David R. Colman

Protein zero of peripheral nerve myelin: Biosynthesis, membrane insertion, and evidence for homotypic interaction

Protein zero (P0), an integral membrane glycoprotein synthesized by Schwann cells, is the major glycoprotein of peripheral nerve myelin. The predicted disposition of P0 with respect to the membrane bilayer postulates the existence of extracellular and intracellular domains, that mediate compaction of the myelin lamellae. We used in vitro translations programmed with sciatic nerve mRNA and cells transfected with a P0 cDNA construct to study the biosynthesis and topology of P0 in the bilayer. The behavior of P0 at the cell surface, when expressed under physiological conditions, was also examined. We have verified the topological predictions of an earlier model, derived from analysis of a P0 cDNA, and provide evidence that the extracellular domain of P0 mediates homotypically cell-cell interactions in the transfectants.

In situ hybridization with digoxigenin-labeled probes: sensitive and reliable detection method applied to myelinating rat brain.

SummaryA method for in situ hybridization of digoxigenin-labeled cDNA and cRNA probes to myelin protein mRNA is described. This technique has dual advantages of high structural resolution and high sensitivity and avoids problems associated with handling of radioactive materials. Furthermore, it can be readily combined in double labeling with immunocytochemical protein detection. We have used this technique to detect and locate mRNA for myelin basic protein (MBP), proteolipid protein (PLP), 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) and myelin-associated glycoprotein (MAG) in oligodendrocytes of 7-day-old and adult rat brains. PLP and MAG mRNA were restricted to the perinuclear cytoplasm, whereas MBP and CNPase mRNA was additionally present in peripheral oligodendrocyte processes.

DM-20 mRNA Is Expressed During the Embryonic Development of the Nervous System of the Mouse

Abstract: We used both the polymerase chain reaction (PCR) and in situ hybridization to search for the presence of proteolipid protein (PLP) gene transcripts in the developing mouse. Total brain RNA extracted from 13‐19‐day embryos, analyzed by PCR, demonstrated the presence of a single transcript that was unambiguously identified with the DM‐20 mRNA. RNA samples from postnatal day 2 animals also showed a signal corresponding to the PLP transcript, in addition to the DM‐20 message. By in situ hybridization of 10‐day embryos using a DM‐20 antisense cRNA probe, we showed that the localization of the DM‐20 message was restricted to the diencephalic basal plate. On the same embryo sections, in addition to the brain localization, an intense hybridizing signal was also detected in the trigeminal and spinal ganglia, the vagal glossopharyngeal ganglion, and the sympathetic ganglion chain. The demonstration of transcription of the PLP gene, long before the beginning of the myelination process, suggests that in addition to a structural function in myelin compaction, some of the products of the PLP gene (DM‐20) may have a role during the compartmentalization and differentiation of the neural tube.

Mechanisms of cell adhesion in the nervous system: role of the immunoglobulin gene superfamily.

The amino acid sequences of a large number of the cell surface proteins known to mediate cell-cell interactions in the nervous system have recently been reported. Many of these proteins are members of the immunoglobulin gene superfamily and have remarkably similar structures and amino acid sequences in their extracellular segments. We have termed this family of immunoglobulin-related cell adhesion molecules the Ig-CAMs. In this article, we review the structural features of the Ig-CAMs, discuss how these features may relate to the role of these proteins in mediating cell adhesion and cell signaling events, and finally consider the evolutionary origins of this family of proteins.

The DM20 Protein of Myelin: Intracellular and Surface Expression Patterns in Transfectants

Abstract: DM20 is an abundant CNS myelin‐specific protein whose role in myelinogenesis is unknown. We have cloned the DM20 cDNA from adult mouse brain total RNA using the polymerase chain reaction and expressed it in HeLa cells. DM20, detected by immunofluorescence in stable transfectants, is present in some cells in large, intensely fluorescent intracellular clumps that probably represent elements of the rough endoplasmic reticulum and Golgi apparatus. Frequently, intense DM20 fluorescence could be detected at the plasma membrane. These findings are consistent with previous studies demonstrating that an intracellular “pool” of DM20 and its larger isoform, proteolipid protein, exists and that a substantial lag occurs between synthesis and insertion of these proteins into the expanding myelin membrane. Permanent DM20 expressors in contact with one another do not display any ultrastructural rearrangements at regions of cell–cell contact, in contrast to what we have previously reported for P0, a PNS‐specific protein shown to mediate adhesion of the extracellular faces of the Schwann cell during PNS myelinogenesis. We believe that these results indicate that if DM20 is indeed an adhesion molecule, this property is likely to be significantly more subtle than P0‐mediated adhesion.

