phosphacan early in CNS development that localizes to extrasynaptic sites prior to synapse formation

Neuroscience 142 (2006) 1055–1069

MONOCLONAL ANTIBODY Cat-315 DETECTS A GLYCOFORM OF RECEPTOR PROTEIN TYROSINE PHOSPHATASE BETA/PHOSPHACAN EARLY IN CNS DEVELOPMENT THAT LOCALIZES TO EXTRASYNAPTIC SITES PRIOR TO SYNAPSE FORMATION M. R. DINO,a S. HARROCH,b S. HOCKFIELDa1 AND R. T. MATTHEWSa*

Key words: extracellular matrix, CSPGs, perineuronal net, RPTP␤, aggrecan, phosphacan.

a

Department of Neurobiology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510, USA

A functioning nervous system requires the precise alignment of pre- and post-synaptic apparatuses. While much progress has been made in defining the molecular composition of synapses in the CNS, little is known about the timing and molecular mechanism that initiate their organization. For example, it is not yet known whether postsynaptic sites are defined prior to contact from the presynaptic cell. Detailed studies of the neuromuscular junction (NMJ) show that a rudimentary prepatterning of synaptic sites is laid down prior to innervation (reviewed in Arber et al., 2002; Kummer et al., 2006). The extracellular matrix (ECM) that surrounds the developing muscle cell has been shown to play a particularly important role in synaptic formation and stabilization of the NMJ. This basal lamina (BL) represents a specialized region of the NMJ matrix that organizes and maintains synaptic specializations during development and regeneration (reviewed in Sanes and Lichtman, 1999, 2001; Goda and Davis, 2003; Patton, 2003). In contrast to the NMJ, central synapses do not have a defined BL and little is known about the role of the ECM in the formation and/or stabilization of central synapses. Furthermore, few of the ECM molecules found in neuromuscular BL are expressed in the CNS, and early work even suggested that the CNS lacked an ECM. More recent studies have demonstrated the presence of an ECM in the CNS, but one distinct from the NMJ (reviewed in Hockfield et al., 1990; Ruoslahti, 1996; Bandtlow and Zimmermann, 2000; Yamaguchi, 2000). The major components of the CNS ECM are proteoglycans, glycoproteins and hyaluronan. The most well-studied ECM structure found in the CNS is the perineuronal net (PN). PNs are lattice-like condensations of the ECM observed on neuronal subpopulations after synapses become functional (reviewed in Celio and Blumcke, 1994; Celio, 1999). Like ECM components in the mature NMJ, ECM components in PNs are in close contact with synapses and play a role in synaptic stabilization (Hockfield et al., 1990; Pizzorusso et al., 2002). Among PN components, chondroitin sulfate proteoglycans (CSPGs) are particularly abundant. CSPGs that have been localized to PNs include neurocan, aggrecan, brevican, and phosphacan (reviewed in Bandtlow and Zimmermann, 2000; Yamaguchi, 2000). Biochemical, in situ hy-

b

Institut Pasteur, Neuroscience Department, 25 Rue du Dr. Roux, Paris 75724, Cedex 15, France

Abstract—Perineuronal nets (PNs) are lattice-like condensations of the extracellular matrix (ECM) that envelop synapses and decorate the surface of subsets of neurons in the CNS. Previous work has suggested that, despite the fact that PNs themselves are not visualized until later in development, some PN component molecules are expressed in the rodent CNS even before synaptogenesis. In the adult mammalian brain, monoclonal antibody Cat-315 recognizes a glycoform of aggrecan, a major component of PNs. In primary cortical cultures, a Cat-315-reactive chondroitin sulfate proteoglycan (CSPG) is also expressed on neuronal surfaces and is secreted into culture media as early as 24 h after plating. In this study, we show that in primary cortical cultures, the Cat-315 CSPG detected in early neural development is expressed in extrasynaptic sites prior to synapse formation. This suggests that ECM components in the CNS, as in the neuromuscular junction (NMJ), may prepattern neuronal surfaces prior to innervation. We further show that while the Cat-315-reactive carbohydrate decorates aggrecan in the adult, it decorates a different CSPG in the developing CNS. Using receptor protein tyrosine phosphatase beta (RPTP␤/protein tyrosine phosphatase zeta) knock-out mice and immunoprecipitation techniques, we demonstrate here that in the developing rodent brain Cat-315 recognizes RPTP␤ isoforms. Our further examination of the Cat-315 epitope suggests that it is an O-mannose linked epitope in the HNK-1 family. The presence of the Cat-315 reactive carbohydrate on different PN components— RPTP␤ and aggrecan—at different stages of synapse development suggests a potential role for this neuron-specific carbohydrate motif in synaptogenesis. © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. 1

Present address: Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139 – 4307, USA. *Correspondence to: R. T. Matthews, Department of Neuroscience and Physiology, WH 3238, State University of New York Upstate Medical University, 750 East Adams Street, Syracuse, NY 13210, USA. Tel: ⫹1-315-464-7766; fax: ⫹1-315-464-7712. E-mail address: [email protected] (R. T. Matthews). Abbreviations: BL, basal lamina; cmd, cartilage matrix deficiency mouse; CSPG, chondroitin sulfate proteoglycan; div, day in vitro; ECM, extracellular matrix; HNK-1, human natural killer cell glycan; mAb 5210, mouse monoclonal anti-phosphacan; NMJ, neuromuscular junction; PN, perineuronal net; PSDs, postsynaptic densities; PSI, phosphacan short isoform; P14, postnatal day 14; P21, postnatal day 21; RPTP␤, receptor protein tyrosine phosphatase beta; SA-PMP, streptavidin paramagnetic particles.

