) according to the manufacturer’s instructions. Transfected cells were selected by growth in medium supplemented with 1 mg/ml G418. FLRT3-transfected cells were analyzed by Western blot and immunostained for FLRT3 to ensure high levels of FLRT3 expression. Cell aggregation assay Aggregation assays were performed using rat FLRT3 transfected and control CHO cells, as described in Araki and Milbrandt (1996). Embryonic rat cortical neuronal culture E15 rat embryos were collected in ice-cold PBS and the brains (eight were sufficient) dissected in cold DMEM medium. First, the meninges were removed. The hippocampus was then discarded and the telencephalic vesicles retained. The brain fragments were incubated at 378C for 15 min in 5 ml DMEM with 10 Al 2.5% trypsin, 500 Al 10 mg/ml collagenase, and 250 Al 1 mg/ml DNase1. The tissue was washed in DMEM plus 10% fetal calf serum, pelleted at 2000 rpm for 2 min, and resuspended in 1 ml of culture medium (Neurobasal medium plus 5% fetal calf serum, 2% B27 supplement, 25 AM glutamic acid, 25 AM l-glutamine, 1:1000 penicillin–streptomycin, and 10 ng/ml BDNF). The tissue was dissociated by trituration. The cells were counted using a

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hemocytometer and plated on 18-mm diameter coverslips coated with 50 Ag/ml poly-d-lysine at a density of 400,000–500,000 cells per coverslip. Biotinylation of cell surface proteins and cellular localization of FLRT3 This study was performed on E15 rat cortical cultures using a procedure based on that used in Mammen et al. (1997). Briefly, cultured cells were washed with PBS containing 1 mM CaCl2 and 0.5 mM MgCl2. They were cooled gradually to 48C, washed twice with cold PBS/Ca2+/Mg2+, and incubated at 48C for 12 min (with gentle agitation) with 2 ml 1 mg/ml biotinylation reagent (EZLinkk Sulfo-NHS-LC-Biotin prepared in PBS/Ca2+/Mg2+, from Pierce Biotechnology, Rockford, IL). The cells were then washed three times (3–4 ml per wash) with cold PBS/Ca2+/Mg2+/0.1% BSA and once with cold PBS/Ca2+/Mg2+. They were harvested in 300 Al of low-salt RIPA buffer (1% NP-40, 20 mM HEPES, 150 mM sodium chloride, 100 mM sodium fluoride, 1 mM sodium orthovanadate, and 5 mM EDTA) plus Protease Inhibitor Cocktail (Sigma, UK) and solubilized for 1 h at 48C on a rotating wheel. Cell debris was pelleted by centrifugation (12,000 rpm at 48C for 10 min). The supernatant was transferred to a fresh microcentrifuge tube. One hundred microliters of streptavidin beads (Neutravidin Beads from Pierce Biotechnology, prepared as a 30% slurry in lowsalt RIPA buffer with Protease Inhibitor Cocktail) was added per microcentrifuge tube and this rotated on the wheel for 2 h at 48C. The beads were pelleted and the supernatant retained. They were then washed twice in 1 ml high-salt RIPA buffer (500 mM sodium chloride) plus Protease Inhibitor Cocktail and twice in 1 ml lowsalt RIPA buffer plus Protease Inhibitor Cocktail. 2 PAGE gel loading buffer (Sambrook et al., 1989) was added directly to the bead pellet/biotinylated proteins and also to a sample of the supernatant/cytosolic proteins. These were denatured at 1008C for 5 min and electrophoresed for Western blotting as described previously. The blot was initially probed with C-terminal rat FLRT3 primary antibody (diluted 1:2000 in 6% dried skimmed milk/PBS/0.1% Tween-20) and then reprobed for GAPDH to check for cytosolic protein contamination in the biotinylated protein sample.

Acknowledgments This work was supported by the MRC and MNDA. We thank Dr. D. Becker for his advice on the antisense knockdown study, Mr. M. Turmaine for his assistance with the electron microscopy, and Dr. A. Moss for his help with the Western blot analyses. We also thank Dr. C. Gadd for his advice on statistical analysis.

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