Lead exposure delays the differentiation of oligodendroglial progenitors in vitro.

Lead (Pb) is an environmental neurotoxicant that can cause hypo- and demyelination. Oligodendrocytes (OLs), the myelin-forming cells in the central nervous system, may be a possible target for Pb toxicity. The present study describes the effect of Pb on the maturation of rat OL progenitor (OP) cells and the developmental expression of myelin-specific galactolipids. Dose-response studies showed that OP cultures were more sensitive to Pb than mature OLs. Pb delayed the differentiation of OL progenitors, as demonstrated by cell morphology and immunostaining with a panel of stage-specific differentiation markers. Pb given prior to and during differentiation caused a decrease in the biosynthesis of galactolipids in both undifferentiated and differentiated OLs, as detected by metabolic radiolabeling with 3H-D-galactose. While the ratios of galacto/gluco-cerebrosides, hydroxy fatty acid/nonhydroxy fatty acid galactolipids, and galactocerebrosides/sulfatides increased in control cultures during cell differentiation, Pb treatment prevented these changes. The results suggest that chronic Pb exposure may impact brain development by interfering with the timely developmental maturation of OL progenitors.

Distinct roles for PI3K in proliferation and survival of oligodendrocyte progenitor cells.

Phosphoinositol 3-kinase (PI3K) is a downstream effector for multiple ligand-activated receptors and modulates cell responses through activation of its target protein kinase B (Akt). We examined the roles of PI3K-Akt signaling in a primary glial (oligodendrocyte) progenitor cell culture system that is ligand-dependent for cell proliferation, survival, and prevention of differentiation. We demonstrate that PI3K and Akt (Ser-473 phosphorylation) are activated in response to platelet-derived growth factor but not basic fibroblast growth factor-2 (FGF2) and that distinct forms of PI3K are activated in early progenitors and later-maturation pro-oligodendroblasts as identified by their sensitivity to wortmannin. By establishing conditions to examine effects on cell proliferation and survival independently, we demonstrate that PI3K is necessary for a full mitogenic response and that PI3K is also necessary for early progenitor survival. Our results therefore demonstrate that PI3K-Akt signaling independently regulates proliferation and survival, that the form of PI3K is distinct in early progenitors and pro-oligodendroblasts, and that FGF2 does not activate this pathway in either primary glial cell population.

Increased levels of surgical adhesions in TGFbeta1 heterozygous mice.

Adhesion formation and fibrosis represent a major complication of surgical intervention. Reducing the morbidity associated with adhesions requires an understanding of the mechanisms underlying their formation. Since increased levels of transforming growth factor-beta1 (TGFbeta1) have been associated with inflammation and adhesion production, we investigated the requirement of TGFbeta1 in peritoneal adhesion formation utilizing mice carrying a targeted disruption of the TGFbeta1 allele. Mice that were either wild-type (+/+), containing two normal alleles of TGFbeta1, or heterozygous (+/-) for the TGFbeta1 null allele received injections of magnesium silicate (talc), and the extent of abdominal adhesions was determined utilizing a standard grading score. Wild-type (+/+) animals had at least twofold more TGFbeta1 protein in peritoneal fluids at 2 h posttrauma compared to heterozygous (+/-) mice (727 vs. 243 pg TGFbeta1/mg protein by enzyme-linked immunosorbent assay (ELISA) in +/+ and +/- mice, respectively), and had significantly less scar and adhesion formation (p < .05) at 7 days posttrauma (1.8 +/- 0.8 vs. 3.4 +/- 1.4, graded from 0 to 5, in +/+ and +/- mice, respectively). These results demonstrate that haploid insufficiency in TGFbeta1 levels can lead to inappropriate matrix and adhesion production during inflammation, and together with previous studies suggest that any perturbation of normal TGFbeta1 levels can modulate the injury response that regulates the extent of adhesion formation.

An inhibitor of cell proliferation associated with adhesion formation is suppressed by N,O-carboxymethyl chitosan.

