Structural Variation of Chondroitin Sulfate Chains Contributes to the Molecular Heterogeneity of Perineuronal Nets

Abstract

Aggrecan, a chondroitin sulfate (CS) proteoglycan, forms lattice-like extracellular matrix structures called perineuronal nets (PNNs). Neocortical PNNs primarily ensheath parvalbumin-expressing inhibitory neurons (parvalbumin, PV cells) late in brain development. Emerging evidence indicates that PNNs promote the maturation of PV cells by enhancing the incorporation of homeobox protein Otx2 and regulating experience-dependent neural plasticity. Wisteria floribunda agglutinin (WFA), an N-acetylgalactosamine-specific plant lectin, binds to the CS chains of aggrecan and has been widely used to visualize PNNs. Although PNNs show substantial molecular heterogeneity, the importance of this heterogeneity in neural plasticity remains unknown. Here, in addition to WFA lectin, we used the two monoclonal antibodies Cat315 and Cat316, both of which recognize the glycan structures of aggrecan, to investigate the molecular heterogeneity of PNNs. WFA detected the highest number of PNNs in all cortical layers, whereas Cat315 and Cat316 labeled only a subset of PNNs. WFA⁺, Cat315⁺, and Cat316⁺ PNNs showed different laminar distributions in the adult visual cortex. WFA, Cat315 and Cat316 detected distinct, but partially overlapping, populations of PNNs. Based on the reactivities of these probes, we categorized PNNs into four groups. We found that two subpopulation of PNNs, one with higher and one with lower WFA-staining are differentially labeled by Cat316 and Cat315, respectively. CS chains recognized by Cat316 were diminished in mice deficient in an enzyme involved in the initiation of CS-biosynthesis. Furthermore, WFA⁺ and Cat316⁺ aggrecan were spatially segregated and formed microdomains in a single PNN. Otx2 co-localized with Cat316⁺ but not with WFA⁺ aggrecan in PNNs. Our results suggest that the heterogeneity of PNNs around PV cells may affect the functional maturation of these cells.

Keywords: Otx2; WFA; aggrecan; chondroitin sulfate; parvalbumin-expressing neuron; perineuronal nets; proteoglycan.

Figure 1

Different laminar distributions of WFA⁺, Cat315⁺ and Cat316⁺ perineuronal nets (PNNs) in the adult visual cortex. (A–C) Immunohistochemical detection of different PNNs in the primary visual cortex of 3-months-old adult mouse using Wisteria floribunda agglutinin (WFA) (A), Cat315 (B), and Cat316 (C). WFA-staining was also observed in the neuropil in layer IV. Cortical layers are indicated in Roman numerals to the left of the panels. WM, white matter. Scale bar, 50 μm. (D) The numbers of WFA⁺ (closed bar), Cat315⁺ (open bar) and Cat316⁺ (gray bar) PNNs. WFA detected the highest number of PNNs. (E) Laminar distributions of PNNs. Bar graphs represent percentage of WFA⁺ (upper), Cat315⁺ (middle) and Cat316⁺ (lower) PNNs in each cortical layer. Cat315⁺ PNNs were most abundant in layers V and VI, whereas Cat316⁺ PNNs were concentrated in layer IV. (F) Percentage of WFA⁺ (closed bar), Cat315⁺ (open bar), and Cat316⁺ (gray bar) PNNs formed around parvalbumin cells (PV cells). n = 327, 131 and 80 cells from three mice for WFA⁺, Cat315⁺ and Cat316⁺ PNNs, respectively. Error bars represent SEM.

Figure 2

Identification of four types of PNNs. (A,B) Triple staining of WFA (red), PV (blue) and Cat315 (green in A) or Cat316 (green in B). Arrowheads and arrows in (A) indicate WFA⁺/Cat315⁺ and WFA⁻/Cat315⁺ PNNs, respectively. Arrows in (B) indicate WFA⁺/Cat316⁺ PNNs, Scale bar, 50 μm. (C) Four types of PNNs, which differ in the glycan structures of aggrecan. (D) Percentage of WFA⁺/Cat315⁺ (closed bar), WFA⁺/Cat316⁺ (gray and Cat315⁺ or Cat316⁺/WFA⁺ (open bar) PNNs (n = 131, 80 and 327 cells, respectively). (E) Fluorescence intensity of WFA-staining (left panels) and PV-staining (right panels) in Cat315⁺ PNNs (closed bar) relative to Cat315⁻ PNNs (closed bar). n = 62 and 52 cells from three mice for Cat315⁻ and Cat315⁺ PNNs, respectively. (F) Fluorescence intensity of WFA-staining and PV-staining in Cat316⁺ PNNs (closed bar) relative to Cat316⁻ PNNs (closed bar). n = 72 and 55 cells from three mice for Cat316⁻ and Cat316⁺ PNNs, respectively. Asterisks denote significant differences (P < 0.00001, Student’s t-test) between two groups. Error bars represent SEM.

