Vilma Schonmann

Central nervous system myelination in mice with deficient expression of Notch1 receptor

Activity of the Notch1 gene is known to inhibit oligodendrocyte (OL) differentiation in vitro. We tested the hypothesis that the Notch1 pathway regulates in vivo myelin formation, by examining brain myelination of Notch1 receptor null heterozygotes mutant animals (Notch1+/–). We show that a deficiency in Notch1 expression leads to increased abundance of products of specific myelin genes in myelinated areas of the brain during the first 2 weeks of postnatal life. We observed increased numbers of myelinated axons in optic nerves and the presence of myelinated fibers in the molecular layer (ML) of the Notch1+/– cerebella. These findings were accompanied by up‐regulation of Mash1 and down‐regulation of Hes5 proteins. In addition, we found expression of Jagged1, one of the Notch1 activators, in unmyelinated axons of the cerebellar ML during normal development. Our findings indicate that the Jagged/Notch signaling pathway might actively participate in the regulation of myelination during central nervous system development and suggest that certain neuronal populations might regulate whether their axons are myelinated by the expression of inhibitory signals such as Jagged1.

Developmental partitioning of myelin basic protein into membrane microdomains

Specific membrane microdomains (including lipid rafts) exist in myelin but have not been fully characterized. Myelin basic protein (MBP) maintains the compactness of the myelin sheath and is highly posttranslationally modified. Thus, it has been suggested that MBP might also have other functions, e.g., in signal transduction. Here, the distribution of MBP and its modified forms was studied, spatially and temporally, by detailed characterization of membrane microdomains from developing and mature bovine myelin. Myelin membranes were extracted with three different detergents (Brij 96V, CHAPS, or Triton X‐100) at 4°C. The detergent‐resistant membranes (DRMs), representing coalesced lipid rafts, were isolated as low‐buoyant‐density fractions on a sucrose density gradient. These myelin rafts were disrupted when cholesterol was depleted with methyl‐β‐cyclodextrin. The use of CHAPS detergent led to enrichment of several myelin proteins, including phospho‐Thr97‐MBP, in the DRMs from mature myelin. Citrullinated and methylated MBP remained in “nonraft” microdomains. In contrast, the DRMs from early myelin were enriched in Golli‐MBP, Fyn, Lyn, and CNP. The localization of various proteins in DRMs was further supported by the colocalization of these lipid raft components in cultured mouse oligodendrocytes. Thus, there is a developmental regulation of posttranslationally modified forms of MBP into specific membrane microdomains.

Expression and regulation of golli products of myelin basic protein gene during in vitro development of oligodendrocytes.

The myelin basic protein (MBP) gene produces two families of proteins, the classic MBPs, important for myelination of the CNS, and the golli proteins, whose biological role in oligodendrocytes (OLs) is still unknown. The goals of this work were to study the in vitro pattern of expression of the golli products during OL differentiation and to compare it with that of the classic MBP products of the gene. Mouse primary glial cultures were analyzed at the mRNA and protein levels with an array of techniques. We found that OLs express golli mRNA primarily during intermediate stages of differentiation, which was confirmed by immunocytochemical analysis. Golli expression was low in proliferating OL progenitors as well as in terminally mature OLs. Golli proteins were found associated with the OL cell soma and nuclei and, to a lesser extent, with the cellular processes. We also found that golli proteins are not targeted to myelin in vitro and in vivo, in contrast to the classic MBPs. Finally, we found that golli expression is regulated during OL development and can be manipulated by growth factors such as basic fibroblast growth factor, neurotrophin‐3, and retinoic acid. J. Neurosci. Res. 66:679–690, 2001.

Thymocytes express the golli products of the myelin basic protein gene and levels of expression are stage dependent

The golli products of the myelin basic protein gene have been shown to be expressed in mouse thymus and brain. The full repertoire of thymic cell types expressing golli products has not yet been determined, although immunoreactivity has been found in some macrophages. We have analyzed the cellular expression of golli mRNAs and proteins in the thymus. The results showed that MTS5+ cortical/MTS10+ medullary epithelial cells and NLDC145+ dendritic cells did not express golli, while some macrophages did exhibit strong immunoreactivity. Golli mRNAs were not detected in macrophages by in situ hybridization. Thymocytes expressed significant levels of golli mRNAs and proteins by in situ hybridization and immunohistochemistry. Interestingly, golli immunoreactivity varied with thymocyte stage of differentiation. For example, CD4−CD8− (double-negative) thymocytes expressed relatively high levels of golli. Upon further differentiation into CD4−CD8− (double-positive) thymocytes, golli protein expression declined dramatically. When thymocytes developed into CD8− or CD4+ (single-positive) thymocytes, golli protein expression increased again, but it never achieved the levels found in double-negative thymocytes. Thus, the altered levels of expression of golli proteins in developing thymocytes correlated with the transitions from double-negative to double-positive and double-positive to single-positive stages. The lack of significant golli expression in thymic stromal cells may offer an alternative explanation for the mechanism of inefficient negative selection of those autoreactive thymocytes with specificity for myelin basic proteins.