0306-4522/06$30.00⫹0.00 © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2006.07.054

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bridization, and immunocytochemical studies have shown that, despite the fact that PNs themselves have not been observed until later in development, some PN component molecules are expressed in the CNS prior to synaptogenesis (Milev et al., 1998; Popp et al., 2003). These studies indicate that, like the NMJ, matrix components may play a role in the earliest stages of cellular development and synapse formation in the CNS. Here we utilized a monoclonal antibody, Cat-315, that detects an epitope highly enriched in adult PNs (Lander et al., 1997; Matthews et al., 2002) to determine the developmental expression pattern of this epitope and the neural ECM early in development. Specifically, we explored the expression of Cat-315 in a cell culture model system that allowed us to investigate the evolution of matrix components during development of central synapses.

EXPERIMENTAL PROCEDURES Antibodies Cat-315 has been previously characterized (Lander et al., 1997). Rabbit polyclonal anti-synaptophysin was a gift from Dr. Pietro de Camilli (Cell Biology Department, Yale University Medical School, New Haven, CT, USA). Mouse monoclonal anti-phosphacan (mAb 5210) and anti-synaptophysin (clone SY35) antibodies were purchased from Chemicon (Temecula, CA, USA). The mouse monoclonal antibody3F8 developed by Dr. Margolis was obtained from the Developmental Hybridoma Bank, developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA, USA). AntiHNK-1 antibodies, CD57 and CBL519, were purchased from Becton Dickinson (San Diego, CA, USA) and from Chemicon, respectively. Anti-receptor protein tyrosine phosphatase beta (RPTP␤) antibody was purchased from Becton Dickinson, and anti-PSD 95/SAP 97/Chapsyn 110 antibody was purchased from Upstate Cell Signaling Solutions (Charlottesville, VA, USA). Rabbit antiGABA was purchased from Sigma (St. Louis, MO, USA) and rabbit anti-glutamate 607 was obtained from the laboratory of Dr. O. P. Ottersen (Oslo, Norway). Alkaline-phosphatase-conjugated and biotin-conjugated secondary antibodies were purchased from Jackson Immunochemicals (West Grove, PA, USA). Alexa-tagged fluorescent secondary antibodies were purchased from Molecular Probes (Eugene, OR, USA).

Preparation of homogenates, soluble and particulate fractions Tissue and cell homogenates and their respective soluble and particulate fractions were prepared as previously described by Viapiano et al. (2003). Briefly, cortices and whole brains were dissected from: (1) embryonic, postnatal and adult Sprague–Dawley rats; (2) P0 and adult aggrecan knockout mice (cartilage matrix deficiency mouse, cmd) and their corresponding isogenic controls, and (3) RPTP␤ knockout mice (generated as described in Harroch et al., 2000) and their corresponding isogenic controls. Dissected samples were homogenized in 20 mM Tris–HCl (pH 7.4) with 250 mM sucrose and a protease inhibitor cocktail (Complete, Roche, Hertfordshire, UK). The homogenates were centrifuged at 900⫻g for 10 min at 4 °C, and the resulting postnuclear supernatant was centrifuged again at 100,000⫻g for 60 min to obtain soluble and particulate fractions.

of 1–2 mg/ml in chondroitinase buffer (40 mM Tris–HCl, 40 mM sodium acetate, 5 mM EDTA, pH 8.0) and treated with 0.25 U of chondroitinase ABC from Proteus vulgaris (Seikagaku America, East Falmouth, MA, USA) for 8 h at 37 °C. Samples were then boiled in 1⫻ sample buffer, electrophoresed on 6.25% reducing SDS–polyacrylamide gels or gradient gels (4 –15% or 3–7%), and then electrophoretically transferred to nitrocellulose. Blots were incubated with one of the following primary antibodies: Cat-315, 3F8, mAb 5210, CBL519 or CD57. In all cases, alkaline phosphatase-conjugated secondary antibodies were employed to detect the primary antibodies. Immunoreactive bands were visualized with nitro blue tetrazolium and 5-bromo-4-chloro3-indoyl phosphate.

Protein deglycosylation Deglycosylations were performed as previously described (Viapiano et al., 2005) Briefly, samples were equilibrated in deglycosylation buffer (20 mM Tris–HCl, 20 mM sodium acetate, 25 mM NaCl, pH 7.0) containing protease inhibitors, at a protein concentration of ⬃1 mg/ml, and treated for 8 h at 37 °C with combinations of the following glycosidases: 0.25 U/ml chondroitinase ABC, 20 mU/ml O-glycosidase (Roche), 100 mU/ml sialidase (Roche) and 100 U/ml glycopeptidase F (PNGase F, Calbiochem). Enzyme digestions were stopped by boiling the samples in 1⫻ gel-loading buffer. For denaturing deglycosylation, required for non-exposed N-linked carbohydrates, samples were first equilibrated in 0.1% w/v SDS/0.1 M 2-mercaptoethanol and heated at 95 °C for 10 min. Subsequently, samples were equilibrated in deglycosylation buffer containing 0.8% v/v Nonidet-P40 and deglycosylation proceeded in the same conditions as indicated above.

Immunoprecipitation Taking advantage of the strong and stable binding between biotin and strepavidin, ⬃100 ␮g/ml of biotinylated secondary antibodies (anti-mouse IgGs and IgMs) were bound to streptavidin paramagnetic particles (SA-PMP, Promega, Madison, WI, USA). Immune complexes were then formed between the appropriate biotinylated secondary antibodies and either one of the following primary antibodies: Cat-315, mAb 5210, 3F8 or CD57. Proteins were extracted from the particulate fractions of E18, E20, P0, and adult rat cortices using a 50 mM Tris–HCl (pH 7.6)/0.6% CHAPS buffer. Immune complexes were then incubated overnight with membrane extracts and soluble fractions brought to a final concentration of 1 mg/ml with the Tris/CHAPS buffer. After washing, bound material was eluted from the SA-PMPs by boiling in 1⫻ sample buffer. Immunoprecipitated and immunodepleted material was resolved in SDS-PAGE and probed using Western blotting.

