Proteolipid Protein Gene Expression Research Articles

Treadmill aerobic training improve beam-walking test, up-regulate expression of main proteins of myelin and myelination in the hippocampus of cuprizone-fed mice

Multiple sclerosis (MS) is a potentially disabling disease of the brain and spinal cord (central nervous system). The aim of this study was to investigate the effect of 6 weeks of aerobic training on the main proteins of myelin including myelin basic protein (MBP), myelin oligodendrocyte (MOG), myelin associated glycoprotein (MAG), and myelin proteolipid protein (PLP) at hippocampus of C57BL/6 mouse model of cuprizone-induced MS. Twenty-eight female C57BL/6 mice (23 ± 3 g) were randomly divided into four groups (n = 7 per group): control, exercise (Exe), cuprizone (CPZ), and cuprizone with exercise (CPZ + Exe). Exercise groups performed treadmill aerobic exercise training 5 days a week, 15–22 m/min, and 15–60 min, during 6 weeks. Cuprizone were fed to mice at CPZ and CPZ + Exe groups for 6 weeks. Animals were sacrificed after 6 weeks. Biochemical and molecular biology analyses were performed. Mice at CPZ group had decreased myelination of nerve cells in the hippocampus. In addition, the use of CPZ in the hippocampus caused a decrease in the MBP, MOG gene expression, as well as a decrease in the MAG and PLP gene and protein expression compared to the healthy control group. However, performing aerobic exercise with CPZ consumption increased MBP gene expression and increased MAG and PLP protein expression, as well as increased myelination of nerve cells in the hippocampus compared to the CPZ group (p < 0.05). It seems that regular aerobic exercise in the MS model controls the destruction of myelin in the nerve cells of hippocampus by upregulating MBP, MAG and PLP, which can have positive effects on cognitive and motor performance.

Expression of Proteolipid Protein Gene in Spinal Cord Stem Cells and Early Oligodendrocyte Progenitor Cells Is Dispensable for Normal Cell Migration and Myelination

Plp1 gene expression occurs very early in development, well before the onset of myelination, creating a conundrum with regard to the function of myelin proteolipid protein (PLP), one of the major proteins in compact myelin. Using PLP-EGFP mice to investigate Plp1 promoter activity, we found that, at very early time points, PLP-EGFP was expressed in Sox2+ undifferentiated precursors in the spinal cord ventricular zone (VZ), as well as in the progenitors of both neuronal and glial lineages. As development progressed, most PLP-EGFP-expressing cells gave rise to oligodendrocyte progenitor cells (OPCs). The expression of PLP-EGFP in the spinal cord was quite dynamic during development. PLP-EGFP was highly expressed as cells delaminated from the VZ. Expression was downregulated as cells moved laterally through the cord, and then robustly upregulated as OPCs differentiated into mature myelinating oligodendrocytes. The presence of PLP-EGFP expression in OPCs raises the question of its role in this migratory population. We crossed PLP-EGFP reporter mice into a Plp1-null background to investigate the role of PLP in early OPC development. In the absence of PLP, normal numbers of OPCs were generated and their distribution throughout the spinal cord was unaffected. However, the orientation and length of OPC processes during migration was abnormal in Plp1-null mice, suggesting that PLP plays a role either in the structural integrity of OPC processes or in their response to extracellular cues that orient process outgrowth.

MicroRNA expression in mouse oligodendrocytes and regulation of proteolipid protein gene expression

