Protein zero is necessary for E-cadherin-mediated adherens junction formation in Schwann cells.

Protein Zero (P0), the major structural protein in the peripheral nervous system (PNS) myelin, acts as a homotypic adhesion molecule and is thought to mediate compaction of adjacent wraps of myelin membrane. E-Cadherin, a calcium-dependent adhesion molecule, is also expressed in myelinating Schwann cells in the PNS and is involved in forming adherens junctions between adjacent loops of membrane at the paranode. To determine the relationship, if any, between P0-mediated and cadherin-mediated adhesion during myelination, we investigated the expression of E-cadherin and its binding partner, beta-catenin, in sciatic nerve of mice lacking P0 (P0(-/-)). We find that in P0(-/-) peripheral myelin neither E-cadherin nor beta-catenin are localized to paranodes, but are instead found in small puncta throughout the Schwann cell. In addition, only occasional, often rudimentary, adherens junctions are formed. Analysis of E-cadherin and beta-catenin expression during nerve development demonstrates that E-cadherin and beta-catenin are localized to the paranodal region after the onset of myelin compaction. Interestingly, axoglial junction formation is normal in P0(-/-) nerve. Taken together, these data demonstrate that P0 is necessary for the formation of adherens junctions but not axoglial junctions in myelinating Schwann cells.

Gtx, an oligodendrocyte-specific homeodomain protein, has repressor activity.

Myelin, a multilamellar membrane structure that facilitates nerve conduction, is synthesized in the central nervous system (CNS) by oligodendrocytes. Gtx, a member of the homeodomain family of transcriptional factors, is a candidate regulator of myelin gene expression, because it is uniquely expressed in myelinating oligodendrocytes in postnatal rodent brain. To analyze the regulatory activity of Gtx, we first identified the optimal Gtx-binding sequence using an in vitro DNA-binding assay. This sequence, (A/T)TTAATGA, contains a TAAT core and is similar, but not identical, to that of other homeodomain protein binding sites. When coexpressed in cultured cells along with a minimal promoter containing five tandem repeats of this optimal Gtx-binding sequence, Gtx demonstrated repressor activity, which was also present when Gtx was tethered to DNA by way of the strong GAL4 DNA-binding domain. Truncations of the GAL4-Gtx fusion identified a portable repressor domain within a relatively proline/alanine-rich region N-terminal to the Gtx homeodomain. Cotransfection of a Gtx expression vector into a variety of cell lines, including oligodendrocytes, along with constructs containing portions of the PLP, MBP, or Gtx promoters fused to a reporter gene, however, did not modulate transcription from any of these promoter constructs. These data support the notion that the oligodendrocyte-specific homeodomain protein Gtx can act as a transcriptional repressor. In addition, they suggest that interaction of Gtx with other, as yet undefined, transcriptional regulators modifies Gtx activity in oligodendrocytes.

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.

GFAP-positive and myelin marker-positive glia in normal and pathologic environments.

The data herein demonstrate that in addition to the well-characterized myelin marker-positive, glial fibrillary acidic protein (GFAP)-negative, membrane sheet-bearing oligodendrocytes, another type of myelin marker-positive, process-bearing glia exists in normal and pathologic conditions. This second type of myelin marker-positive glia expresses GFAP, and therefore these cells have been referred to as mixed phenotype glia. Although mixed phenotype glia have been documented previously, their identity and function have remained a mystery. The goal of this immunocytochemical study was to further characterize these cells. Using the MBPlacZ transgenic mouse in which beta-galactosidase is under the control of the myelin basic protein (MBP) gene promoter, GFAP-positive/beta-galactosidase-positive and myelin/oligodendrocyte-specific protein (MOSP)-positive/beta-galactosidase-positive cells were detected in subcortical white matter and in perivascular locations within cerebral white and gray matter. In cultures prepared from highly enriched myelin marker-positive immature glia, mixed phenotype glia were detected that were GFAP-positive and either MOSP-, MBP-, O1-, and O4-positive. The expression of multiple myelin markers by mixed phenotype glia may suggest that these cells are of oligodendrocyte origin. Increased numbers of MOSP-positive/GFAP-positive mixed phenotype glia were detected in sections from adult hypomyelinated brain from shiverer, quaking, and PKU mice compared to myelinated control adult mouse brain. Similarly, cultures from control brain exposed to elevated pH for 2-3 weeks showed dramatically increased numbers of mixed phenotype glia (80%) compared to control (<10%). Increased numbers of mixed phenotype glia also were detected in shiverer cultures (40%). Since increases in the number of mixed phenotype glia occur in shiverer, quaking, and PKU mouse brain, these data suggest that mixed phenotype glia contribute to gliosis in pathologic white matter.

Regulation of myelin-specific gene expression. Relevance to CMT1.

