Proteolipid Protein Promoter Research Articles

Hyperphosphorylation and insolubility of alpha-synuclein in transgenic mouse oligodendrocytes.

(Oligodendro)glial cytoplasmic inclusions composed of alpha-synuclein (alpha SYN) characterize multiple system atrophy (MSA). Mature oligodendrocytes (OLs) do not normally express alpha SYN, so MSA pathology may arise from aberrant expression of alpha SYN in OLs. To study pathological deposition of alpha SYN in OLs, transgenic mice were generated in which human wild-type alpha SYN was driven by a proteolipid protein promoter. Transgenic alpha SYN was detected in OLs but no other brain cell type. At the light microscopic level, the transgenic alpha SYN profiles resembled glial cytoplasmic inclusions. Strikingly, the diagnostic hyperphosphorylation at S129 of alpha SYN was reproduced in the transgenic mice. A significant proportion of the transgenic alpha SYN was detergent insoluble, as in MSA patients. The histological and biochemical abnormalities were specific for the disease-relevant alpha SYN because control green fluorescent protein was fully soluble and evenly distributed throughout OL cell bodies and processes. Thus, ectopic expression alpha SYN in OLs might initiate salient features of MSA pathology.

Myelin transcription factor 1 function in oligodendrocyte development

Myelin transcription factor 1 (Myt1) is a CCHC zinc‐finger protein that is expressed in oligodendrocyte progenitors (OP) and is down‐regulated as oligodendrocytes differentiate and accumulate proteolipid protein (PLP). Myt1 contains six zinc‐finger DNA‐binding domains, a putative acidic transcriptional activation domain, and an alpha‐helical protein–protein interaction domain. Myt1 has two zinc‐fingers near the N‐terminus and four zinc‐fingers (4F) near the C‐terminus. Each set of zinc‐fingers can bind to the PLP promoter. The objective of this study was to determine the function of Myt1 by expressing the 4F domain of Myt1, which lacks the putative transcriptional activation domain and protein–protein interaction domain. We predicted the 4F domain would compete for Myt1 response elements, and lacking other functional domains would interfere with endogenous Myt1 function. A retroviral expression system that included a FLAG epitope tag was used to ectopically express the 4F domain of Myt1 in cultured rat neonatal OPs. OPs and their progeny exhibited nuclear immunoreactivity for the 4F‐FLAG fusion protein. Expression of 4F in OPs grown with PDGF and FGF mitogens reduced proliferation as compared to controls. In the absence of mitogens, expression of 4F inhibited the differentiation of OPs as assessed by morphological criteria, O1 immunostaining, and PLP mRNA expression. This 4F inhibition of OP differentiation along the oligodendrocyte pathway was not due to alternative differentiation along an astrocytic pathway. These data suggest that Myt1 regulates a critical transition in oligodendrocyte lineage development in modulating the OP proliferative response relative to terminal differentiation and myelin gene expression.Acknowledgements: Supported by USUHS grant RO70IE.

Control of oligodendrocyte differentiation by theNkx2.2homeodomain transcription factor

Oligodendrocytes are derived from glial precursors that arise from the ventral neural tube early in development. In the developing chicken CNS, oligodendrocyte progenitors selectively express Nkx2.2 homeodomain transcription factor, raising the possibility that Nkx2.2 may directly regulate oligogliogenesis. In this study, we have examined Nkx2.2 expression in rodent glial precursors and studied the effect of a loss of Nkx2.2 on oligodendrocyte and astrocyte differentiation. We show that Nkx2.2 is also expressed in mammalian oligodendrocyte progenitors and that the differentiation of MBP-positive and PLP-DM20-positive oligodendrocytes is dramatically retarded in Nkx2.2-null mutants along the entire rostrocaudal axis. In contrast, no effect is seen on astrocytic differentiation. Interestingly, absence of Nkx2.2 expression leads to a ventral expansion of the Olig1/Olig2 expression in neuroepithelial cells into the Nkx2.2 domain and a consequent increase in the production of Olig1/Olig2-positive and platelet-derived growth factor receptor alpha-positive oligodendrocyte progenitors. These results strongly suggest that Nkx2.2 regulates the differentiation and/or maturation, but not the initial specification, of oligodendrocyte progenitors. Consistent with this suggestion, overproduction of Nkx2.2 protein in fibroblast cells can induce gene expression from the proteolipid protein promoter.

The transcriptional regulator Yin Yang 1 activates the myelin PLP gene.