Platelet-derived growth factor and basic fibroblast growth factor regulate cell proliferation and the expression of notch-1 receptor in a new oligodendrocyte cell line

We generated a new cell line, N38, by conditionally immortalizing mouse oligodendrocytes (OLs) at early stages of maturation. The morphology and marker expression pattern suggest N38 cells are similar to immature OLs. N38 cells were sensitive to changes in serum concentrations, and forcing the cells to differentiate in low serum at 39°C significantly decreased the survival of the cells. Importantly, addition of PDGFaa, bFGF or astrocyte‐conditioned medium had protective effects on the cells, by increasing cell proliferation but not cell differentiation. This effect was receptor‐mediated. Exposure of N38 cells to differentiating signals such as retinoic acid did not cause further differentiation of the cells. The N38 cell line expresses the vertebrate homolog of the Drosophila notch‐1 receptor, a molecule that appears to regulate OL differentiation. Notch‐1 receptor was homogeneously distributed in the somas of N38 cells. Incubation of N38 cells with either PDGFaa or bFGF, however, induced a polarized distribution of the receptor in the majority of the cells as well as an upregulation of receptor protein levels. The upregulation of molecules, such the notch‐1 receptor, in pathways that control differentiation might be an important mechanism for keeping OL precursors in an undifferentiated state during their exit of the germinal layer and migration in the developing central nervous system. This OL cell line might constitute a suitable model for studies of regulatory mechanisms at this stage of OL differentiation. J. Neurosci. Res. 62:319–328, 2000.

TWO NEURONAL CELL LINES EXPRESSING THE MYELIN BASIC PROTEIN GENE DISPLAY DIFFERENCES IN THEIR IN VITRO SURVIVAL AND IN THEIR RESPONSE TO GLIA

We have generated two conditionally immortalized neuronal cell lines from primary cultures of embryonic day 13 (E13) and postmitotic (postnatal day 0; P0) cortical neurons transformed with the temperature‐sensitive SV‐40 large‐T antigen. Two clonal cell lines (CN1.4 from E13 cultures and SJ3.6 from P0 cultures) were isolated and stable maintained in vitro. Both cell lines expressed a number of neuronal markers such as the neurofilaments, glutamic acid decarboxylase 67, neuron‐specific enolase, and the BG21 isoform of the myelin basic protein gene. At 34°C, the CN1.4 cell line had elaborated short processes, whereas the SJ3.6 cell line produced long processes that formed a delicate network. When these cell lines were cultured at 39°C, some of the cellular processes grew longer, adopting a more mature neuronal morphology. Interestingly, at 39°C, the in vitro survival of these cell lines differed significantly. Whereas the survival of CN1.4 cell line was greatly unaffected, SJ3.6 cells died soon after they were cultured at 39°C. The cell death of SJ3.6 cells was accompanied by fragmentation and condensation of DNA in their nuclei, indicative of an apoptotic event. Under these conditions, SJ3.6 showed an upregulation of the p75 receptor. When this cell line was cocultured with oligodendrocytes, astrocytes, or glial conditioned media (GCM), there was a marked increase in survival. In contrast, little effect of glial cells or GCM was observed on the CN1.4 cell line. These lines appear to be useful models to study neuronal–glial interactions in addition to neuronal cell death and the effects of glial factors that promote the survival of neurons. J. Neurosci. Res. 54:309–319, 1998.

Identification of a protein that interacts with the golli-myelin basic protein and with nuclear LIM interactor in the nervous system.

The myelin basic protein (MBP) gene encodes the classic MBPs and the golli proteins, which are related structurally to the MBPs but are not components of the myelin sheath. A yeast two‐hybrid approach was used to identify molecular partners that interact with the golli proteins. A mouse cDNA was cloned that encoded a protein of 261 amino acids and called golli‐interacting protein (GIP). Database analysis revealed that GIP was the murine homolog of human nuclear LIM interactor‐interacting factor (NLI‐IF), a nuclear protein whose function is just beginning to be understood. It is a member of a broad family of molecules, found in species ranging from yeast to human, that contain a common domain of ∼100 amino acids. Immunocytochemical and Northern blot analyses showed co‐expression of GIP and golli in several neural cell lines. GIP and golli also showed a similar developmental pattern of mRNA expression in brain, and immunohistochemical staining of GIP and golli showed co‐expression in several neuronal populations and in oligodendrocytes in the mouse brain. GIP was localized predominantly in nuclei. GIP co‐immunoprecipitated with golli in several in vitro assays as well as from PC12 cells under physiologic conditions. GIP was the first member of this family shown to interact with nuclear LIM interactor (NLI). NLI co‐immunoprecipitated with GIP and golli from lysates of N19 cells transfected with NLI, further confirming an interaction between golli, GIP, and NLI. The ability of GIP to interact with both golli and NLI, and the nuclear co‐localization of GIP and golli in many cells, indicates a role for the golli products of the MBP gene in NLI‐ associated regulation of gene expression.

