Cynthia S. Duchala

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.

The toppler mouse: A novel mutant exhibiting loss of Purkinje cells

We describe the genetic and neurological features of toppler, a spontaneous autosomal mutation that appeared in a colony of FVB/N mice and that manifests as severe ataxia appearing at around 12 days of age, worsening with age. The lifespan of affected mice is 8–12 months, with occasional mice living longer. Both homozygous males and females are fertile, and females are able to nurture litters. Histological examination of brain revealed no striking abnormalities other than the loss of cerebellar Purkinje cells. The toppler mutation was mapped to mouse chromosome 8, and to assess whether it was novel or a recurrence of a previously described chromosome 8 mouse mutant, toppler mice were crossed with the nervous and tottering mouse mutants. These studies demonstrate that toppler is a unique mouse mutation. Purkinje cell abnormalities in toppler mice were obvious around postnatal day (P) 14, i.e., toppler Purkinje cells already exhibited abnormal morphology. Staining for calbindin, a calcium binding protein enriched in Purkinje cells, showed altered dendritic morphology. Between P14 and P30, dramatic Purkinje cell loss occurred, although there were differences in the degree of Purkinje cell loss in each lobule. At P30, the surviving Purkinje cells expressed zebrin II. From P30 through 6 months, many of the remaining Purkinje cells gradually degenerated. Purkinje cell loss was analyzed by terminal deoxynucleotidyl transferase‐mediated biotinylated UTP nick end labeling (TUNEL), and Purkinje cells were TUNEL‐positive most abundantly at P21. In addition, Bergmann glia were TUNEL positive at P21, and they expressed activated caspase‐3 at earlier time points. Interestingly, despite the apparent death of some Bergmann glia, there was up‐regulation of glial fibrillary acidic protein, expressed in astrocytes as well as Bergmann glia. Given the changes in both Purkinje cells and glia in toppler cerebellum, this may be a very useful model in which to investigate the developmental interaction of Purkinje cells and Bergmann glia. J. Comp. Neurol. 476:113–129, 2004.

Proteolipid Protein mRNA Stability Is Regulated by Axonal Contact in the Rodent Peripheral Nervous System

Proteolipid protein (PLP) and its alternatively spliced isoform, DM20, are the main intrinsic membrane proteins of compact myelin in the CNS. PLP and DM20 are also expressed by Schwann cells, the myelin-forming cells in the PNS, and are necessary for normal PNS function in humans. We have investigated the expression of PLP in the PNS by examining transgenic mice expressing a LacZ transgene under the control of the PLP promoter. In these animals, myelinating Schwann cells expressed beta-galactosidase more prominently than nonmyelinating Schwann cells. PLP/DM20 mRNA levels, but not those of LacZ mRNA, increased during sciatic nerve development and decreased after axotomy, with resultant Wallerian degeneration. PLP/DM20 transcription rates, in nuclear run off experiments, however, did not increase in developing rat sciatic nerve despite robust increases in PLP/DM20 mRNA levels during the same period. In RNAse protection studies, PLP mRNA levels fell to undetectable levels following nerve transection whereas levels of DM20 were essentially unchanged despite both being transcribed from the same promoter. Finally, cotransfection studies demonstrated that PLP-GFP, but not DM20-GFP mRNA is down-regulated in Schwann cells cultured in the absence of forskolin. Taken together these data demonstrate that steady state levels of PLP mRNA are regulated at a posttranscriptional level in Schwann cells, and that this regulation is mediated by Schwann cell-axonal contact. Since the difference between these two mRNAs is a 105-bp sequence in PLP and not in DM20, this sequence is likely to play a role in the regulation of PLP mRNA.

