Wendy B. Macklin

An AG→GG transition at a splice site in the myelin proteolipid protein gene in jimpy mice results in the removal of an exon

The myelin proteolipid protein gene was characterized in jimpy mice to identify the specific mutation that produces dysmyelination, oligodendrocyte cell death, and death of the animal by 30 days of age. Exon 5 and flanking intron segments were isolated from jimpy proteolipid protein genomic clones and sequenced. A single nucleotide difference was noted between the normal and jimpy proteolipid protein genes: the conversion of an AG/GT to a GG/GT in the splice acceptor signal preceding exon 5, which apparently destroys the splice signal. Thus, exon 5 of the mouse myelin proteolipid protein gene is skipped during the processing of mRNA, producing a shortened proteolipid protein mRNA.

Myelin proteolipid protein gene expression in Jimpy and JimpyMSD mice

Abstract: Proteolipid protein (PLP) gene expression was studied in the dysmyelinating mouse mutant jimpymsd (jpmsd; myelin synthesis deficient) and compared with that in wild‐type mice and the allelic mutant, jimpy (jp). Southern analyses of genomic DNA from jpmsd mice revealed no major rearrangements of the PLP gene relative to the wild‐type mouse PLP gene. PLP‐specific mRNA levels were significantly reduced in these mutant mice, although both the 3.2‐ and 2.4‐kilobase PLP‐specific mRNAs were seen. Also, no size differences in either PLP or DM20 mRNAs were found by S1 nuclease assays of brain RNA from either jpmsd or wild‐type mice. Both PLP and DM20 protein were detectable at low levels in jpmsd brain homoge‐nates, and these proteins comigrated with PLP and DM20 protein from normal mice. Western analyses showed an altered PLP:DM20 ratio in jpmsd mice relative to wild‐type mice; DM20 levels exceeded PLP levels. It is surprising that a similar pattern of expression was seen in normal mice at <10 days of age: DM20 protein expression preceding PLP expression. Thus, jpmsd mice are capable of synthesizing normal PLP and DM20 protein; however, the PLP gene defect has affected the normal developmental pattern of expression for these two proteins.

Characterization of myelin proteolipid mRNAs in normal and jimpy mice.

A clone specific for the rat myelin proteolipid protein (PLP) was isolated from a cDNA library made in pUC18 from 17-day-old rat brain stem mRNA. This clone corresponded to the carboxyl-terminal third of the PLP-coding region. The clone was used to identify PLP-specific mRNAs in mouse brain and to establish the time course of PLP mRNA expression during mouse brain development. Three PLP-specific mRNAs were seen, approximately 1,500, 2,400, and 3,200 bases in length, of which the largest was the most abundant. During brain development, the maximal period of PLP mRNA expression was from 14 to 25 days of age, and this was a similar time course to that for myelin basic protein mRNA expression. When the jimpy mouse, an X-linked dysmyelination mutant, was studied for PLP mRNA expression, low levels of PLP mRNA were seen which were approximately 5% of wild-type levels at 20 days of age. When jimpy brain RNA was analyzed by Northern blotting, the PLP-specific mRNA was shown to be 100 to 200 bases shorter than the wild-type PLP-specific mRNA. This size difference was seen in the two major PLP mRNAs, and it did not result from a loss of polyadenylation of these mRNAs.

Expression of Cell Surface Markers and Myelin Proteins in Cultured Oligodendrocytes from Neonatal Brain of Rat and Mouse: A Comparative Study (Part 1 of 2)

Dissociated brain cell cultures are a useful model for investigating development and differentiation of oligodendrocytes in vitro. The current studies compare the developmental patterns of expression for oligodendrocyte lineage/myelin markers in both primary and secondary oligodendrocyte cultures derived from mouse and rat neonates. The rat and mouse dissociated brain cell cultures express the same myelin-specific antigens, but mouse oligodendrocytes produce a larger and more elaborate sheet-like membrane than rat oligodendrocytes, and some of the myelin markers (O4, GC, and MBP) show more intense membrane staining in mouse cultures. GD3 appears to be a good oligodendrocyte marker for rat cells, but it is nonspecific in mouse cells. There are fewer oligodendrocytes in mouse cultures, and they appear to require a longer differentiation time than rat oligodendrocytes. These same results are also observed in secondary oligodendrocyte cultures, although in general late myelin markers such as MBP and PLP are expressed at a much lower level in mouse cells than rat cells.

Mutations in the Myelin Proteolipid Protein Gene Alter Oligodendrocyte Gene Expression in Jimpy and Jimpymsd Mice

Abstract: The mouse myelin proteolipid protein (PLP) gene has been studied in normal and jimpymsd mice. Potential upstream regulatory regions of the normal gene have been cloned and mapped, but when these regions were studied in jimpymsd mice by Southern blots, no alterations were observed, relative to the normal gene. To assess whether the low ratio of PLP to DM20 proteins in this mutant reflected an altered PLP/DM20 ratio mRNAs, S1 nuclease analyses were undertaken, which demonstrated that at all ages studied in both jimpy and jimpymsd mice, PLP mRNA was elevated above DM20 mRNA. When exon 3 (the site of the alternative splice signal for DM20 mRNA) of the jimpymsd PLP gene was sequenced, no mutation was identified. The transcription of the PLP gene in normal and mutant animals was studied. The transcription rate increases in normal animals with development, and in very young jimpymsd or jimpy mice, the transcription rate of the PLP gene was close to that of agematched normal animals. However, by 10 days of age, the transcription rate of this gene in both mutants was significantly below that of age‐matched controls. The transcription rate of the myelin basic protein (MBP) gene was also reduced, indicating that expression of both genes is affected by this mutation. In contrast, the transcription rate of the glycerol phosphate dehydrogenase (GPDH) gene, an early marker of oligodendrocytes, is equal to or greater than normal in both mutants. We have confirmed an earlier report of a point mutation in exon 6 of the jimpymsd PLP gene, which converts an alanine to a valine. This mutation apparently alters oligodendrocyte metabolism such that the cell can differentiate to express early oligodendrocyte genes such as GPDH, but it cannot differentiate to a stage where it expresses the PLP and MBP genes at normal high levels.

