F. Arthur McMorris

Insulin-like growth factor I increases brain growth and central nervous system myelination in tTransgenic mice

Insulin-like growth factor I (IGF-I) is a potent regulator of oligodendrocyte development and myelination in vitro, but its effect on myelination in vivo has never been tested directly. Therefore, we examined brain growth and myelination in a transgenic mouse line that overexpresses IGF-I. By postnatal day 55, when brain growth and myelination are essentially complete in normal mice, the brains of transgenic mice were 55% larger than those of controls owing to an increase in cell size and apparently in cell number. Most or all brain structures appeared to be affected. At the same time, total myelin content of the transgenic mice was 130% greater than that of controls. Oligodendrocyte number as a percentage of total cell number was not increased in the transgenic mouse brains; the increase in myelin content was primarily the result of an increase in myelin production per oligodendrocyte. These findings indicate that IGF-I is a potent inducer of brain growth and myelination in vivo.

Retinal ganglion cells lose trophic responsiveness after axotomy.

Whereas PNS neurons in culture are intrinsically responsive to peptide trophic factors, retinal ganglion cells (RGCs) are not unless they are depolarized, or their intracellular levels of cyclic AMP (cAMP) are elevated. We show here that depolarization increases cAMP in cultured RGCs sufficiently to enhance their responsiveness and that the trophic responsiveness of developing RGCs in intact retinas depends on physiological levels of activity and cAMP elevation. Responsiveness is lost after axotomy but is restored by cAMP elevation. The death of axotomized RGCs can be prevented if they are simultaneously stimulated by several trophic factors together with cAMP elevation. Thus, the death of RGCs after axotomy is not caused solely by the loss of retrograde trophic stimuli but also by a profound loss of trophic responsiveness.

Cyclic AMP Induction of the Myelin Enzyme 2′,3′-Cyclic Nucleotide 3′-Phosphohydrolase in Rat Oligodendrocytes

Abstract: Cyclic AMP (cAMP) is known to induce the activity of the myelin enzyme 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase (CNP; EC 3.1.4.37) in C6 rat glioma cells. This report shows that CNP is also inducible in oligodendrocytes explanted from 1‐day‐old rat cerebrum and grown in tissue culture. Induction was observed after a 1‐day treatment with 1 mM N6, O2′ ‐dibutyryl cyclic AMP (dbcAMP) and was maximal after 5 days, reaching 200–240% of control. Induction was observed both in mixed cerebral cell cultures containing oligodendrocytes and astrocytes, and in purified cultures of oligodendrocytes prepared by a differential shakeoff procedure. Addition of dbcAMP to the cultures 3–9 days after the cells were explanted from rat brain induced CNP activity, but no induction was observed when dbcAMP treatment was begun 13 or more days after explantation. These results demonstrate that one component of myelin, CNP, is inducible in oligodendrocytes by a cAMP‐mediated mechanism, and suggest a role for cAMP in the regulation of the myelin‐associated functions of oligodendrocytes.

Regulation of Oligodendrocyte Development and Central Nervous System Myelination by Insulin‐like Growth Factors

In summary, our studies show that IGFs are potent regulators of oligodendrocyte development and myelination in vitro and in vivo. IGFs act at several levels: by promoting proliferation of oligodendrocytes and oligodendrocyte precursors, by inducing immature oligodendrocyte precursors to develop into oligodendrocytes, and by regulating myelin gene expression and the amount of myelin produced per oligodendrocyte. Our findings indicate that IGFs play a crucial role in normal oligodendrocyte development and myelination, and suggest that IGFs may have applications for the promotion of remyelination in myelin disorders such as MS.

