Fetal Neuronal Cells Research Articles

Biosynthesis of the D2-cell adhesion molecule: post-translational modifications, intracellular transport, and developmental changes.

Posttranslational modifications and intracellular transport of the D2-cell adhesion molecule (D2-CAM) were examined in cultured fetal rat neuronal cells. Developmental changes in biosynthesis were studied in rat forebrain explant cultures. Two D2-CAM polypeptides with Mr of 187,000-210,000 (A) and 131,000-158,000 (B) were synthesized using radiolabeled precursors in cultured neurons. A and B were found to contain only N-linked complex oligosaccharides, and both polypeptides appeared to be polysialated as determined by [14C]mannosamine incorporation and precipitation with anti-polysialic acid antibody. The two polypeptides were sulfated in the trans-Golgi compartment and phosphorylated at the plasma membrane. D2-CAM underwent rapid intracellular transport, appearing at the cell surface within 35 min of synthesis. A and B were shown to be integral membrane proteins as seen by radioiodination by photoactivation employing a hydrophobic labeling reagent. In rat forebrain explant cultures, D2-CAM was synthesized as four polypeptides: A (195,000 Mr), B (137,000 Mr), C (115,000 Mr), and a group of polypeptides in the high molecular weight region (HMr) between 250,000 and 350,000. Peptide maps of the four polypeptides yielded similar patterns. Biosynthesis of C and HMr increased with age, relative to A and B. A and B were sulfated in embryonic brain, however, sulfation was not noticeable at postnatal ages. Phosphorylation, on the other hand, of A and B was observed at all ages examined. We suggest that D2-CAM function may be modified during development by changes in the relative synthesis of the different polypeptides, as well as by changes in their glycosylation and sulfation.

Biosynthesis of the D2 cell adhesion molecule: pulse-chase studies in cultured fetal rat neuronal cells.

D2 is a membrane glycoprotein that is believed to function as a cell adhesion molecule (CAM) in neural cells. We have examined its biosynthesis in cultured fetal rat brain neurones. We found D2-CAM to be synthesized initially as two polypeptides: Mr 186,000 (A) and Mr 136,000 (B). With increasing chase times the Mr of both molecules increased to 187,000-201,000 (A) and 137,000-158,000 (B). These were similar to the sizes of D2-CAM labeled with [14C]glucosamine, [3H]fucose and [14C]mannosamine, indicating that the higher Mr species are glycoproteins. In the presence of tunicamycin, which specifically blocks the synthesis of high mannose cores, Mr were reduced to 175,000 (A) and 124,000 (B). Newly synthesized A and B are susceptible to degradation by endo-beta-N-acetyl-glucosaminidase H, which specifically degrades high mannose cores, but they are resistant to such degradation after 150 min of posttranslational processing. Hence, we deduce that A and B are initially synthesized with four to five high mannose cores which are later converted into N-linked complex oligosaccharides attached to asparagine residues. However, no shift of [35S]methionine radioactivity between A and B was detected with different pulse or chase times, showing that these molecules are not interconverted. Thus, our data indicate that the neuronal D2-CAM glycoproteins are derived from two mRNAs.

Cytodifferentiation and synaptogenesis in the neostriatum of fetal and neonatal rhesus monkeys.

Cytodifferentiation and synaptogenesis in the neostriatum (caudate nucleus and putamen) were analyzed by the Golgi impregnation method and electron microscopy in 14 fetuses and 8 postnatal rhesus monkeys. During the second fetal month the neostriatum consists primarily of simple, mostly bipolar, immature cells and a small number of undefined profiles ending with growth cones. The first morphologically defined synapses appear in the putamen at embryonic day 60 ( E60 ) and in the head of the caudate nucleus at E65 . Synaptic density in both structures is less than one per 1000/micron2 of neuropil at this stage; synapses are characterized by asymmetric junctions between axonal profiles and immature dendritic shafts, accumulation of an intermembrane web and aggregation of round clear vesicles in presynaptic profiles. During the third fetal month neuronal cell bodies and glial cells enlarge, and axonal and dendritic processes in Golgi preparations become more complex. Although the basic morphology of synapses remains unchanged, their density increases to 9/1000 micron2 in the putamen and 3.7/1000 micron2 in the caudate. During the fourth fetal month the four principal cell classes of the neostriatum emerge. Spines on the shafts of dendrites are followed closely by the appearance of axospinous synapses. Synaptic density in the putamen is still significantly higher (10.1/1000 micron2) than in the caudate (5.4/1000 micron2), but by the end of the fifth fetal month ( E150 ) it is the same (80/1000 micron2) in both structures. A dramatic increase in synaptic density to 125/1000 micron2 occurs before term ( E165 ) with the emergence of the first asymmetric synapses as well as symmetric synapses with flat or pleomorphic vesicles that terminate predominately on dendritic shafts. Synaptic density continues to increase after birth, reaching a plateau of approximately 190/1000 micron2 at the end of the first postnatal month. Throughout postnatal development the proportions of symmetric and asymmetric synapses on the smooth dendritic shafts undergo systematic fluctuations which may reflect the ingrowth of various afferents as well as local cytological differentiation including the formation of cellular compartments.