Neuronal cultures Neuronal cultures were prepared as described by Quinn et al. (2003), with minor modifications. Briefly, cortices from E15 or E18 rats were dissected, trypsinized, dissociated and then plated at a density of 200 – 400 cells/mm2 in coverslips or Petri dishes coated with poly-L-lysine (100 ␮g/ml, ⬎70,000 MW). Culture media (Neurobasal supplemented with 2% B27, 1 mM pyruvate, 1 mM glutamine, 50 U penicillin and 50 ␮g streptomycin, all purchased from Invitrogen, Carlsbad, CA, USA) initially contained 5% fetal bovine serum, (Hyclone, Logan, UT, USA) but was changed into serumfree medium 4 h after plating. Thereafter, half the volume of the medium in each plate was replaced every 2–3 days.

Immunocytochemistry SDS-PAGE and Western blotting Prior to protein electrophoresis, aliquots of homogenates, soluble and particulate fractions were equilibrated at a final concentration

Cultures at different time points were fixed with 4% paraformaldehyde for 30 min at room temperature, rinsed and then incubated in primary antibodies overnight at 4 °C. Primary antibodies used

M. R. Dino et al. / Neuroscience 142 (2006) 1055–1069 were Cat-315, anit-PSD 95/SAP 97/Chapsyn 110, anti-GABA, anti-glutamate 607 and anti-synaptophysin. After rinsing, the cultures were incubated in the appropriate species- and subclassspecific Alexa-tagged secondary antibodies and mounted in Prolong Anti-Fade (Molecular Probes). In addition, live staining, as previously described by Lander et al. (1998), was done on a separate set of cultures at the same time points. Briefly, each coverslip was incubated for 30 min at 4 °C in 500 ␮l of a primary antibody solution containing Cat-315 hybridoma medium (diluted 1:5). After several rinses, cells were fixed with 4% paraformaldehyde in 1⫻ PBS for 30 min at room temperature. Cells were then incubated in the appropriate Alexa dyetagged secondary antibody for 1–3 h at room temperature. Some coverslips were then double stained with rabbit polyclonal antisynaptophysin

RESULTS Neuronal cell-surface expression of an early-expressed Cat-315 proteoglycan precedes the development of synaptic contacts Our previous work has shown that, while the monoclonal antibody Cat-315 detects PNs in the adult cerebral cortex, it also detects a cell-surface CSPG far in advance of net development. In E19 and P0 rodent brain, Cat-315 staining is diffuse in the cortical parenchyma but is prominent in fiber tracts (data not shown). To further investigate the early expression of Cat-315, we investigated cultured cortical neurons prepared from E16 or P0 rats. As had been previously demonstrated, we found punctate cell-surface Cat-315 staining after only 1 day in culture (Lander et al., 1998). The early expression of Cat-315, prior to synaptic input, led us to ask whether expression of this ECM molecule may play a role in cortical neuron development and connectivity. In particular, since previous work has demonstrated that ECM molecules in PNs are expressed adjacent to sites of synaptic contact, we asked whether the early expression of Cat-315 might correlate with synaptic development. To further characterize the early developmental expression pattern of Cat-315 in primary cortical neurons, we followed Cat-315, PSD 95/SAP 97/Chapsyn 110 and synaptophysin expression in primary cortical cultures prepared from rat cortices at two different ages. Primary neuronal cultures from rat cortex at embryonic days 15 and 18 were maintained for a range of days in vitro (div), and then stained for Cat-315. As in our previous study (Lander et al., 1998), we used live staining to verify the cell surface expression of Cat-315 in cultures at various time points. Primary cultures of E15 cortices were used because at this age cortical neurons have received little, if any, synaptic input (Konig et al., 1975). These cultures were compared with primary cultures of E18 cortices to determine whether Cat-315 expression in vitro would be affected by previously established synaptic input. Primary cortical cultures prepared from E18 rats are a well-established in vitro system for studying synaptogenesis, i.e. hippocampal and cortical primary neuronal cultures (Banker and Goslin, 1988; de Lima et al., 1997), and have been used to establish a time course of synaptogenesis.

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The spatiotemporal expression of Cat-315 was similar in cultures prepared from the two embryonic ages. In cultures from both E15 and E18 cortices we detected Cat-315 on the surface of neurons in advance of the establishment of synapses. As early as 1 div, primary cortical cultures from E15 rats showed neuritic processes studded with Cat-315 immunoreactive puncta (Fig. 1A). At 1 div most, but not all, neurons (approximately 80%) were immunoreactive for Cat-315. Immunoreactivity was detected on neuronal cell bodies and on neurites (Fig. 1A). Cat-315 immunoreactivity increased in intensity over the first several div. By 4 div (Fig. 1B), most of the neurons remained Cat-315positive. At 7 div, E15 cultures (Fig. 2A) showed an increase in the density of Cat-315-positive puncta on the cell bodies and neuritic processes. Like the E15 primary cortical cultures, in cultures from E18 cortex Cat-315-positive puncta were detected as early as 1 div and increased in density and intensity of staining up to 15 div (Fig. 2D and 2G), after which no further increase in immunoreactivity was observed. Synaptophysin is a major presynaptic protein that has been used to follow synaptogenesis (Ehrhart-Bornstein et al., 1991; de Lima et al., 1997). In contrast to the early expression of Cat-315 immunoreactivity, synaptophysinpositive synaptic contacts were not detected until after several days in culture. As shown in Fig. 1C, and as previously described (de Lima et al., 1997), synaptophysin immunoreactivity in primary cortical cultures after 1– 4 div was distributed in neuronal cell bodies and along the proximal regions of the developing neurites; synaptic-like structures were not observed at early days in culture. Over time, synaptophysin staining became more punctuate, and neuritic processes appeared more beaded, assuming the staining pattern characteristic of synapses (compare Figs. 1C and 2B). In E18 cultures, the density of synaptophysin puncta on cell bodies and neuronal processes increased dramatically after 4 div, and peaked at 15 div (Fig. 2E and 2H). Like synaptophysin, immunostaining with the anti-PSD 95/SAP 97/Chapsyn 110 at 1 div and 4 div was mostly intracellular (data not shown). At 7 div, PSD-positive puncta localized to the neuronal cell bodies and primary neuritic processes (Fig. 3, 7 div). As the neuritic processes grew in length, the number and density of PSD-positive puncta increased concurrently (Fig. 3, 21 div). Thus, in cultures from both E15 and E18, Cat-315 was detected on the surface of neurons in advance of the establishment of synapses. As described above, Cat-315 immunoreactivity appeared on neurons as soon after plating as we could examine them, at 1 div. In contrast, PSD 95/SAP 97/Chapsyn 110-immunoreactive and synaptophysin-immunoreactive puncta were first observed on cell bodies and neurites only after several days in culture. In the mature brain, we and others have demonstrated that a component of the neuronal ECM, the PN, is distributed over neuronal cell bodies, proximal dendrites and the initial segment of axons, but is excluded from regions of synaptic contact (Hockfield and McKay, 1983; Hockfield et al., 1983; Zaremba et al., 1989). To determine whether

