Plp Gene Expression Research Articles

Neuronal expression of the proteolipid protein gene in the medulla of the mouse

The proteolipid protein (PLP) gene (Plp) encodes the major myelin proteins, PLP and DM20. Expression of Plp occurs predominantly in oligodendrocytes, but evidence is accumulating that this gene is also expressed in neurons. In earlier studies, we demonstrated that myelin-deficient (MD) rats, which carry a mutation in the Plp gene, exhibit lethal hypoxic ventilatory depression. Furthermore, we found that, in the MD rat, PLP accumulated in neuronal cell bodies in the medulla oblongata. In the current study, we sought to determine which neurons expressed the Plp gene in the medulla oblongata and whether Plp gene expression changed in neurons with maturation. A transgenic mouse expressing the Plp promoter driving expression of enhanced green fluorescent protein (Plp-EGFP) was used to identify neurons expressing this gene. Plp expression in neurons was confirmed by immunostaining EGFP-positive cells for NeuN and by in situ hybridization for PLP mRNA. The numbers of neurons expressing Plp-EGFP and their distribution increased between P5 and P10 in the medulla. Immunostaining for surface receptors and classes of neurons expressing Plp-EGFP revealed that Plp gene expression in brainstem neurons was restricted to neurons expressing specific ligand-gated channels and biosynthetic enzymes, including glutamatergic NMDA receptors, GABA(A) receptors, and ChAT in defined areas of the medulla. Plp gene expression was rarely found in interneurons expressing GABA and was never found in AMPA receptor- or tyrosine hydroxylase-expressing neurons. Thus, Plp expression in the mouse caudal medulla was found to be developmentally regulated and restricted to specific groups of neurons.

Characterization of an intronic enhancer that regulates myelin proteolipid protein (Plp) gene expression in oligodendrocytes

The myelin proteolipid protein (Plp) gene is expressed in oligodendrocytes and encodes the most abundant protein (approximately 50%) present in mature myelin from the central nervous system (CNS). Plp gene activity is low to nonexistent early in development but sharply increases, concurrently with the active myelination period of CNS development. Work from our laboratory suggests that the temporal regulation of Plp gene expression in mice is mediated by a positive regulatory element located within Plp intron 1 DNA. We have termed this regulatory element/region ASE (for antisilencer/enhancer). The ASE is situated approximately 1 kb downstream of exon 1 DNA and encompasses nearly 100 bp. To understand the mechanisms by which the ASE augments Plp gene expression in oligodendrocytes, Plp-lacZ constructs were generated and transfected into a mouse oligodendroglial cell line (N20.1). Results presented here demonstrate that upstream regulatory elements in the Plp promoter/5'-flanking DNA are not required for ASE activity; the ASE worked perfectly well when the thymidine kinase (TK) promoter was substituted for the Plp promoter. However, the relative location of the ASE appears to be important. When placed upstream of 2.4 kb of Plp 5'-flanking DNA, or downstream of the lacZ expression cassette, the ASE was no longer effective. Thus, the ASE might have to be in the context of the intron in order to function. To begin to identify the crucial nucleotides within the ASE, orthologous sequences from rat, human, cow, and pig Plp genes were swapped for the mouse sequence. Results presented here demonstrate that the orthologous sequence from rat can substitute for the mouse ASE, unlike those from human, cow, or pig.

Where, when and how much: regulation of myelin proteolipid protein gene expression.

The myelin proteolipid protein (PLP) gene ( Plp) encodes the most abundant protein found in myelin from the central nervous system (CNS). Expression of the gene is regulated in a spatiotemporal manner with maximal levels of expression occurring in oligodendrocytes during the active myelination period of CNS development, although other cell types in the CNS as well as in the periphery can express the gene to a much lower degree. In oligodendrocytes, Plp gene expression is tightly regulated. Underexpression or overexpression of the gene has been shown to have adverse effects in humans and other vertebrates. In light of this strict control, this review provides an overview of the current knowledge of Plp gene regulation.

Myelin proteolipid protein (Plp) intron 1 DNA is required to temporally regulate Plp gene expression in the brain.

