CNS integrins switch growth factor signalling to promote target-dependent survival.

Depending on the stage of development, a growth factor can mediate cell proliferation, survival or differentiation. The interaction of cell-surface integrins with extracellular matrix ligands can regulate growth factor responses and thus may influence the effect mediated by the growth factor. Here we show, by using mice lacking the alpha(6) integrin receptor for laminins, that myelin-forming oligodendrocytes activate an integrin-regulated switch in survival signalling when they contact axonal laminins. This switch alters survival signalling mediated by neuregulin from dependence on the phosphatidylinositol-3-OH kinase (PI(3)K) pathway to dependence on the mitogen-activated kinase pathway. The consequent enhanced survival provides a mechanism for target-dependent selection during development of the central nervous system. This integrin-regulated switch reverses the capacity of neuregulin to inhibit the differentiation of precursors, thereby explaining how neuregulin subsequently promotes differentiation and survival in myelinating oligodendrocytes. Our results provide a general mechanism by which growth factors can exert apparently contradictory effects at different stages of development in individual cell lineages.

Control of cell survival and proliferation of postnatal PSA-NCAM(+) progenitors.

In the present work, we studied the effects of several growth factors on survival and proliferation of freshly isolated neural progenitors expressing the polysialylated form of neural cell adhesion molecule (PSA-NCAM). Cells were obtained from postnatal day 2 rat forebrain, using isolation method. We found that (1) insulin-like growth factor 1 (IGF-1) exerts a powerful survival effect by inhibiting apoptotic cell death, (2) epidermal growth factor (EGF) strongly increases cell proliferation, (3) the combination of IGF-1 plus EGF promotes cellular expansion, (4) basic fibroblast growth factor displays only a weak mitogenic effect, and (5) platelet-derived growth factor-AA (PDGF-AA) has no effect on cell survival and proliferation. These results suggest that the postnatal PSA-NCAM(+) progenitors characterized in the present work may represent a transitional stage, between the embryonic EGF-responsive neural progenitors and the postnatal PSA-NCAM(+) progenitors already described that are PDGF-responsive. For these "early PSA-NCAM(+) progenitors," insulin-like growth factor 1 and EGF seem to play a pivotal role in the control of cell death and cell proliferation.

Fibroblast growth factor-2 (FGF-2) and platelet-derived growth factor AB (PDGF AB) promote adult SVZ-derived oligodendrogenesis in vivo.

The capacity of multipotential progenitor cells of the adult mammalian forebrain to generate myelin-forming oligodendrocytes was tested by grafting fragments of different regions of the subventricular zone (SVZ) of the lateral ventricle and the striatum of 6-month-old wild-type mice into the brain of neonate shiverer and wild-type mice. Without growth factor treatment, only few cells of the rostral SVZ survived and formed myelin after engraftment. Treating donors prior to transplantation with a single intraperitoneal injection of epidermal growth factor, basic fibroblast growth factor 2 (FGF-2), and platelet-derived growth factor AB (PDGF(AB)) vigorously promoted the survival, migration, and differentiation of the grafted SVZ cells into myelin-forming oligodendrocytes. In situ, both growth factors expanded the constitutively proliferative PSA-NCAM+ population and favored their differentiation toward the neuronal and oligodendroglial cell fate. The adult central nervous system thus harbors a focal reservoir of FGF-2 and PDGF(AB)-responsive cells which are able to generate substantial amounts of myelin-forming oligodendrocytes in vivo, opening a new prospective area for therapy in demyelinating diseases.

3alpha,5alpha-Tetrahydroprogesterone (allopregnanolone) and gamma-aminobutyric acid: autocrine/paracrine interactions in the control of neonatal PSA-NCAM+ progenitor proliferation.