Surgical adhesions are a major cause of morbidity and mortality. The ideal barrier agent will both minimize adhesions and provide a milieu for the regeneration of the mesothelium lining of the abdominal and thoracic cavities. N,O-Carboxymethylchitosan (NOCC), a derivation of chitin that markedly reduces adhesions, may function to modulate intracellular signals such as growth factors and cytokines in the inflammatory exudate. Since transforming growth factor-beta is implicated in the fibrotic process, we investigated the possibility that NOCC's effects on adhesion formation reflects a modulation of TGF-beta activity. Using a biological assay for inhibition of cell proliferation to detect TGF-beta activity, we demonstrate that NOCC suppresses the levels of an inhibitor of cell proliferation released into serum and peritoneal exudates after cecal abrasion in the rat. However, this activity was distinct from known forms of TGF-beta as determined using both TGF-beta-neutralizing antisera and a TGF-beta-resistant cell proliferation assay. Thus at least one potential effect of NOCC involves a mechanism distinct from TGF-beta inhibition.

Transplanted oligodendrocyte progenitor cells expressing a dominant-negative FGF receptor transgene fail to migrate in vivo.

The proliferation, migration, survival, and differentiation of oligodendrocyte progenitor cells, precursors to myelin-forming oligodendrocytes in the CNS, are controlled by a number of polypeptide growth factors in vitro. The requirement and roles for individual factors in vivo, however, are primarily unknown. We have used a cell transplantation approach to examine the role of fibroblast growth factor (FGF) in oligodendrocyte development in vivo. A dominant-negative version of the FGF receptor-1 transgene was introduced into oligodendrocyte progenitors in vitro, generating cells that were nonresponsive to FGF but responsive to other mitogens. When transplanted into the brains of neonatal rats, mutant cells were unable to migrate and remained within the ventricles. These results suggest a role for FGF signaling in establishing a motile phenotype for oligodendrocyte progenitor cell migration in vivo and illustrate the utility of a somatic cell mutagenesis approach for the study of gene function during CNS development in vivo.

Regulation of oligodendrocyte development and CNS myelination by growth factors: prospects for therapy of demyelinating disease.

Multiple sclerosis (MS), the most common neurological disorder diagnosed in young adults, is characterized by autoimmune demyelination in the central nervous system (CNS). Promotion of remyelination in the brain and spinal cord is a potential strategy for therapeutic intervention in MS and other demyelinating diseases. Recent studies have shown that the development of oligodendrocytes, the myelin-forming cells of the CNS, is extensively controlled by growth factors. These factors regulate the proliferation, migration, differentiation, survival and regeneration of oligodendroglial cells and the synthesis of myelin, and often interact in a complex manner. Moreover, insulin-like growth factor I (IGF-I) has proven effective for therapy of experimental autoimmune encephalomyelitis (EAE), an animal model of autoimmune demyelination. In this review we summarize recent findings on the regulation of oligodendrocyte development and CNS myelination by growth factors, and discuss these findings in the context of possible clinical application for the therapy of neurological disease in humans.

Prevention of postoperative adhesions with the chitin derivative N-O-carboxymethylchitosan.

The ideal barrier agent for the prevention of surgical adhesions has remained elusive. We have examined the ability of a new hydrogel N-O-carboxymethylchitosan, a derivative of chitin with properties similar to the extracellular matrix, to prevent adhesions when applied topically to traumatized mesothelial surfaces. In two rodent adhesion models (pericardial and peritoneal), the application of N-O-carboxymethylchitosan significantly prevented or minimized the formation of scar and fibrosis. According to a scoring system from 0 to 3 (0 = no adhesions and 3 = severe dense adhesions), control groups in each model consistently produced severe dense adhesions (2.9 +/- 0.2, 2.7 +/- 0.3). All treated groups consistently scored less than 1.0, indicating minimal or no fibrosis. The differences between the control and treated groups were statistically significant (p < 0.05). Thus, the application of N-O-carboxymethylchitosan to traumatized mesothelial surfaces may have significant potential in the prevention of postoperative adhesion formation.

Motor function analysis of myelin mutant mice using a rotarod.

