The fundamental mechanism that underlies the degeneration of dopaminergic neurons, leading to the neurologic syndrome of Parkinson's disease remains unresolved, despite three decades of intense research. We have focused on glial sources of dopaminergic neurotrophic factors (DNTFs). The oligodendrocyte type-2 astrocyte (0-2A) progenitor cell and type-1 astrocytes (T1-As), proliferated from the striatum and ventral mesencephalon of the E16 rat brain respectively, are two major sources of DNTFs. We prepared 15L of conditioned media from T1-As, and partially- purified two glycosylated proteins of molecular mass 15 and 50 Kd, that retained dopaminergic neurotrophic activity. However, we failed, for technical reasons, to obtain useful sequence data. A major effort is currently being devoted by several leading laboratories, including our own, to testing the following members of the transforming growth factor (TGF) family of compounds as possible DNTFs, and determining their cellular sources: glial cell line-derived neurotrophic factor (GDNF), TGF-beta1, TGF-beta2 and TGF-beta3. Using RT-PCR, we have identified mRNA for GDNF and TGF-beta1, -2 and -3 in T1-As and B49 cells (source of GDNF), and mRNA for TGF-beta1 and -3 in 0-2A progenitors. Our bioassay data on the efficacy of these growth factors on the survival of dopaminergic neurons in culture show that GDNF is effective, TGF-beta1 is ineffective, and TGF-beta2 and -3 may be toxic. These results differ markedly from recent reports suggesting the possible use of members of the GF family to treat neurodegenerative diseases. There is a critical need for a standardized bioassay to test putative DNTFs. We have developed such an assay, based on a precise, microdissection technique, and the use of microisland cultures and imaging methods. We have developed a new and more efficient method for proliferating 0-2A progenitor cells to 95% purity, and avoids contamination with microglia. The 5% of contaminating cells are mainly T1-As. This pure 0-2A cell preparation will facilitate our research on the isolation of putative DNTFs. It will also greatly facilitate research on the biology of oligodendrocyte precursors, particularly paradigms related to remyelinating studies in the CNS. In collaboration with the Laboratory of Cellular and Molecular Neurobiology (LMCN), we have identified a minor ganglioside that is recognized by the A2B5 antibody, and which may be uniquely localized to oligodendrocyte precursors. This finding may provide the basis for the production of an antibody specific for 0-2A progenitor cells of cortical and striatal origin, and CG4 cells.