The myelin proteolipid protein (Plp) gene encodes the most abundant protein found in mature CNS myelin. Expression of the gene is regulated spatiotemporally, with maximal expression occurring in oligodendrocytes during the myelination period of CNS development. Plp gene expression is tightly controlled. Misregulation of the gene in humans can result in the dysmyelinating disorder Pelizaeus-Merzbacher disease, and in transgenic mice carrying a null mutation or extra copies of the gene can result in a variety of conditions, from late onset demyelination and axonopathy, to severe early onset dysmyelination. In this study we have examined the effects of Plp intron 1 DNA in mediating proper developmental expression of Plp-lacZ fusion genes in transgenic mice. Our results reveal the importance of Plp intron 1 sequences in instigating the expected surge in Plp-lacZ gene activity during (and following) the active myelination period of brain development. Transgene expression was also detected in the testis (Leydig cells), however, the presence or absence of Plp intron 1 sequences had no effect on the temporal profile in the testis. Surprisingly, expression of the transgene missing Plp intron 1 DNA was always higher in the testis, as compared to the brain, in all of the transgenic lines generated.

Repression of myelin proteolipid protein gene expression is mediated through both general and cell type-specific negative regulatory elements in nonexpressing cells.

The myelin proteolipid protein gene (Plp ) is expressed primarily in oligodendrocytes. Yet how the gene remains repressed in nonexpressing cells has not been defined, and potentially could cause adverse effects in an organism if the mechanism for repression was impaired. Previous studies suggest that the first intron contains element(s), which suppress expression in nonexpressing cells, although the identity of these elements within the 8 kb intron was not characterized. Here we report the localization of multiple negative regulatory elements that repress Plp gene expression in nonexpressing cells (+/+ Li). Two of these elements (regions) correspond to those used by Plp expressing cells (N20.1), whilst another acts in a cell type-specific manner (i.e. operational in +/+ Li liver cells, but not N20.1 cells). By gel-shift and DNase I footprinting analyses, the factor(s) that bind to the cell type-specific negative regulatory region appear to be far more abundant in +/+ Li cells than in N20.1 cells. Thus, Plp gene repression is mediated through the combinatorial action of both "general" and cell type-specific negative regulatory elements. Additionally, repression in +/+ Li cells cannot be overcome via an antisilencer/enhancer element, which previously has been shown to function in N20.1 cells.

Myelin proteolipid protein (Plp) intron 1 DNA is required to temporally regulate Plp gene expression in the brain

The myelin proteolipid protein (Plp) gene encodes the most abundant protein found in mature CNS myelin. Expression of the gene is regulated spatiotemporally, with maximal expression occurring in oligodendrocytes during the myelination period of CNS development. Plp gene expression is tightly controlled. Misregulation of the gene in humans can result in the dysmyelinating disorder Pelizaeus–Merzbacher disease, and in transgenic mice carrying a null mutation or extra copies of the gene can result in a variety of conditions, from late onset demyelination and axonopathy, to severe early onset dysmyelination. Previous transfection studies suggest that the first intron contains elements that are vital for directing Plp gene activity, spatiotemporally. Here we verify, in vivo, the importance of these regulatory elements by the use of a Plp‐lacZ transgene [PLP(–)Z], which does not contain any Plp intron 1 sequence. The developmental profile of PLP(–)Z expression in transgenic mice is flat throughout the active myelination period of brain development, whereas mice which harbor a related transgene [PLP(+)Z] that contains the first intron, demonstrate the expected surge in oligodendroglial expression during this period. Although lacZ expression was extremely low in the brains of PLP(–)Z mice (and could not be detected in oligodendrocytes by X‐gal staining), expression in the testes was far more abundant and restricted to Leydig cells. Similar levels of lacZ expression were detected in the testes of PLP(+)Z mice. Thus, intron 1 DNA contains regulatory sequences critical for Plp gene expression in oligodendrocytes, but may not be required for expression in other cell types.

Antisilencing: myelin proteolipid protein gene expression in oligodendrocytes is regulated via derepression.

Antisilencer or antirepressor elements have been described, thus far, for only a few eukaryotic genes and were identified by their ability not to augment gene expression per se but to override repression mediated via negative transcription regulatory elements. Here we report the first case of antisilencing for a neural-specific gene, the myelin proteolipid protein (PLP) gene (Plp). PLP is the most abundant protein found in CNS myelin. The protein is synthesized in oligodendrocytes, and its expression is regulated developmentally. Previously we have shown that a PLP-lacZ transgene (which includes the entire sequence for Plp intron 1) is regulated in mice, in a manner consistent with the spatial and temporal expression of the endogenous Plp gene. In the present report, we demonstrate by transfection analyses, using various PLP-lacZ deletion constructs, that Plp intron 1 DNA contains multiple elements that collectively regulate Plp gene expression in oligodendrocytes. One of these regulatory elements functions as an antisilencer element, which acts to override repression mediated by at least two negative regulatory elements located elsewhere within Plp intron 1 DNA. The mechanism for antisilencing appears to be complex as the intragenic region that mediates this function binds multiple nuclear factors specifically.

