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

Retinas can be protected from subsequent severe ischemic injury by ischemic preconditioning. Ischemic preconditioning is dependent on gene expression and protein synthesis; however, it is not clear which genes are important in this process. In this study, we have identified and characterized the rat homolog of yeast Apg3p/Aut1p, an important autophagy protein encoded by the autophagy 3-like (APG3L) gene. We have also further characterized the homologous human APG3L gene. A fragment of the rat Apg3 cDNA was identified by mRNA differential display from hypoxia-treated E1A-NR3, an immortalized cell line derived from rat retinal cells that manifests phenotypes of retinal neurons. The full length of rat Apg3 (rApg3) cDNA sequence (about 1.4 kb) encoding 341 amino acids was cloned from a rat retinal cDNA library and characterized using Southern and northern blot analysis, and a global GenBank search. Protein expression was determined by western blotting, and immunohistochemistry. Ischemic preconditioning was achieved by ligation of the retinal arteries of the right eye for 5 min followed by 5 h reperfusion. The prolonged retinal ischemia was induced by ligation of the retinal arteries for 45 min followed by 5 h reperfusion. The full-length homologous human APG3L gene was cloned and sequenced from a human genomic DNA library. The combination of genomic Southern blot analysis and a global 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 also detected in other tissues. In the process of comparing the rat and human APG3L genes we showed that the organization of the human APG3L gene includes a unique transcriptional start site, a coding region with 12 translated exons and 11 introns and is located on human chromosome 3q13.1. Subcellular localization studies showed that recombinant rat autophagocytosis protein (Apg3p) is a cytosolic protein. Rat Apg3 mRNA level was upregulated by ischemic preconditioning but downregulated by prolonged ischemia. Our results suggest that the upregulation of rApg3 is a specific response to ischemic preconditioning rather than to retina ischemia, and autophagy may contribute to the neuroprotective effect of ischemic preconditioning in the retina.