Oligodendrogenesis is differentially regulated in gray and white matter of jimpy mice

The factors that regulate oligodendrogenesis have been studied extensively in optic nerve, where oligodendrocyte production and myelination quickly follow colonization of the nerve by progenitor cells. In contrast, oligodendrocyte production in the cerebral cortex begins approximately 1 week after progenitor cell colonization and continues for 3–4 weeks. This and other observations raise the possibility that oligodendrogenesis is regulated by different mechanisms in white and gray matter. The present study examined oligodendrocyte production in the developing cerebral cortex of jimpy (jp) and jimpymsd (msd) mice, which exhibit hypomyelination and oligodendrocyte death due to mutations in and toxic accumulations of proteolipid protein, the major structural protein of CNS myelin. Proliferation of oligodendrocyte progenitors and production of myelinating oligodendrocytes was reduced in jp cerebral cortex when compared to wild‐type (wt) and msd mice. The incidence of oligodendrocyte cell death was similar in jp and msd cortex, but total dying oligodendrocytes were greater in msd. We confirm previous reports of increased oligodendrocyte production in white matter of both jp and msd mice. The jp mutation, therefore, reduces oligodendrocyte production in cerebral cortex but not in white matter. These data provide additional evidence that oligodendrogenesis is differentially regulated in white matter and gray matter and implicate PLP/DM20 as a modulator of these differences.

Expression of molecular chaperones and vesicle transport proteins in differentiating oligodendrocytes

The major stages of oligodendrocyte differentiation are defined by the presence or absence of certain myelin‐specific proteins. Events leading to the successful processing of these proteins, such as the folding, assembly, and trafficking of these proteins through the biosynthetic pathway, are largely undefined. In the present study, we have examined both cultured primary oligodendrocytes and immortalized oligodendrocyte cell lines for the presence of molecular chaperones and/or vesicle transport proteins. We find that a select set of these proteins are expressed relatively early in oligodendrocyte differentiation, whereas a characteristically different set of proteins are expressed at later stages of oligodendrocyte differentiation. In other systems, these proteins participate in the folding and assembly of protein complexes, in the prevention of protein aggregation, as well as the trafficking of proteins via vesicles to specific subcellular destinations including the plasma membrane. Some of the chaperones and/or vesicle transport proteins investigated in this study may play a pivotal role in the certain aspect of myelin biogenesis. J. Neurosci. Res. 50:769–780, 1997. © 1997 Wiley‐Liss, Inc.

Expression of a Myelin Proteolipid Protein (Plp)-lacZ Transgene is Reduced in both the CNS and PNS of Plp jp Mice

Jimpy (Plpjp) is an X-linked recessive mutation in mice that causes CNS dysmyelination and early death in affected males. It results from a point mutation in the acceptor splice site of myelin proteolipid protein (Plp) exon 5, producing transcripts that are missing exon 5, with a concomitant shift in the downstream reading frame. Expression of the mutant PLP product in Plpjp males leads to hypomyelination and oligodendrocyte death. Expression of our Plp-lacZ fusion gene, PLP(+)Z, in transgenic mice is an excellent readout for endogenous Plp transcriptional activity. The current studies assess expression of the PLP(+)Z transgene in the Plpjp background. These studies demonstrate that expression of the transgene is decreased in both the central and peripheral nervous systems of affected Plpjp males. Thus, expression of mutated PLP protein downregulates Plp gene activity both in oligodendrocytes, which eventually die, and in Schwann cells, which are apparently unaffected in Plpjp mice.

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.

Expression of the PLP Gene and a PLP-LacZ Transgene in Oligodendrocytes

Myelination is essential for normal nervous system development, and a wide variety of environmental and genetic factors lead to dysmyelination, which is a serious neurobiological problem (Raine, 1984). Central nervous system (CNS) myelin is a differentiation of the oligodendrocyte plasma membrane, which is produced during a relatively narrow time period in the early postnatal rodent brain. This differentiation program can be studied extensively in primary culture since cultured oligodendrocyte progenitors can differentiate and express myelin genes, even in the absence of neurons or astrocytes (Dubois-Dalcq et al., 1986: Campagnoni and Macklin, 1988).