Phosphorylation and lipid raft association of fibroblast growth factor receptor-2 in oligodendrocytes.

Fibroblast growth factors (FGFs) and their receptors (FGFRs) initiate diverse cellular responses that contribute to the regulation of oligodendrocyte (OL) function. To understand the mechanisms by which FGFRs elicit these cellular responses, we investigated the phosphorylation of signal transduction proteins and the role of cholesterol-glycosphingolipid-enriched "lipid raft" microdomains in differentiated OLs. Surprisingly, we found that the most abundant tyrosine-phosphorylated protein in OLs was the 120-kd isoform of FGFR2 and that it was phosphorylated even in the absence of FGF2, suggesting a potential ligand-independent function for this receptor. Furthermore, FGFR2, but not FGFR1, was associated with lipid raft microdomains in OLs and myelin (but not in astrocytes). This provides the first evidence for the association of FGFR with TX-100-insoluble lipid raft fractions. FGFR2 phosphorylated the key downstream target, FRS2 in OLs. Raft disruption resulted in loss of phosphorylated FRS2 from lipid rafts, coupled with the loss of Akt but not of Mek or Erk phosphorylation. This suggests that FGFR2-FRS2 signaling in lipid rafts operates via the PI3-Kinase/Akt pathway rather than the Ras/Mek/Erk pathway, emphasizing the importance of microenvironments within the cell membrane. Also present in lipid rafts in OLs and myelin, but not in astrocytes, was a novel 52-kd isoform of FGFR2 that lacked the extracellular ligand-binding region. These results demonstrate that FGFR2 in OLs and myelin possess unique characteristics that are specific both to receptor type and to OLs and provide a novel mechanism to elicit distinct cellular responses that mediate both FGF-dependent and -independent functions.

Myelin associated glycoprotein cross-linking triggers its partitioning into lipid rafts, specific signaling events and cytoskeletal rearrangements in oligodendrocytes.

Myelin-associated glycoprotein (MAG) has been implicated in inhibition of nerve regeneration in the CNS. This results from interactions between MAG and the Nogo receptor and gangliosides on the apposing axon, which generates intracellular inhibitory signals in the neuron. However, because myelin-axon signaling is bidirectional, we undertook an analysis of potential MAG-activated signaling in oligodendrocytes (OLs). In this study, we show that antibody cross-linking of MAG on the surface of OLs (to mimic axonal binding) leads to the redistribution of MAG into detergent (TX-100)-insoluble complexes, hyperphosphorylation of Fyn, dephosphorylation of serine and threonine residues in specific proteins, including lactate dehydrogenase and the beta subunit of the trimeric G-protein-complex, and cleavage of alpha-fodrin followed by a transient depolymerization of actin. We propose that these changes are part of a signaling cascade in OLs associated with MAG function as a mediator of axon-glial communication which might have implications for the mutual regulation of the formation and stability of axons and myelin.

Antibody cross-linking of myelin oligodendrocyte glycoprotein leads to its rapid repartitioning into detergent-insoluble fractions, and altered protein phosphorylation and cell morphology.

Myelin oligodendrocyte glycoprotein (MOG) is, quantitatively, a relatively minor component of the myelin membrane. Nevertheless, peritoneal administration of MOG evokes potent cellular and humoral immunoreactivity, resulting in an experimental allergic encephalitis with immunopathology similar to multiple sclerosis. Moreover, antibodies against MOG cause myelin destruction in situ. Therefore, it appears that MOG-related demyelination is dependent on anti-MOG antibody, but the mechanism(s) by which it occurs is unclear. Of potential significance are observations that some proteins are selectively partitioned into specialized plasma membrane microdomains rich in glycosphingolipids and cholesterol ("lipid rafts"). In particular, during ligand or antibody cross-linking, various plasma membrane receptors undergo enhanced partitioning into rafts as an obligatory first step toward participation in early signal transduction events. In contrast to mature myelin, in oligodendrocytes (OLs) in culture MOG is not raft associated [Triton X-100 (TX-100) soluble, 4 degrees C]. However, in this study we show that antibody cross-linking (anti-MOG plus secondary antibody) of MOG on the surface of OLs results in the repartitioning of approximately 95% of MOG into the TX-100-insoluble fraction. This repartitioning of MOG is rapid (

Changes in the expression of cytoskeletal proteins in the CNS of transferrin transgenic mice.

