This competitive renewal application continues to focus on the search for polypeptide factors regulating growth and survival of differentiating oligodendrocytes (OCs). Dr. Gard has identified two modes of trophic support for cultures of oligodendroblasts purified from postnatal brain by antibody capture. Murine astrocytes specifically release into non-supportive, serumless culture medium a powerful oligodendrogiotrophic stimulus (ACM), polypeptide activity significantly more efficacious than other tested cytokines with OC survival-promoting capacity. The astroglial stimulus effecting long- term OC survival is related immunochemically to leukemia inhibitory factor (LIF) but appears distinct from LIF itself. Hypothesizing that another gp130 transducer-activating cytokine is responsible, LIF homologues produced by astrocytes in addition to astrocyte- derived inflammatory cytokines and neurotrophins will be systematically bioassayed in Aim 1 by Dr. Gard as potential gliotrophins for differentiating OBs, and cultured human astrocytes screened for the release for similar activity.
Aim 2 proposes to purify the LIF-like stimulus from rat astrocyte-conditioned culture medium by FPLC and its subsequent cloning if amino acid sequencing indicates a novel factor.
In Aim 3, the developmental expression of LIF receptors by antigenically distinct OBs and OCs developing in vitro and in vivo will be examined, and their activation by ACM assessed. Cultured OBs deprived of diffusible growth factors can survive differentiation if presented with specific cell adhesion molecules in the immunoglobulin superfamily, the myelin- associated glycoprotein (MAG) and NCAM, which also promote growth by augmenting myelin-like membrano-genesis. The novel concept of cell contact-mediated signaling at the OC surface contributing oligodendrogliotrophic support will be tested in Aim 4 by quantifying OC death occurring (a) naturally in brains of pre-myelinating mice deficient in MAG or its associated non-receptor tyrosine kinase Fyn, and in (b) response to MAG antibody perturbation in vitro and in vivo. Other in vitro studies will characterize the growth, and response to MAG/NCAM, of immunopurified MAG- and Fyn-deficient OBs and begin to address the possible convergence of cytokine-and cell recognition molecule-mediated oligodendrogliotrophic signaling by MAG- and LIF-receptor activation. Dr. Gard's studies are expected to increase knowledge of the changing scope of myelinogenic signals engaged during OC maturation and should provide insight concerning the etiology of dysmyelinating diseases and prospects for trophic factor therapy.