Fyn tyrosine kinase regulates oligodendroglial cell development but is not required for morphological differentiation of oligodendrocytes

The non‐receptor protein tyrosine kinase Fyn, which is a member of the Src family of kinases, has been shown to be essential for normal myelination and has been suggested to play a role in oligodendrocyte development. However, oligodendrocyte development has not been studied directly in cells lacking Fyn. Additionally, because Fyn is expressed in neurons as well as oligodendrocytes, it is possible that normal myelination requires Fyn expression in neurons but not in oligodendrocytes. To address these issues, we analyzed the development of oligodendrocytes in neuron‐free glial cell cultures from fyn−/− mice that express no Fyn protein. We observed that oligodendrocytes develop to the stage where they elaborate an extensive network of membranous processes and express the antigenic components of mature oligodendrocytes in the complete absence of Fyn. However, as compared with fyn+/+ controls, fewer oligodendroglia developed in fyn−/− cell cultures, and a smaller proportion of them matured to the stage characterized by a high degree of morphological complexity. In addition, we found that insulin‐like growth factor‐I, a potent stimulator of oligodendrocyte development, failed to stimulate morphological maturation of fyn−/− oligodendroglia. The pyrazolopyrimidine PP2, believed to be a selective inhibitor of Fyn, did not prevent the development of morphologically complex oligodendrocytes. Unexpectedly, however, it was toxic to both fyn+/+ and fyn−/− glial cells, indicating that this class of inhibitors can have significant effects that are independent of Fyn. J. Neurosci. Res. 63:303–312, 2001.

The Growth Arrest-Specific Gene Product Gas6 Promotes the Survival of Human Oligodendrocytes via a Phosphatidylinositol 3-Kinase-Dependent Pathway

Microarray analysis revealed that transcripts for the Axl and Mer receptor tyrosine kinases are expressed at high levels in O4+-immunopanned oligodendrocytes isolated from second trimester human fetal spinal cord. In humans the sole known ligand for the Axl/Rse/Mer kinases is growth arrest-specific gene 6 (Gas6), which in the CNS is secreted by neurons and endothelial cells. We hypothesized that Gas6 is a survival factor for oligodendrocytes and receptor activation signals downstream to the phosphatidylinositol 3 (PI3)-kinase/Akt pathway to increase cell survival in the absence of cell proliferation. To test this hypothesis, we grew enriched human oligodendrocytes for 6 d on a monolayer of NIH3T3 cells stably expressing Gas6. CNP+ oligodendrocytes on Gas6-secreting 3T3 cells had more primary processes and arborizations than those plated solely on 3T3 cells. Also, a twofold increase in CNP+ and MBP+ oligodendrocytes was observed when they were plated on the Gas6-secreting cells. The effect was abolished in the presence of Axl-Fc but remained unchanged in the presence of the irrelevant receptor fusion molecule TrkA-Fc. A significant decrease in CNP+/TUNEL+ oligodendrocytes was observed when recombinant human Gas6 (rhGas6) was administered to oligodendrocytes plated on poly-l-lysine, supporting a role for Gas6 signaling in oligodendrocyte survival during a period of active myelination in human fetal spinal cord development. PI3-kinase inhibitors blocked the anti-apoptotic effect of rhGas6, whereas a MEK/ERK inhibitor had no effect. Thus Gas6 sustains human fetal oligodendrocyte viability by receptor activation and downstream signaling via the PI3-kinase/Akt pathway.

Expression of biochemical properties of oligodendrocytes in oligodendrocyte x glioma cell hybrids proliferating in vitro

Abstract In order to generate cell lines that grow continuously in tissue culture and that express the biochemical properties of oligodendrocytes (the cells which produce myelin in the central nervous system), we isolated oligodendrocytes from calf brain and fused them with C6 rat glioma cells. Of the 60 hybrid clones tested, several expressed oligodendroglial properties at levels comparable to isolated oligodendrocytes. In particular, hybrid clone CO-13-7 showed a high level of expression of all six oligodendroglial properties tested: 2′ : 3′-cyclic nucleotide 3′-phosphohydroiase, glycero-3-phosphate dehydrogenase, and induction of GPDH by hydrocortisone, all of which were also found in C6 cells and in oligodendrocytes; and galactocerebroside, sulfatide, and myelin basic protein, which were found in normal oligodendrocytes but not in C6 glioma cells. Therefore, the hybrids express a spectrum of oligodendrocyte biochemical properties that is not found in any other cell line that can be maintained continuously in tissue culture.