Late-onset neurodegeneration in mice with increased dosage of the proteolipid protein gene.

Mutations of the proteolipid protein (Plp) gene cause a generalized central nervous system (CNS) myelin deficit in Pelizaeus-Merzbacher disease of man and various tremor syndromes in animal models. X-linked spastic paraplegia is also due to Plp gene mutations but has a different clinical profile and more restricted pathology involving specific tracts and regions. We have shown previously that PLP overexpression in mice homozygous for a Plp transgene results in premature arrest of CNS myelination and premature death. Here, we demonstrate that a low-level increase in Plp gene expression in transgenic mice causes significant axonal degeneration and demyelination with predilection for specific tracts. Following normal motor development, aged mice develop progressive myelin loss, axonal swellings with resultant Wallerian degeneration, and marked vacuolation of the neuropil associated with ataxia, tremor, and seizures. The age of onset and severity of the phenotype is a function of Plp gene dosage. The corticospinal tracts, optic nerve, fasciculus gracilis cerebellum, and brainstem are particularly involved. Although oligodendrocyte cell bodies show little abnormality, their inner adaxonal tongue is often abnormal, suggesting a perturbation of the axon/glial interface that may underlie the axonal changes. We conclude that abnormal expression of an oligodendrocyte-specific gene can cause axonal damage, a finding that is relevant to the pathogenesis of PLP-associated disorders and probably to other myelin-related diseases.

Demyelination induced by protein kinase C-activating tumor promoters in aggregating brain cell cultures

The plasticity of mature oligodendrocytes was studied in aggregating brain cell cultures at the period of maximal expression of myelin marker proteins. The protein kinase C (PKC)-activating tumor promoters mezerein and phorbol 12-myristate 13-acetate (PMA), but not the inactive phorbol ester analog 4alpha-PMA, caused a pronounced decrease of myelin basic protein (MBP) content and 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) activity. In contrast, myelin/oligodendrocyte protein (MOG) content was affected relatively little. Northern blot analyses showed a rapid reduction of MBP and PLP gene expression induced by mezerein, and both morphological and biochemical findings indicate a drastic loss of compact myelin. During the acute phase of demyelination, only a relatively small increase in cell death was perceptible by in situ end labeling and in situ nick translation. Basic fibroblast growth factor (bFGF) also reduced the levels of the oligodendroglial differentiation markers and enhanced the demyelinating effects of the tumor promoters. The present results suggest that PKC activation resulted in severe demyelination and partial loss of the oligodendrocyte-differentiated phenotype.

Evidence that some oligodendrocyte progenitors in the developing optic pathway express the plp gene.

DM-20, a product of the proteolipid protein (plp) gene, has been demonstrated in the spinal cord of the mouse embryo as early as embryonic day 12 (E12) in certain cells, some of which are identifiable as oligodendrocyte progenitors. The present work uses optic pathways of rat and mouse as well-characterized systems for the study of gliogenesis. plp gene expression was monitored with a combination of reverse transcriptase polymerase chain reaction, in situ hybridization, and immunostaining with antibodies to different PLP peptide sequences, combined with O-2A lineage markers. In tissue sections, hybridizing cells were detected initially in the proximal optic tracts between E18 and birth and thereafter progressively in the chiasm and optic nerves. Small unbranched cells expressing DM-20 but not myelin basic protein (MBP) and probably representing progenitors were detectable by immunostaining in similar locations. With increasing postnatal ages, cells representing maturing oligodendrocytes which co-label for PLP and MBP are present in the optic pathways. In vitro analysis of freshly dissociated cells from premyelinated optic nerve demonstrated that the plp gene is expressed in some O-2A progenitor cells as well as mature oligodendrocytes. We also present evidence that increase in expression of the plp gene along the O-2A lineage differentiation is not progressive but that downregulation at the proligodendroblast (O4+/O1-) stage probably occurs. We suggest that progenitors express the dm-20 isoform while oligodendrocytes express predominantly the plp isoform. Not all progenitors express the plp gene at the times studied, indicating that the presence of DM-20 is either transitory in individual cells or that only a sub-population is involved. The function of DM-20 at this early stage of the oligodendrocyte lineage has yet to be determined.