Schwann cells, the myelinating cells of the peripheral nervous system, are derived from the neural crest. Once neural crest cells are committed to the Schwann cell fate, they can take on one of two phenotypes to become myelinating or nonmyelinating Schwann cells, a decision that is determined by interactions with axons. The critical step in the differentiation of myelinating Schwann cells is the establishment of a one-to-one relationship with axons, the so-called "promyelinating" stage of Schwann cell development. The transition from the promyelinating to the myelinating stage of development is then accompanied by a number of significant changes in the pattern of gene expression, including the activation of a set of genes encoding myelin structural proteins and lipid biosynthetic enzymes, and the inactivation of a set of genes expressed only in immature or nonmyelinating Schwann cells. These changes are regulated mainly at the transcriptional level and also require continuous interaction between Schwann cells and their axons. Two transcription factors, Krox 20 (EGR2) and Oct 6 (SCIP/Tst1), are necessary for the transition from the promyelinating to the myelinating stage of Schwann cell development. Krox 20, expressed in myelinating but not promyelinating Schwann cells, is absolutely required for this transition, and myelination cannot occur in its absence. Oct 6, expressed mainly in promyelinating Schwann cells and then down-regulated before myelination, is necessary for the correct timing of this transition, since myelination is delayed in its absence. Neither Krox 20 nor Oct 6, however, is required for the initial activation of myelin gene expression. Although the mechanisms of Krox 20 and Oct 6 action during myelination are not known, mutation in Krox 20 has been shown to cause CMT1, further implicating this protein in the pathogenesis of this disease. Identifying the molecular mechanisms of Krox 20 and Oct 6 action will thus be important both for understanding myelination and for designing future treatments for CMT1. Point mutlations in the genes encoding the myelin proteins PMP22 and P0 cause CMT1A without a gene duplication and CMT1B, respectively. Although the clinical and pathological phenotypes of CMT1A and CMT1B are similar, their molecular pathogenesis is quite different. Point mutations in PMP22 alter the trafficking of the protein, so that it accumulates in the endoplasmic reticulum (ER) and intermediate compartment (IC). Mutant PMP22 also sequesters its normal counterpart in the ER, further reducing the amount of PMP22 available for myelin synthesis at the membrane, and accounting, at least in part, for its severe effect on myelination. Mutant PMP22 probably also activates an ER-to-nucleus signal transduction pathway associated with misfolded proteins, which may account for the decrease of myelin gene expression in Schwann cells in Trembler mutant mice. In contrast, absence of expression of the homotypic adhesion molecule, P0, in mice in which the gene has been inactivated, produces a unique pattern of Schwann cell gene expression, demonstrating that P0 plays a regulatory as well as a structural role in myelination. Whether this role is direct, through a P0-mediated adhesion pathway, or indirect, through adhesion pathways mediated by cadherins or integrins, however, remains to be determined. The molecular mechanisms underlying dysmyelination in CMT1 are thus complex, with pleitropic effects on Schwann cell physiology that are determined both by the type of mutation and the protein mutated. Identifying these molecular mechanisms, however, are important both for understanding myelination and for designing future treatments for CMT1. Although demyelination is the hallmark of CMT1, the clinical signs and symptoms of this disease are probably produced by axonal degeneration, not demyelination. (ABSTRACT TRUNCATED)

A minimal human MBP promoter-lacZ transgene is appropriately regulated in developing brain and after optic enucleation, but not in shiverer mutant mice.

Previous studies, both in vitro and in vivo, suggest that small portions of the mouse myelin basic protein (MBP) promoter are sufficient to activate regulated expression of MBP. To confirm our previous in vitro studies, we prepared transgenic mice with short regions of the human MBP promoter fused to the lacZ reporter gene. We found that 750 nucleotides of the proximal human MBP promoter is sufficient to activate oligodendrocyte-specific, developmentally regulated expression of lacZ in three independent lines. This promoter, however, does not activate expression of lacZ in Schwann cells in peripheral nerve or in adult mouse brain. The relative levels of beta-galactosidase specific activity, mRNA, and transcription parallel those of MBP mRNA during myelinogenesis. Thus, we exploited this transgene as a quantitative tool to evaluate the response to stimuli known to affect myelination. Transgene expression is reduced 75 % after optic enucleation, as previously reported for levels of MBP mRNA, indicating that axons signal to this portion of the proximal MBP promoter to fully activate MBP expression during myelinogenesis. Instead, in adult shiverer mice, another setting in which MBP transcription is modulated, transgene expression is not increased, in contrast to the increased transcriptional activation of MBP previously reported in these mice. These data suggest that the regulatory region that mediates transcriptional activation of the MBP gene is modular, since discrete subregions are required for activation in Schwann cells, during myelinogenesis in oligodendrocytes, during maintenance myelination in adult brain, and in the dysmyelinating mutant shiverer mouse.

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.