Pharmacological manipulation of rhodopsin retinitis pigmentosa.

Mutations in rhodopsin cause autosomal dominant retinitis pigmentosa. The majority of these mutations (class II) lead to protein misfolding. The misfolded protein is retained in the ER then retrotranslocated into the cytoplasm for degradation by the proteasome. If degradation fails, the protein can aggregate to form intracellular inclusions. In addition, the mutant rod opsin exerts a dominant negative effect on the wild-type protein. Here, we review these pathways and how different drug treatments can affect mutant rod opsin. Interestingly, drugs targeted at general protein stability (kosmotropes) or improving the cellular folding and degradation machinery (molecular chaperone inducers and autophagy induction) reduced P23H rod opsin aggregation and inclusion formation together with associated caspase activation and cell death, but did not enhance mutant protein processing or reduce the dominant negative effects. In contrast, pharmacological chaperones (retinoids) enhanced P23H folding and reduced the dominant negative effects, as well as reducing the other gains of function. Therefore, targeting the toxic gain of function did not require improved folding, whereas reducing the dominant negative effects required improved folding. These studies suggest that some forms of rhodopsin retinitis pigmentosa could be treated by targeting protein folding and/or reducing protein aggregation.

Pax6p46 binds chromosomes in the pericentromeric region and induces a mitosis defect when overexpressed.

PURPOSE: Haploinsufficiency and overexpression of the Pax6 gene are responsible for defective central nervous system development. The purpose of the current work was to identify and characterize a new potential role for the Pax6 transcription factor in cellular proliferation in addition to its role at the level of gene expression. METHODS: Expression vectors encoding tagged Pax6p46 protein were used to observe directly protein localization during the cell cycle in cells lines. Three dimensional (3-D) fluorescence microscopy imaging was used to observe in vivo mitotic progression and chromosome dynamics to define the mitotic step affected by p46DsRed as well as to validate endogenous p46 localization on chromosomes in quail retinal cells. Video imaging was used to identify the precise moment of onset of effects related to p46 overexpression in living cells. A pulldown assay in HEK cells was used to identify a specific partner of p46. RESULTS: Pax6p46 protein in transfected cells is localized on the chromosomes, predominantly in a pericentromeric area, and its localization changes as mitosis progresses. Overexpression of p46 protein induces incomplete chromatid separation, resulting in defective mitosis at the onset of the anaphase. A physical interaction between p46 and ESPL1 was identified. CONCLUSIONS: The results suggest that Pax6 exerts an effect on mitosis through protein-protein interactions with proteins localized on chromosomes. Supported by the observation that p46 interacts with separase, an enzyme required for chromatid separation, the authors propose that this interaction is responsible for the mitosis defect observed in cells overexpressing Pax6.

OTX2 activates the molecular network underlying retina pigment epithelium differentiation.

The retina pigment epithelium (RPE) is fundamental for the development and function of the vertebrate eye. Molecularly, the presumptive RPE can be identified by the early expression of two transcription factors, Mitf and Otx. In mice deficient for either gene, RPE development is impaired with loss of melanogenic gene expression, raising the possibility that in the eye OTX proteins operate either in a feedback loop or in cooperation with MITF for the control of RPE-specific gene expression. Here we show that Otx2 induces a pigmented phenotype when overexpressed in avian neural retina cells. In addition, OTX2 binds specifically to a bicoid motif present in the promoter regions of three Mitf target genes, QNR71, TRP-1, and tyrosinase, leading to their transactivation. OTX2 and MITF co-localize in the nuclei of RPE cells and physically interact, and their co-expression results in a cooperative activation of QNR71 and tyrosinase promoters. Collectively, these data suggest that both transcription factors operate at the same hierarchical level to establish the identity of the RPE.