| CERKL is a novel gene cloned in2004, which is highly homologous with caramide kinase (CERK), therefore named the ceramide kinase-like protein. Mutations in CERKL are associated with severe retinal degeneration diseases, including retinitis pigmentosa (RP) and cone rod dystrophy (CRD). However, the exact function of CERKL in the retina remains unknown.In this thesis, we design experiments to investigate the pathogenic molecular mechanism of CERKL. I find that CERKL can interact with TRX2in mitochondria by yeast two-hybrid, co-immunoprecipitation, etc. CERKL protects cells from oxidative stress-induced apoptosis by interacting with thioredoxin2(TRX2) and modulating its redox state in mitochondria, whereas two known causative human CERKL mutations R106S and C125W cannot. Conversely, suppressing CERKL expression increases ROS accumulation and cell apoptosis. And the importance of TRX2in the cytoprotective function of CERKL is confirmed by using TRX2siRNA and a pharmacological inhibitor of TRX2reductase (TrxR2), auranofin. Both ways result in abolishment of CERKL mediated cytoprotection.In zebrafish, CERKL is mainly expressed in the cone photoreceptor. Knockdown of CERKL in zebrafish by morpholino leads to appear obvious symptoms of photoreceptor degeneration disease. The photoreceptor layer are significantly shorter and irregular in arrangement. And TUNEL positive cells are detected in the photoreceptor layer and inner nuclear layer. The TRX2oxidized level is increased with signs of oxidative damage to marcomolecules in CERKL deficient zebrafish. These results are in agreement with the data in cell.Our results show that CERKL is a novel key player in the regulation of the TRX2antioxidant pathway and, for the first time, provide a mechanistic explanation of how mutations in CERKL may lead to photoreceptor death through oxidative damage. This new knowledge may suggest rational therapeutic targets for CRD and a broad category of inherited and age-related retinal and neural degenerations. |
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