Discordant localization of WFA reactivity and brevican/ADAMTS-derived fragment in rodent brain
© Ajmo et al; licensee BioMed Central Ltd. 2008
Received: 22 May 2007
Accepted: 25 January 2008
Published: 25 January 2008
Proteoglycan (PG) in the extracellular matrix (ECM) of the central nervous system (CNS) may act as a barrier for neurite elongation in a growth tract, and regulate other characteristics collectively defined as structural neural plasticity. Proteolytic cleavage of PGs appears to alter the environment to one favoring plasticity and growth. Brevican belongs to the lectican family of aggregating, chondroitin sulfate (CS)-bearing PGs, and it modulates neurite outgrowth and synaptogenesis. Several ADAMTSs (a disintegrin and metalloproteinase with thrombospondin motifs) are glutamyl-endopeptidases that proteolytically cleave brevican. The purpose of this study was to localize regions of adult CNS that contain a proteolytic-derived fragment of brevican which bears the ADAMTS-cleaved neoepitope sequence. These regions were compared to areas of Wisteria floribunda agglutin (WFA) reactivity, a common reagent used to detect "perineuronal nets" (PNNs) of intact matrix and a marker which is thought to label regions of relative neural stability.
WFA reactivity was found primarily as PNNs, whereas brevican and the ADAMTS-cleaved fragment of brevican were more broadly distributed in neuropil, and in particular regions localized to PNNs. One example is hippocampus where the ADAMTS-cleaved brevican fragment is found surrounding pyramidal neurons, in neuropil of stratum oriens/radiatum and the lacunosum moleculare. The fragment was less abundant in the molecular layer of the dentate gyrus. Mostly PNNs of scattered interneurons along the pyramidal layer were identified by WFA. In lateral thalamus, the reticular thalamic nucleus stained abundantly with WFA whereas ventral posterior nuclei were markedly immunopositive for ADAMTS-cleaved brevican. Using Western blotting techniques, no common species were reactive for brevican and WFA.
In general, a marked discordance was observed in the regional localization between WFA and brevican or the ADAMTS-derived N-terminal fragment of brevican. Functionally, this difference may correspond to regions with varied prevalence for neural stability/plasticity.
Wisteria floribunda agglutinin (WFA) is a lectin that binds to terminal N-acetylgalactosamine residues  and decorates various structures, including PNNs in the CNS, where its reactivity has been well-documented [5–7]. Various rostral-caudal layers of rat cerebral cortex, and particularly the retrosplenial cortex, thalamus, cerebellum and brain stem are regions that contain numerous PNNs prominently labeled by WFA . In rat neocortex, several types of functional morphology are associated with WFA-reactive PNNs [5, 8]. Some data indicate that lectin binding identifies terminal N-acetylgalactosamine present on neuronal cell surface glycoproteins , whereas others suggest the reactivity seen with WFA may detect CS directly. Importantly, WFA binding in nervous tissue co-localizes with signal from antibodies raised against CSs , and signal is lost when tissue sections are pre-treated with Chase , suggesting that WFA binds indirectly or directly to CS. Thus, WFA reactivity has become a standard method of identifying CS-containing subsets of neurons in the CNS that are surrounded by PNNs.