Apotransferrin injected intracranially into young rats has been shown in our laboratories to induce an early differentiation of oligodendroglial cells and an increased deposition of myelin. The expression of some myelin-specific proteins such as myelin basic protein (MBP) and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) and of their mRNAs were significantly increased in these animals. Also, in the cytoskeleton obtained from isolated myelin, it was found that several microtubule associated proteins (MAPs), particularly the stable tubule only peptide (STOP) and MAP 1B, as well as actin and tubulin were markedly increased. In the present paper, we compare the changes in expression of brain and myelin cytoskeletal proteins in a newly generated transferrin transgenic mouse (Tg), overexpressing the human transferrin gene, with the results obtained in aTf-injected rats. In the myelin cytoskeletal fraction of Tg mice there was a significant increase in the expression of MBP, tubulin, tau and STOP, similarly to what was previously found in the aTf-injected rats. Immunohistochemical studies showed that a variance with what occurs in the aTf-injected model, in which the above mentioned changes were limited to the corpus callosum, in the Tg mice the changes in expression of cytoskeletal proteins were observed in the various anatomical areas studied such as cerebral cortex, brain stem and cerebellum. There was also an increased expression of neurofilaments in the Tg animals, in contrast with results obtained in aTf-injected rats, suggesting that in the Tg mice, the continuous overexpression of Tf might also induce some neuronal changes. Changes in tau, total and acetylated tubulin and MAP 1B were observed in both neurons and OLGc. The increase in STOP was more significant in OLGc while the changes in MAP2 were exclusively found in neurons.

Anxiolytic-like behavior in rats is induced by the neonatal intracranial injection of apotransferrin.

To determine whether neonatal intracranial injection of apotransferrin (aTf), which increases myelin deposition, has behavioral effects in rats, 3-day-old rats were intracranially injected with 350 ng of aTf and tested at 25 and 60 days of age. An anxiolytic-like behavior was observed in aTf-treated rats, evidenced by an increase in the exploration of open arms in the plus maze test without changes in the locomotor activity. This behavioral profile persists until adulthood. Intraperitoneal injection of 0.75 mg/kg of picrotoxin, a GABA(A) receptor channel antagonist, abolished this anxiolytic-like behavior, indicating that neonatal aTf induces a long-lasting increase in GABA(A) receptor functionality.

Relationship between the ubiquitin-dependent pathway and apoptosis in different cells of the central nervous system: effect of thyroid hormones.

We have recently shown that sustained neonatal hyperthyroidism in the rat activates apoptosis of oligodendroglial cells (OLGc) and that inhibition of the proteasome-ubiquitin (Ub) pathway by lactacystin produces increased apoptosis in cerebellar granule cells (CGC). In the present study we have analyzed the relationship between the activation of the Ub-dependent pathway, the expression of the Ub genes and programmed cell death in neurons of the rat cerebellum and cerebral cortex and in OLGc. This study was carried out in normal animals, in rats submitted to sustained neonatal hyperthyroidism and in cell cultures treated with an excess of thyroid hormones. In neurons of the cerebral cortex, thyroid hormone produces an increase of Ub-protein conjugates, an enhancement in the expression of the Ub genes and an increase in apoptosis, while the opposite results are obtained in CGC. These results indicate that in neurons, the changes in the cell death program produced by thyroid hormone run in parallel with those occurring in the Ub-dependent pathway. In OLGc, thyroid hormone increases apoptosis but does not produce changes in the Ub pathway. Preliminary studies indicate that in coincidence with what occurs in optic nerves, the sciatic nerves both in controls and in hyperthyroid animals are unable to form Ub-protein conjugates. These results indicate that in cells of the CNS such as neurons, in which the Ub-dependent pathway is actively expressed, it appears to be closely correlated with apoptosis.

