Influences of retinal axons on the cultural substrate containing biotin-conjugated chondroitin sulfate in vitro

Abstract

Although chondroitin sulfate (CS) is known to act as an inhibitory axon guidance cue, retinal axons show substantial growth on a culture substrate containing CS. Thus, the question arises as to how retinal axons elongate on CS-containing culture substrates. To elucidate the effects of retinal axons on a substrate containing CS, we synthesized biotinylated CS (biotin-CS) and developed a culture substrate with streptavidin-conjugated biotin-CS (complex between streptavidin and biotin-CS) to culture retinal axons. The effects of retinal axons on the streptavidin-biotin-CS complex were analyzed immunocytochemically using antibodies against CS and streptavidin, which recognize the carbohydrate and protein portions of the complex, respectively. After the axons were cultured on the substrate, areas that were CS-immunonegative but streptavidin-immunopositive were observed on the surface, corresponding to areas with or without axons, respectively. Absence of CS immunostaining was considered to be caused by structural alterations in the carbohydrate chains of the CS under the influence of the axons.

Moderate repulsive effects of E-unit-containing chondroitin sulfate (CSE) on behavior of retinal growth cones

“…Transmembrane protein tyrosine phosphatase (PTPσ) and contactin-1 were shown to be the functional receptors of CSPG and CSE, respectively (Mikami et al, 2009;Shen et al, 2009;Coles et al, 2011); it would be intriguing if this is the case in pathway formation of retinal axons. It has been reported in vitro, using CSbiotin-streptavidin complex, that some retinal axons growing on CS-containing substrate reduce the MO-225 staining along axons (Ando 2010), which is thought to be an adaptation for the retinal axons to alter their microenvironment for growth. Alteration of the CS microenvironment may reduce sensitivity to CSE-PE beads and lead to diverse behaviors.…”

Application of Chondroitin Sulfate Derivatives for Understanding Axonal Guidance in the Nervous System during Development

“…A broad band stained with both SYPRO Ruby and Alcian blue was observed, indicating the formation of a complex between CS-biotin and streptavidin ( Figure 3C). Unlike the sharp band for streptavidin, the band for the complex was broad in the higher molecular-weight range because of the variation in the molecular weights and anionic charges of the CS with varying lengths [46]. Because it is known that 1 biotin molecule binds to a streptavidin and streptavidin molecules form a tetramer, it is thought that the complex consists of a tetramer: 4 CS-biotins and 4 streptavidins [47].…”

“…Ando et al [46] generated complex of CS-biotin and streptavidin. The CS-biotin and streptavidin were mixed and electrophoresed on non-denaturing polyacrylamide gels.…”

“…Ando et al [46] used CS-biotin in investigating axonal elongation in vitro, which is a successful example of application of the CS derivative ( Figure 3D); thus, here we describe their approach. They used CS-biotin to generate the CS-biotin-streptavidin complex as an artificial CSPG, and further examined the interaction between the retinal axons and CS.…”