The lecticans, including brevican, aggrecan and the V2 isoform of versican, are highly expressed in the adult brain. The deposition of these lecticans is heterogeneous in the complex ECM of PNNs and in the neuropil  (Fig. 1E). Functionally, CS side chains of the lecticans inhibit neurite elongation and even may stabilize synapses in neural networks [3, 12, 13]. Among the lecticans, brevican is highly abundant in brain, where various isoforms are found including a > 145 kD molecule that carries 1–3 CS chains (Fig. 1A), a core 145 kD protein without CS (Fig. 1B), a 120 kD glycosylphosphatidylinositol-linked membrane-bound form, and 55 kD N-terminal, and 80 kD C-terminal fragments (Fig. 1C, 1D) that are the result of endopeptidase cleavage of the holoprotein. The proteases mainly responsible for cleavage of brevican are glutamyl-endopeptidases, the ADAMTSs (a disintegrin and metalloproteinase with thrombospondin motifs) (Fig. 1E, 1F). Several of these multi-domain proteases  are expressed in brain (ADAMTS1, 4, 5, and 9) [15–17] (our unpublished observations) and cleave aggrecan , versican  and brevican  (for review, see ). ADAMTS-cleaved fragments of each lectican are found in untreated nervous tissue extracts [16, 19, 20, 22–24], indicating that ADAMTSs are active proteases capable of cleaving lecticans in a "normal" nervous system. Fragments of lecticans may be localized in brain tissue sections by using antibodies raised against the terminal, neoepitope sequence of the core protein that is exposed after ADAMTS cleavage [20, 21, 25, 26]. Using an antibody that recognizes the C-terminal residues (EAVESE) that are uncovered after ADAMTS-induced release of the N-terminal fragment of brevican , we noted that the distribution of this immunoreactivity in rat hippocampus was markedly different from WFA reactivity in the same region. We expected that the distribution of the signal from both reagents would be similar, since both N- and C-terminal fragments of brevican appear to be stable after cleavage , and the preponderance of the C-terminal fragments bear CS chains. Thus, the purpose of this study was to describe the distribution and characteristic immunoreactivity for the ADAMTS-cleaved fragment of brevican, and compare this with WFA binding in the rodent CNS. The results show a marked discordance between the two, with the breadth of distribution of the ADAMTS-derived brevican fragment being much wider than that of WFA reactivity.
Recent evidence indicates that ECM molecules are important regulators of structural neural plasticity, and regional expression in the CNS may be indicative of their role. Localization of brevican by immunohistochemistry [7, 28] and in situ hybridization [29, 30] within the CNS revealed high expression in cerebellar and cerebral cortex, hippocampus and thalamic nuclei and brain stem. It is localized perisynaptically, it inhibits neurite outgrowth  and it is thought to stabilize neural networks in the adult . In contrast, conditions that augment the proteolytic cleavage of brevican, especially ADAMTS-induced cleavage, are associated with enhanced neural plasticity [16, 24, 32]. Here, an antibody which selectively recognizes the neoepitope sequence of brevican that is uncovered upon proteolysis by an ADAMTS was used to map the distribution of this fragment in untreated rodent CNS. This pattern was compared to the distribution of PNNs and neuropil stained by the classical reagent WFA. By both Western blot and immunochemistry, the ADAMTS-derived fragment of brevican appeared to be stable, abundant and widely-distributed. There was a discordance between the regional and local expression of classical PNNs identified by WFA and regions where the proteolytic fragment of brevican was most highly expressed. In addition, there appeared to be an association between regions with significant deposition of the brevican fragment and areas known to be involved in neural plasiticity (eg. hippocampus), supporting the involvement of ADAMTSs in neural plasticity mechanisms [16, 21, 24]. WFA staining in these regions was notably low.
A significant proportion of total brevican immunoreactivity in brain extracts was found as a fragment formed by proteolytic cleavage of the intact holoprotein. In fact, all antibodies raised against the brevican holoprotein, intended only to detect the holoprotein, inherently recognize either the N- or C-terminal proteolytically-cleaved fragment. Proteolysis of lecticans may be an important mechanism by which the nervous system overcomes inhibition exerted by PGs during periods of neural plasticity. After systemic injection of the excitotoxin, kainic acid  or after discrete, unilateral lesion of the entorhinal cortex , there was an increase in the abundance of an ADAMTS-derived brevican fragment in the dentate gyrus, the target of projections from entorhinal cortex and a region where sprouting occurs in response to the lesion. Thus, proteolytic cleavage of PGs may be a key mechanism involved in sprouting and reorganization of the dentate gyrus, whereas intact PGs may promote neural stabilization.
There is debate in the literature about which molecule(s) in the CNS are labeled by WFA, and here we investigated whether WFA binds to the CS-bearing lectican, brevican. WFA is a lectin that binds to N-acetylgalactosamine-linked α or β to the 3 or 6 position of galactose . WFA is often used as marker for PNNs that contain CS chains, yet only indirect evidence suggests that WFA binds to CS polymers. Our data show that WFA clearly did not recognize CS-containing brevican on Western blot of soluble brain extracts, although there was at least one high molecular weight WFA reactive band that was diminished upon treatment with Chase. No corresponding bands were observed at the molecular weights representing any isoform of brevican. In addition, the preponderance of brevican was found in the soluble fraction of brain extract, whereas WFA reactivity was mainly located in the particulate, insoluble fraction after differential centrifugation of brain extract. These results reveal that WFA binds molecules that differ from the lectican, brevican, although the possibility that WFA has a differential affinity for selected sulfated forms of CS cannot be excluded. Murakami et al has gathered convincing data that indicates that WFA binds to cell surface glycoproteins. This group showed that terminal N-acetylgalactosamine residues, which are present on neuronal cell surface glycoproteins, are responsible for the PNN reactivity seen with WFA lectin binding [9, 34]. Based on a series of studies using degradative enzymes to define the presence or absence of polysaccharides bound to ECM proteins, their model suggests that perineuronal proteoglycans, such as brevican, bind to these cell surface glycoproteins. Chase treatment removes the terminal N-acetylgalactosamine from these glycoproteins, thereby releasing the lectican from its binding partner on the cell surface. This may, in part, account for the discordance in reactivity seen here between brevican and WFA.
