The expression of myelin protein genes in Schwann cells has been studied in situ hybridization. 35S-UTP-labelled, antisense and sense RNA probes to the major protein Po, myelin basic protein (MBP), myelin-associated glycoprotein (MAG) and proteolipid protein (PLP) were employed with paraffin-embedded sections, teased fibres and dissociated Schwann cells from sciatic nerves of rats. Teased fibres were also prepared from cervical sympathetic trunks. Po mRNA was strongly expressed in the mid-internodal perinuclear area of Schwann cell cytoplasm. The degree of signal appeared to be related to fibre size. MBP mRNA showed a diffuse pattern along the Schwann cell internode with a marked increase in grains at the paranodal cytoplasm, particularly in larger fibres. This distribution suggests that the paranodal area is a major site of insertion of MBP into myelin membrane. The expression of MAG and PLP mRNA was markedly lower than Po and MBP. Both mRNAs were localized in the perinuclear cytoplasm and showed a dependence on fibre size. No significant signal was present in Schwann cells associated with unmyelinated axons. In addition to providing data on the cellular expression of myelin protein genes, these studies have shown that teased fibres are invaluable in allowing the localization of low abundance mRNAs.

Rumpshaker (rsh) is an X-linked mutation causing hypomyelination of the CNS of mice and has recently been identified as an allele of jimpy (jp). The mutation (known as jprsh) differs in several respects from other X-linked myelin mutants, including jp, in that mice have normal longevity, oligodendrocyte numbers are not decreased, and cell death is not a feature. Myelin sheaths are deficient in immunostainable PLP protein. The present study examines the developmental expression of the major myelin protein genes and translatability of PLP and MBP mRNA. Differences between the spinal cord and brain of mutants are evident in that mRNA levels are more markedly decreased in the brain. Protein levels are severely reduced in both locations and to a proportionately greater extent than the mRNA, particularly in the spinal cord where PLP RNA and protein are approximately 80% and 10-20%, respectively, of age-matched wild type mice. DM-20 protein, the other major product of the PLP gene, is disproportionately expressed in rumpshaker as is a 10 kDa proteolipid. In vitro translation studies indicate a marked decrease in PLP translation products from mutant RNA. There is no deficiency in the number of PLP mRNA-expressing oligodendrocytes although the abundance per cell is reduced. The data suggest that the phenotypic effects of the mutation may be associated with reduced translation of major myelin proteins, in particular PLP and its incorporation into compact myelin. However, the mutation is compatible with survival of oligodendrocytes and their differentiation to the stage of expressing PLP/DM-20 mRNA.

The expression of many myelin-specific molecules in Schwann cells is profoundly decreased following denervation. This study examines the early reexpression of myelin protein genes associated with reinnervation. Following sciatic nerve crush, the distal, regenerated nerve was divided into appropriate (2.5 or 5 mm) consecutive lengths in which gene expression was monitored using Northern blotting, in situ hybridization, and immunostaining. The spatial separation of the distal axon tip and the more proximally located Schwann cells showing initial upregulation of P0 mRNA was constant over the period of 5-13 days after crush at approximately 3-4 mm in fixed, processed material. Axons associated with Schwann cells showing the initial upregulation were completely or partially enveloped in Schwann cell cytoplasm, with very few having any degree of ensheathment. It is probable that only a limited axon-Schwann cell contact is required for induction of the myelin protein genes. Myelin-associated glycoprotein mRNA was upregulated prior to those for P0 and myelin basic protein which had similar time courses. Reexpression of galactocerebroside also preceded that for P0 mRNA. Signal abundance for all myelin proteins decreased in a proximal to distal direction from the crush site, and with time the "wave" of upregulation moved distally down the nerve. In the more proximal, remyelinating zones, the signal intensity exceeded that of the contralateral normal nerve. Signal intensity also varied considerably between adjacent, expressing Schwann cells. The data provide further evidence of the strong temporospatial relationship between axons and the regulation of myelin protein genes in Schwann cells.

The effect of optic nerve transection on myelin protein gene expression was studied in rats following axotomy at two ages: during active myelination (17 days of age) and after peak expression of the genes (35 days of age). mRNA levels for proteolipid protein, myelin basic protein and myelin-associated glycoprotein were assessed by northern and dot blotting and by in situ hybridization using tissue sections and cultured individual oligodendrocytes. Transection at 17 days caused down-regulation of mRNAs for proteolipid protein, myelin basic protein and myelin-associated glycoprotein by 5 days after axotomy with an increase in GFAP mRNA. A more protracted change followed axotomy at 35 days of age. The abundance of mRNAs for proteolipid protein and myelin basic protein was significantly reduced by 28 days after transection in the affected nerve. Quantification of proteolipid protein mRNA expression in individual oligodendrocytes confirmed the down-regulation. However, in contrast to the effects on the major myelin proteins, the abundance of myelin-associated glycoprotein mRNA increased in the affected nerve for at least the initial month after lesioning at 35 days. The results show that optic nerve transection has significant effects on myelin protein mRNA expression in oligodendrocytes of optic nerve. However, the changes in myelin protein gene activity are relatively small and more protracted than those seen in Schwann cells after peripheral nerve section. Because axotomy also causes marked changes in the glial population of the optic nerve it is not possible unequivocally to ascribe the alteration in gene expression to loss of axons. However, the data may provide evidence that axons do influence myelin protein genes in oligodendrocytes and are necessary for them to develop their full expression.

This study examines the expression of the major myelin protein gene P0 in cultured Schwann cells, grown on their own or in association with neurons. Many freshly dissociated Schwann cells from actively myelinating nerves express Po mRNA in high abundance. If neurons are not present, signal intensity falls markedly with time so that by 7 days in culture only a basal expression is evident which is negligible compared to the level in vivo. Dorsal root ganglia from embryo day 16 (E16) rats contain no significant levels of Po mRNA but when grown in full myelinating medium (containing serum and embryo extract) increasing expression is seen from 4 to 5 days onward even though myelination does not occur until after the second week. In this intervening period the intensity of P0 mRNA expression is lower than that found in the actively myelinating cell. Neurons from sympathetic ganglia are also capable of inducing P0 mRNA expression. Schwann cells in dorsal root ganglia explants grown in serum-free defined medium do not assemble a basal lamina and will not wrap or myelinate axons. Nevertheless P0 mRNA, but not protein, is expressed in levels similar to those found in full myelinating medium prior to myelination. Such Schwann cells also exhibit galactocerebroside and the sulphatide recognised by the 04 antibody. It appears that in defined medium or in myelinating medium prior to myelination axonal signals can induce P0 mRNA expression to a certain degree. However, full up-regulation is usually associated with the rapid membrane expansion accompanying myelination. Whether this augmented up-regulation is due to further axonal signalling or events in the Schwann cell is unknown, but the results suggest that P0 expression can be regulated at several stages of synthesis.