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Fig. 1. Monoclonal antibody Cat-315 decorates the surface of neurons in advance of the establishment of synapses. Primary cortical cultures were prepared from E15 rats, when few synapses have been established on cortical neurons. (A, B) At 1 div, neuritic processes are studded with Cat-315 immunoreactive puncta. Approximately 80% of neurons are Cat-315-positive, including both large and small (inset) diameter neurons. (C) At 4 div, immunoreactivity for synaptophysin, a marker of synaptic proteins, is found in cell bodies and neurites, but has not yet coalesced into synaptic boutons. (D) Merging the images in (B) and (C) shows that Cat-315 expression on the neuronal surface precedes the expression of synaptophysin at synaptic boutons. Scale bar⫽10 ␮m.

developing neurons show a similar, reciprocal distribution of ECM proteoglycans and early synapses, we used dou-

ble label immunocytochemistry with confocal microscopy to study the relationship between Cat-315-immunoreactive

M. R. Dino et al. / Neuroscience 142 (2006) 1055–1069

regions and synaptophysin-positive synaptic puncta. As shown in Fig. 2F, 2I, and Fig. 3, in primary neuronal cultures pre- and post synaptic markers were interspersed between Cat-315 puncta on neuronal cell bodies and neurites. A reciprocal distribution of Cat-315-positive regions and synaptic profiles was observed as early as synaptic puncta were observed, and persisted through the entire culture period examined. These results illustrate that an ECM component, the Cat-315 CSPG, accumulates at extrasynaptic sites before the formation of presynaptic specializations, perhaps delineating sites of future synapses prior to synaptogenesis. Neurotransmitter content of Cat-315-positive neurons in primary cortical cultures In the rat cortex, cells expressing PNs include interneurons immunoreactive for GABA, the calcium-binding protein parvalbumin, and the potassium channel Kv3.1 (Brückner et al., 2004, 1994; Wegner et al., 2003; Pizzorusso et al., 2002; Haunso et al., 2000, 1999; Wintergerst et al., 1996). In addition, Brückner et al. (1994, 2004) also described PNs on pyramidal cells immunoreactive for glutamate and the GABAA receptor ␣1 subunit. To determine which subtypes of cortical cells express Cat-315 in vitro, we doublelabeled 21-day old primary cortical cultures with anti-GABA or anti-glutamate antibodies (Fig. 4A–C). In cultures double-labeled with GABA, approximately 75% of Cat-315positive cells (40/54) were double-labeled. These numbers are similar to those described by others: Pizzorusso et al. (2002) found that 77% of the cortical neurons possessing PNs use GABA as a neurotransmitter, while Wegner et al. (2003) reported that GABA colocalized in 100% of nonpyramidal PN-expressing interneurons in the rat parietal cortex. While the vast majority of cells decorated with Cat-315 are GABAergic, only 60% (42/70) of GABA positive cells in our cultures expressed the Cat-315 epitope. Because both our anti-GABA and anti-glutamate antibodies were made in rabbit, we could not triple-label our cultures. However co-labeling with anti-glutamate and Cat315 revealed that approximately 30% (20/62) of Cat-315 positive cells contained glutamate. These included densely and lightly-stained pyramidal cells (see Fig. 4D–F), similar to those described by Wegner et al. (2003). Among glutamatergic cells in 21-day old cultures, approximately 40% (21/51) were also positive for Cat-315. Monoclonal antibody Cat-315 recognizes distinct CSPGs in the developing and adult CNS We have previously shown that the antigen recognized by Cat-315 in 1-day old primary cortical cultures (Lander et al., 1998), in P1 cortical homogenates (Lander et al., 1998), and in adult rat (Matthews et al., 2002) and cat (Lander et al., 1997) brain homogenates are all CSPGs. As shown in Fig. 5A, the Cat-315 antigen ran as a diffuse band on Western blots of embryonic and adult rat cortices. Digestion with chondroitinase shifted the Cat-315 band to a lower molecular mass (Fig. 5A), confirming that the Cat-315 antigen at all ages is a CSPG. At postnatal day 14 (P14) the molecular weight of the Cat-315 CSPG shifts