No direct evidence exists, to our knowledge, of WFA binding to any specific CS-containing PG, including the lecticans. However, the reactivity observed with WFA and antibodies generated against CS chains are similarly distributed in rat brain  (our unpublished observations), such as in PNNs, and binding of WFA to fixed brain sections is diminished or lost after Chase treatment [35, 11]. Nonetheless, there may be a host of proteins present in ECM complexes and it is possible, in fact likely, that WFA binds to a molecule that may be indirectly bound to CS. In any case, no biochemical evidence is available to show that WFA binds to large polymeric chains of repeating disaccharides that contain N-acetylgalactosamine.
The differential distribution between WFA and the fragment of brevican may relate to their functional environment, i.e. greater abundance of intact CS-bearing PGs chains in regions stained with WFA (more stable environment) whereas increased in proteolytic cleavage stained the neoepitope fragment in regions that are capable of undergoing neural plasticity. These findings are intriguing since we observe intense immunoreactivity for the ADAMTS-derived brevican fragment in areas thought to be highly plastic such as the hippocampus. Therefore, the relative abundance of cleaved proteoglycans, such as brevican, in a particular region suggest a functional change in surrounding ECM complexes which may contribute to overall neural plasticity. However, the data presented here only begin to uncover the intricate molecular environment around individual neurons that modulate their function and structure.
A marked discordance was observed in the regional localization between WFA and brevican or the proteolytically-derived N-terminal fragment of brevican. Functionally, this difference may correspond to regions with varied prevalence for neural stability/plasticity.
All animal procedures described in this manuscript were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of South Florida. Adult male C57BL/6 mice (23 g – 27 g; Harlan, Indianapolis, IN) and adult male Sprauge-Dawley rats (250 g – 300 g; Harlan, Indianapolis, IN) were housed under a 12 hour light cycle with regulated temperature and humidity. Mice were housed 3 to 4 per cage and rats were housed individually with both having free access to food and water. Brain tissue was collected from animals between 3 and 4 months of age: For biochemical analysis n = 4 and immunohistochemistry n = 6.
For collection of tissue immunoblotted with antibodies and biotin-WFA, animals were euthanatized by exposure to excess CO2 until death and immediately decapitated. Various brain regions were rapidly dissected and extracted with a teflon-glass homogenizer in 5 volumes of Triton-X-100-containing buffer (20 mM Tris-HCl at pH 7.4, 10 mM EDTA, 1% Triton-X-100, and 1:100 protease inhibitor cocktail [Calbiochem type III, LaJolla, CA]) for 2 minutes. The homogenate was centrifuged in a microcentrifuge at 6800 × g for 5 minutes, and the supernatant collected and stored at -80°C.
In some experiments, brain tissue extract was treated with Chase prior to Western blot to determine whether WFA recognized CS-containing proteins. Thus, 25 μl of sodium acetate buffer (50 mM sodium acetate, 1 M Tris, 10 mM EDTA) containing 10 mU of chondroitinase ABC (Sigma-Aldrich, St. Louis, MO) was added to 25 μl of brain tissue extract and incubated for 1.5 h at 37°C. To determine whether there was protease contamination in the Chase preparation, samples underwent Chase digestion in the presence of a protease inhibitor cocktail. All samples were reduced (mercaptoethanol-containing, SDS-PAGE sample buffer), denatured for 4 minutes at 95°C, and subjected to SDS-PAGE.
