Final Report Summary - RP11MOUSE (Validation of the disease model and therapy for retinitis pigmentosa 11)
With a prevalence of 1 in 3500 retinitis pigmentosa (RP) is a significant cause of blindness in the Western world. RP is a group of hereditary retinal disorders, for which there is no effective treatment. It is mostly caused by mutations in genes related to the biochemistry of photoreceptors. PRPF31, however was the first ubiquitously expressed gene implicated in this disorder. Mutations in this gene are responsible for one of the commoner forms of autosomal dominant RP (RP11) with implications for a significant proportion of patients. Overall it has been noted that patients who manifest the disease present a severe form of retinal degeneration, often losing vision completely by the third decade. With the cloning of PRPF31, a novel class of genes, i. e. splicing factor genes, causing RP was revealed (PRPF3, PRPF8, RP9, SNRNP200 and PRPF6). These six genes are associated with a common splicing complex, U4/U6. U5 snRNP, and they are likely to share the molecular mechanism of the pathology. It has been noted that in families affected by PRPF31 mutations, manifestation of the disease is also dependent on the expression level of the wild-type PRPF31 allele. Thus, high levels of wild-type PRPF31 protein, can override the effect of mutant copy of the PRPF31 gene. This is known as partial penetrance and can be viewed as nature's way of curing the disease. The goal of this project was to establish a mouse model with retinitis pigmentosa phenotype due to the mutations in Prpf31 and the subsequent rescue of this phenotype.
Initially two Prpf31 mouse models were established, a knock-in model harbouring p. A216P change and a knock-out model, where exon 7 of Prpf31 was deleted, which lead to the degradation of the Prpf31 transcript by nonsense mediated decay. Both mouse models were embryonic lethal in the homozygous state and therefore the retinal phenotypes of the heterozygous mice were investigated. These mice have not shown an apparent retinal phenotype up to 18 months of age as investigated by histological sections, examination of the eye fundus and electroretinography, which records electrophysiological responses of the retina. These results were published in a peer-reviewed journal (Bujakowska et al. 2009).
Lack of the clear phenotype in the Prpf31 mice gave rise to a question of why the relatively severe phenotype in human is not reproduced in mice. One of the possible explanations could be that the highly expressing wild-type allele rescues the potential deleterious effect of the mutant allele. It has been hypothesized that as in human subjects, in mice there might be a variation of the expression of the Prpf31 alleles, which can be verified by considering different mouse strains. Using quantitative real-time PCR on cDNA obtained from different mouse strains, we were able to show that there is no significant variation of the Prpf31 expression between the investigated mouse strains.
One of the unresolved questions about the splicing factor RP is why mutations in the ubiquitously expressed genes cause a very specific and severe retinal phenotype without affecting other organs. One of the hypotheses was that since retina is a very metabolically active tissue, with a very significant daily protein turnover (10 % of rhodopsin renewed) there is a high demand for these splicing factors. In line with this hypothesis even a small alteration in the quantity of these proteins may have significant effects leading to photoreceptor cell death. It was expected that retina would have a high expression of the splicing factor genes compared to other tissues. The experiment proved the opposite; comparing Prpf31, Prpf8 and Prpf3 levels in brain, retina, kidney, spleen and testis demonstrated that the expression of these splicing factor genes is comparable between the brain, retina and kidney. Four to nine fold higher expression was observed in spleen and testis, which are highly active in protein synthesis and cell division, requiring efficient splicing machinery. A possible explanation for these results is that the highly specialised photoreceptor cells are programmed to maximise synthesis of rhodopsin (90 % of all proteins in the photoreceptor outer segments) and other proteins of the visual system with a cost of reducing expression of the housekeeping genes such as splicing factors. Therefore a small imbalance of the latter may lead to cell death in the retina and spare other organs.
During this project, a collaboration was established with Professor Eric Pierce and Dr Michael Farkas from Harvard University, who have developed mouse models for Prpf3 and Prpf8 genes. Further in depth analysis of Prpf31 and Prpf3 and Prpf8 mouse models was done in parallel between the two laboratories. The findings were subject of a common peer reviewed paper (Graziotto et al. 2010) and were presented as posters at the annual meeting of The Association for Research in Vision and Ophthalmology (ARVO 2010). In all three mouse models anomalies of retinal pigment epithelium (RPE) cells were observed compared to the wild-type litter mate controls. These changes included loss of basal infoldings and accumulation of amorphous material between Bruch's membrane and RPE. Moreover, using electroretinogram it has been observed that in Prpf3 mice the rod photoreceptor function is decreased significantly in 2-year-old mice. These changes were not significant in Prpf8 and Prpf31 mutant mice (Graziotto et al. 2010, unpublished data).
