Characterization of the mitotic Stil gene in dopaminergic cell survival

The study of Parkinson's disease (PD) has mainly included mammalian models treated with neurotoxins that selectively destroy dopaminergic (DA) cells. We previously isolated the zebrafish night blindness b (nbb) mutation, which causes a progressive loss of DA cells in the heterozygous adult retina and small eyes with disorganized retinas in the embryonic lethal homozygous mutants. In this study, brain DA neurons that were comparable to the human nigral striatal system affected in PD were examined in aged heterozygous nbb mutants. Variation in the number of TH-positive cell numbers was seen in the diencephalic posterior tuberculum in both aged wild-type and mutant fish, but no significant overall decreases in average cell number were found in the mutants. Identification of individual mutant fish with cell numbers lower than wild-type range or with higher frequency of low cell count may imply incomplete gene penetrance. Previous cloning of the nbb locus revealed an intron-splicing point mutation in the SCL/TAL1 interrupting locus (Stil). Complementation tests with nbb and another zebrafish Stil mutant, cassiopeia (cspcz65), showed that the two mutant alleles did not complement.

STIL is involved in embryonic development and cancer cell proliferation. It serves dual functions as a binding partner to SUFU in the SHH pathway, and as a checkpoint protein in spindle pole formation during mitotic entry. To further evaluate novel mechanisms of STIL in DA cells, the catecholaminergic mammalian PC12 line was used to examine its role in DA proliferation, differentiation and survival. It was hypothesized that STIL would have a multi-functional role in PC12 cells. RNAi knockdown of STIL yielded a decreased proliferation rate, whereas overexpression of the human full-length STIL transcript increased proliferation. The differentiation of PC12 cells by NGF stimulated STIL expression in low-serum conditions. Knockdown or over-expression of STIL did not affect neurite extension during differentiation. In cell survival assays with 6-OHDA, , knockdown of STIL did not affect toxin sensitivity in undifferentiated cells, but over-expression of STIL augmented toxin sensitivity. In NGF-induced cells, STIL knockdown increased drug sensitivity, whereas over-expression of STIL did not have an effect.

The up- and down-regulation of the SHH pathway by pharmacological approaches (e.g., cyclopamine and purmorphamine) demonstrated that STIL may function in this pathway for PC12 proliferation. Results from this study suggests a novel role for the oncogene STIL in progenitor cell proliferation and survival of differentiated cells, and further introduces STIL as a genetic bio-marker for catecholiminergic (e.g., DA) cells.