Tissue extracts were loaded (equal amounts of protein) onto pre-cast, 1.5 mm, 4–20% gradient SDS-PAGE gels (Novex Tris-glycine, Invitrogen, Carlsbad, CA). Separated proteins were electrophoretically transferred to a polyvinylidine difluoride membrane (PVDF, Immobilon, Millipore, Billerica, MA). For brevican and EAV(M)ESE immunoblotting, the membranes were washed with Buffer B (10 mM phosphate buffered saline, pH 7.4 containing 0.05% Tween 20) for 5 minutes, blocked for 1 h in 5% non-fat dry milk diluted in Buffer B and probed for 2 hours using primary antibodies against mouse anti-brevican (1:1000, BD Transduction Labs, San Jose, CA), rabbit anti-EAMESE (1:1000)  (the neoepitope sequence for mouse brevican fragment), or rabbit anti-EAVESE (1:500) [23, 36] (the neoepitope sequence for rat brevican fragment). For WFA blotting, the membranes were washed with Buffer B for 5 minutes, blocked in 1% bovine serum albumin diluted in Buffer B for 1 hour and probed for 2 hours using biotinylated Wisteria floribunda lectin (1:10,000 in 1% BSA, Vector Laboratories, Burlingame, CA) as the primary binding reagent. Primary antibodies and biotinylated Wisteria floribunda lectin were detected with corresponding secondary antibodies including anti-mouse, anti-rabbit and streptavidin conjugated to horse radish peroxidase (Chemicon, Temecula, CA), respectively. Antigens were visualized using a chemiluminescence developing system (SuperSignal, Pierce, Rockford, IL), and equal protein loading was verified by examining the Commassie blue stained membrane. It should be noted that ADAMTS-derived fragment antibodies were raised against the species-specific neoepitopes for rat and mouse, since they show limited cross-reactivity with one another, ie. anti-EAVESE (rat sequence) does not effectively recognize the C-terminus of the N-terminal, ADAMTS-cleaved fragment EAMESE (murine sequence) of mouse brevican.
Isolation of Membrane Fractions
Whole rat brain was collected as descibed above and homogenized for 1 minute with a Glas-Col (Terre Haute, IN) motorized (low speed, 333 rpm), teflon-glass homogenizer in 10 volumes of 50 mM Tris-HCl, pH 7.4, 1 mM EDTA, containing 1:100 protease inhibitor cocktail (Calbiochem type III, LaJolla, CA). The homogenate was centrifuged at 500 × g for 5 minutes, the supernatant collected and centrifuged at 40,000 × g to obtain soluble and insoluble fractions. The supernatant, "soluble" fraction, was removed immediately, aliquoted and stored at -80°C. The insoluble "membrane" fraction was resuspended with buffer, centrifuged again (40,000 × g for 30 min) and reconstituted in detergent-containing RIPA buffer (50 mM Tris base, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton-X-100, 1% sodium deoxycholate, 1% SDS, pH = 7.4), aliquoted and stored at -80°C.
Rats and mice were euthanatized with excess Nembutal, and the brains fixed via cardiac perfusion as described . Brains were cleared using phosphate buffered saline (PBS; pH 7.4), fixed with fresh 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4), collected, post-fixed overnight in 4% paraformaldehyde and cryoprotected with 15% and 30% sucrose (in PBS) for 24 h each. The individual brains were mounted on a cryostat chuck at -20°C and sectioned at 30 μm. Sections were stored freely floating in antifreeze solution (50 mM sodium phosphate, pH 7.4, 30% ethylene glycol and 30% glycerol) at -20°C.
For Chase treated tissue, matched sections were selected, washed three times with PBS and incubated in 500 mU of Chase in 0.5 ml of sodium acetate buffer for 1.5 hours at 37°C. Selected sections to be used for immunohistochemistry were washed in PBS for 15 minutes, blocked and permeabilized in 10% normal goat serum, 3% 1 M lysine and 3% Triton-X-100 for 1 h and incubated overnight in primary antibodies anti-EAMESE (1:1000) (Mayer et al., 2005), EAVESE (1:500) [16, 36], brevican (1:1000, N-terminal (G1); Transduction Labs, San Jose, CA), RB18 (1:500, a generous gift from Yu Yamaguchi, Burnham Institute, La Jolla, CA, an antibody that recognizes an epitope in the G3 domain of rat brevican) and Wisteria floribunda lectin (1:1000) at 4°C. Doubly probed sections were washed and incubated in anti-rabbit IgG conjugated to Alexa-Fluor 488 (Molecular Probes, Eugene, OR) and streptavidin conjugated to Alexa-Fluor 594 (Molecular Probes, Eugene, OR) for 1 hr at room temperature. The sections were washed for 15 minutes, wet mounted on glass slides, and coverslipped with VectaShield mounting medium (Vector Labs, Burlingame, CA).
