Miquel Tuson, Gemma Marfany, and Roser González-Duarte
Departament de Genética, Facultat de Biologia, Universitat de Barcelona, Barcelona
Abstract
Retinitis pigmentosa (RP), the main cause of adult blindness, is a genetically heterogeneous disorder characterized by progressive loss of photoreceptors through apoptosis. Up to now, 39 genes and loci have been implicated in nonsyndromic RP, yet the genetic bases of 150% of the cases, particularly of the recessive forms, remain unknown.
Previous linkage analysis in a Spanish consanguineous family allowed us to define a novel autosomal recessive RP (arRP) locus, RP26, within an 11-cM interval (17.4 Mb) on 2q31.2-q32.3. In the present study, we further refine the RP26 locus down to 2.5 Mb, by microsatellite and single-nucleotide polymorphism (SNP) homozygosity mapping.
After unsuccessful mutational analysis of the nine genes initially reported in this region, a detailed gene search based on expressed-sequence-tag data was undertaken.We finally identified a novel gene encoding a ceramide kinase (CERKL), which encompassed 13 exons. All of the patients from the RP26 family bear a homozygous mutation in exon 5, which generates a premature termination codon. The same mutation was also characterized in another, unrelated, Spanish pedigree with arRP. Human CERKL is expressed in the retina, among other adult and fetal tissues. A more detailed analysis by in situ hybridization on adult murine retina sections shows expression of Cerkl in the ganglion cell layer. Ceramide kinases convert the sphingolipid metabolite ceramide into ceramide-1-phosphate, both key mediators of cellular apoptosis and survival. Ceramide metabolism plays an essential role in the viability of neuronal cells, the membranes of which are particularly rich in sphingolipids. Therefore, CERKL deficiency could shift the relative levels of the signaling sphingolipid metabolites and increase sensitivity of photoreceptor and other retinal cells to apoptotic stimuli. This is the first genetic report suggesting a direct link between retinal neurodegeneration in RP and sphingolipid-mediated apoptosis.

Alejandro Garanto,1,2,3,4 Marina Riera,1,2,3,4 Esther Pomares,1,2,3 Jon Permanyer,1,2 Marta de Castro-Miro´,1 Florentina Sava,1 Josep F. Abril,1,2 Gemma Marfany,1,2,3 and Roser Gonza`lez-Duarte1,2,3

PURPOSE. To shed light on the pathogenicity of the mutations in the retinitis pigmentosa gene CERKL, the authors aimed to characterize its transcriptional repertoire and focused on the use of distinct promoters and alternative splicing in human and mouse tissues. METHODS. In silico genomic and transcriptomic computational customized analysis, combined with experimental RT-PCRs on different human and murine tissues and cell lines and immunohistochemistry, have been used to characterize the transcriptional spectrum of CERKL. In the mouse retina, Cerkl is detected primarily in ganglion cells and cones but can also be observed in rods. Cerkl is mainly cytosolic. It localizes in the outer segments of photoreceptors and in the perinuclear regions of some cells. RESULTS. An unexpected multiplicity of CERKL transcriptional start sites (four in each species) plus a high variety of alternative splicing events primarily affecting the 5 half of the gene generate 20 fully validated mRNA isoforms in human and 23 in mouse. Moreover, several translational start sites, compatible with a wide display of functional domains, contribute to the final protein complexity. CONCLUSIONS. This combined approach of in silico and experimental characterization of the CERKL gene provides a comprehensive picture of the species-specific transcriptional products in the retina, underscores highly tuned gene regulation in different tissues, and establishes a framework for the study of CERKL genotype-phenotype correlations. (Invest Ophthalmol Vis Sci. 2011;52:5202–5214) DOI:10.1167/iovs.10-7101

