The Role of Akt/Protein Kinase B Subtypes in Retinal Ischemic Preconditioning

Retinal ischemia is associated with vascular diseases that cause visual loss. The retina's blood and oxygen supply may be decreased in diabetic retinopathy, central retinal artery occlusion, carotid artery stenosis, and sickle cell retinopathy. Previously we demonstrated that a profound endogenous protective capacity in the rat retina can be activated (ischemic preconditioning, IPC) to attenuate ischemic injury (Roth et al. 1998; Zhang et al. 2002; Roth et al. 2006). Such protection offers a model for uncovering pathways and agents to treat retinal ischemia. Ischemic preconditioning (IPC) protects the rat retina against the deleterious effects of a severe ischemic event (Roth et al. 1998; Li and Roth 1999; Li et al. 2000; Li et al. 2003). The cytoplasmic proto-oncogene 57 kD serine/threonine protein kinase Akt (protein kinase B, PKB), is one of the most important kinases, and at the core of numerous and diverse physiological functions. It is activated by receptor tyrosine kinases, integrins, G-protein coupled receptors, and other stimuli, with over 100 reported non-redundant Akt substrates (Manning and Cantley 2007). The Akt pathway has been identified as a cellular survival signal in diverse neuronal cell types and in protection from oxidative stress (Chong et al. 2005). Akt phosphorylates Ser136 on Bad, thus interfering with mitochondrial cytochrome C release (Datta et al. 2000), and it inhibits caspase-9 by phosphorylating Ser196 (Cardone et al. 1998). Via phosphorylation of transcription factors including FOXO and p53, FOXO-mediated transcription of apoptosis-promoting proteins is inhibited (Greer and Brunet 2005). Inhibition of Akt has received increasing attention as a potential anti-cancer treatment (Robey and Hay 2006). The three mammalian enzymatic isoforms, Akt1, 2, and 3, have extensive homology to protein kinases A, G, and C. Isotypes are similar in structure, size, and substrate specificity, but differ in organ distribution (Manning and Cantley 2007). Knockouts differentially result in developmental defects (Akt1), insulin-resistance (Akt2), and defective brain development (Akt3) (Fayard et al. 2005). These distinct phenotypes suggest cellular differences in functionality. Few data are available, however, on the role of Akt subtypes in disease states. Upregulation of Akt has ameliorative effects upon ischemic organs (Mullonkal and Toledo-Pereyra 2007). Recent evidence supports a role for Akt in survival of retinal cells under diverse conditions impacting retinal survival, including chronic ocular hypertension, (Huang et al. 2008) diabetic retinopathy, (Liu et al. 2008) photoreceptor light damage, (Li et al. 2007; Ueki et al. 2008), angiogenesis (Kitamura et al. 2008), retinal development, (Chavarria et al. 2007) and growth hormone action. (Sanders et al. 2006) Vascular endothelial growth factor protected axotomized retinal ganglion cells (RGCs) by activation of Akt. (Kilic et al. 2006) Moreover, neurotrophin signaling complex LINGO-1 antagonists increased Akt activation, and rescued RGCs from cell death after optic nerve transaction or ocular hypertension, an effect eliminated by PI-3 kinase blockade.(Fu et al. 2008) Because of its involvement in cell survival, Akt is a logical candidate as a critical mediator in the ischemic tolerance induced by IPC. Indeed, pharmacological blockade of Akt attenuated hypoxic preconditioning in human brain endothelial cells (Zhang et al. 2007) and in the brains of post-natal rats (Yin et al. 2007). Results were conflicting when PC12 cells were exposed to oxygen and glucose deprivation (Hillion et al. 2006). The findings suggest that Akt may be an essential mediator in the endogenous protection provided by IPC, and also that enhancing Akt-mediated mechanisms of survival may decrease neuronal ischemic injury. We hypothesized differential actions for Akt subtypes in retinal IPC. In this study we examined the effects of the Akt isoforms in vivo, using a well described model of retinal IPC. We also examined the relationship between Akt's role as a mediator in IPC and some of the significant IPC initiators we previously uncovered including adenosine receptors and mitochondrial KATP channels.