The earliest identified neonatal neural progenitors are cells that express the polysialylated form of the neural cell adhesion molecule (PSA-NCAM). One of these progenitors is the early PSA-NCAM+ progenitor (ePSA-NCAM+ progenitor; Gago et al. [2003] Mol Cell Neurosci 22:162-178), which corresponds to a multipotential cell with a default differentiation through glial lineages. The ePSA-NCAM+ progenitor can synthesize the neurosteroid progesterone (PROG) and its reduced metabolite 3alpha,5alpha-tetrahydroprogesterone (3alpha,5alpha-THP, or allopregnanolone; Gago et al. [ 2001] Glia 36:295-308). The latter is a potent positive allosteric modulator of gamma-aminobutyric acid type A (GABAA) receptors. In the present work, we demonstrate that PROG and 3alpha,5alpha-THP both stimulate ePSA-NCAM+ progenitor proliferation. PROG exerted its mitogenic effect indirectly, through its conversion to 3alpha,5alpha-THP, since it could be abolished by an inhibitor of the 5alpha-reductase (L685-273) and mimicked by 3alpha,5alpha-THP. A dose-response curve revealed a bell-shaped effect of 3alpha,5alpha-THP on ePSA-NCAM+ progenitor proliferation, with greatest stimulation at nanomolar concentrations. The mitogenic effect of 3 alpha,5 alpha-THP was mediated by GABAA receptors, insofar as it could be blocked by the selective antagonist bicuculline. ePSA-NCAM+ progenitors indeed expressed mRNAs for GABAA receptor subunits, and GABA enhanced cell proliferation, an effect that was also bicuculline sensitive. Moreover, these cells synthesized GABA, which was involved in a tonic stimulation of their proliferation. These results reveal complex autocrine/paracrine loops in the control of ePSA-NCAM+ progenitor proliferation, involving both neurosteroid and GABA signaling, and suggest a novel key role for 3alpha,5alpha-THP in the development of the nervous system.

Inducible expression of FGF2 by a rat oligodendrocyte precursor cell line promotes CNS myelination in vitro.

Transplantation of glial cells into the central nervous system (CNS) may be a promising approach for the treatment of myelin disorders such as multiple sclerosis (MS). Myelination by transplantation of oligodendrocyte precursors has been obtained in different animal models of demyelination. A strategy to favor CNS remyelination is to enrich the lesioned areas in growth factors to stimulate the quiescent population of oligodendrocyte precursors. In this context, we have developed a genetically modified CG4 cell line (CG4-FGF2), which are able to release significant amounts of fibroblast growth factor 2 (FGF2) in a controlable fashion in vitro. The data presented here demonstrate that upon induction with Dox, CG4-FGF2 cells retain their capacity to differentiate in vitro. Additionally, we provide evidence that FGF2 release by engineered cells enhance proliferation and migration of cells of the oligodendrocyte lineage without preventing them to differentiate and myelinate axons in vitro.

Osteopontin is upregulated during in vivo demyelination and remyelination and enhances myelin formation in vitro.

We have used in vitro oligodendrocyte differentiation and the in vivo remyelination model, the cuprizone model, to identify genes regulating oligodendrocyte function and remyelination. One of the genes we identified, osteopontin (opn), is a secreted glycoprotein with cytokine-like, chemotactic, and anti-apoptotic properties that contains an Arg-Gly-Asp (RGD) cell adhesion motif-mediating interactions with several integrins. Both microglia and astrocytes in demyelinating brain regions of cuprizone-fed mice expressed OPN protein. Recombinant OPN protein produced in a baculovirus expression system induced proliferation of both the rat CG-4 and the mouse Oli-neu oligodendrocyte precursor (OLP)-like cell lines in a dose-dependent manner. In addition, recombinant OPN treatment stimulated both myelin basic protein (MBP) synthesis and myelin sheath formation in mixed cortical cultures from embryonic mouse brain, an in vitro primary culture model of myelination. Interestingly, myelinating mixed cultures prepared from OPN(-/-) mice contained significantly less MBP compared to wild-type cultures after 17 days in culture. We propose that in the central nervous system, OPN may act as a novel regulator of myelination and remyelination.