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slightly higher and at postnatal day 21 (P21) becomes a doublet (Fig. 5B). As previously shown by Matthews et al. (2002), Cat-315 reactive aggrecan in adult rat brain homogenates runs as a doublet. Matthews et al. (2002) have shown that in the adult rat cortex, antibody Cat-315 recognizes a carbohydrate epitope on specific glycoforms of the CSPG, aggrecan. They also reported that, while Cat-315 also recognizes a CSPG in the embryonic brain, the early-expressed Cat315 proteoglycan is distinct from aggrecan. We previously examined Cat-315 immunoreactivity in the brain of the cmd, a mouse strain that carries a deletion in the aggrecan gene and showed that, consistent with the identification of the adult Cat-315 antigen as aggrecan, Cat-315 immunoreactivity is reduced in adult cmd heterozygotes (Matthews et al., 2002). To confirm that the CSPG recognized by Cat-315 in perinatal rodent brain is not aggrecan, we took advantage of cmd mice and their corresponding isogenic littermates. Here, we reasoned that if Cat-315 does not recognize aggrecan in the perinatal rodent brain, then Western blots of brain from P0 cmd animals would not show a reduction in Cat-315 immunoreactivity. In contrast with the adult, the Cat-315 immunoreactive band from P0 animals was expressed at approximately equal levels in the ⫺/⫺, ⫺/⫹, and ⫹/⫹ cmd brains (Fig. 5C). This observation provides strong evidence that the Cat-315-reative CSPG in the P0 brain is not aggrecan. Monoclonal antibody Cat-315 recognizes RPTP␤ in embryonic rodent cortex To identify the proteoglycan recognized by Cat-315 in the embryonic CNS, we used Cat-315 to immunoprecipitate its antigen from the soluble and particulate fractions of E18 rat cortices. Western blots of the Cat-315 immunoprecipitated material were then probed with antibodies against proteoglycans that have been reported previously to be expressed in the embryonic brain and that have a molecular mass in the range of 300 –500 kDa. Of the antibodies tested, an antibody that recognizes phosphacan (mAb 5210) stained Cat-315 immunoprecipitated material from the soluble fraction (Fig. 6A). Cat-315 immunoprecipitation depleted mAb 5210-reactive material from the soluble fraction and, reciprocally, immunoprecipitation with mAb 5210 depleted Cat-315 from the soluble fraction of E18 rat cortices. These results suggested that Cat-315 recognizes phosphacan in the soluble fraction of embryonic rat brain. Phosphacan is a secreted isoform of RPTP␤, also known as protein tyrosine phosphatase zeta (PTP␨). Alternative mRNA splicing produces four isoforms of RPTP␤ (Margolis et al., 1996; Oohira et al., 2000; Garwood et al., 2003; reviewed in Sugahara et al., 2003): two isoforms, the long and short receptor forms, are membrane bound and contain cytoplasmic tyrosine phosphatase domains; the other two, phosphacan and the phosphacan short isoform, PSI, are secreted into the ECM. Of the two forms secreted into the ECM, the PSI is not a proteoglycan (Garwood et al., 2003).

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Fig. 2. Cat-315 immunoreactivity is distributed in extra-synaptic zones. Double label confocal microscopy illustrates the relationship between Cat-315-immunoreactive regions and synaptophysin-positive synaptic puncta. (A–C) In cultures from E15 animals after 7 div, Cat-315-positive puncta (A) on the cell bodies are more numerous and extend along neuritic processes while synaptophysin staining (B) has become more punctate, and labeled neuritic processes appeared more beaded, assuming the staining pattern characteristic of synapses. (C) Merged images show that

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All four isoforms possess an N-terminal carbonic-anhydrase domain followed by a fibronectin type III domain, and a long cysteine-free, serine-glycine rich sequence termed the spacer domain. Based on their primary protein sequence, the calculated molecular weights of the long receptor isoform, phosphacan, the short receptor isoform and PSI are predicted to be 250, 175, 164, and 67 kDa respectively (Garwood et al., 2003). However, due to extensive post-translational modifications, the molecular masses of phosphacan and the long receptor isoform are ⬎800 kDa, and those of the short receptor isoform and PSI are 190 and 90 kDa respectively (Garwood et al., 2003). Chondroitinase ABC digestion shifts the molecular weights of phosphacan and the long receptor isoform to approximately 350 – 400 kDa, while N-glycosidase F treatment increases the mobility of the short receptor and PSI isoforms (Garwood et al., 2003). Immunoprecipitation of Cat315 from the soluble fraction strongly suggests that Cat315 recognizes phosphacan, the secreted isoform of RPTP␤. To determine if Cat-315 recognizes the long or short receptor forms of RPTP␤, immunoprecipitates of Cat-315 and mAb 5210 from the particulate fraction of E18 cortices were treated with chondroitinase ABC, and then resolved using SDS-PAGE. Cat-315 immunoprecipitation from E18 particulate/membrane fractions diminished, but did not completely remove the mAb 5210 antigen, whereas mAb 5210 immunoprecipitation depleted Cat-315 from the starting material (Fig. 6A). In both the starting and immunoprecipitated materials, the Cat-315 band was always in the 350 – 400 kDa range. The absence of a Cat-315 band at approximately 190 –205 kDa in both the starting and Cat-315-immunoprecipitated material suggests that the Cat-315 antibody recognizes the long, but not the short receptor isoform. To verify that this is indeed the case, we immunoblotted the Cat-315 immunoprecipitates from E18 cortices with a commercially available antibody generated against the second cytoplasmic phosphatase domain. As shown in Fig. 6A, the anti-RPTP␤ antibody detected a band corresponding in molecular weight to the long receptor form of the protein but did not detect a band corresponding to the short-receptor form in Cat-315 immunoprecipitates from the particulate fraction. In contrast the anti-RPTP␤ antibody detected bands corresponding to both the long and short receptor forms in material immunoprecipitated with mAb 5210. In primary neuronal cultures, Cat-315 reactive material was found in the media (not shown) and in cell lysates (Fig. 6B). In both cases, the Cat-315 ran at an apparent molecular mass consistent with phosphacan and the long receptor form of the RPTP␤. While phosphacan was clearly detected in the media we further investigated if Cat-315 could detect the receptor forms of RPTP␤ in the cell lysates. Cell lysates precipitated with Cat-315 and detected

