Suppression of nidogen-1 translation by antisense targeting affects the adhesive properties of cultured astrocytes.

The multidomain glycoprotein nidogen-1 is a common component of basal membranes. Nidogen-1 is produced by the endothelial cells and the mesenchymal cells of the developing central nervous system. Recent results give evidence that nidogen-1 may also be secreted by cultured Schwann cells to basement membranes of peripheral nerves. We were interested in ascertaining whether astrocytes, which have the capacity to produce laminin and fibronectin and are an important source of extracellular matrix (ECM) molecule secretion in the brain, might also produce nidogen-1. Immunocytochemistry, in combination with polymerase chain reaction and in situ hybridization techniques, revealed that astrocytes in culture synthesize nidogen-1. To show the functional significance of the nidogen-1 secretion by astrocytes, antisense targeting techniques were applied. These experiments showed that nidogen-1 may be an essential modulator of astrocytic adhesion to the substrate. The suppression of nidogen-1 synthesis by the application of antisense oligonucleotides induced a morphological transition from a flat, polygonal to a round cell and was accompanied by the detachment of the astrocytes from the substrate. Hence, nidogen-1 might be an important component of the ECM secreted by astrocytes. The suppression of nidogen-1 synthesis may disturb the aggregation of ECM molecules to a functional basement membrane and thus reduce the astrocytic adhesion to the substrate. Nidogen-1 secretion to basement membranes by astrocytes may have important functional implications during blood-brain barrier and scar formation.

Structure and chromosomal localization of the mouse neurocan gene.

Cosmid clones containing the mouse neurocan gene were isolated from a genomic library using rat neurocan cDNA fragments as probe. The murine gene has a size of approximately 25 kb and contains the coding sequence for the mRNA on 15 exons. The exon-intron structure reflected the structural organization of neurocan, which is a multidomain protein belonging to the aggrecan/versican proteoglycan family. All introns between conserved modular protein domains are phase I introns. Primer extension experiments indicate a transcriptional start point 28 bases downstream of a consensus TATA sequence. Further analysis of 1 kb of 5' flanking sequence revealed in addition to AP1, AP2, and SP1 consensus binding sites multiple E-box elements and a glucocorticoid responsive element. Single-strand conformation polymorphism was used to map neurocan to chromosome 8 between the microsatellite markers D8Mit29 and D8Mit78. Among mouse mutants that have been mapped around this region are the three allelic neurological diseases tottering, leaner, and rolling. The multidomain structure and the preferential expression of neurocan in the brain suggest a potential involvement in these diseases.