Oligodendroglial cell differentiation in rat brain is accelerated by the intracranial injection of apotransferrin.

In the present paper we first studied the brain distribution and the time and dose dependent effects of apotransferrin, after its intracranial injection into young rats and at different post-natal ages. Its action upon the transferrin receptor (TfR) and upon the expression of brain transferrin, as well as its effect on the proliferation and differentiation of oligodendroglial cells (OLGc) was one of the main objectives of our investigation. Total DNA and BrdU labeling, as an index of cellularity and proliferation, respectively, were the same in the control and experimental groups of rats. A significant increase in the MBP+ and CA II+ OLGc, and a decrease in the more immature (A2B5+) OLGc were found in the aTf injected rats. At 10 and 17 days of age, Tf-mRNA decreased to around 20% of the amount present in control animals. The TfR-mRNA in the animals receiving a single dose of aTf at 3 days of age showed an increase in its expression at 10 and 17 days of age, coincident with a higher immunoreactivity of the TfR itself of neurons, choroid plexus and brain capillaries in different brain areas. Although TfR+ OLGc were present up to 7 days of age in controls and in the Tf injected rats, no positive cells were observed at 17 days of age, even in the aTf injected rats. Our results give support to the hypothesis that aTf is an important factor necessary for the maturation of the OLGc, and that the effects that it produces in the OLGc-myelin unit after its intracranial injection in young rats are not due to an increase in proliferation, but to an accelerated differentiation of Tf-sensitive OLGc.

Sustained neonatal hyperthyroidism in the rat affects myelination in the central nervous system.

We have carried out a study of the effects of sustained neonatal hyperthyroidism on myelin and on the oligodendroglial cells, in an effort to obtain further insight into the molecular mechanisms underlying the action of thyroid hormones on the central nervous system (CNS). Expression of the mRNAs of myelin basic protein (MBP) myelin proteolipid protein (PLP), 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), transferrin, and c-Jun was investigated in 10- and 17-day-old normal and hyperthyroid rats, using Northern blot analysis. At 10 days of age, the levels of all the explored mRNAs were markedly higher in the experimental animals. The mRNA of transferrin showed a ninefold increase over control values, suggesting the possibility that this putative trophic factor might act as one of the mediators in the action of thyroid hormones. At 17 days of age on the other hand, the levels of all the mRNAs decreased markedly, reaching values below control, except for c-Jun, which remained higher than in normals. At 70 days of age, hyperthyroid rats showed clear evidence of myelin deficit, in agreement with previous results of our laboratories (Pasquini et al.: J Neurochem 57: Suppl S124, 1991). Immunocytochemistry of 70-day-old rat brain tissue sections showed a substantial reduction in the amount of MBP-reacting structures and a marked decrease in the number of oligodendroglial cells. Although the above-mentioned results could be the consequence, as proposed by Barres et al. (Development 120:1097-1108, 1994) and Baas et al. (Glia 19:324-332, 1997) of a premature arrest in oligodendroglial cell proliferation followed by early differentiation, the persistent high levels of expression of c-Jun, together with the dramatic decrease in the number of oligodendrocytes, suggested the possibility that prolonged hyperthyroidism could activate apoptotic mechanisms in the myelin forming cells. Using propidium iodide-labeled isolated oligodendroglial cells, we found, by flow cytometry, a significant increase in the number of apoptotic/hypo-diploid propidium iodide-positive cells. These results indicate that one of the actions of sustained levels of thyroid hormones in the neonate rat is to increase oligodendroglial cell death by apoptosis.