Cortical plasticity is most evident during a critical period in early life, but the mechanisms that restrict plasticity after the critical period are poorly understood. We found that a developmental increase in the 4-sulfation/6-sulfation (4S/6S) ratio of chondroitin sulfate proteoglycans (CSPGs), which are components of the brain extracellular matrix, leads to the termination of the critical period for ocular dominance plasticity in the mouse visual cortex. Condensation of CSPGs into perineuronal nets that enwrapped synaptic contacts on parvalbumin-expressing interneurons was prevented by cell-autonomous overexpression of chondroitin 6-sulfation, which maintains a low 4S/6S ratio. Furthermore, the increase in the 4S/6S ratio was required for the accumulation of Otx2, a homeoprotein that activates the development of parvalbumin-expressing interneurons, and for functional maturation of the electrophysiological properties of these cells. Our results indicate that the critical period for cortical plasticity is regulated by the 4S/6S ratio of CSPGs, which determines the maturation of parvalbumin-expressing interneurons.

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Extracellular factors that inhibit axon growth and intrinsic factors that promote it affect neural regeneration. Therapies targeting any single gene have not yet simultaneously optimized both types of factors. Chondroitin sulphate (CS), a glycosaminoglycan, is the most abundant extracellular inhibitor of axon growth. Here we show that mice carrying a gene knockout for CS N-acetylgalactosaminyltransferase-1 (T1), a key enzyme in CS biosynthesis, recover more completely from spinal cord injury than wild-type mice and even chondroitinase ABC-treated mice. Notably, synthesis of heparan sulphate (HS), a glycosaminoglycan promoting axonal growth, is also upregulated in TI knockout mice because HS-synthesis enzymes are induced in the mutant neurons. Moreover, chondroitinase ABC treatment never induces HS upregulation. Taken together, our results indicate that regulation of a single gene, T1, mediates excellent recovery from spinal cord injury by optimizing counteracting effectors of axon regeneration—an extracellular inhibitor of CS and intrinsic promoters, namely, HS-synthesis enzymes.

Ocular dominance plasticity is easily observed during the critical period in early postnatal life. Chondroitin sulfate (CS) is the most abundant component in extracellular structures called perineuronal nets (PNNs), which surround parvalbumin-expressing interneurons (PV-cells). CS accumulates in PNNs at the critical period, but its function in earlier life is unclear. Here, we show that initiation of ocular dominance plasticity was impaired with reduced CS, using mice lacking a key CS-synthesizing enzyme, CSGalNAcT1. Two-photon in vivo imaging showed a weaker visual response of PV-cells with reduced CS compared to wild-type mice. Plasticity onset was restored by a homeoprotein Otx2, which binds the major CS-proteoglycan aggrecan and promotes its further expression. Continuous CS accumulation together with Otx2 contributed bidirectionally to both onset and offset of plasticity, and was substituted by diazepam, which enhances GABA function. Therefore, CS and Otx2 may act as common inducers of both onset and offset of the critical period by promoting PV-cell function throughout the lifetime.

A novel alpha2,9-linked polysialic acid (polySia)-containing glycoprotein of sea urchin sperm flagella was identified and named "flagellasialin." Flagellasialin from Hemicentrotus pulcherrimus shows a diverse relative molecular mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of 40-80 kDa. Flagellasialin is a 96-amino acid, threonine-rich, heavily O-glycosylated (80-90% by weight) glycoprotein with a single transmembrane segment at its C-terminus and no apparent cytosolic domain. Of 12 extracellular Thr residues, eight are O-glycosylated and three are nonglycosylated. Flagellasialin is highly expressed in the testis but cannot be detected in the ovary. The amino acid sequences of flagellasialin from three sea urchin species (H. pulcherrimus, Strongylocentrotus purpuratus, and Strongylocentrotus franciscanus) are identical, but some species differences exist in the three core glycan structures to which the sulfated alpha2,9-linked polyNeu5Ac chain is linked. Finally, the treatment of sperm with a specific antibody against the alpha2,9-linked polyNeu5Ac structure results in the elevation of intracellular Ca(2+) and inhibition of sperm motility and fertilization, implicating flagellasialin as a regulator of these critical processes.

A new type of polysialic acid (polySia) structure was demonstrated to occur in a major unknown sialoglycoprotein with a diverse molecular mass of 40-80 kDa in sea urchin sperm. The polySia-containing glycan structure was determined to be HSO(3)-->8Neu5Acalpha2-->9(Neu5Acalpha2-->9)(n-2) Neu5Acalpha2-->6GalNAcalpha1-->Ser/Thr (n, on average 15), based on carbohydrate analysis of the sialoglycopeptide obtained by an exhaustive protease digestion of whole sperm, fluorometric anion-exchange high-performance liquid chromatography, and methylation analysis. The sulfate group was predominantly localized to the nonreducing terminus of the polySia chain. This is the first example of an alpha2,9-linked polySia structure in animal sperm. The polySia-containing sialoglycoprotein was present in sperm flagellum but not in the head. Furthermore, this sialoglycoprotein localized in the sperm lipid raft, which contains an enriched ganglioside (Neu5Acalpha2-->8Neu5Acalpha2-->6GlcCer), a receptor for sperm-activating peptide (speract), and its associated guanylate cyclase.

Chondroitin sulphate proteoglycans (CSPGs), consisting of core proteins linked to one or more chondroitin sulphate (CS) chains, are major extracellular matrix (ECM) components of the central nervous system (CNS). Multi-functionality of CSPGs can be explained by the diversity in structure of CS chains that undergo dynamic changes during development and under pathological conditions. CSPGs, together with other ECM components, form mesh-like structures called perineuronal nets around a subset of neurons. Enzymatic digestion or genetic manipulation of CSPGs reactivates neural plasticity in the adult brain and improves regeneration of damaged axons after CNS injury. Recent studies have shown that CSPGs not only act as non-specific physical barriers that prevent rearrangement of synaptic connections but also regulate neural plasticity through specific interaction of CS chains with its binding partners in a manner that depends on the structure of the CS chain.

Perineuronal nets (PNNs) are lattice-like extracellular matrix structures composed of chondroitin sulfate proteoglycans (CSPGs). The appearance of PNNs parallels the decline of neural plasticity, and disruption of PNNs reactivates neural plasticity in the adult brain. We previously reported that sulfation patterns of chondroitin sulfate (CS) chains on CSPGs influenced the formation of PNNs and neural plasticity. However, the mechanism of PNN formation regulated by CS sulfation remains unknown. Here we found that overexpression of chondroitin 6-sulfotransferase-1 (C6ST-1), which catalyzes 6-sulfation of CS chains, selectively decreased aggrecan, a major CSPG in PNNs, in the aged brain without affecting other PNN components. Both diffuse and PNN-associated aggrecans were reduced by overexpression of C6ST-1. C6ST-1 increased 6-sulfation in both the repeating disaccharide region and linkage region of CS chains. Overexpression of 6-sulfation primarily impaired accumulation of aggrecan in PNNs, whereas condensation of other PNN components was not affected. Finally, we found that increased 6-sulfation accelerated proteolysis of aggrecan by a disintegrin and metalloproteinase domain with thrombospondin motif (ADAMTS) protease. Taken together, our results indicate that sulfation patterns of CS chains on aggrecan influenced the stability of the CSPG, thereby regulating formation of PNNs and neural plasticity.