STIL: a multi-function protein required for dopaminergic neural proliferation, protection, and regeneration.

Degeneration of dopaminergic (DA) neurons in the brain is the major cause for Parkinson's disease (PD). While genetic loci and cellular pathways involved in DA neuron proliferation have been well documented, the genetic and molecular and cellular basis of DA cell survival remains to be elucidated. Recently, studies aimed to uncover the mechanisms of DA neural protection and regeneration have been reported. One of the most recent discoveries, i.e., multi-function of human oncogene SCL/TAL interrupting locus (Stil) in DA cell proliferation, neural protection, and regeneration, created a new field for studying DA cells and possible treatment of PD. In DA neurons, Stil functions through the Sonic hedgehog (Shh) pathway by releasing the inhibition of SUFU to GLI1, and thereby enhances Shh-target gene transcription required for neural proliferation, protection, and regeneration. In this review article, we will highlight some of the new findings from researches relate to Stil in DA cells using zebrafish models and cultured mammalian PC12 cells. The findings may provide the proof-of-concept for the development of Stil as a tool for diagnosis and/or treatment of human diseases, particularly those caused by DA neural degeneration.

A novel function of the human oncogene Stil: Regulation of PC12 cell toxic susceptibility through the Shh pathway.

The human oncogene SCL/TAL1 interrupting locus (Stil) is highly conserved in vertebrate species. Here, we report new findings of Stil in the regulation of toxic susceptibility in mammalian dopaminergic (DA)-like PC12 cells. RNAi-mediated knockdown of Stil expression did not affect the survival of proliferating PC12 cells but caused a significant amount of cell death in differentiated neurons after toxic drug treatment. In contrast, overexpression of Stil increased toxic susceptibility only in proliferating cells but produced no effect in mature neurons. Exogenetic inactivation or activation of the Sonic hedgehog (Shh) signaling transduction mimicked the effect of Stil knockdown or overexpression in regulation of PC12 cell toxic susceptibility, suggesting that Stil exerts its role through the Shh pathway. Together, the data provide evidence for novel functions of the human oncogene Stil in neural toxic susceptibility.

Characterization of the human oncogene SCL/TAL1 interrupting locus (Stil) mediated Sonic hedgehog (Shh) signaling transduction in proliferating mammalian dopaminergic neurons.

The human oncogene SCL/TAL1 interrupting locus (Stil) is highly conserved in all vertebrate species. In humans, the expression of Stil is involved in cancer cell survival, apoptosis and proliferation. In this research, we investigated the roles of Stil expression in cell proliferation of mammalian dopaminergic (DA) PC12 cells. Stil functions through the Sonic hedgehog (Shh) signal transduction pathway. Co-immunoprecipitation tests revealed that STIL interacts with Shh downstream components, which include SUFU and GLI1. By examining the expression of Stil, Gli1, CyclinD2 (cell-cycle marker) and PCNA (proliferating cell nuclear antigen), we found that up-regulation of Stil expression (transfection with overexpression plasmids) increased Shh signaling transduction and PC12 cell proliferation, whereas down-regulation of Stil expression (by shRNA) inhibited Shh signaling transduction, and thereby decreased PC12 cell proliferation. Transient transfection of PC12 cells with Stil knockdown or overexpression plasmids did not affect PC12 cell neural differentiation, further indicating the specific roles of Stil in cell proliferation. The results from this research suggest that Stil may serve as a bio-marker for neurological diseases involved in DA neurons, such as Parkinson's disease.

Transcription of the SCL/TAL1 interrupting Locus (Stil) is required for cell proliferation in adult Zebrafish Retinas.

The human oncogene SCL/TAL1 interrupting locus (Stil) is highly conserved in vertebrate species. Previously, we identified a homolog of the Stil gene in zebrafish mutant (night blindness b, nbb), which showed neural defects in the retina (e.g. dopaminergic cell degeneration and/or lack of regeneration). In this research, we examined the roles of Stil in cell proliferation after degeneration in adult zebrafish retinas. We demonstrated that knockdown of Stil gene expression or inhibition of Sonic hedgehog (Shh) signaling transduction decreases the rate of cell proliferation. In contrast, activation of Shh signal transduction promotes cell proliferation. In nbb(+/-) retinas, inhibition of SUFU (a repressor in the Shh pathway) rescues the defects in cell proliferation due to down-regulation of Stil gene expression. The latter data suggest that Stil play a role in cell proliferation through the Shh signal transduction pathway.

