Partial purification of a pramipexole-induced trophic activity directed at dopamine neurons in ventral mesencephalic cultures

We previously demonstrated that media conditioned by exposure to ventral mesencephalic (VM) cultures in the presence of pramipexole (PPX) and other drugs with dopamine (DA) D₃ properties, increased the growth and survival of DA neurons in recipient VM cultures. This trophic activity was heat-labile and not present in parietal cortex cultures or cultures pretreated with the DA neuron toxin MPP⁺. In an effort to begin to identify the protein(s) responsible for this trophic effect, we compared the conditioned media from normal VM cultures, VM cultures incubated with PPX, and VM cultures pretreated with MPP⁺ and treated with PPX. Neutralization studies using anti-GDNF and anti-BDNF failed to reduce the conditioned media transfer effect, and Millipore Ultrafree centrifugation studies placed the mol.wt. of the activity around 30 kDa. SDS separation revealed three potential bands of interest. A 35-kDa band was present in normal cultures, increased in PPX-incubated cultures, and absent in MPP⁺-pretreated/PPX-incubated cultures. This conforms to the effect the protein concentrates used to produce these gels had on the growth of DA neurons in VM cultures. Since VM cultures grown in neural basal media, which inhibits the growth of glia, still responded to PPX in a dose-dependent fashion, the trophic activity may be a DA autotrophic factor. However, the gels also revealed two bands at 31 and 55 kDa that were reduced by exposure to PPX and present in MPP⁺-pretreated cultures. The possibility that these are neuroinhibitory factors that are also regulated by PPX therefore cannot be ruled out.