We have examined motor control in normal and shiverer mutant mice using the rotarod assay, a forced motor activity which tests for balance and co-ordination. Shiverer mice carry a deletion of the myelin basic protein (MBP) gene, resulting in CNS dysmyelination and characteristic motor dysfunction. Homozygous mutant mice had a significant increase in cumulative falls from the rotarod relative to heterozygous mice. Non-acclimated animals of both genotypes showed progressive improvement in performance when tested on successive days. The rotarod test also discriminated shiverer mutants from animals that received gene therapy intervention. Shiverer animals carrying an MBP transgene showed gene-dosage-dependent improvements in motor function, and mutants which received thalamic transplants of wild type oligodendrocyte precursor cells showed improvement relative to sham operated and non-transplanted controls. Thus the rotarod is a sensitive measure of motor function in hypomyelinated mice, and may be useful for assessing the results of experimental manipulations including transgenic gene therapy and cell transplantation.

Expression and regulation of a glutamate receptor subunit by bFGF in oligodendrocyte progenitors.

Oligodendrocyte progenitor cells (O-2A) express both kainate-preferring and AMPA-preferring glutamate receptors (Gallo et al., Soc. Neurosci. Abstr., 18:653, 1992b; Gallo et al., 1994). The expression and regulation of the GluR-4c, AMPA-preferring subunit by growth factors was studied by Northern blot analysis of total RNA from purified rat cortical O-2A progenitors. Differently from cortical neurons, O-2A progenitors only expressed two high molecular weight GluR-4c transcripts (6.2 and 4.2 kb), as observed also in cultured astrocytes. Basic fibroblast growth factor (bFGF) increased GluR-4c transcript levels in O-2A progenitors and its effects were not mimicked by platelet derived growth factor (PDGF) or fetal calf serum. Therefore, bFGF may regulate O-2A progenitor responsiveness to glutamate during development through the expression of glutamate receptor subunits.

Expression of the receptor tyrosine kinase c-kit in oligodendrocyte progenitor cells.

The growth and differentiation of neural precursor cells in the central nervous system (CNS) are regulated by their response to polypeptide growth factors which interact with specific transmembrane receptor tyrosine kinases (RTKs). We demonstrate that rat oligodendrocyte-type 2 astrocyte (O-2A) glial progenitor cells, precursors of the myelin-forming cells in the CNS, express the transmembrane RTK c-kit, the gene product of the murine dominant white spotting (W) locus and receptor for stem cell factor. Expression of c-kit transcripts and immunoreactive protein is lost when O-2A progenitors differentiate into post-mitotic oligodendrocytes. Analysis of developing rat brain revealed an increase in the expression of c-kit transcripts between postnatal days 10 and 12, a window of time preceding the emergence of oligodendrocytes and the onset of myelination in vivo. Expression of c-kit in vitro and in vivo suggests a role for this receptor and its ligand during oligodendrocyte development.

A role for TGF-beta in oligodendrocyte differentiation.

Oligodendrocyte-type-2 astrocyte (O-2A) glial progenitor cells undergo a limited number of mitotic divisions in response to PDGF before differentiating into oligodendrocytes, the myelin-forming cell of the CNS. We examined the mechanism limiting O-2A proliferation, and demonstrate that these cells secrete an inhibitor of cell proliferation that can be neutralized with antibodies to TGF-beta. O-2A cells also secrete an inhibitory activity that cannot be neutralized with TGF-beta antibodies. O-2A progenitor cultures express TGF-beta 1 isoform and its transcript, while oligodendrocyte cultures express TGF-beta 1, beta-2, and beta-3 isoforms. Both recombinant TGF-beta 1 and O-2A conditioned medium inhibit the proliferation of O-2A progenitor cells cultured in the presence of PDGF, and this inhibition can be partially neutralized with polyclonal TGF-beta antibodies. Thus, TGF-beta produced by O-2A cells may limit PDGF-driven mitosis and promote oligodendrocyte development. TGF-beta is a less potent inhibitor of O-2A proliferation when these cells are cultured in the presence of bFGF, suggesting that bFGF interferes with TGF-beta signaling. Thus, the production of TGF-beta by cells in the O-2A lineage may account for the distinct effects of PDGF and bFGF on O-2A progenitor cell proliferation. Moreover, our results suggest that TGF-beta may be an important mediator of oligodendrocyte differentiation.

Distinct effects of bFGF and PDGF on oligodendrocyte progenitor cells.