Figure 3

WFA⁺ and Cat316⁺ chondroitin sulfate (CS) chains form microdomains in PNNs. (A) Enzymatic digestion of PNNs. The reactivities of WFA and Cat316, but not Cat315 and PV, were diminished by treatment with chondroitinase ABC (Chase). WFA- and Cat316-staining were also markedly decreased upon treatment with β-N-acetylhexosaminidase (HexNAcase). Scale bar, 50 μm. (B) The three-dimensional reconstruction showed a meshwork of WFA⁺ CS chains (red). Cat316⁺ CS chains (green) showed a punctate pattern and only partially overlapped with WFA⁺ CS chains. Orthogonal views of the XZ plane (bottom panel) and YZ plane (right panel) revealed distinct distribution patterns of WFA⁺ and Cat316⁺ CS chains on the surface of PV cell (gray). (C,D) Otx2 (green) co-localized with Cat316⁺ (red in C), but not with WFA⁺ CS chains (red in D). Scale bar, 2 μm.

Figure 4

Reduced level of Cat316⁺ CS chains in ChGn-1 KO mice. (A) Expression of Cat316⁺ CS chains in the mouse brain increased during postnatal development. Brain lysates prepared from 1-week-old (1W) and 3-month-old (Adult) mice were detected by Cat316. The reactivity was lost after treatment with chondroitinase ABC (Chase) as indicated by an arrow. Arrowheads indicate chondroitinase ABC-insensitive non-specific bands. (A′,A″) Cat316⁺ CS chains were markedly diminished in the brain of ChGn-1 KO (A″), but not in C6ST-1 TG mice (A′). Data are representative of at least two mice per group. (B) Quantification of total amount of CS chains in the cerebrum of wild-type (WT; closed bar) and ChGn-1 KO mice (open bar). Asterisks denote significant differences (P < 0.05, Student’s t-test) between two groups. Error bars represent SEM. n = 3 mice for each group. (C) Immunohistochemical detection of Cat316⁺ PNNs (red) and Otx2 (green) in the adult brain of WT and ChGn-1 KO mice. Arrowheads indicate co-localization of Cat316⁺ PNNs and Otx2. Magnified views are shown in (C′). Scale bars, 50 μm in (C) and 2 μm in (C′). (D) CS chains isolated from WT (closed square) and ChGn-1 KO (open square) mice were biotinylated and immobilized on a streptavidin-coated plate (1–100 ng/well). Wells were incubated with Cat316 antibody, followed by peroxidase-conjugated secondary antibody. The color was developed using ABTS substrate kit and the optical density was measured at 415 nm. Absorbance was expressed as relative values to control (0 ng/well of CS). Data are means of duplicate wells and are representative of two independent experiments.

Figure 5

Proposed biosynthetic pathways of Cat315⁺, Cat316⁺ and WFA⁺ aggrecan. The glycosaminoglycan-protein linkage region is assembled on specific Ser residues of aggrecan. Although aggrecan core protein contains more than 100 potential CS attachment sites, only one attachment site is shown in the figure for simplicity. (A) HNK-1ST transfer a sulfate to the non-reducing terminal GlcA residue of the linkage region, generating a Cat315⁺ truncated CS chain that prevents further elongation of the repeating disaccharides. (B,C) Transfer of a GalNAc to the terminal GlcA residue in the linkage region by ChGn-1, 2 or chondroitin polymerases triggers the elongation of a CS chain. Cat316⁺ CS chains are mainly produced by a ChGn-1-dependent pathway (B). WFA⁺ and Cat316⁺ CS chains may have different sulfation pattern and/or chain length (C). Otx2 may selectively bind to Cat316⁺ CS chains.