Background Retinal and macular diseases are a major cause of untreatable vision impairment. Progressive neurodegenerative diseases, such as retinitis pigmentosa (RP), age-related macular degeneration (AMD), diabetic retinopathy and glaucoma have distinct etiology but ultimately lead to the death of rod and cone photoreceptors. Identification of disease causing mutations and elucidation of underlying mechanisms and pathways of photoreceptor degeneration constitute the first steps in developing targeted therapeutic strategies. Results 1. Linkage studies in human families with retinal diseases 1.1. We had mapped a new genetic locus for autosomal dominant RP on chromosome 7p15. We then identified three different disease-causing mutations in KLHL7 gene in six independent families (EY000473-01). A long-term follow-up study of a Scandinavian family, in collaboration with S Andreasson, revealed a phenotype characterized by varying degree of retinal photoreceptor dysfunction and, in some family members, by late onset degeneration, with preserved rod function and night vision until late in life (4). 1.2. Linkage analysis in collaboration with R Gonzalez-Duarte on a Spanish pedigree with autosomal recessive RP determined a peak LOD score of 2.532 on chromosome 4p15.31. Mutation analysis revealed a homozygous c.869delG mutation in PROM1 in this family (15). 1.3 In a comprehensive functional analysis of variants and disease-causing mutations in photoreceptor-specific nuclear receptor NR2E3, we determined that a majority of mutations lead to mislocalization of NR2E3 and that mutations in the DNA-binding domain (DBD) and in the ligand-binding domain (LBD) of NR2E3 alter its interaction with NRL as well as with CRX and reduce DNA-binding and transcriptional activation of the rhodopsin promoter (7). 1.4. With other collaborators, we assessed patients with X-linked retinitis pigmentosa (XLRP) carrying RP2 mutations and generated a genotype-phenotype correlation that will facilitate clinical diagnosis and targeted genetic screening in these patients (6). 1.5. In a long-term follow-up of a previously described family with XLRP we documented the progression of disease in male and female members caused by a de novo insertion after nucleotide 173 in exon ORF15 of RPGR (16). 1.6. We established that a common allele in RPGRIP1L is a modifier of retinal degeneration in ciliopathies (8). 1.7. In collaboration with Dr. Brian Brooks, we reported that two novel CRX mutations causing autosomal dominant Leber congenital amaurosis (LCA) interact differentially with NRL (14). 2. Animal models of human retinal diseases 2.1. We have initiated a comprehensive survey of our wide collection of mouse rd mutants and are generating comparative data, including fundoscopy, histology, immunofluorescence, ERG and gene profiling of each mutant. We plan to expand this approach to include more transgenic mice with rd, with a goal to generate a web-based resource on mouse models of retinal degeneration. 2.2. Altered or loss of function of Cep290 results in syndromic phenotypes including retinal degeneration. The mouse mutant, rd16, mimics mutations in CEP290 observed in about 25% of LCA patients. CEP290, CC2D2a, and several BBSome proteins have key functions in ciliogenesis. They are essential for photoreceptor development or maturation. We are applying comprehensive approaches to determine the role of CEP290 and its molecular network in photoreceptors and RPE development and function. We have recently discovered the interaction between Cep290rd16 and BBS6 (MKKS) and are exploring its physiological relevance in cilia biogenesis and/or transport. We have generated Cep290-knockout and genetrap mouse lines to further elucidate CEP290 function. We are characterizing compound mouse mutants with different Cep290 and Mkks alleles as well. 2.3. In collaboration with S. Jacobson, we are comparing phenotypes of human LCA patients with CEP290 and NPHP5 mutations with those of rd16 mouse mutant. Our studies show that mice lacking Nrl and carrying the Cep290rd16 mutation exhibit significantly prolonged cone survival and residual ERG response despite lacking outer segments. This is similar to what is observed in CEP290-LCA patients where cones in the foveal region survive for a long time despite the lack of outer segments and phototransduction. 2.4. We are also investigating molecular mechanisms underlying photoreceptor disease using zebrafish as a model system. We have established a role for BBS9 in ciliogenesis in IMCD3 cells using RNAi. Rescue of zebrafish morphants using human BBS9 mRNA suggests functional conservation. 2.5. We have defined a role for CC2D2a in eye development using morpholinos in zebrafish. Rescue experiments using human CC2D2a mRNAs (WT or mutant) are underway. CC2D2aKO mice have been generated and are being crossed to homozygosity to turther investigate CC2D2a role in eye development. 2.6 rd11 and 2845 mouse lines develop retinal degeneration early in life and can be useful models of human retinal disease. We performed a mutation screen of genes in the critical interval determined by our collaborator, B. Chang. We determined that both lines carry mutations in the gene encoding the lipid remodeling enzyme, LPCAT1. The content in the retina of the downstream product of this enzyme, dipalmitoylphosphatidylcholine (DPPC), is reduced in affected mice (2). 2.7. The Nrl-KO mouse carries the unique phenotype of a cone-only retina. We showed that cone cell death in Nrl-KO mice is transitory and occurs between 1 and 4 months. During this time, microglia and Muller glia cell activation are observed as well as blood vessels leakage. By microarray analysis and RNAseq with NGS (Illumina) we are investigating the molecular cues of cone cell death in Nrl-KO mice. 2.8. We have identified a novel autosomal dominant mutant mouse line that exhibit abnormal photoreceptor development, including absence of detectable ERG, no rod photoreceptors, and lack of expression of most visual transduction proteins. ONL nuclei resemble cone nuclei but have no outer segments. NRL and NR2E3 are not expressed. Despite the lack of phototransduction, a discrete number of photoreceptor nuclei persist even by 10 months of age. Mating with Crxp-NRL mice (rod-only retina) resulted in partial rescue of rhodopsin expression, but neither of outer segment deficiency nor of a-wave abnormality. By linkage analysis in collaboration with B. Chang, we have found that the genetic modification is located on Chr7 in mouse, in a region syntenic with Chr19 in human and associated with autosomal dominant macular degeneration. We are actively pursuing the identification of the genetic defect in this mutant strain. Significance Our work in animal models of retinal degeneration has highlighted an interesting convergence on biogenesis and transport functions associated with primary cilia and revealed novel pathways associated with protein degradation and lipid metabolism that contribute to photoreceptor homeostasis. Our results will have significance for treating patients with LCA and retinal degeneration.
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