Alihamze Fathinajafabadi1., Eva Pe´rez-Jime´nez1., Marina Riera2,3¤, Erwin Knecht1,3*",
Roser Gonza` lez-Duarte2,3"
1 Laboratorio de Biologı´a Celular, Centro de Investigacio´n Prı´ncipe Felipe, Valencia, Spain, 2 Departament de Gene` tica, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain, 3 Centro de Investigacio´n Biome´dica en Red de Enfermedades Raras (CIBERER), Spain
Abstract
The function of CERKL (CERamide Kinase Like), a causative gene of retinitis pigmentosa and cone-rod dystrophy, still awaits characterization. To approach its cellular role we have investigated the subcellular localization and interaction partners of the full length CERKL isoform, CERKLa of 532 amino acids, in different cell lines, including a photoreceptor-derived cell line.
We demonstrate that CERKLa is a main component of compact and untranslated mRNPs and that associates with other RNP complexes such as stress granules, P-bodies and polysomes. CERKLa is a protein that binds through its N-terminus to mRNAs and interacts with other mRNA-binding proteins like eIF3B, PABP, HSP70 and RPS3. Except for eIF3B, these interactions depend on the integrity of mRNAs but not of ribosomes. Interestingly, the C125W CERKLa pathological mutant does not interact with eIF3B and is absent from these complexes. Compact mRNPs containing CERKLa also associate with microtubules and are found in neurites of neural differentiated cells. These localizations had not been reported previously for any member of the retinal disorders gene family and should be considered when investigating the pathogenic mechanisms and therapeutical approaches in these diseases.

Alihamze Fathinajafabadi1., Eva Pe´rez-Jime´nez1., Marina Riera2,3¤, Erwin Knecht1,3*",Roser Gonza` lez-Duarte2,3"
1 Laboratorio de Biologı´a Celular, Centro de Investigacio´n Prı´ncipe Felipe, Valencia, Spain, 2 Departament de Gene` tica, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain, 3 Centro de Investigacio´n Biome´dica en Red de Enfermedades Raras (CIBERER), Spain
Abstract
The function of CERKL (CERamide Kinase Like), a causative gene of retinitis pigmentosa and cone-rod dystrophy, still awaits characterization. To approach its cellular role we have investigated the subcellular localization and interaction partners of the full length CERKL isoform, CERKLa of 532 amino acids, in different cell lines, including a photoreceptor-derived cell line. We demonstrate that CERKLa is a main component of compact and untranslated mRNPs and that associates with other RNP complexes such as stress granules, P-bodies and polysomes. CERKLa is a protein that binds through its N-terminus to mRNAs and interacts with other mRNA-binding proteins like eIF3B, PABP, HSP70 and RPS3. Except for eIF3B, these interactions depend on the integrity of mRNAs but not of ribosomes. Interestingly, the C125W CERKLa pathological mutant does not interact with eIF3B and is absent from these complexes. Compact mRNPs containing CERKLa also associate with microtubules and are found in neurites of neural differentiated cells. These localizations had not been reported previously for any member of the retinal disorders gene family and should be considered when investigating the pathogenic mechanisms and therapeutical approaches in these diseases.

Chang Li a,1, LeiWanga,1, Jing Zhanga,b,1, Mi Huanga, Fulton Wong c,d, Xuexue Liua, Fei Liu a, Xiukun Cui a, Guohua Yang e, Jiaxiang Chen a, Ying Liua, JiuxiangWang a, Shengjie Liao a, Meng Gao a, Xuebin Hu a, Xinhua Shu f, QingWang a, Zhan Ying, Zhaohui Tang a,⁎⁎, Mugen Liu a,⁎

a Key Laboratory ofMolecular Biophysics of theMinistry of Education, Center for Human Genome Research, College of Life Science and Technology, Huazhong University of Science and Technology,
Wuhan, Hubei, PR China
b Key Laboratory of Cellular and Molecular Immunology, Institute of Immunology, Medical College of Henan University, Kaifeng, Henan 475004, PR China
c Department of Ophthalmology, Duke University School of Medicine, Durham, NC 27710, USA
d Department of Neurobiology, Duke University School of Medicine, Durham, NC 27710, USA
e Department of Medical Genetics, Wuhan University School of Medicine, Wuhan, Hubei, PR China
f MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, Edinburgh, UK
g Institute of Hydrobiology, Chinese Academy of Sciences,Wuhan, Hubei, PR China