Overexpression of the major myelin proteolipid protein (PLP) is detrimental to brain development and function and is the most common cause of Pelizaeus-Merzbacher disease. microRNA (miRNA), small, noncoding RNAs, have been shown to play critical roles in oligodendrocyte lineage. In this study, we sought to investigate whether miRNAs control PLP abundance. To identify candidate miRNAs involved in this regulation, we have examined differentiation-induced changes in the expression of miRNAs in the oligodendroglial cell line Oli-neu and in enhanced green fluorescent protein positive oligodendrocytes ex vivo. We have identified 145 miRNAs that are expressed in oligodendrocyte cell lineage progression. Dicer1 expression decreases in differentiated oligodendrocytes, and knock down of Dicer1 results in changes in miRNAs similar to those associated with differentiation. To identify miRNAs that control the PLP expression, we have selected miRNAs whose expression is lower in differentiated vs. undifferentiated Oli-neu cells and that have one or more binding site(s) in the PLP 3'-untranslated region (3'UTR). The PLP 3'UTR fused to the luciferase gene reduces the activity of the reporter, suggesting that it negatively regulates message stability or translation. Such suppression is relieved by knock down of miR-20a. Overexpression of miR-20a decreases expression of the endogenous PLP in primary oligodendrocytes and of the reporter gene. Deletion or mutation of the putative binding site for miR-20a in the PLP 3'UTR abrogated such effects. Our data indicate that miRNA expression is regulated by Dicer1 levels in differentiated oligodendrocytes and that miR-20a, a component of the cluster that controls oligodendrocyte cell number, regulates PLP gene expression through its 3'UTR.

Mice with Altered Myelin Proteolipid Protein Gene Expression Display Cognitive Deficits Accompanied by Abnormal Neuron-Glia Interactions and Decreased Conduction Velocities

Conduction velocity (CV) of myelinated axons has been shown to be regulated by oligodendrocytes even after myelination has been completed. However, how myelinating oligodendrocytes regulate CV, and what the significance of this regulation is for normal brain function remain unknown. To address these questions, we analyzed a transgenic mouse line harboring extra copies of the myelin proteolipid protein 1 (plp1) gene (plp1(tg/-) mice) at 2 months of age. At this stage, the plp1(tg/-) mice have an unaffected myelin structure with a normally appearing ion channel distribution, but the CV in all axonal tracts tested in the CNS is greatly reduced. We also found decreased axonal diameters and slightly abnormal paranodal structures, both of which can be a cause for the reduced CV. Interestingly the plp1(tg/-) mice showed altered anxiety-like behaviors, reduced prepulse inhibitions, spatial learning deficits and working memory deficit, all of which are schizophrenia-related behaviors. Our results implicate that abnormalities in the neuron-glia interactions at the paranodal junctions can result in reduced CV in the CNS, which then induces behavioral abnormalities related to schizophrenia.

Analyses of Proteolipid Protein Mutants Show Levels of Proteolipid Protein Regulate Oligodendrocyte Number and Cell Death in vitro and in vivo

Previous tissue culture studies indicate that the level of native proteolipid protein (PLP) or mutated PLP regulates the number of oligodendrocytes (Olgs). The regulation of Olg number is most likely due to toxicity of over-expression of native PLP or mis-sense mutations of PLP. We tested, in vivo and in vitro, the hypothesis that the absence of native PLP or reduced amounts of mutated PLP leads to an increase in numbers of Olgs and a corresponding decrease in the number of apoptotic Olgs. In cultures derived from PLP deficient mice, the number of Olgs is twofold greater than in wild-type mice. In primary glial cultures or in enriched OLG cultures, in which the synthesis of native PLP is blocked using antisense technology, the number of apoptotic cells is several-fold reduced. Injection of PLP antisense oligodeoxynucleotides into jimpy (jp) mice reduces the number of dying glia in spinal cord 3x compared to controls, and increased the number of myelinated fibers. These studies demonstrate that inhibition of native or mutant PLP synthesis directly reduces apoptosis. The regulation of apoptosis by PLP gene expression occurs independently of myelination, indicating that the PLP gene has multiple primary functions.

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.

