Discordant localization of WFA reactivity and brevican/ADAMTS-derived fragment in rodent brain

Joanne M Ajmo,Autumn K Eakin,Paul E Gottschall,Michelle G Hamel

doi:10.1186/1471-2202-9-14

Abstract

BackgroundProteoglycan (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.ResultsWFA 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.ConclusionIn 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.

Highlights

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
To verify that this antibody recognizes ADAMTS-cleaved brevican, the chondroitin sulfate (CS)-bearing form of brevican was partially purified on a DEAE anion exchange matrix, and the PG-containing eluant was incubated with active human recombinant ADAMTS4
To verify that the effect of Chondroitinase ABC (Chase) on the high molecular weight, Wisteria floribunda agglutinin (WFA) reactive smear was due solely to degradation of polysaccharides, samples were treated with Chase in the absence and presence of protease inhibitor cocktail (Fig. 3E, left panel). In both rat and mouse samples, the pattern of WFA reactivity was identical, whether or not the samples contained protease inhibitor cocktail. These results suggest that WFA does bind to a high molecular weight, CS moiety that is removed after Chase treatment, but that is clearly different from brevican

Summary

Introduction

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. 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. Extracellular matrix (ECM) in the central nervous system (CNS) is deposited in the extracellular space of the neuropil and around a subset of neurons in the form of distinctive perineuronal nets (PNNs), coverings of matrix that ensheath perikarya, proximal dendrites and axon initial segments The components of this matrix are aggregating proteoglycans (PGs), termed lecticans, that interact with tenascin and hyaluronan to form complexes which maintain an anionic environment in the extracellular milieu of the CNS [1,2] (Fig. 1D).

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