Abstract:We have conditionally immortalized oligodendl-ocytes isolated from normal and shiverer primary mouse brain cultures through the use of the retroviral vector ZIPSVtsA58. This vector encodes an immortalizing thermolabile simian virus 40 large T antigen (Tag) and allows for clonal selection by conferring neomycin (C4 18) resistance. We isolated 14 shiverer and 10 normal lines that expressed the early oligodendrocyte marker 2',3'-cyclic nucleotide 3'-phosphodiesterase mRNA. These cell lines grew continuously at the permissive temperature (34°C) and displayed Tag nuclear immunostaining. On shifting to nonpermissive temperatures (39"C), the cells showed rapid arrested cell growth and loss of Tag staining. One line (N20.1) engineered from normal oligodendrocytes also expressed myelin basic protein (MBP) and proteolipid protein (PLP) mRNAs, genes normally expressed by mature, differentiated oligodendrocytes. No differences in any of the myelin-specific protein mRNA levels were observed in N20.1 cells grown at 39°C for >9 days compared with cells maintained at 34°C. Immunocytochemical staining revealed N20. I cells to be positive for the oligodendrocyte surface markers-galactocerebroside, A007, and AZB5. However, MBP and PLP polypeptides could not be detected by western blot or immunocytochemical staining at either the permissive or nonpermissive temperature. Cell-free protein synthesis experiments indicated that the MBP m R N A s isolated from N20.1 cells were translatable and directed the synthesis of the 17-, 1 8 5 , and 2 1.5-kDa MBP isoforms. Analysis of the PLP/DM20 gene splice products by polymerase chain reaction indicated that the expression of DM20 mRNA predominated over that of PLP mRNA in this cell line. Because the cell line expressed the MBP and PLP genes, it represents a "mature" oligodendrocyte, but the splicing patterns of these genes indicate that it is at an early stage of "maturation." This cell line has now been passaged >40 times with fidelity of phenotype and genotype.
Interleukin‐6 (IL‐6) was produced by the spontaneously immortal Schwann cell clone, iSC, when cocultured with PC12 cells. The iSC cell‐derived IL‐6 in coculture conditioned media caused the neuronal differentiation of naive PC12 cells and this bioactivity was neutralized by preincubation of conditioned media with antisera to IL‐6. Cocultured iSC transcribe IL‐6 message as confirmed by northern analysis. Stimuli that induce IL‐6 production in the hematopoietic lineage induced transcription and production of IL‐6 by iSC cells. Lipopolysaccharide, tumor necrosis factor‐α, IL‐1α, IL‐6, and serum withdrawal induced iSC cell IL‐6 mRNA. The kinetics of IL‐6 production was confirmed in the mouse IL‐6‐dependent B9 bioassay and that activity could be neutralized with antisera to IL‐6. Expression of both the IL‐6 receptor and the gp130 signal transduction component by iSC as determined by northern analysis suggests an autocrine regulatory mechanism. The observed iSC production of IL‐6 in vitro led to an investigation of the sciatic nerve crush model of Schwann cell activation in vivo. In the initial 12 h after crush injury, IL‐6 message is induced. IL‐6 mRNA expression was highest distal to the crush injury. Our in vitro data demonstrate that iSC cells produce IL‐6 in response to PC12 cell coculture and to stimuli that induce IL‐6 production in the hematopoietic lineage. The induction of IL‐6 message distal to a crush injury suggests another mechanism by which Schwann cells facilitate peripheral nerve regeneration.
Expression of mRNAs for the two major myelin proteins, myelin basic protein (MBP) and proteolipid protein (PLP), was examined in a number of regions of the developing mouse brain using in situ hybridization. In general, MBP and PLP mRNAs were observed to be coexpressed during ontogeny, prior to the histological appearance of myelin. Expression of both mRNAs was detected as early as 6 hours postpartum in the medulla oblongata and, with development, expression of these mRNAs progressed in a caudal to rostral direction. Peak expression occurred at approximately postnatal day 20 in most regions examined, regardless of time of onset of expression. As myelination proceeded, two different labeling patterns were observed with the PLP and MBP 35S-labeled cDNA probes. In the earliest stages of myelinogenesis MBP mRNA labeling was restricted to oligodendrocyte cell bodies, but shortly after the gene began to be expressed the labeling became more diffuse. In contrast, PLP mRNA labeling remained over or surrounding oligodendrocyte cell bodies at all stages of myelinogenesis. These two distinctly different patterns of labeling are consistent with alternative intracellular trafficking of MBP and PLP mRNAs, in which PLP mRNAs remain associated with ribosomes within the cell soma and MBP mRNAs move from the cell soma to the oligodendrocyte processes at a specific stage early in myelinogenesis. However, there appeared to be a clear time lag between the onset of MBP mRNA expression and the movement of ribosomes carrying MBP mRNAs into the oligodendrocyte processes. Additionally, the in situ hybridization studies revealed a population of unidentified cells residing in cortical molecular layers that express PLP mRNA in the absence of MBP mRNA.
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