EVIDENCE FOR A NOVEL NEUROTROPHIC FACTOR FOR DOPAMINERGIC-NEURONS SECRETED FROM MESENCEPHALIC GLIAL-CELL LINES

Our previous studies have shown that primary mesencephalic glia secret e factors that promote dopaminergic cell survival and differentiation in vitro, To obtain enough starting material to identify the neurotrop hic activity, embryonic day (E) 14.5 rat mesencephalic glia were stimu lated with acidic fibroblast growth factor to increase number of cells , These cells were replated in the absence of neurons and immortalized by transfection with the SV 40 large T-antigen, Clonal cell lines wer e established and characterized for immunoreactivity (IR) to various g lial and non-glial markers, Media conditioned by these cell lines were tested for survival-promoting effects on dopaminergic neurons in seru m-free cultures of the dissociated E14.5 rat mesencephalon. All cell l ines expressed IR for the astrocytic marker, GFAP, the oligodendroglia l marker, CNP, and for A2B5, a marker for 0-2A progenitor cells, but w ere negative for the neuronal marker, microtubule associated protein-2 , and the fibroblast marker, fibronectin, Moreover, treatment of serum -free cultures of the dissociated E14.5 mesencephalon with glial cell line-conditioned medium (CM) delayed dopaminergic cell death in a dose -dependent manner, resulting in a maximal twofold to sixfold increase in the number of surviving tyrosine hydroxylase-IR neurons at various days in vitro, This increase in dopaminergic cell survival was not mim icked by GDNF, BDNF or NT-3 within the initial 3 days of cultivation, Moreover, initial biochemical characterization demonstrated that the n eurotrophic activity is restricted to the high MW fraction of > 50 kD of glial cell line-CM, Since the apparent MW of this factor exceeds th e size of most known growth factors, it may represent a novel dopamine rgic neurotrophic factor. (C) 1996 Wiley-Liss, Inc.