Inauguration of the myelin program in developing oligodendrocytes requires the activation of those genes that encode the myelin proteins and the enzymes responsible for the synthesis and degradation of myelin lipids. An activator of the most abundantly expressed myelin protein, proteolipid protein (PLP), has been identified in a yeast one-hybrid system. The ubiquitously expressed zinc finger protein Yin Yang 1 (YY1) recognizes the myelin PLP promoter in vitro and in vivo. When overexpressed in an oligodendrocyte cell line, YY1 enhances transcription of the PLP promoter. A truncated version of YY1 that includes only the four zinc finger domains has little effect. The binding site for YY1 in the PLP promoter (site 3) fits the YY1 consensus site and DNA-protein complexes containing site 3 can be supershifted with an antibody directed against YY1 protein. Moreover, oligonucleotides with a mutated version of the PLP promoter site 3 are unable to bind to nuclear proteins or to compete for binding in a gel shift system. Finally, mutation of this site greatly reduces the activity of a 1-kb PLP promoter region in transfected glial cells. Our results suggest that PLP is a target gene for the transcriptional regulator YY1. This versatile transcription factor and nuclear matrix protein may boost transcription of the PLP gene to meet the demands of actively myelinating oligodendrocytes.

Purification and Analysis of in Vivo-Differentiated Oligodendrocytes Expressing the Green Fluorescent Protein

A complete understanding of the molecular mechanisms involved in the formation and repair of the central nervous system myelin sheath requires an unambiguous identification and isolation of in vivo-differentiated myelin-forming cells. In order to develop a novel tool for the analysis of in vivo-differentiated oligodendrocytes, we generated transgenic mice expressing a red-shifted variant of the green fluorescent protein under the control of the proteolipid protein promoter. We demonstrate here that green fluorescent protein-derived fluorescence in the central nervous system of 9-day- to 7-week-old mice is restricted to mature oligodendrocytes, as determined by its spatiotemporal appearance and by both immunocytochemical and electrophysiological criteria. Green fluorescent protein-positive oligodendrocytes could easily be visualized in live and fixed tissue. Furthermore, we show that this convenient and reliable identification now allows detailed physiological analyses of differentiated oligodendrocytes in situ. In addition, we developed a novel tissue culture system for in vivo-differentiated oligodendrocytes. Initial data using this system indicate that, for oligodendrocytes isolated after differentiation in vivo, as yet unidentified factors secreted by astrocytes are necessary for survival and/or reappearance of a mature phenotype in culture.

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.

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.

Evidence That the Homeodomain Protein Gtx Is Involved in the Regulation of Oligodendrocyte Myelination

We have investigated the patterns of postnatal brain expression and DNA binding of Gtx, a homeodomain transcription factor. Gtx mRNA accumulates in parallel with the RNAs encoding the major structural proteins of myelin, myelin basic protein (MBP), and proteolipid protein (PLP) during postnatal brain development; Gtx mRNA decreases in parallel with MBP and PLP mRNAs in the brains of myelin-deficient rats, which have a point mutation in the PLP gene. Gtx mRNA is expressed in differentiated, postmitotic oligodendrocytes but is not found in oligodendrocyte precursors or astrocytes. These data thus demonstrate that Gtx is expressed uniquely in differentiated oligodendrocytes in postnatal rodent brain and that its expression is regulated in parallel with the major myelin protein mRNAs, encoding MBP and PLP, under a variety of physiologically relevant circumstances. Using a Gtx fusion protein produced in bacteria, we have confirmed that Gtx is a sequence-specific DNA-binding protein, which binds DNA sequences containing a core AT-rich homeodomain binding site. Immunoprecipitation of labeled DNA fragments encoding either the MBP or PLP promoter regions with this fusion protein has identified several Gtx-binding fragments, and we have confirmed these data using an electrophoretic mobility shift assay. In this way we have identified four Gtx binding sites within the first 750 bp of the MBP promoter and four Gtx binding sites within the first 1. 3 kb of the PLP promoter. In addition, inspection of the PLP promoter sequence demonstrates the presence of six additional Gtx binding sites. These data, taken together, strongly suggest that Gtx is important for the function of differentiated oligodendrocytes and may be involved in the regulation of myelin-specific gene expression.

Usefulness of a mouse myelin basic protein promoter for gene therapy of malignant glioma: myelin basic protein promoter is strongly active in human malignant glioma cells.

We have searched for suitable promoters to regulate the expression of suicide genes for use in gene therapy. We have shown that the 1.3‐kb fragment of the mouse myelin basic protein (MBP) promoter region initiates transcription in mouse glioma cells more efficiently than glial flbrillary acidic protein (GFAP) or myelin proteolipid protein (PLP) promoter. Among three different lengths of the MBP promoter, the shortest (256‐bp) core promoter region initiates transcription as efficiently as 650‐bp or 1.3‐kh MBP promoter lengths in RSV‐M glioma cells. To assess the suitability of the MBP promoter for use in clinical trials of malignant glioma gene therapy, we also had to show that it (the 1.3‐kb length in this case) Is effective in human glioma cells, as well as in murine glioma cells. The activity of the MBP promoter is much higher than that of GFAP or PLP promoter in most human glioma cells, suggesting that the MBP promoter would be best for directing toxic gene expression in gene therapy for patients with malignant glioma. Human glioma cells in which the MBP promoter was strongly active were sensitive to ganciclovir when they were transduced with MBP promoter/herpes simplex virus thymidine kinase gene‐bearing retroviruses. In conclusion, retrovirus‐targeted gene therapy for malignant glioma using this MBP promoter is a promising candidate for clinical trials.