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with the anti-RPTP␤ antibody indicated that in addition to detecting phosphacan, it also detected the long-receptor form of the protein. We have no evidence that Cat-315 detected the short receptor form of RPTP␤ in primary cultures but Cat-315 does not allow us to differentiate between phosphacan and long receptor form. Consistent with our immunochemical observations, immunostaining of E18 and P0 rat brains with Cat-315 (unpublished observations) are similar to the pattern observed after staining with 3F8, an antibody that recognizes phosphacan (Maurel et al., 1994; Meyer-Puttlitz et al., 1995): both Cat-315 and 3F8 intensely stain fiber tracts, the marginal zone and the cortical parenchyma in perinatal cortex. Moreover, when 7d primary cultures prepared from E18 rat cortices were immunostained with either Cat-315 or mAb 5210 (not shown), numerous Cat-315-positive and mAb 5210-positive puncta were observed on neuritic processes and cell bodies. Because both antibodies belong to the mouse IgM class, we could not unequivocally prove colocalization of the two antibodies. To verify that Cat-315 recognizes phosphacan/RPTP␤ in the perinatal rodent brain, we examined Cat-315 expression in the brains of RPTP␤ knockouts. In Western blots of P1 and adult brain extracts probed with Cat-315, the Cat315 band was absent in P1⫺/⫺ samples, but was present in P1⫹/⫹, adult ⫹/⫹, and adult ⫺/⫺ lanes (Fig. 6C). These results provided unequivocal proof that Cat-315 recognizes phosphacan/RPTP␤ in the perinatal, but not in the adult, rodent brain. Further support for this identification was obtained by immunoblotting RPTP␤ ⫺/⫺ material with 3F8 (Fig., 6C), an antibody that also recognizes phosphacan (Maurel et al., 1994; Meyer-Puttlitz et al., 1995). As expected, the 3F8 band was absent in P1 and adult knockout brains, but was present in the wild types of the same ages. Unlike the Cat-315 band, which was most prominent in embryonic and early postnatal ages, the 3F8 band was less pronounced in the P1 wild type brain than in the adult wild type. Given that previous work has demonstrated that Cat-315 likely detects a carbohydrate epitope (Matthews et al., 2002), these results suggest that phosphacan glycoforms may be differentially regulated during development. The Cat-315 epitope is a subset of the HNK-1 antigen As described above, Cat-315 specifically detects two distinct CSPGs at different developmental time points. However, interestingly, at both time points the distinct CSPGs are localized in perisynaptic or extrasynaptic domains on the neuronal cell surface. This coincident localization led us to ask if the epitope detected by Cat-315 may itself have been an important structural and functional feature of these CSPGs. To begin to investigate a role for this

synaptophysin profiles intermingle with Cat-315 puncta, but Cat-315 positive zones do not co-localize with synaptophysin-positive boutons. (D–I) The number of synaptophysin-positive profiles and processes increased dramatically after 4 div, and peaked at 15 div. In cultures from E18 cortex after 15 div, Cat-315-positive puncta and synaptophysin-positive boutons never overlap. Synaptophysin-positive boutons (E and H) along neurites and on neuronal cell bodies are excluded from regions of Cat-315 (D and G) staining, as seen in the merged images (F and I). At 15 div and longer, synaptophysin-labeled processes were interspersed between Cat-315 puncta. Scale bar⫽10 ␮m.

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Fig. 3. Cat-315 and PSD-95 are expressed in a complementary manner. Double labeling with Cat-315 and an antibody that recognizes PSD 95/SAP 97/Chapsyn 110 reveals a reciprocal distribution between Cat-315 and these postsynaptic markers. At 7 div, PSD-immunoreactive profiles are distributed in primary processes and in neuronal cell bodies. Over time, as the neuritic processes increase in length, so do the number of PSD-positive puncta (21 div). Similar to the findings with synaptophysin staining, PSD-95 is interspersed with Cat-315-reactive puncta.

epitope in brain development we identified the Cat-315 epitope. We have previously shown that Cat-315 recognizes an oligosaccharide epitope in the adult CNS that is resistant to chondroitinase, sialidase, N-glycanase and O-

glycanase enzymatic treatments, but is abolished by ␤-elimination with mild alkaline borohydride, a treatment that specifically removes O-linked glycans (Matthews et al., 2002). Similarly, treatment of P0 brain soluble fraction

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Fig. 4. Cat-315 is present on the surfaces of GABAergic interneurons and glutamatergic pyramidal cells. Double labeling with anti-GABA antibody and Cat-315 (A–C) reveals that a subset of GABA-immunoreactive interneurons expresses the Cat-315 epitope. In addition, Cat-315 is also found on the surface of glutamate-positive cells with a pyramidal morphology (D–F). Scale bar⫽10 ␮m A–C; D–F⫽20 ␮m.

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Fig. 5. Cat-315 recognizes a CSPG distinct from aggrecan in the developing brain. Homogenate from cerebral cortex at different ages was analyzed for Cat-315 reactivity by Western blot. (A) Cat-315 recognizes a CSPG at all ages as shown by the shifting of the Cat315-reactive band to a lower apparent molecular mass after digestion with chondroitinase in both the developing (embryonic day 18, e18, shown) and adult cortex. (B) Chondroitinase-treated samples were analyzed across development. Notice that at P14 the Cat-315 band shifted to a slightly higher molecular mass, and at P21 became a doublet (arrows). This represents the expression of distinct Cat-315reactive proteoglycans over development. (C) Previously, we showed that Cat-315 immunoreactivity is reduced in adult cmd heterozygotes (aggrecan ⫹/⫺ mice) (Matthews et al., 2002). To determine if the Cat-315 antigen in the perinatal brain is aggrecan, Western blots of P0 cmd brains were stained with Cat-315. No reduction in Cat-315 immunoreactivity was detected in brain from ⫺/⫺ or ⫺/⫹ cmd animals compared with wild type (⫹/⫹) brains, demonstrating that Cat-315 does not recognizes aggrecan in P0 brain.

with the combination of chondroitinase, sialidase, N-glycanase and O-glycanase enzymes increased the mobility of the Cat-315 bearing proteoglycan but did not affect