Cleavage of PGs with human recombinant ADAMTS4
PGs present in whole rat brain extracts were bound to and eluted from a DEAE Sepharose Fast Flow cation exchange matrix (Pharmacia, Pfizer, New York, NY) as described  with modifications. All procedures were carried out at 4°C unless otherwise stated. Briefly, rat brain tissue (1 g) was placed in 10 ml, ice cold, 4 mM HEPES pH 8.0, 0.15 mM NaCl, 0.1% Triton-X-100 containing 2 mM 1,10 phenanthroline (Sigma, St. Louis, MO) and protease inhibitor cocktail (set III, Calbiochem, San Diego, CA). The tissue was disrupted in a Teflon-glass homogenizer and the extract centrifuged at 30,000 × g for 30 min. The supernatant was removed, diluted 1:1 with 50 mM Tris-HCl, 0.15 M NaCl, 0.1% Triton-X-100, and applied to a DEAE column pre-equilibrated with the same buffer at a flow rate of less than 0.5 ml per minute. The flow through was collected, passed over the column again, and bound proteins were eluted with 5 column volumes of consecutive buffers containing 50 mM Tris-HCl pH 8.2, 0.15 M NaCl, 0.1% Triton-X-100, then 50 mM Tris-HCl pH 8.2, 0.25 M NaCl, 6 M urea, 0.1% Triton-X-100, and fractions containing PGs were eluted with 50 mM Tris-HCl pH 8.2, 1.0 M NaCl. PG (protein)-containing fractions were dialyzed against water for 24 h in SpectraPor 6000–8000 MWCO (Millipore, Billerica, MA) membrane at 4°C, the samples concentrated on an Eppendorf "speed-vac" and aliquoted. Total protein was measured in the samples (1.3 μg/μl). DEAE-purified PG samples were incubated with 25 nM human recombinant ADAMTS4, (a gift of Carl Flannery, Wyeth Pharmaceuticals, Collegeville, PA) diluted in 10 mM Tris-HCl, 0.15 M NaCl, and 10 mM CaCl2 for two hours at 37°C. After the incubation period, beta-mercaptoethanol-containing, SDS-PAGE sample buffer was added to the samples, the samples were heated at 95°C for 4 minutes, subjected to SDS-PAGE, and electrically transferred to Immobilon PVDF membrane (Millipore, Bedford, MA). Membranes were probed with mouse anti-brevican (BD Biosciences, San Jose, CA) at 1:1000 primary antibody detected with anti-mouse conjugated to horse-radish peroxidase (Chemicon, Temecula, CA), and signal detected using SuperSignal chemiluminescence substrate (Pierce, Rockford, IL). The membrane was probed a second time with anti-EAVESE (1:100) , as described under the Western blot section above.
Microscopy and image acquisition
Single and multi-labeled, epifluorescent tissue sections were viewed using a Zeiss Axioskop microscope, interfaced with an Axiocam and images acquired with Openlab software. Confocal images (Fig. 8) were attained using a Leica SP-2 confocal microscope and Leica LCS software. Controls for each immunomarker included secondary antibody in the absence of a primary antibody, in which the staning in control sections was minimal to absent. Exposure times and aperture opening were constant for each magnification and antibody used. Some images were minimally and equally modified (contrast and brightness) using Abobe photoshop.
List of abbrevications
PG – proteoglycan, ECM – extracellular matrix, CNS – central nervous system, CS – chondroitin sulfate, ADAMTS – a disintegrin and metalloproteinase with thrombospondin motifs, WFA – Wisteria floribunda, PNN – perineuronal net, Chase – Chondroitinase ABC, IACUC – Institutional animal care and use committee, PVDF – polyvinylidine difluoride, MWCo – molecular weight cutoff.
This work was supported in part by National Institutes of Health (AG022101), Alzheimer's Association (grant #IIRG-02-3758), and Shriner's of North America (grant #8560), The authors would like to thank Dr. Carl Flannery for providing human recombinant ADAMTS4 and Dr. Yu Yamguchi for the brevican antibody, RB18.
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