The rat Apg3p/Aut1p homolog is upregulated by ischemicpreconditioning in the retina

[摘要] Purpose: Retinas can be protected from subsequent severe ischemicinjury by ischemic preconditioning. Ischemic preconditioning isdependent on gene expression and protein synthesis; however, it is notclear which genes are important in this process. In this study, we haveidentified and characterized the rat homolog of yeast Apg3p/Aut1p, animportant autophagy protein encoded by the autophagy 3-like (APG3L)gene. We have also further characterized the homologous human APG3Lgene.Methods: A fragment of the rat Apg3 cDNA was identified by mRNAdifferential display from hypoxia-treated E1A-NR3, an immortalized cellline derived from rat retinal cells that manifests phenotypes of retinalneurons. The full length of rat Apg3 (rApg3) cDNA sequence(about 1.4 kb) encoding 341 amino acids was cloned from a rat retinalcDNA library and characterized using Southern and northern blotanalysis, and a global GenBank search. Protein expression was determinedby western blotting, and immunohistochemistry. Ischemic preconditioningwas achieved by ligation of the retinal arteries of the right eye for 5min followed by 5 h reperfusion. The prolonged retinal ischemia wasinduced by ligation of the retinal arteries for 45 min followed by 5 hreperfusion. The full-length homologous human APG3L gene was clonedand sequenced from a human genomic DNA library.Results: The combination of genomic Southern blot analysis and aglobal GenBank search indicated that rat APG3L is a single copy gene.Rat Apg3 mRNA is expressed in the retina at a high level but is alsodetected in other tissues. In the process of comparing the rat and humanAPG3L genes we showed that the organization of the human APG3Lgene includes a unique transcriptional start site, a coding region with12 translated exons and 11 introns and is located on human chromosome3q13.1. Subcellular localization studies showed that recombinant ratautophagocytosis protein (Apg3p) is a cytosolic protein. Rat Apg3mRNA level was upregulated by ischemic preconditioning but downregulatedby prolonged ischemia.Conclusions: Our results suggest that the upregulation of rApg3is a specific response to ischemic preconditioning rather than to retinaischemia, and autophagy may contribute to the neuroprotective effect ofischemic preconditioning in the retina.

[学科分类] 生物化学/生物物理