Cat-315 reactivity (Fig. 7A). Since the Cat-315 carbohydrate is expressed in a remarkably specific manner on distinct proteoglycans early and late in development, we next identified the carbohydrate detected by Cat-315. A number of lines of evidence raised the possibility that Cat-315 detects a human natural killer cell glycan (HNK-1) related epitope. HNK-1 is a 3=-sulfated lactosamine that can be found on N- and O-linked carbohydrates on proteins and on glycolipids (Kleene and Schachner, 2004). HNK-1 epitopes have been reported in aggrecan (Domowicz et al., 2003) and RPTP␤ isoforms (Maeda et al., 1994; Garwood et al., 2003). We also previously found that the ability of Cat-315 to detect its epitope was dependent on sulfation (data not shown). However, Cat-315 does not appear to recognize N-linked sugars or glycolipids in the brain, so it certainly could only be detecting a subset of HNK-1 epitopes, if at all. Interestingly, previous work has indicated that all O-linked HNK-1 in the brain is exclusively O-mannosyl linked, making it resistant to enzymatic deglycosylation but releasable by mild alkaline borohydride treatment (Yuen et al., 1997), which we also have previously demonstrated for Cat-315 (Matthews et al., 2002). Together these data led us to investigate whether Cat-315 detects this subset of the HNK-1 family of epitopes. Immunoblotting of adult brain homogenates demonstrated the presence of several HNK-1 bands in the ⬎125 kDa MW range (Fig. 7B). Consistent with previous reports that HNK-1 determinants include both N-linked and Olinked oligosaccharides (Yuen et al., 1997), treatment with N-glycanase removed three HNK-1 bands between 125 and 200 kDa MW range (Fig. 6B, arrows), but not those ⬎200 kDa. Interestingly, N-glycanase digestion left an HNK-1 band of the same size as that visualized by Cat-315 (Fig. 7B). To determine if the early-expressed Cat-315 oligosaccharide is a subset of the HNK-1 determinant, we performed immunoprecipitation studies using the soluble fraction of P0 rodent cortices. HNK-1 immunoprecipitation depleted all Cat-315-reactive bands (Fig. 7C). In addition to the Cat-315 band, HNK-1 immunoprecipitated material detected two other bands in the 150 –180 kDa MW range (arrows, Fig. 7C), which likely represent N-glycanase sensitive HNK-1 bands. To determine if this is indeed the case, Western blots of P0 soluble fraction were treated with N-glycanase and chondroitinase. N-glycanase digestion removed these two HNK-1 bands, leaving a band that was the same size as Cat-315 (Fig. 7D). Taken together, these results suggest that the Cat-315 detects at least a subset of O-linked HNK-1 epitopes.

DISCUSSION We demonstrate here that Cat-315-reactive puncta decorate the surface of primary neuronal cultures within 24 h after plating, well before the formation of synapses. Interestingly, as synapses develop in culture, synaptic markers are never co-localized with Cat-315 puncta. Instead, Cat315 puncta are always found adjacent to sites of synaptic contact. The finding that the Cat-315 puncta are detected

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Fig. 6. Monoclonal antibody Cat-315 detects two isoforms of RPTP␤ in embryonic and early postnatal brain. To determine if Cat-315 detects particular isoforms of RPTP␤ and phosphacan we did cross-immunoprecipitation (ip) studies with Cat-315 and a known RPTP␤/phosphacan antibody (mAb 5210). (A) Cat-315 ip depleted (d) phosphacan/mAb 5210-reactive material from the soluble fraction starting material (s) and, reciprocally, ip with mAb 5210 depleted Cat-315 from the soluble fraction of E18 rat cortices, suggesting that Cat-315 recognizes phosphacan, the secreted isoform of RPTP␤. To determine if Cat-315 recognizes specific receptor forms of RPTP␤, immunoprecipitates of Cat-315 and mAb 5210 from the solubilized particulate fraction of E18 cortices were probed with both antibodies and an antibody against the cytoplasmic phosphatase domain of RPTP␤ (RPTP␤). Cat-315 ip from E18 membrane fractions diminished, but did not remove completely the mAb 5210 antigen, whereas mAb 5210 ip depleted Cat-315 from the starting material. We detected no RPTP␤ positive bands in the soluble material, which is not surprising since this antibody specifically detects domains associated with membrane forms of RPTP␤. However material immunoprecipitated with Cat-315 showed a single reactive band with the RPTP␤ antibody, consistent with the long receptor form of the protein. In contrast, material precipitated with mAb 5210 showed two reactive bands consistent with the short and long receptor forms of RPTP␤. Cat-315 does not appear to detect the short receptor form or RPTP␤. (B) Cell lysates from primary neuronal cultures and Cat-315 immunoprecipitates from solubilized cell lysates contain a single prominent Cat-315-reactive band greater than 220 kDa, and two mAb 5210-reactive bands, one greater than 220 kDa and one in the 160 kDa range. Immunoprecipitated material detected with the RPTP␤ antibody showed a single band that corresponds in molecular weight to the long receptor form of RPTP␤. In this culture system Cat-315 likely detects both phosphacan and the long receptor form of RPTP␤ on the cell surface (C) To verify if Cat-315 recognizes RPTP␤, Western blots of P1 and adult RPTP␤ ⫹/⫹ and ⫺/⫺ brains were probed with Cat-315 and 3F8, another antibody that recognizes phosphacan (Maurel et al., 1994). The Cat-315 band disappeared only in P1 ⫺/⫺ and was present in P1 and adult ⫹/⫹ and adult ⫺/⫺ (arrows), confirming that in early postnatal, but not in adult brain, Cat-315 recognizes the RPTP␤ isoforms. As expected, 3F8 bands were absent in P1 and adult RPTP␤ knockouts.

early in development and are found exclusively in extra- or non-synaptic sites, suggests a prepatterning of the neuronal surface prior to synaptic innervation.