Regulation of murine myelin proteolipid protein gene expression

To identify putative sequences that direct cell type‐specific expression and/or enhance proteolipid protein (PLP) gene expression, glial or nonglial cells were transfected with various PLP‐luciferase constructs that collectively span the entire mouse PLP‐specific DNA present in a transgene known to direct cell type specificity in transgenic mice. These constructs were transfected into murine oligodendrocyte cell lines that transcribe the PLP gene and, hence, should contain the requisite trans‐acting factors necessary for PLP gene expression. Mouse NIH/3T3 fibroblasts were used as a nonglial model. We have finely mapped the PLP promoter region for transcriptional regulatory elements and demonstrate both positive and negative elements, none of which appear to extinguish expression in nonglial cells. The 5′‐flanking PLP DNA tested did not enhance the basal herpes simplex‐1 virus thymidine kinase (TK) promoter, nor did PLP sequences present in the distal half of intron 1. The 5′ portion of intron 1 did enhance TK promoter activity, suggesting that this region of the gene may contain enhancer elements that modulate PLP gene expression; however, the enhancement did not appear to be cell type‐specific. Intriguingly, a 541 bp region of the intron that significantly enhanced TK promoter activity contains multiple JC virus repeated elements and other elements known to be important in restricting the virus to oligodendrocytes. These results suggest that intron 1 sequences may modulate expression of the PLP gene. J. Neurosci. Res. 50:917–927, 1997.

Transgenic Models for Investigating Oligodendrocyte Differentiation and Myelin Formation

The myelin membrane is a highly specialized extension of the oligodendroglial plasma membrane, which surrounds axons and forms a tightly compacted multilamellar structure1. Myelin has a relatively simple protein composition, with the proteolipid (PLP) and DM20 proteins comprising approximately 50% of adult CNS myelin protein2,3. These proteins are translated from alternatively spliced mRNAs encoded by the PLP gene4,5,6. Thus, a major portion of the oligodendrocyte differentiation program is dedicated to the expression of the PLP gene. Mutations in this gene are devastating, and animals such as the jimpy mouse or the md rat are severely compromised, dying within the first postnatal month. CNS changes have been noted in these animals, at ages prior to the time of oligodendrocyte differentiation7,8,9.10, 11. In earlier studies, we and others demonstrated that the DM20 mRNA and protein appear prior to PLP in the developing nervous system12, 13, 14, 15. In addition, it has now been shown that the DM20 mRNA is expressed in the developing embryonic nervous system16, 17. Thus, a number of studies have suggested that in addition to the production of the most abundant proteins of the myelin membrane, the PLP gene may encode a protein(s), perhaps the DM20 protein, which is expressed in cells in the developing nervous system prior to oligodendrocyte differentiation.

Developmental Expression of the Myelin Proteolipid Protein Gene

Myelinogenesis is a highly regulated developmental process, which is necessary for normal CNS development. Oligodendrocyte differentiation and its subsequent elaboration of a myelin membrane that ensheaths certain axons occur within a very precise time frame — for rodents, from 15 to 25 days of age. Until recently, studies had been restricted to ultrastuctural and biochemical analyses of oligodendrocytes and myelin. Early studies identified the proteolipid proteins (PLP, DM20 protein) and myelin basic proteins (MBP) as two classes of structural proteins which comprise 80% of all proteins found in myelin membranes (Eng et al., 1968; Agrawal et al., 1972; Norton and Poduslo, 1973). Thus during the period of active myelination, the protein synthetic machinery of oligodendrocytes is dedicated largely to the production of these two classes of myelin proteins.

MYELIN PROTEOLIPID PROTEIN EXPRESSION IN NORMAL AND JIMPY MICE

The myelin membrane is a highly specialized extension of the oligo-dendroglial plasma membrane, which surrounds axons and forms a tightly compacted multilamellar structure (Peters et al., 1970). Myelin integrity is essential for normal nervous system function, and understanding the mechanism of myelin formation is important because of the large number of clinical problems observed when normal myelin formation is altered (Adams and Victor, 1970). Oligodendrocyte differentiation and myelin formation have become important developmental problems to investigate at the molecular level for several reasons. Oligodendrocyte differentiation occurs over a narrow time period during brain development, indicating that it is tightly developmentally regulated. Since the myelin membrane has a somewhat simple protein composition, with the proteolipid protein (PLP) and myelin basic protein (MBP) comprising 70–80% of adult CNS myelin protein (Eng et al., 1968; Norton and Poduslo, 1973) a major portion of the oligodendrocyte differentiation program is dedicated to the synthesis of these two groups of proteins.