Changes in oligodendrocytes and myelin gene expression after radiation in the rodent spinal cord

Purpose The aim of this study was to assess changes in oligodendrocytes (OL), myelin gene expression, and their relationships with late demyelination after irradiation. Methods and materials Adult rats were given single doses of 8 or 22 Gy to the cervical spinal cord. Immunohistochemistry for APC or GST-π was used to identify OL. Changes in myelin gene expression were assessed using RT-PCR for proteolipid protein (PLP). Luxol fast blue staining was used to assess demyelination. CNP-βgeo transgenic mice were used to confirm some of the results of the rat model. Cells of the oligodendroglial lineage in these animals express β-galactosidase (β-gal). Results Early apoptosis of APC, GST-π, and β-galactosidase positive cells was observed in the spinal cord of rats and CNP-βgeo mice. At 24 h after 22 Gy, there was a significant decrease in OL density. Cell density continued to decline thereafter after both 8 and 22 Gy, and a reduction in PLP expression was observed at 4–5 weeks. A further decrease in PLP expression was seen beginning at 18 weeks after 22 Gy only. Demyelination was observed at 19 weeks after 22 Gy. Conclusions Apoptosis of OL and changes in OL density and PLP gene expression were observed early after both 8 and 22 Gy. This suggests that these early changes are unlikely to be directly related to the late demyelination observed.

Stimulation of myelin proteolipid protein gene expression by eicosapentaenoic acid in C6 glioma cells

In this study, the role of exogenous fatty acids in the regulation of proteolipid protein (PLP) gene expression was investigated using the following model culture system: C6 glioma cells expressing the green-fluorescent protein (eGFP) driven by different segments of PLP promoter. Eicosapentanoic acid (EPA; 20:5 n-3), but not arachidonic acid (AA; 20:4 n-6), induced a significant increase in medium fluorescence intensity (MFI) determined by fluorescence-activated cell sorting (FACS). The induction of PLP promoter was time-dependent showing maximal activity between 24 and 48 h after EPA exposure. PLP promoter activation was dependent on fatty acid concentration, with maximum activation at 200 μM. Northern blot analysis confirmed the fluorescence data in C6 cells incubated with EPA. Furthermore, this treatment increased the adenylyl cyclase-cyclic AMP (cAMP) levels and the mitogen-activated protein kinase (MAPK) activation in C6 cells. PLP promoter activity was inhibited by pre-treatment with H89 (protein kinase A (PKA) inhibitor), but not with PD98059 (MAPK inhibitor), suggesting that EPA stimulates the expression of PLP via cAMP-mediated pathways.

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.

Proteolipid Protein Gene Modulates Viability and Phenotype of Neurons

Overexpression or lack of expression of proteolipid protein (PLP) gene by oligodendrocytes causes axonal pathology. It is unclear whether dysfunction of the PLP gene mediates its effects directly on neurons or indirectly by abnormal formation of myelin sheaths. We performed experiments using cocultures and conditioned media (CM) to test the direct effect of PLP gene expression on neurons. Non-glial cell lines were stably transfected with PLP or DM20 (an alternate splice variant of PLP) cDNAs. Immunocytochemistry and enhanced green fluorescent protein expression showed that translated products were synthesized and inserted into the plasma membrane in proper conformation. The number of surviving dorsal root ganglion (DRG) neurons was significantly less than controls when cocultured for 5 d with PLP-expressing cells. The number of degenerating neurons increased in a dose-dependent manner corresponding to increasing numbers of PLP-expressing cells. However, the number of surviving DRG neurons cocultured with DM20-expressing cells was comparable to that of controls, indicating that PLP-specific products contributed to decreased neuron survival. When DRG neurons were cultured with CM from PLP- or DM20-expressing cells, significantly fewer neurons survived with CM of PLP- but not DM20-expressing cells. This suggests that secreted factors from PLP-expressing cells contribute to neuronal death. Increased neuronal death found with PLP-expressing cells cannot be attributed to density-dependent artifacts, because in each experiment the density of different cell lines was similar. This effect of CM may be mediated by a negative pH shift elicited from PLP but not DM20 expression. These results indicate that PLP gene products directly modulate neuron viability.