Chromosomal Location of the Mouse Gene that Encodes the Myelin-Associated Glycoproteins

The two myelin‐associated glycoprotein (MAG) species, designated large MAG (L‐MAG) and small MAG (S‐MAG), are believed to be generated by differential splicing from a single RNA transcript. We have now defined the genetic locus encoding the two MAG proteins in the mouse. Analysis of a panel of interspecies somatic cell hybrids indicated that all MAG coding sequences reside on chromosome 7. Following the inheritance of a restriction fragment length polymorphism associated with MAG coding sequences allowed the locus to be positioned 0.5 centimorgans from the locus Abpa (androgen binding protein α) on proximal chromosome 7. These data strongly support the hypothesis that a single gene encodes the two MAG proteins, and we propose the name Mag for this locus. This localization places Mag in close proximity to the neurological mutant locus qv (quivering) and raises the possibility of a functional relationship or identity between Mag and qv. However, an analysis of the MAG gene, its RNA transcripts, and its protein products revealed no abnormalities in homozygous qv mutant mice, suggesting that this chromosomal linkage is not etiologically significant.

Distribution of Myelin Basic Protein and P2 mRNAs in Rabbit Spinal Cord Oligodendrocytes

Abstract: Myelin basic protein (MBP) and P2 protein are small positively charged proteins found in oligodendrocytes of rabbit spinal cord. Both proteins become incorporated into compact myelin. We have begun investigations into the mechanisms by which MBP and P2 become incorporated into the myelin membrane. We find that P2, like the MBPs, is synthesized on free polysomes in rabbit spinal cord. Cell fractionation experiments reveal that rabbit MBP mRNAs are preferentially segregated to the peripheral myelinating regions whereas P2 mRNAs are predominantly localized within the perikaryon of the cell. In vitro synthesized rabbit MBP readily associates with membranes added to translation mixtures, whereas P2 protein does not. It is possible that P2 requires a “receptor” molecule, perhaps a membrane‐anchored protein, for association with the cytoplasmic face of the myelin membrane.

Biosynthesis of the Myelin 2′,3′‐Cyclic Nucleotide 3′‐Phosphodiesterases

We have investigated the site of synthesis of the 2′,3′‐cyclic nucleotide 3′‐phosphodiesterases (CNPs I and II) in rat brain. Rapid kinetics of incorporation of CNPs into oligodendrocyte plasma membrane in the intact brain are consistent with their synthesis on free polysomes. This hypothesis was confirmed by the translation in vitro of RNA isolated from free and bound polysomes, respectively. Unlike myelin basic protein (MBP) mRNAs, CNP mRNAs are not enriched in a myelin‐associated pool of RNA. MBPs, but not CNPs, were found to readily associate in vitro with membrane vesicles derived from rough endoplasmic reticulum. The avidity of MBPs in binding to membranes is probably related to the previously observed spatial segregation of MBP mRNAs into actively myelinating cellular processes of the oligodendrocyte. Such a segregation would ensure that newly synthesized MBPs are immediately incorporated into myelin. In contrast, the CNPs probably associate with the cytoplasmic surface of the oligodendrocyte plasma membrane through interaction with a membrane‐bound receptor.

Functional properties of adhesion molecules in myelin formation.

Abstract Several adhesion/recognition molecules appear to play distinct roles in the inductive events leading to the initial ensheathment of axons by myelinating cells, and in the subsequent formation of compact myelin.

Physiologic properties of myelin proteins revealed by their expression in nonglial cells.

The transfection paradigm described herein can be used to investigate the functional properties of individual nervous system proteins in ways that have not been explored before. In particular, observations on the structural proteins of myelin are being made that have already yielded certain unique insights into the physiologic properties of these polypeptides. The ease with which site-directed mutagenesis procedures can be applied to these systems should eventually enable us to define with great precision the functional domains within each myelin protein.