Induction of Myelin Components: Cyclic AMP Increases the Synthesis Rate of 2′,3′-Cyclic Nucleotide 3′-Phosphohydrolase in C6 Glioma Cells

Abstract: In an effort to determine the factors that stimulate myelin synthesis, we investigated the mechanism by which dibutyryl cyclic AMP induces the activity of the myelin enzyme, 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase (CNP; EC 3.1.4.37), in C6 glioma cells. Immunotitration experiments and measurements of the accumulation of [35S]methionine‐labeled CNP showed that dibutyryl cyclic AMP increased the amount of CNP in the cells but not the catalytic activity per molecule of the enzyme. Moreover, inhibition of protein synthesis with cycloheximide abolished induction of enzyme activity. Dibutyryl cyclic AMP doubled the rate of CNP synthesis but had no effect on the half‐life of the enzyme (∼33 h). The induction was partially blocked by the inhibitors of mRNA synthesis, cordycepin or α‐amanitin. Thus, cyclic AMP induces the synthesis of CNP.

Expression of insulin-like growth factor-binding protein messenger RNAs in developing rat oligodendrocytes and astrocytes

Insulin‐like growth factors, IGF‐I and IGF‐II, are potent regulators of oligodendrocyte development. Most of the IGF present in vivo is bound to members of a family of six high‐affinity IGF‐binding proteins (IGFBPs), which can either potentiate or inhibit IGF action, depending on other conditions. Additionally, serum contains a structurally unrelated protein, acid‐labile sub‐unit (ALS), which forms a ternary complex with IGF and IGFBP3. In this study, we used reverse‐transcriptase polymerase chain reaction (RT‐PCR) to examine the expression of mRNAs for IGFBP 1–6 and ALS in purified populations of oligodendroglial cells and astrocytes. We found that astrocytes express all six IGFBPs. A2B5+/O4− oligodendrocyte precursors, O4+/O1− intermediate precursors, and O1+ oligodendrocytes express IGFBP3, 5, and 6, while IGFBP4 is expressed in oligodendrocyte precursors but not at more mature stages. We were unable to detect ALS mRNA in whole brain or in cultured oligodendroglial cells. The presence of differentially expressed IGFBPs in developing oligodendrocytes and astrocytes could significantly affect the biological activity of IGF‐I and IGF‐II in the central nervous system and the IGF‐responsiveness of the IGFBP‐expressing cells. J. Neurosci. Res. 50:721–728, 1997. © 1997 Wiley‐Liss, Inc.

Differential expression of 2′∶3′-cyclic nucleotide 3′-phosphodiesterase in cultured central, peripheral, and extraneural cells

The relative levels of the central nervous system myelin marker enzyme 2′:3′-cyclic nucleotide 3′-phosphodiesterase (EC 3.1.4.37, CNPase) were determined in neuroblastoma, astrocyte, oligodendrocyte and Schwann cell cultures and in freshly isolated human lymphocytes and platelets. The highest specific activities were associated with the cells that elaborate myelin membrane in the central and peripheral nervous system, oligodendrocytes and Schwann cells, respectively. Antiserum to bovine CNPase recognized both CNP1 and CNP2 in CNS myelin and human oligodendroglioma. In addition, a 53,000 dalton protein was evident on autoradiographs of immunoblotted PNS myelin and human oligodendroglioma proteins. Cultured rat oligodendrocyte, C6 and mouse NA neuroblastoma CNPase appear to share common determinants with the corresponding normal rat CNS enzyme.