Proteolipid Promoter Activity Distinguishes Two Populations of NG2-Positive Cells throughout Neonatal Cortical Development

Transgenic mice expressing enhanced green fluorescent protein (EGFP) driven by the mouse myelin proteolipid protein (PLP) gene promoter have been developed to investigate cells in the oligodendrocyte lineage. Transgene expression is consistent with the developmental expression of PLP, with cells at all stages of oligodendrocyte differentiation clearly visualized. These animals were analyzed to establish the time course of oligodendrocyte progenitor migration, proliferation, and differentiation in neonatal cortex. In these animals, two populations of NG2 proteoglycan-positive oligodendrocyte progenitor cells were identified that exist in postnatal subventricular zone and cortex. These two populations are distinguished by the presence or absence of PLP gene expression. Thus, PLP gene expression defines a subpopulation of NG2-positive cells from very early postnatal ages, which migrates toward the pial surface and proliferates in situ. EGFP(+)/NG2(+) cells are present in the cortex from postnatal day 1, and they remain in the cortex as undifferentiated oligodendrocyte progenitors for up to 3 weeks before myelination begins. These data could be explained by the presence of an important inhibitor of oligodendrocyte differentiation in the cortex during this period, which is downregulated in a region-specific manner to allow myelination. On the other hand, it is possible that oligodendrocyte progenitor cells remain undifferentiated in cortex until an essential signal is produced in situ to induce differentiation.

Hypoxic–ischemic injury results in acute disruption of myelin gene expression and death of oligodendroglial precursors in neonatal mice

Studies of ischemic brain injury in neonatal rodents have focused upon the pathophysiology of neuronal damage. Much less consideration has been given to white matter injury, even though it is a major contributor to chronic neurological dysfunction in children. In the human neonate, particularly in those born prematurely, periventricular white matter is highly susceptible to hypoxic–ischemic (H–I) injury. To understand the basis for this selective vulnerability, we examined myelin gene expression and cell death in the subventricular layer and the surrounding white matter of neonatal mice following H–I insult. Using an in situ hybridization technique that gives high resolution and is very sensitive, we examined myelin basic protein and proteolipid protein gene expression three and twenty-four hours after a H–I insult. To elicit unilateral forebrain hypoxic and ischemic injury, 9–10-day-old mice underwent right carotid artery ligation followed by timed (40–70 min) exposure to 10% oxygen. Twenty-four hours following H–I, myelin basic protein and proteolipid protein transcripts were markedly reduced in striatum, external capsule, fornix, and corpus callosum in the injured side. Three hours after lesioning (ligation+70 min hypoxic exposure) myelin basic protein gene transcripts were visibly reduced in the ipsilateral white matter tracts. Interestingly, some cells in the subventricular layer expressed proteolipid protein transcripts, and 3 h after a H–I insult they were degenerating in the injured but not contralateral side. TUNEL staining showed an increase in the number of positive cells in the injured subventricular layer and corpus callosum but the adjacent striatum did not show a corresponding change in the number of TUNEL labeled cells. Ultrastructural studies of the subventricular zone and corpus callosum 3 h after H–I revealed that many subventricular cells, glial cells in the corpus callosum, and callosal axons in the injured side had already degenerated. However, the subventricular cells, glia and axons in the contralateral corpus callosum were spared. Many cells in the injured corpus callosum exhibited a apoptotic morphology; yet more mature oligodendrocytes in this region appeared normal. Our results show that a H–I insult causes a surprisingly swift and dramatic degenerative response in the subventricular layer and adjacent white matter. Within 3 h after H–I, the programmed cell death cascade was initiated; internucleosomal DNA degradation took place in subventricular and glial cells; oligodendrocyte progenitors died and axonal degeneration in the ipsilateral corpus callosum was extensive. The swiftness of the subventricular and glial cell degeneration suggests the H–I insult directly targets glia, as well as neurons, and raises the provocative question of whether glia exert damaging effects upon neurons and axons. Since the severity of the H–I insult can be modulated by varying the duration of hypoxia, the model is ideal to study whether oligodendrocyte progenitors are more susceptible to death than mature oligodendrocytes, whether mature oligodendrocytes de-differentiate and then are induced to remyelinate surviving axons, and/or whether oligodendrocyte progenitors in the subventricular layer can be stimulated to proliferate, migrate, and remyelinate the surviving axons.

Functional characterization of a cis-acting DNA antisilencer region that modulates myelin proteolipid protein gene expression.

