Axonal proteins involved in myelination: characterization of a collagen-like protein.

An anti-axolemma monoclonal antibody, designated G21.3, has been isolated in order to understand molecular mechanisms involved in myelination. Both biochemical and morphological studies showed that the monoclonal antibody inhibits myelin production by oligodendrocytes in cerebellar slice cultures. On Western blots of axolemma preparations, the antibody recognized 140- and 120-kD proteins. The present study involves the isolation and characterization of the G21.3 antigen. The G21.3-immunoreactive proteins of 140 and 120 kD were purified from the adult rat sciatic nerve and amino acid sequencing of these proteins revealed significant homology to alpha I and alpha II chains of collagen type I. Biochemical and Western blot analysis using pure collagen, collagen I antibody and collagenase D suggest that the antigen isolated from sciatic nerve is collagen. However, immunofluorescence studies using the G21.3 antibody, collagen I antibody, collagenase D and Northern blot analysis using a collagen probe do not fully support the view that the G21.3 antigen in the CNS is also a collagen. We conclude that the G21.3 antigen is a collagen-like protein involved in CNS myelination.

Developmental expression of neurofilament and glial filament proteins in rat cerebellum.

Neurofilament protein (NFP) consists of three subunits: NF200, NF150 and NF68. Several studies on expression of NFP in developing brain have shown that NF200 appears later than NF150 and NF68. However, there are some reports on simultaneous appearance of these subunits in development. The present study is an attempt to resolve this controversy. Rat cerebellum was chosen as most of its development takes place during the first three weeks of postnatal period. Cytoskeletal and NFP preparations from newborn (P0), postnatal day 8 (P8), P15, P21, P30 and adult (3 months) rat cerebella were subjected to electrophoresis on 7.5% SDS-PAGE. All the NFP subunits were present from P0 onwards and there was an increase in NFP content and glial fibrillary acidic protein (GFAP) with age as revealed by the densitometric scanning. Immunoblots of NFP preparations confirmed the presence of NF200 in the early postnatal cerebellum. In vivo phosphorylation studies indicated the presence of phosphorylated NF subunits from P8 onwards, which was confirmed by staining in immunoblots by SMI31. Immunohistochemical studies on Bouin's fixed tissues revealed that in P0 cerebella, the deeper neurones (soma and processes) expressed all the NFP subunits while from P8 onwards they were negative for NF200. Similarly, Purkinje cells (soma) expressed transiently NF200 subunits on P8 and ceased to express them from P15 onwards. The white matter was immunopositive for NF200 and NF150 on P0 and the intensity of staining increased progressively. Astrocytes expressing GFAP were seen in cerebellar white matter from P8 onwards and the staining in radial glia could be detected from P15 onwards.(ABSTRACT TRUNCATED AT 250 WORDS)