We have compared the effects of platelet-derived (PDGF) and basic fibroblast (bFGF) growth factors on the shape, migration, and differentiation of oligodendrocyte progenitor cells, the precursors of myelin-forming cells in the CNS. In the presence of bFGF, oligodendrocyte progenitors purified from rat neonatal brain cultures were stellate, non-motile, and had a morphological complexity of 1.26 +/- 0.03 as measured by fractal dimension (D). These cells expressed transcripts encoding the POU-homeodomain transcription factor Oct-6, but not myelin genes. Upon addition of PDGF, bFGF-treated cells became motile and twofold less complex in shape (D = 1.19 +/- 0.03). These changes occurred within 6 +/- 4 h and were dependent on de novo transcription and translation, but not DNA synthesis. Upon removal of PDGF the cells reverted to their stellate shape (D = 1.26). Removal of both bFGF and PDGF resulted in oligodendrocyte differentiation after 3 days, with a fourfold increase in complexity of shape (D = 1.55 +/- 0.08), loss of Oct-6 transcripts, and gain of myelin transcripts. Thus PDGF is both necessary and sufficient to induce a motile state in progenitor cells growing in the presence of bFGF. Together with our previous data (McKinnon et al.: Neuron 5:603, 1990), our results suggest that bFGF and PDGF may control distinct phases of proliferation and migration of oligodendrocyte progenitor cells in vivo.

A role for fibroblast growth factor in oligodendrocyte development.

The differentiation of oligodendrocyte-type 2 astrocyte (O-2A) glial progenitor cells into myelin-forming oligodendrocytes is influenced by several polypeptide growth factors. Exposure of O-2A progenitors to basic fibroblast growth factor (bFGF) promotes their sustained proliferation, blocks their differentiation, and maintains both a high level of platelet-derived growth factor (PDGF) alpha-receptors and PDGF sensitivity. Exposure to PDGF, in contrast, promotes only a limited number of cell divisions prior to their differentiation and triggers progenitor cell migration. In the continued presence of bFGF the cells have a stellate morphology with short processes. Upon addition of PDGF these stellate cells become bipolar with long processes, and on removal of PDGF their morphology reverts back to stellate. Thus the phenotype of O-2A progenitor cells in response to these growth factors is plastic. Our studies suggest that bFGF (or a related ligand) in the CNS may sensitize O-2A progenitors to PDGF and thereby initiate their ability to migrate into white matter tracts prior to the onset of myelination.

FGF modulates the PDGF-driven pathway of oligodendrocyte development.

PDGF promotes the growth of oligodendrocyte type-2 astrocyte (O-2A) glial progenitor cells and allows their timely differentiation into oligodendrocytes, the CNS myelin-forming cells. We demonstrate that basic FGF is a potent mitogen for brain O-2A progenitor cells, but blocks their differentiation into oligodendrocytes. Treatment with basic FGF also influences the level of expression of PDGF receptors on O-2A progenitor cells. These cells express only the alpha chain PDGF receptor, and the levels of PDGF alpha receptors decrease as the cells differentiate. In contrast, basic FGF maintains a high level of functionally responsive PDGF alpha receptors in O-2A progenitors. Thus basic FGF activates a signaling pathway that can positively regulate PDGF receptors in O-2A progenitor cells. In this way basic FGF or an FGF-like factor may modulate the production of myelin-forming cells in the CNS.

Expression of small cytoplasmic transcripts of the rat identifier element in vivo and in cultured cells.

We examined the level of expression of small RNA transcripts hybridizing to a rodent repetitive DNA element, the identifier (ID) sequence, in a variety of cell types in vivo and in cultured mammalian cells. A 160-nucleotide (160n) cytoplasmic poly(A)+ RNA (BC1) appeared in late embryonic and early postnatal rat brain development, was enriched in the cerebral cortex, and appeared to be restricted to neural tissue and the anterior pituitary gland. A 110n RNA (BC2) was specifically enriched in brain, especially the postnatal cortex, but was detectable at low levels in peripheral tissues. A third, related 75n poly(A)- RNA (T3) was found in rat brain and at lower levels in peripheral tissues but was very abundant in the testes. The BC RNAs were found in a variety of rat cell lines, and their level of expression was dependent upon cell culture conditions. A rat ID probe detected BC-like RNAs in mouse brain but not liver and detected a 200n RNA in monkey brain but not liver at lower hybridization stringencies. These RNAs were expressed by mouse and primate cell lines. Thus, tissue-specific expression of small ID-sequence-related transcripts is conserved among mammals, but the tight regulation found in vivo is lost by cells in culture.