Prepatterning has not been thoroughly studied in the CNS, but several lines of evidence indicate that, like the NMJ, central synapses also establish preferred synaptic

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Fig. 7. Cat-315 carbohydrate epitope is a subset of the HNK-1 determinant. To determine the nature of the Cat-315 epitope, deglycosylation experiments were conducted. (A) As we have previously shown on aggrecan (Matthews et al., 2002), while the apparent molecular mass of the Cat-315 reactive protein shifts when subjected to enzymatic deglycosylation, the Cat-315 carbohydrate epitope on RPTP␤ is not removed with chondroitinase, N- and O-glycanase, and sialidase treatment. Our preliminary studies suggested that Cat-315 may detect a carbohydrate related to the HNK-1 family. (B) Interestingly, immunostaining of homogenate from adult brain with an anti-HNK-1 antibody reveals several reactive bands below 200 kDa that were not detected with Cat-315. However, treatment with N-glycanase, which had no effect on Cat-315 reactivity, completely eliminated the HNK-1 reactive bands below 200 kDa. The remaining HNK-1 positive bands look remarkably similar to Cat-315 reactivity. (C) To determine if HNK-1 and Cat-315 detect related epitopes we performed immunoprecipitation studies. Soluble fraction from P0 brains were immunoprecipitated with anti-HNK-1 and the starting material (s), depleted material (d) and precipitated material (ip) was detected with either Cat-315 or anti-HNK-1. Notice that anti-HNK-1 virtually completely depleted Cat-315 reactive material. (D) Like the adult brain, HNK-1 bands present in P0 brain include N-glycanase-sensitive bands (arrows) and bands not sensitive to N-glycanase are identical in MW to Cat-315 reactivity.

regions prior to innervation by autonomously aggregating postsynaptic proteins. For example, Hinds and Hinds (1976) found that before the start of synapse formation, the olfactory bulb at E14 has structures that resemble postsynaptic densities (PSDs) facing extracellular space or unmodified processes. In addition, Frosch and Dichter (1992) demonstrated that in cortical cultures prepared from 15 to 16 day old rat embryos, neurons devoid of synaptic contacts had regions of high GABAA receptor density 24 h after plating. We previously reported that the Cat-315-reactive CSPG detected early in development is not aggrecan (Matthews et al., 2002), and here, using immunoprecipitation and genetic techniques we now show that the early Cat-315 CSPGs are the secreted and long-receptor isoforms of RPTP␤. Immunostaining of embryonic and early postnatal rodent brain with Cat-315 is very similar to that of phosphacan, as described by Meyer-Puttlitz et al. (1995). RPTP␤ knockout mice are viable and have no obvious brain malformations (Harroch et al., 2000), leading to the suggestion that the mechanisms involved in synapse formation and stabilization are modulated by multiple redundant pathways. Such pathways may involve carbohydrate motifs shared by different CSPGs. The HNK-1 carbohydrate epitope, for example, has been detected on aggre-

can (Domowicz et al., 2003) and RPTP␤ isoforms (Maeda et al., 1994; Garwood et al., 2003). Previous work in the adult rodent brain has demonstrated that the monoclonal antibody Cat-315 detects a carbohydrate on the CSPG aggrecan that is resistant to treatment with O-glycanase, but is removed by ␤-elimination with mild alkaline borohydride, a treatment that specifically removes O-linked glycans (Matthews et al., 2002). In this study, we have shown that N-glycanase and Oglycanase digestion does not release the Cat-315 epitope from RPTP␤. Margolis and co-workers (Finne et al., 1979; Krusius et al., 1986, 1987; Chai et al., 1999) have previously shown that the majority of O-linked sugars released by alkaline borohydride treatment contain mannose at their proximal ends and that over half of the carbohydrate-peptide linkages in brain proteoglycans are of the mannosyl-O-serine/ threonine type. Using a combination of fractionation, neoglycolipid and mass spectrometry techniques, Yuen et al. (1997) showed that HNK-1 reactive epitopes obtained by reductive alkaline hydrolysis are linked to the protein core via O-mannose residues. The evidence we have presented here and in previous studies indicates that the Cat-315 subset of the HNK-1 determinant is linked to aggrecan and phosphacan through O-mannose linkages.

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O-mannose saccharides were originally thought to exist only in yeast but have since been found in mammalian proteins such as dystroglycan (reviewed in Endo and Toda, 2003; Haltiwanger and Lowe, 2004). Importantly, recent studies have demonstrated that O-mannosyl glycans are critical for proper formation and maintenance of the NMJ (Ibraghimov-Beskrovnaya et al., 1992; Zaccaria et al., 1998; Heathcote et al., 2000; Grady et al., 2000) and that defects in O-mannosylation machinery are responsible for some forms of muscular dystrophy (Yoshida et al., 2001; Michele et al., 2002; Michele and Campbell, 2003; Kim et al., 2004). It is interesting that Cat-315 positive, and thus O-mannosylated, phosphacan and aggrecan are localized perisynaptically, perhaps suggesting a role for these molecules in synapse formation and stabilization in the CNS in a fashion similar to the NMJs. In fact, some patients with defects in O-mannosylation also show CNS deficits (Yoshida et al., 2001; Michele et al., 2002; BeltranValero et al., 2002; reviewed in Grewal and Hewitt, 2003; Endo, 2004). Presently, the O-mannosylated proteins responsible for these deficits have not been identified. Like the Cat-315 epitope, carbohydrate modifications found on the RPTP␤ isoforms, including chondroitin and keratan sulfates, and the Lewis X carbohydrate epitope, are expressed in a developmentally-regulated manner (Rauch et al., 1991; Maeda et al., 1994; Allendoerfer et al., 1995). It has been suggested that the constantly changing carbohydrate modifications on RPTP␤ during development may alter binding affinities with different ligands. Indeed, Maeda et al. (2003) have recently shown that the changes in the structure of chondroitin sulfate on RPTP␤ during brain development are accompanied by changes in its binding affinity for pleiotrophin. Unlike many other carbohydrate modifications, however, Cat-315 glycosylation is neuron specific. Matthews et al. (2002) and Lander et al. (1997, 1998) have previously shown that neurons express the Cat-315 antigen, but that glial cells do not. Here we show that the Cat-315 glycan is present on two different proteins—RPTP␤ and aggrecan— during different periods of synaptic development. Future work will determine whether the Cat-315 glycan serves similar roles during early and late stages of synaptogenesis. Acknowledgments—This work was supported by NIH grant EY06511 (to S.H.) and NSF postdoctoral minority fellowship (to M.R.D.). We thank Smaragda Lamprianou for preparation of tissue samples from the RPTP␤ knockout mice. We also thank Mariano Viapiano and Gail Kelly for advice, discussion, and comments during the preparation of this manuscript.

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(Accepted 14 July 2006) (Available online 20 September 2006)