Final Activity Report Summary - RETNET (European retinal research training network)
RETNET has promoted training in visual neuroscience, molecular biology of the retina and ophthalmo-genetics and improved the knowledge of degenerative retinal diseases. During the 4-year project period the following outstanding results were achieved:
The work performed provided new insights into the degeneration mechanisms of Retinitis Pigmentosa (RP) and suggests the involvement of two enzyme systems, calpain and poly(ADP-ribose) polymerase. A new gene for dominant RP (adRP) known as TOPORS on Human Chromosome 9 was identified. In another family a new mutation was found in exon 39 of the splicing factor gene PRPF8. A mouse model carrying a deficient PRPF31 gene as found in human adRP patients was created. On preliminary evaluation this mouse does not show retinal disease. Nevertheless, the mutation in homozygous state is lethal. Additionally it was identified that mutations in the CSPG2/Versican gene are responsible for Wagner disease, an eye disease characterised by abnormal vessel architecture and variable vision impairment, in most cases due to retinal detachment.
The expression profile of 7 miRNAs in the vertebrate eye was determined by in situ hybridisation in various developmental stages. These miRNAs exhibit very similar expression patterns to that obtained for the corresponding host genes and the targeting of the transcription factor MITF was validated in vitro by eye-expressed miRNAs. In parallel the first expression atlas of RP genes in the human and murine retina was generated in the form of a publicly available database. Using both in silico and experimental analysis the Transcription Start Sides for 32 out of 48 investigated retinal genes were mapped, most of which led to the identification of additional 5' transcribed sequences including distant first exons in some of the genes. Promoters for OPA1, CNGA3 and RDH12 genes were cloned and analysed using reporter gene constructs in retinal and non-retinal cell lines.
Phenotypes associated with degenerative retinopathies were investigated in murine model systems and approaches to gene therapy for autosomal dominant retinopathies were systemically developed. This has involved the development of an ultra-high titre AAV (adeno-associated virus) delivery platform and the development of processes for the suppression of mutations within the rhodopsin, RDS-peripherin and inosine monophosphate dehydrogenase 1 genes. Gene silencing technologies have been assessed in vitro, in retinal explants and in animal models mimicking disease.
In another RETNET project the therapeutic potential of Rod-derived Cone Viability Factor (RdCVF) for its support of cone survival during retinal degeneration was investigated, e.g. by using transgenic RdCVF-knockout mice. Transcriptomic and functional characterisation of this model has demonstrated that the retina is compromised by the absence of RdCVF. The phenotype is further exacerbated in stess-induced conditions (light damage, hypoxia), supporting a role of RdCVF in cell survival signalling based on redox balance mechanisms.
Another focus has been the analysis of the photoreceptor-specific role of the small GTPase Rac1. It was found that Rac1 specifically interacts with the poorly characterised SON DNA-binding protein. In collaboration between Munich and Dublin expression of a wild-type form of rac, a dominant negative form of rac and a constitutively active form of rac was achieved in mouse photoreceptors in situ. Rac dominant negative and constitutive active expression affected the selectivity of protein transport between the inner and outer segment visualised by coexperessed GFP as well as rhodopsin. As such, rac is a candidate for regulating protein transport between rod outer and inner segments, most likely by affecting cytoskeletal architecture and actin dynamics.
Finally a dedicated transcriptomics database and analysis system (Retinobase) was constructed and made available, which was accompanied with a set of powerful query and data analysis tools. Additionally novel data-mining approaches and a novel platform for automatic statistical analysis were developed.
The work performed provided new insights into the degeneration mechanisms of Retinitis Pigmentosa (RP) and suggests the involvement of two enzyme systems, calpain and poly(ADP-ribose) polymerase. A new gene for dominant RP (adRP) known as TOPORS on Human Chromosome 9 was identified. In another family a new mutation was found in exon 39 of the splicing factor gene PRPF8. A mouse model carrying a deficient PRPF31 gene as found in human adRP patients was created. On preliminary evaluation this mouse does not show retinal disease. Nevertheless, the mutation in homozygous state is lethal. Additionally it was identified that mutations in the CSPG2/Versican gene are responsible for Wagner disease, an eye disease characterised by abnormal vessel architecture and variable vision impairment, in most cases due to retinal detachment.
The expression profile of 7 miRNAs in the vertebrate eye was determined by in situ hybridisation in various developmental stages. These miRNAs exhibit very similar expression patterns to that obtained for the corresponding host genes and the targeting of the transcription factor MITF was validated in vitro by eye-expressed miRNAs. In parallel the first expression atlas of RP genes in the human and murine retina was generated in the form of a publicly available database. Using both in silico and experimental analysis the Transcription Start Sides for 32 out of 48 investigated retinal genes were mapped, most of which led to the identification of additional 5' transcribed sequences including distant first exons in some of the genes. Promoters for OPA1, CNGA3 and RDH12 genes were cloned and analysed using reporter gene constructs in retinal and non-retinal cell lines.
Phenotypes associated with degenerative retinopathies were investigated in murine model systems and approaches to gene therapy for autosomal dominant retinopathies were systemically developed. This has involved the development of an ultra-high titre AAV (adeno-associated virus) delivery platform and the development of processes for the suppression of mutations within the rhodopsin, RDS-peripherin and inosine monophosphate dehydrogenase 1 genes. Gene silencing technologies have been assessed in vitro, in retinal explants and in animal models mimicking disease.
In another RETNET project the therapeutic potential of Rod-derived Cone Viability Factor (RdCVF) for its support of cone survival during retinal degeneration was investigated, e.g. by using transgenic RdCVF-knockout mice. Transcriptomic and functional characterisation of this model has demonstrated that the retina is compromised by the absence of RdCVF. The phenotype is further exacerbated in stess-induced conditions (light damage, hypoxia), supporting a role of RdCVF in cell survival signalling based on redox balance mechanisms.
Another focus has been the analysis of the photoreceptor-specific role of the small GTPase Rac1. It was found that Rac1 specifically interacts with the poorly characterised SON DNA-binding protein. In collaboration between Munich and Dublin expression of a wild-type form of rac, a dominant negative form of rac and a constitutively active form of rac was achieved in mouse photoreceptors in situ. Rac dominant negative and constitutive active expression affected the selectivity of protein transport between the inner and outer segment visualised by coexperessed GFP as well as rhodopsin. As such, rac is a candidate for regulating protein transport between rod outer and inner segments, most likely by affecting cytoskeletal architecture and actin dynamics.
Finally a dedicated transcriptomics database and analysis system (Retinobase) was constructed and made available, which was accompanied with a set of powerful query and data analysis tools. Additionally novel data-mining approaches and a novel platform for automatic statistical analysis were developed.