Figure 2.

Removal of PSA moieties increases myelination. (A) Anti-PSA-NCAM mAb (α-PSA) or endo-N were added to the culture medium between 10 and 18 DIV, and myelination was quantified at 22–24 DIV by counting the number of MBP-positive myelinated internodes per coverslip. Controls of specificity of anti-PSA mAb were cultures treated with either K5-2 mAb, an irrelevant mouse IgM used at the same concentration as anti-PSA, or A2B5 mAb, an IgM that binds to axons, but not to PSA-NCAM, or anti-PSA mAb adsorbed with 1 mM colominic acid, a polymer of α2,8-linked sialic acid. Anti-PSA mAb and endo-N-treated cultures showed a 5.2- and 4.8-fold increase in myelination, respectively. Results are expressed as the mean ± SEM of three experiments in quintuplicate. (**, P < 0.001; Student's t test). (B) At the ultrastructural level, the myelin formed in anti-PSA-treated cultures appears normal and well compacted. (C and D) Anti-PSA-NCAM mAb treatment induced an internalization of the PSA-anti-PSA-NCAM complex. Anti-PSA-NCAM mAb were added to culture medium between 10 and 18 DIV. Cultures were then either fixed (C) in 4% PFA or maintained alive (D) before being incubated with the fluorescein-conjugated secondary antibody alone. Staining was only visible on fixed cultures (C), demonstrating internalization of anti-PSA-NCAM mAb. (Magnification: B, ×35,000; and C and D, ×350.)