These finding opened a new area of investigation of the RPE cells in these three mouse models. This new research theme incited collaboration with Dr Emeline Nandrot from the Institut de la Vision in Paris, who is a specialist in the RPE cell biology. The subsequent experiments were designed to characterise different aspects of the biology of the RPE cells, which can differ between wild-type and mutant mice. Such differences were observed in in-vitro phagocytosis. Preliminary results obtained form this study lead to a successful grant application to the US National Institute of Health (NIH). The proposed work aims at further characterisation of the RPE phenotypes of the splicing factor RP mouse models.
Initially two Prpf31 mouse models were established, a knock-in model harbouring p. A216P change and a knock-out model, where exon 7 of Prpf31 was deleted, which lead to the degradation of the Prpf31 transcript by nonsense mediated decay. Both mouse models were embryonic lethal in the homozygous state and therefore the retinal phenotypes of the heterozygous mice were investigated. These mice have not shown an apparent retinal phenotype up to 18 months of age as investigated by histological sections, examination of the eye fundus and electroretinography, which records electrophysiological responses of the retina. These results were published in a peer-reviewed journal (Bujakowska et al. 2009).
Lack of the clear phenotype in the Prpf31 mice gave rise to a question of why the relatively severe phenotype in human is not reproduced in mice. One of the possible explanations could be that the highly expressing wild-type allele rescues the potential deleterious effect of the mutant allele. It has been hypothesized that as in human subjects, in mice there might be a variation of the expression of the Prpf31 alleles, which can be verified by considering different mouse strains. Using quantitative real-time PCR on cDNA obtained from different mouse strains, we were able to show that there is no significant variation of the Prpf31 expression between the investigated mouse strains.
One of the unresolved questions about the splicing factor RP is why mutations in the ubiquitously expressed genes cause a very specific and severe retinal phenotype without affecting other organs. One of the hypotheses was that since retina is a very metabolically active tissue, with a very significant daily protein turnover (10 % of rhodopsin renewed) there is a high demand for these splicing factors. In line with this hypothesis even a small alteration in the quantity of these proteins may have significant effects leading to photoreceptor cell death. It was expected that retina would have a high expression of the splicing factor genes compared to other tissues. The experiment proved the opposite; comparing Prpf31, Prpf8 and Prpf3 levels in brain, retina, kidney, spleen and testis demonstrated that the expression of these splicing factor genes is comparable between the brain, retina and kidney. Four to nine fold higher expression was observed in spleen and testis, which are highly active in protein synthesis and cell division, requiring efficient splicing machinery. A possible explanation for these results is that the highly specialised photoreceptor cells are programmed to maximise synthesis of rhodopsin (90 % of all proteins in the photoreceptor outer segments) and other proteins of the visual system with a cost of reducing expression of the housekeeping genes such as splicing factors. Therefore a small imbalance of the latter may lead to cell death in the retina and spare other organs.
During this project, a collaboration was established with Professor Eric Pierce and Dr Michael Farkas from Harvard University, who have developed mouse models for Prpf3 and Prpf8 genes. Further in depth analysis of Prpf31 and Prpf3 and Prpf8 mouse models was done in parallel between the two laboratories. The findings were subject of a common peer reviewed paper (Graziotto et al. 2010) and were presented as posters at the annual meeting of The Association for Research in Vision and Ophthalmology (ARVO 2010). In all three mouse models anomalies of retinal pigment epithelium (RPE) cells were observed compared to the wild-type litter mate controls. These changes included loss of basal infoldings and accumulation of amorphous material between Bruch's membrane and RPE. Moreover, using electroretinogram it has been observed that in Prpf3 mice the rod photoreceptor function is decreased significantly in 2-year-old mice. These changes were not significant in Prpf8 and Prpf31 mutant mice (Graziotto et al. 2010, unpublished data).
These finding opened a new area of investigation of the RPE cells in these three mouse models. This new research theme incited collaboration with Dr Emeline Nandrot from the Institut de la Vision in Paris, who is a specialist in the RPE cell biology. The subsequent experiments were designed to characterise different aspects of the biology of the RPE cells, which can differ between wild-type and mutant mice. Such differences were observed in in-vitro phagocytosis. Preliminary results obtained form this study lead to a successful grant application to the US National Institute of Health (NIH). The proposed work aims at further characterisation of the RPE phenotypes of the splicing factor RP mouse models.