Regulation of myelin proteolipid protein (PLP:) gene expression is tightly controlled, both spatially and temporally. Previously, we have shown with transgenic mice that a PLP:-lacZ fusion gene (which includes the entire sequence for PLP: intron 1 DNA) is regulated in a similar manner to endogenous PLP: gene expression. Furthermore, by deletion-transfection analyses using assorted PLP:-lacZ constructs with partial deletion of PLP: intron 1 sequences, we have shown that the first intron possesses an antisilencer region that is capable of over-coming repression mediated by two distinct regions located elsewhere within intron 1 DNA. Here, we report the ability of various fragments encompassing the antisilencer region to restore beta-galactosidase activity when inserted into PLP:-lacZ constructs, which originally exhibited low levels of beta-galactosidase activity. Additional constructs were generated to test the effects of these antisilencer-containing fragments in constructs that are missing either one or both of the negative regulatory regions that are overridden during antisilencing. Transfection analyses, in conjunction with protein-DNA binding assays, suggest that several nuclear factors are necessary for derepression of PLP: gene activity in an oligodendroglial cell line. Moreover, either the "core" or complete antisilencing region can act in an additive or synergistic fashion when multiple copies are inserted into the Plp-lacZ constructs.

Proteolipid Protein Gene Product Can Be Secreted and Exhibit Biological Activity during Early Development

A gene encoding myelin proteolipid protein (PLP) and its smaller isoform DM20 is expressed at least 1 week before myelination. Mutations within the gene cause abnormalities in the development of premyelinating oligodendrocytes, resulting in hypomyelinating disorders. These findings suggest a premyelinating function of the PLP gene products. We previously demonstrated that PLP gene expression is directly associated with secretion of a factor that increases the number of oligodendrocytes. Here we show that this activity is mediated by a secreted fragment containing the C-terminal portion of PLP. This factor increased the bromodeoxyuridine incorporation rate in both oligodendrocyte and astrocyte lineage cells; a synthetic peptide (PLP 215-232) exhibited a similar activity. Dose-response curves of PLP and PLP peptide showed maximum activities at a concentration in the picomolar range, which decreased at higher concentrations. These observations demonstrate that a secreted PLP gene product exerts biological activity at a premyelinating stage before the major induction of the gene.

Myelin Proteolipid Protein Intron 1 Sequences Do Not Appear to Enhance Myelin Proteolipid Protein Gene Transcription

Sequences from the first intron of the mouse myelin proteolipid protein (PLP) gene were examined for their ability to modulate PLP gene expression. Glial (N20.1) or nonglial (NIH 3T3) cells were transiently transfected with constructs that contained 1.4 kb of PLP promoter sequence driving luciferase reporter gene expression, as well as various portions of PLP intron 1 DNA. Although these same PLP intron 1 fragments enhanced reporter gene expression from a heterologous basal promoter in a previous study, the results reported here demonstrate that they do not augment PLP promoter activity. Thus, the regulation of PLP cell-type-specific expression, conferred by the first intron, appears to be mediated by an enhancer-independent mechanism.

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.

The first intron of the myelin proteolipid protein gene confers cell type-specific expression by a transcriptional repression mechanism in non-expressing cell types

Chimeric genes containing portions of the mouse myelin proteolipid protein (PLP) gene fused to the lacZ reporter gene were used to detect the effect of PLP intron 1 sequences on cell type-specific expression. A transfected fusion gene containing PLP intron 1 sequences was expressed in an oligodendrocyte cell line but not in a liver cell line, consistent with endogenous PLP gene expression. However, an analogous fusion gene missing the first intron was expressed in either oligodendrocyte or liver transfected cells. These studies suggest that transcriptional repressor element(s) located in PLP intron 1 are important in extinguishing expression in non-glial cell types and that the promoter alone functions in an indiscriminate manner. This moderately large intron (>8 kb) was sequenced to aid in future fine mapping of these cell-specific regulatory element(s).

Oligodendrocyte development in PLP "pt" mutant rabbits: glycolipid antigens and PLP gene expression.