The human oncogene SCL/TAL1 interrupting locus is required for mammalian dopaminergic cell proliferation through the Sonic hedgehog pathway.

The human oncogene SCL/TAL1 interrupting locus (Stil) is highly conserved in all vertebrate species. In humans, the expression of Stil regulates cancer cell proliferation and survival. In this study, we examined the function of Stil in neural progenitor cell proliferation and neural differentiation using the mammalian dopaminergic (DA) PC12 cells. Stil is expressed in both proliferating and differentiated PC12 cells. The RNAi-mediated knockdown of Stil expression yielded a decreased proliferation rate of PC12 cells, whereas the overexpression of Stil transcript increased PC12 cell proliferation. The up- and down-regulation of the Sonic hedgehog (Shh) pathway by pharmacological approaches targeting Smoothened (Smo) demonstrated that Stil functions in the Shh pathway for PC12 proliferation. Smo antagonist cyclopamine decreased the proliferation rate of PC12 cells, whereas the overexpression of Stil rescued the cyclopamine-induced decrease in cell proliferation. Oppositely, the application of Smo agonist purmorphamine increased the rate of PC12 cell proliferation. However, the proliferation defect caused by Stil knockdown remained evident after activating the Shh pathway by purmorphamine. The expression of Stil is not required for PC12 cell neural differentiation. In PC12 cells transfected with Stil shRNA plasmids, the outgrowth of neurites persisted after treatment with nerve growth factor (NGF), whereas overexpression of Stil did not increase neurite growth in response to NGF induction. Together, the results from this study suggest a novel role for the oncogene Stil in neural progenitor cells through the Shh pathway, and further introduces Stil as a bio-marker for DA cells.

Functional expression of SCL/TAL1 interrupting locus (Stil) protects retinal dopaminergic cells from neurotoxin-induced degeneration.

We previously isolated a dominant mutation, night blindness b (nbb), which causes a late onset of retinal dopaminergic cell degeneration in zebrafish. In this study, we cloned the zebrafish nbb locus. Sequencing results revealed that nbb is a homolog of the vertebrate SCL/TAL1 interrupting locus (Stil). The Stil gene has been shown to play important roles in the regulation of vertebrate embryonic neural development and human cancer cell proliferation. In this study, we demonstrate that functional expression of Stil is also required for neural survival. In zebrafish, decreased expression of Stil resulted in increased toxic susceptibility of retinal dopaminergic cells to 6-hydroxydopamine. Increases in Stil-mediated Shh signaling transduction (i.e. by knocking down the Shh repressor Sufu) prevented dopaminergic cell death induced by neurotoxic insult. The data suggest that the oncogene Stil also plays important roles in neural protection.

CLOCK is required for maintaining the circadian rhythms of Opsin mRNA expression in photoreceptor cells.

In zebrafish, the expression of long-wavelength cone (LC) opsin mRNA fluctuated rhythmically between the day and night. In a 24-h period, expression was high in the afternoon and low in the early morning. This pattern of fluctuation persisted in zebrafish that were kept in constant darkness, suggesting an involvement of circadian clocks. Functional expression of Clock, a circadian clock gene that contributes to the central circadian pacemaker, was found to play an important role in maintaining the circadian rhythms of LC opsin mRNA expression. In zebrafish embryos, in which the translation of Clock was inhibited by anti-Clock morpholinos, the circadian rhythms of LC opsin mRNA expression diminished. CLOCK may regulate the circadian rhythms of LC opsin mRNA expression via cyclic adenosine monophosphate (cAMP)-dependent signaling pathways. In control retinas, the concentration of cAMP was high in the early morning and low in the remainder of the day and night. Inhibition of Clock translation abolished the fluctuation in the concentration of cAMP, thereby diminishing the circadian rhythms of opsin mRNA expression. Transient increase of cAMP concentrations in the early morning (i.e. by treating the embryos with 8-bromo-cAMP) restored the circadian rhythms of LC opsin mRNA expression in morpholino-treated embryos. Together, the data suggest that Clock plays important roles in regulating the circadian rhythms in photoreceptor cells.