We have scaled up the production of type-1 astrocytes, and after partial purification based on ion exchange chromatography followed by gel filtration, identified two glycosylated proteins of molecular mass 15 kD and 50 kD that retain activity for promoting survival of fetal, dopaminergic neurons in culture. We will also attempt to purify the potent dopaminergic, neurotrophic factor present in the conditioned medium from O-2A progenitor cells. The O-2A progenitors can be easily expanded in culture in protein-free, defined medium from a relatively small number of E16 striatal cells, using bFGF (10 ng/ml) as the mitogen, versus ventral mesencephalon or cerebral cortex of the same age. A reasonable working hypothesis is that an O-2A-like cell might be present in the striatum, and could be the source of the target (striatum)-derived dopaminergic neurotrophic factor we have identified in the conditioned medium. Regulating production of this putative neurotrophic factor is of considerable physiological importance, since it could provide a mechanistic explanation of the cause of idiopathic Parkinson's disease (PD). We have tested the three major monoamines, all at 1.0 mu M, for their effect on survival of striatal O-2A progenitors in culture. Dopamine (DA) was severely toxic, norepinephrine (NE) had no effect, and 5-hydroxytryptamine (5-HT) caused marked increased survival of O-2A progenitors. We are currently trying to determine if 5-HT is mitogenic, or whether it acts by promoting survival of O-2A progenitors generated under the influence of bFGF. The function of the dense 5-HT innervation of the striatum is presently unknown. Our preliminary results suggest that it might be involved in regulating production of a dopaminergic neurotrophic factor. Dopaminergic neurons are present in the midbrain, diencephalon, olfactory bulb, retina, spinal cord and dorsal root ganglia. Of these, the highly organized nigrostriatal, DA system is the one that is most severely compromised in PD. Idiopathic PD should now be regarded as a neurotrophic deficiency disease caused by reduced production of a target-derived, dopaminergic neurotrophic factor. We have standardized a bioassay method to test dopaminergic neurotrophic activity reliably, based on the use of microcultures and an imaging method. B49 neuroblastoma cells are the source of GDNF, the most potent DA neurotrophic factor isolated to date. Conditioned medium from O-2A progenitors contains much more DA neurotrophic factor than that from type-1 astrocytes which in turn contain more than that from B49 neuroblastoma cells.
