IMMUNOCYTOCHEMICAL LOCALIZATION OF CHONDROITIN AND CHONDROITIN 4-SULFATES AND 6-SULFATES IN DEVELOPING RAT CEREBELLUM

Monoclonal antibodies specific for unsulfated, 4-sulfated, and 6-sulfated disaccharide "stubs" that remain attached to the core protein after chondroitinase ABC digestion of chondroitin/dermatan sulfate proteoglycans have been used to study the localization of chondroitin and the two isomeric chondroitin sulfates in developing rat cerebellum. At 1-2 weeks postnatal, unsulfated chondroitin is present in the granule cell layer, molecular layer, and prospective white matter, but there was no staining of the external granule cell layer other than light staining of Bergmann glia fibers. By 3 weeks postnatal, staining of the molecular layer has disappeared and has diminished in the white matter, whereas in adult cerebellum only the granule cell layer remains stained. The staining pattern of chondroitin 4-sulfate is similar to that for chondroitin at 1-2 weeks postnatal, but in contrast to chondroitin, chondroitin 4-sulfate increases in the molecular layer at 3 weeks, and this becomes the most densely stained region of adult cerebellum. Chondroitin 6-sulfate is present predominantly in the prospective white matter of 1-2 week postnatal cerebellum, although significant staining of the granule cell layer is also seen. By 3 weeks postnatal the granule cell staining of chondroitin 6-sulfate has decreased, and in adult cerebellum staining is seen only in the white matter and to a lesser extent in the granule cell layer. Electron microscopy confirmed the presence of chondroitin sulfate in the cytoplasm of neurons and glia of adult brain. HPLC of chondroitin sulfate proteoglycan disaccharides demonstrated that the significant amounts of chondroitin (29%) and chondroitin 6-sulfate (19%) present at 7 days are replaced almost entirely by chondroitin 4-sulfate in adult brain, and immunoblots of chondroitinase-treated proteoglycans from 7-day and adult brain revealed a distinctive pattern of major core proteins that stained with each of the three antibodies. The different localizations and developmental changes of chondroitin, chondroitin 4-sulfate, and chondroitin 6-sulfate suggest that sulfation may be an important factor in specifying the biological functions of chondroitin sulfate proteoglycans in nervous tissue.