The myelin proteolipid protein (PLP) gene (i.e., the PLP/DM20 gene) has been of some interest because of its role in certain human demyelinating diseases, such as Pelizaeus-Merzbacher disease. A substantial amount of evidence, including neuronal pathology in knock-out and transgenic animals, suggests the gene also has functions unrelated to myelin structure, but the products of the gene responsible for these putative functions have not yet been identified. Here we report the identification of a new exon of the PLP/DM20 gene and at least two new products of the gene that contain this exon. The new exon, located between exons 1 and 2, is spliced into PLP and DM20 mRNAs creating a new translation initiation site that generates PLP and DM20 proteins with a 12 amino acid leader sequence. This leader sequence appears to target these proteins to a different cellular compartment within the cell bodies of oligodendrocytes and away from the myelin membranes. Furthermore, these new products are also expressed in a number of neuronal populations within the postnatal mouse brain, including the cerebellum, hippocampus, and olfactory system. We term these products somal-restricted PLP and DM20 proteins to distinguish them from the classic PLP and DM20 proteolipids. They represent putative candidates for some of the nonmyelin-related functions of the PLP/DM20 gene.

Expression of the dopamine D3 receptor (D3r) was found in primary mixed glial cultures from newborn brain and in the corpus callosum in vivo during the peak of myelination. Expression of the D3r mRNA, but not D2r mRNA, was detected as early as 5 d in vitro (DIV) by RT-PCR. Immunoblot studies revealed D3r protein was also expressed in the cultures. Double immunofluorescence analysis for the D3r and for surface markers of specific stages of oligodendrocyte development indicated that D3r expression occurred in precursors and in immature oligodendrocytes but not in mature oligodendrocytes (i.e. , A2B5(+) 007(-) 01(-) and A2B5(+) 007(+) 01(-) cells but not A2B5(-) 007(+) 01(+) cells). Confocal microscopic analysis indicated that D3r was associated with cell bodies and cell membranes but not with the processes emanating from cell somas. Immunohistochemistry of brain sections revealed the presence of D3r in some oligodendrocytes located mainly within the genu and radiato of the corpus callosum during the active period of myelination. Treatment of cultures with 20 microM quinpirole led to decreased numbers of O1(+) oligodendrocytes possessing myelin-like membranes as well as an increase in the number of precursors in 14 DIV cultures. This effect was prevented by the dopamine antagonist haloperidol. These results show that the D3r expression is not restricted to neurons but it is also expressed in differentiating oligodendrocytes before terminal maturation. It also suggests that dopamine or some other D3r ligand may play a role in oligodendrocyte differentiation and/or the formation of myelin by mature oligodendrocytes.

Activity of the Notch1 gene is known to inhibit oligodendrocyte (OL) differentiation in vitro. We tested the hypothesis that the Notch1 pathway regulates in vivo myelin formation, by examining brain myelination of Notch1 receptor null heterozygotes mutant animals (Notch1(+/-)). We show that a deficiency in Notch1 expression leads to increased abundance of products of specific myelin genes in myelinated areas of the brain during the first 2 weeks of postnatal life. We observed increased numbers of myelinated axons in optic nerves and the presence of myelinated fibers in the molecular layer (ML) of the Notch1(+/-) cerebella. These findings were accompanied by up-regulation of Mash1 and down-regulation of Hes5 proteins. In addition, we found expression of Jagged1, one of the Notch1 activators, in unmyelinated axons of the cerebellar ML during normal development. Our findings indicate that the Jagged/Notch signaling pathway might actively participate in the regulation of myelination during central nervous system development and suggest that certain neuronal populations might regulate whether their axons are myelinated by the expression of inhibitory signals such as Jagged1.

Specific membrane microdomains (including lipid rafts) exist in myelin but have not been fully characterized. Myelin basic protein (MBP) maintains the compactness of the myelin sheath and is highly posttranslationally modified. Thus, it has been suggested that MBP might also have other functions, e.g., in signal transduction. Here, the distribution of MBP and its modified forms was studied, spatially and temporally, by detailed characterization of membrane microdomains from developing and mature bovine myelin. Myelin membranes were extracted with three different detergents (Brij 96V, CHAPS, or Triton X-100) at 4 degrees C. The detergent-resistant membranes (DRMs), representing coalesced lipid rafts, were isolated as low-buoyant-density fractions on a sucrose density gradient. These myelin rafts were disrupted when cholesterol was depleted with methyl-beta-cyclodextrin. The use of CHAPS detergent led to enrichment of several myelin proteins, including phospho-Thr97-MBP, in the DRMs from mature myelin. Citrullinated and methylated MBP remained in "nonraft" microdomains. In contrast, the DRMs from early myelin were enriched in Golli-MBP, Fyn, Lyn, and CNP. The localization of various proteins in DRMs was further supported by the colocalization of these lipid raft components in cultured mouse oligodendrocytes. Thus, there is a developmental regulation of posttranslationally modified forms of MBP into specific membrane microdomains.