The 3' long terminal repeat of a transcribed yet defective endogenous retroviral sequence is a competent promoter of transcription.

Although actively transcribed and present as multiple genomic copies, a distinct class of endogenous murine leukemia virus-related sequence does not give rise to infectious virus. Since the long terminal repeat at the 3' terminus provides the transcriptional start site after reintegration, we determined the structure and potential promoter activity of that sequence obtained from cDNA of endogenous retroviral transcripts. These studies demonstrate that the distinctive 3' long terminal repeat sequence of these transcripts could serve as an effective promoter of transcription and, therefore, may not be the primary defect in the infectious cycle of retroviral replication but may result in the propagation of these endogenous retroviral sequences in the genome as retrotransposons.

The neuronal identifier element is a cis-acting positive regulator of gene expression.

A middle-repetitive DNA element termed the identifier (ID) sequence, located in introns of postnatal-onset neuronal-specific genes, is transcribed in early postnatal rats by RNA polymerase III (Pol III) specifically in the brain. We show that these Pol III transcripts, although brain specific in vivo, are also expressed in immortalized rodent cell lines in culture. We demonstrate that the ID sequence can act as a positive regulator or enhancer of RNA polymerase II gene expression in cell lines that express these RNAs but not in primary cells in which Pol III transcription of the ID sequence is absent. Thus, ID elements may be positive regulators of postnatal-onset neuronal-specific gene expression.

Nonrandom insertion of Tn5 into cloned human adenovirus DNA.

The bacterial transposable element Tn5 displays regional selectivity in target sites for transposition. To examine this integration specificity of Tn5, we have mapped 57 insertion events in a plasmid pXC1 containing a eukaryotic viral DNA fragment as a target for Tn5 insertional mutagenesis. We found a nonrandom distribution of integration sites in pXC1, suggesting preferred targets for transposition. However, DNA sequence analysis of seven mutants revealed no target site sequence specificity for Tn5 insertion. We demonstrated that the majority of these insertions mapped downstream from a fortuitous promoter sequence which was present and active in this cloned insert in pXC1. Furthermore, when this promoter region was removed, Tn5 was able to transpose into previously unused upstream target sequences. Our data suggest that transcriptional activity may influence Tn5 transposition.

Tn5 mutagenesis of the transforming genes of human adenovirus type 5.

We have cloned the entire human adenovirus type 5 (Ad5) genome into the pBR322 plasmid in two segments: the BamHI-A fragment (21 kb) and the BamHI-B fragment (15 kb). We have also generated a series of clones with smaller Ad5 DNA inserts, all containing the left-end of the viral genome. One such clone, pXCl, containing the left 16% of the Ad5 DNA molecule, has been shown to transform rodent cells by DNA transfection. We have used the transposable element Tn5 as an insertion mutator to isolate pXCl mutants containing Tn5 inserted at a large number of sites. By assaying transforming activity of selected pXC::Tn5 plasmids we have identified Ad5 sequences which are essential for DNA-mediated transformation. Our results with these mutants and with a plasmid pCD1, containing a deletion within the Ad5-transforming region, indicate that sequences present in both early region 1a and the N-terminal region of early region 1b are essential for DNA-mediated transformation.

The role of the guanine insertion enzyme in O-biosynthesis in Drosophila melanogaster.

Drosophila tRNA can be guanylated by a crude enzyme from rabbit reticulocytes. Guanylating activity is also present in crude extracts of adult Drosophila. A major product of this reaction as well as several minor ones were resolved by RPC-5 chromatography. The main substrate of both the Drosophila and rabbit reticulocyte enzymes was the non-Q-containing aspartic acid tRNA, tRNA2gammaAsp. The QU-lacking (gamma) forms of asparagine, histidine and tyrosine tRNAs were also substrates and gave rise to the minor products of the reaction. In contrast, the Q- or Q*-containing (delta) forms of these tRNAs appear not to be substrates. The evidence strongly suggests that the guanyating enzyme is involved in Q biosynthesis and would be better termed a guanine replacement or pre-Q insertion enzyme.