Notch1 and Numb genes are inversely expressed as oligodendrocytes differentiate.

The Notch1 pathway plays a fundamental role during the establishment of cell fates in the central nervous system (CNS) by regulating neural cell differentiation. In oligodendrocytes (OLs), Notch1 activity prevents these cells from becoming terminally mature, thereby influencing CNS myelination. Little is known of how OLs regulate the expression of this receptor at the gene level or if OLs have mechanisms to control the level of intracellular activity of the Notch1 pathway. In this study, we have found that Notch1 gene expression was higher in proliferative OL progenitor cells (OPCs) and was reduced when cells were forced to withdraw from the cell cycle and became mature, indicating that Notch1 gene expression is developmentally regulated in OLs. We observed that the blockade of terminal differentiation of OPCs by incubation with Delta1, an activator of Notch1, was a dominant process and OL-differentiating signals such as thyroid hormone could not overcome this inhibition in culture. This suggests that a downregulation of the Notch1 pathway might be required to allow OPCs to enter terminal differentiation. We also provide evidence that OPCs and OLs express the Numb gene, a known negative regulator of Notch1 activity. In vivo, Numb was found in postnatal OLs from cerebellar and cerebral white matter. In vitro, Numb expression showed to be inversely correlated to that of Notch1, with higher levels of Numb proteins in mature OLs, in association with myelin-like membranes.

Neuronal regulation of myelin basic protein mRNA translocation in oligodendrocytes is mediated by platelet-derived growth factor.

Translocation of mRNAs has emerged as an important form of protein targeting. In oligodendrocytes, the myelin-forming cells of the central nervous system, myelin basic protein (MBP) mRNAs are transported into cell processes and the cytoplasmic channels that infiltrate the myelin sheath. This mRNA movement is important for myelination and occurs in purified oligodendrocytes in vitro, but not in oligodendrocytes grown on bed layers of astrocytes. We have shown previously that this astrocytic inhibition depends on cell-cell contact and is partially relieved in primary cultures that contain some neurons in addition to oligodendrocytes and astrocytes. We report here that soluble factors, secreted by neurons, are responsible for relieving the astrocytic inhibition of MBP mRNA translocation. Of several growth factors tested, only platelet-derived growth factor (PDGF) was effective in alleviating the astrocytic inhibition. Double immunofluorescence analysis demonstrated the presence of PDGF alpha-receptors in oligodendrocytes. PDGF appears to mediate its effect via its alpha-receptors and receptor tyrosine kinases. This interaction among the three neural cell types may play an important role in regulating remyelination after injury.

Differential sensitivity in the survival of oligodendrocyte cell lines to overexpression of myelin proteolipid protein gene products.

The proteolipid (PLP) gene encodes at least four proteins, including the classic PLP and DM20, which are important components of the myelin sheath, and the recently identified soma‐restricted (sr) isoforms, srPLP and srDM20. The classic PLP and DM20 gene products have been implicated in oligodendrocyte survival by overexpression studies in vitro and in vivo. The classic and sr proteolipids are targeted to different cellular compartments in the oligodendrocyte, suggesting different cellular functions. Accordingly, we examined the effects of in vitro overexpression of the sr‐PLP/DM20 isoforms on the survival of stably transfected, conditionally immortalized, oligodendroglial cell lines and compared this to overexpression of the classic and the jimpy‐mutated proteolipids. The results indicate that overexpression of either normal or jimpy classic PLP/DM20 resulted in a dramatic reduction in the survival of the oligodendrocyte cell lines at the nonpermissive temperature, but not the COS‐7 cell line, a cell line expressing the same oncogene constitutively. Survival of the oligodendrocyte cell lines was significantly less affected when either the sr‐PLP/DM20 or the dopamine D‐2 receptor, another cell membrane protein, was overexpressed in the cell lines. These results suggest that overexpression of the “classic” PLP or DM20 can compromise the survival of oligodendrocytes whether or not they are mutated. Furthermore, they suggest that the internal mechanisms for normal targeting of the PLP/DM20 isoforms of either the “classic” or the “sr” types influence the oligodendrocytes ability to survive when these proteolipids are overexpressed. J. Neurosci. Res. 65:485–492, 2001.