Paralytic tremor (pt), a hereditary neurological disorder of rabbits is a recessive, X-linked point mutation of the gene for proteolipid protein (PLP) biosynthesis. This mutation results in substitution of histidine by glutamine in the PLP molecule and produces severe hypomyelination. In the present study, we investigated the developmental expression of myelin-oligodendrocyte-specific glycolipid markers by means of ELISA assay. While immunoreactivity with antibodies recognising proligodendroblast (POA) antigen was unchanged, only minute amounts of the other glycolipid markers characteristic for more advanced stages of OLs maturation, such as 04 and 01 antigens, were expressed in pt brain. The degree of down-regulation was similar to that for MBP. Concomitantly, the level of in situ expression of the mutated PLP gene mRNA in glial cells of 14 day old pt brain was found to be as high as in age-matched controls. Northern blot analysis of developmental PLP gene expression showed a significant deficit of this message in pt brain, but only at more advanced developmental stages. However, aside from changes in myelin structure, no changes in glial cell number or morphology were evident by light microscopic examination of pt mutants. In contrast, electron microscopy revealed substantial abnormalities in pt oligodendrocyte cytoarchitecture, indicating functional impairment of intracellular transport and utilisation of myelin constituents. Thus, only POA expression is positively correlated with the unchanged content of OLs in pt brain, whereas decreases of 04 and 01 antigens, together with MBP immunoreactivity, are indicators of the degree of hypomyelination. Furthermore, oligodendrocyte differentiation appears to proceed normally in pt mutant brain up to the stage of PLP gene expression. Then, due to intracellular accumulation of this abnormal gene product, synthesis of PLP as well as the other myelin-specific constituents is inhibited by a "feed-back" control mechanism.

Embryonic development of central nervous system myelination in a reptilian species, Eumeces fasciatus

The myelin proteolipid proteins are a vital component of the vertebrate central nervous system (CNS), contributing essential functions to the development of the myelinating cells of the CNS and to the structure of CNS myelin. Alternative splicing of the proteolipid protein (PLP) gene to produce two related isoforms occurs in Mammalia, Aves, and Reptilia, but not Amphibia. As part of a long-term investigation into the function of the different isoforms of PLP, embryonic development, myelination, and PLP gene expression in reptilian CNS were examined. PLP gene expression was already substantial by day 19 (stage 39) of the 27-day Eumeces fasciatus egg incubation period. By day 21 of incubation, also stage 39, PLP mRNA was at peak levels; there was a significant amount of CNS myelination as demonstrated by electron microscopy of the spinal cord; and the reflexive motor response was evident. Although most axons were myelinated by the time of hatching, myelin sheaths continued to increase in size and compactness after hatching. The correlation of physiological development, CNS myelination, and expression of the PLP gene in the lizard corresponded well with the developmental pattern seen in mammals.

Expression of proteolipid protein gene is directly associated with secretion of a factor influencing oligodendrocyte development.

Oligodendroglial cell death in the myelin proteolipid protein (PLP) mutants can be partially rescued by the environment factor(s) supplied by the wild-type cells in vivo and in vitro. It is possible that the presence of PLP or DM-20 results in secretion of a factor or factors in the CNS influencing oligodendrocyte development. We previously showed that DM-20 mRNA is produced in G26 mouse oligodendroglioma, B104 rat neuroblastoma, and B16 mouse melanoma but not in NIH3T3 mouse fibroblast cell lines. Culture supernatants from these cell lines were added to primary glial cell cultures from embryonic day 17 mouse brain. After 4 days, the number of oligodendrocytes present in cultures with supernatants from DM-20-producing cells (G26, B104, and B16) was significantly higher than that of control cultures but not with the NIH3T3 supernatant. To investigate more directly whether the PLP gene expression is involved in this process, NIH3T3 cells (nonneural cells) were forced to produce PLP or DM-20. By addition of the supernatants from the PLP/DM-20 transformants, the number of oligodendrocytes in the mixed glial cell cultures increased. This clearly demonstrates that the expression of the PLP gene is sufficient for and directly associated with secretion of a factor, which influences the oligodendrocyte development.