Abstract
Mutations in the ceramide kinase-like gene (CERKL) are associatedwith severe retinal degeneration. However, the exact function of the encoded protein (CERKL) remains unknown. Here we show that CERKL interacts with mitochondrial thioredoxin 2 (TRX2) and maintains TRX2 in the reduced redox state. Overexpression of CERKL protects cells from apoptosis under oxidative stress, whereas suppressing CERKL renders cells more sensitive to oxidative stress. In zebrafish, CERKL protein prominently locates in the outer segment and inner segment of the photoreceptor of the retina. Knockdown of CERKL in the zebrafish leads to an increase of retinal cell death, including cone and rod photoreceptor degeneration. Signs of oxidative damage to macromolecules were also detected in CERKL deficient zebrafish retina. Our results show that CERKL interacts with TRX2 and plays a novel key role in the regulation of the TRX2 antioxidant pathway and, for the first time, provides an explanation of how mutations in CERKL may lead to retinal cell death.

Shanshan Yu1,†, Chang Li1,†, Lincoln Biswas2, Xuebin, Hu1, Fei Liu1, James Reilly2, Xiliang Liu1, Ying Liu1, Yuwen Huang1, Zhaojing Lu1, Shanshan Han1, Lei Wang3, Jing Yu Liu1, Tao Jiang1, Xinhua Shu2,Fulton Wong4, Zhaohui Tang1,* and Mugen Liu1,*

1Key Laboratory of Molecular Biophysics of Ministry of Education, Department of Genetics and Developmental Biology, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, Hubei 430074, P.R. China, 2Department of Life Sciences, Glasgow Caledonian University, Glasgow G4 0BA, UK, 3Department of Pathology & Lab Medicine, University of Cincinnati Medical Center, Cincinnati, OH 45267, USA and 4Department of Ophthalmology, Duke University School of Medicine, Durham, NC 27710, USA

*To whom correspondence should be addressed at: Department of Genetics and Developmental Biology, College of Life Science and Technology, 1037 Luoyu Road, Wuhan, P. R. China. Tel: 86 27 87794549; Fax 86 27 87794549; Email: lium@hust.edu.cn(M.L.); zh_tang@mail.hust.edu.cn(Z.T.)

Abstract
In humans, CERKL mutations cause widespread retinal degeneration: early dysfunction and loss of rod and cone photoreceptors in the outer retina and, progressively, death of cells in the inner retina. Despite intensive efforts, the function of CERKL remains obscure and studies in animal models have failed to clarify the disease mechanism of CERKL mutations. To address this gap in knowledge, we have generated a stable CERKL knockout zebrafish model by TALEN technology and a 7bp deletion in CERKL cDNA that caused the premature termination of CERKL. These CERKL-/- animals showed progressive degeneration of photoreceptor outer segments (OSs) and increased apoptosis of retinal cells, including those in the outer and inner retinal layers. Additionally, we confirmed by immunofluorescence and western-blot that rod degeneration in CERKL-/- zebrafish occurred earlier and was more significant than that in cone cells. Accumulation of shed OSs in the interphotoreceptor matrix was observed by transmission election microscopy (TEM). This suggested that CERKL may regulate the phagocytosis of OSs by the retinal pigment epithelium (RPE). We further found that the phagocytosis-associated protein MERTK was significantly reduced in CERKL-/- zebrafish. Additionally, in ARPE-19 cell lines, knockdown of CERKL also decreased the mRNA and protein level of MERTK, as well as the ox-POS phagocytosis. We conclude that CERKL deficiency in zebrafish may cause rod-cone dystrophy, but not cone-rod dystrophy, while interfering with the  hagocytosis function of RPE associated with down-regulation of the expression of MERTK.