Proteolipid/DM-20 proteins bearing the paralytic tremor mutation in peripheral nerves and transfected Cos-7 cells

Paralytic tremor (Plp-pt) is a missense mutation of the myelin proteolipid gene (Plp) in rabbits. The myelin yield in the Plp-pt brain is reduced and the protein and lipid composition of central nervous system (CNS) myelin is abnormal. We studied the intracellular transport of the normal and Plp-pt mutant PLP and DM-20 in transiently transfected Cos-7 cells. While the mutant PLP accumulates in the rough endoplasmic reticulum and does not reach the plasma membrane, the spliced isoform of PLP, mutant DM-20, is normally transported to the cell surface and integrated into the membrane. Analysis of rabbit sciatic nerves revealed that concentration of peripheral nervous system (PNS) myelin proteins is normal in Plp-pt myelin. In the PNS like in the CNS, the level of Plp gene products is subnormal. But this does not affect myelination in the PNS where PLP, present in low concentration, is not a structural component of compact myelin. The normal level of Plp gene expression in Schwann cells is low and these results suggest that, in the Plp-pt PNS, Schwann cell function is not affected by the deficiency in PLP and/or the impairment of intracellular PLP transport.

Glial Transplants: An in vivo Analysis of Extrinsic and Intrinsic Determinants of Dysmyelination in Genetic Variants

Myelination in the CNS depends on the ability of oligodendrocytes (Ols) to efficiently colonize the brain, differentiate, and express a precise balance of specific genes necessary for myelin synthesis. Mutations in these genes produce different types of dysmyelination in animal as in human. Defects in the synthesis of myelin constituents usually lead to mild dysmyelinations. IN contrast, mutations affecting the gene encoding the proteolipid, another major protein of myelin, produce various perturbations of Ols biology suggesting a pleiotropic effect of the gene in the development of the CNS. Studies on expansion of cell population and survival have provided contradictory information on the extrinsic and intrinsic action of the gene on Ols biology. On one hand, in vitro studies using conditioned media as in vivo studies on heterozygotes, and transplantations experiments suggest that excess of programmed cell death in these mutants is ruled out by intrinsic factors which could act during embryonic life. On the other hand, attempts to compensate the gene defect by transgenic correction demonstrate a dominant negative effect of the jp mutation on both survival and functional potential of Ols. Finally, total suppression of PLP gene expression has a restricted effect on myelin structure without excess of cell death. These contradictory results are discussed in the perspective of regulation of cell death by competition for growth factors in limiting amount. The proposed model suggests that this contradiction is only apparent, and that excess of cell death in PLP/DM20 mutant is intrinsically determined by diminished competitivity of the mutant Ols for limited amounts of environmental factors.

Glial cell degeneration and hypomyelination caused by overexpression of myelin proteolipid protein gene

Myelin proteolipid protein (PLP), the major myelin protein in the CNS, has been thought to function in myelin assembly. Thus, mutations within the gene coding for PLP ( Plp) cause hypomyelination, such as the jimpy phenotype in mice and Pelizaeus-Merzbacher disease in humans. However, these mutants often exhibit premature death of oligodendrocytes, which form CNS myelin. To elucidate the functional roles of Pip gene products in the maturation and/or survival of oligodendrocytes, we produced transgenic mice overexpressing the Plp gene by introducing extra wild-type mouse Pip genes. Surprisingly, transgenic mice bearing 4 more Plp genes exhibited dysmyelination in the CNS, whereas those with 2 more Plp genes showed normal myelination at an early age (3 weeks after birth), but later developed demyelination. Overexpression of the Plp gene resulted in arrested maturation of oligodendrocytes, and the severity of arrest was dependent on the extent of overexpression. Overexpression also led to oligodendrocyte cell death, apparently caused by abnormal swelling of the Golgi apparatus. Thus, tight regulation of Plpgene expression is necessary for normal oligodendrocyte differentiation and survival, and its overexpression can be the cause of both dys- and demyelination.