Use of Transgenic Systems to Investigate Oligodendrocyte Differentiation and Function

Transgenic techniques are generating new strains of animals that are of great importance for many neurological research projects. This includes new animal models of human diseases that should allow analysis of disease etiology and treatment. The insertion of new genetic material into the mouse genome enables the investigator to study the effects of overexpression of normal or mutated genes under a variety of experimental conditions. The use of cell-specific and/or developmentally regulated promoters permits studies on the expression of the specific DNA in selected cells within the nervous system at important developmental stages. This article focuses on the techniques for generating transgenic mice, noting specific advantages or problems that should be considered when designing a transgenic project. The use of reporter genes such as the LacZ gene is discussed, using the particular example of the myelin proteolipid protein promoter directing expression of the LacZ gene in differentiating oligodendrocytes.

Transcriptional regulation of the rat PLP promoter in primary cultures of oligodendrocytes.

Proteolipid protein (PLP) is the major intrinsic membrane protein of CNS myelin and is expressed in oligodendrocytes as part of a coordinate program of myelin-specific gene activation. In order to identify the DNA sequences and proteins involved in the regulation of PLP transcription, we have analyzed the 5' flanking sequences of the rat PLP gene by transient transfections into primary cultures of developing oligodendrocytes, the glial tumor line, C6, and L cells. High levels of expression of the CAT reporter gene in oligodendrocytes and C6 cells were obtained with constructs containing both 4270 and 225 bp of PLP promoter. A fusion construct containing 1061 bp of the PLP promoter, however, showed two-fold lower CAT expression. In addition, the activity of these promoter fusion constructs in oligodendrocytes was 2.5-4.6 higher than that observed in C6 cells, while very little expression was found in L cells. These data suggest that 225 bp of PLP promoter is sufficient for oligodendrocyte-specific regulation of PLP expression. Furthermore, both positive and negative elements within the PLP promoter are involved in this process. Finally, primary cultures of developing oligodendrocytes are a useful model system for the analysis of myelin-specific gene activation.

Characterization of a brain-specific Sp1-like activity interacting with an unusual binding site within the myelin proteolipid protein promoter.

The proteolipid protein (PLP) gene encodes the main integral protein of the myelin membrane of the central nervous system. The expression of the gene is regulated in a cell- and development-specific manner. Comparison of approximately 1.5 kb of the upstream noncoding region from man, mouse, and rat gene revealed an extensive sequence identity of about 95% between -250 and +100 (the most upstream transcription start site is defined as +1) but only about 50% identity further upstream. To define potential cis-acting elements in the promoter of the mouse PLP gene the upstream region was studied by transfection of C6 glioblastoma cells and CHO fibroblasts with various 5' deletion constructs fused to the reporter gene luciferase. We localized a promoter at position -184 to +90, which is active in both cell lines. Analysis of this region by DNase I foot-printing experiments and band shift analysis with nuclear extracts from myelinating brain, liver, C6, and CHO cells shows the binding of several different proteins to the promoter region. One brain-specific and two ubiquitous factors bound to the sequence AAGGGGAGGAG (DR1/2 box). This motif is also present in the upstream region of other myelin-specific genes and in some variants of the glia cell-specific virus JC. The factors bound with similar affinity to a Sp1-binding site. Therefore one of the ubiquitous factors seems to be Sp1 suggesting that Sp1 may play a role in the transcriptional regulation of the PLP gene. It has been shown that the DR1/2 box-binding factors are Zn(2+)-dependent. By Southwestern blotting it has been demonstrated that the DR1/2 box binds a protein of about 66 kDa that is enriched in brain.

Identification of cis-regulatory elements in the myelin proteolipid protein (PLP) gene.

Regulatory elements of the proteolipid protein (PLP) gene were identified physically by footprinting and gel mobility shift assays and functionally by transfecting glial cell lines with PLP-chloramphenicol acetyltransferase chimeric genes. In both human and rat glial cells, only several hundred base pairs of upstream sequence were sufficient for high level activity of the human PLP promoter. This region contains five sites that contact nuclear proteins in vitro. More distal recognition sites may exist, as regions upstream of -524 displayed silencing activity indicative of a negative regulatory element. A series of site directed mutations revealed one essential positive element (ATGGA at -118) which is found in other genes encoding myelin proteins. Our combined biochemical and functional analyses indicate that the key cis sites for maximal tissue-specific expression of PLP in cultured glial cells are clustered near the promoter. Within this cluster are several conserved motifs that may coordinate the regulation of myelin-specific genes.