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Autophagy is the major intracellular degradation pathway that regulates long-lived proteins and organelles turnover. This process occurs at basal levels in all cells but it is rapidly upregulated in response to starvation and cellular stress. Although being recently implicated in neurodegeneration, it remains still unclear whether autophagy has a detrimental or protective role. In this study, we investigated the dynamics of the autophagic process in retinal tissue that has undergone transient ischemia, an experimental model that recapitulates features of ocular pathologies, including glaucoma, anterior ischemic optic neuropathy and retinal vessels occlusion. Retinal ischemia, induced in adult rats by increasing the intraocular pressure, was characterized by a reduction in the phosphatidylethanolamine-modified form of LC3 (LC3II) and by a significant decrease in Beclin-1. The latter event was associated with a proteolytic cleavage of Beclin-1, leading to the accumulation of a 50-kDa fragment. This event was prevented by intravitreal treatment with the non-competitive N-methyl-D-aspartate antagonist MK801 and calpain inhibitors or by calpain knockdown. Blockade of autophagy by pharmacological inhibition or Beclin-1 silencing in RGC-5 increased cell death, suggesting a pro-survival role of the autophagic process in this neuronal cell type. Altogether, our results provide original evidence for calpain-mediated cleavage of Beclin-1 and deregulation of basal autophagy in the rat retina that has undergone ocular ischemia/reperfusion injury.

AKAP121 focuses distinct signaling events from membrane to mitochondria by binding and targeting cAMP-dependent protein kinase (PKA), protein tyrosine phosphatase (PTPD1), and mRNA. We find that AKAP121 also targets src tyrosine kinase to mitochondria via PTPD1. AKAP121 increased src-dependent phosphorylation of mitochondrial substrates and enhanced the activity of cytochrome c oxidase, a component of the mitochondrial respiratory chain. Mitochondrial membrane potential and ATP oxidative synthesis were enhanced by AKAP121 in an src-and PKA-dependent manner. Finally, siRNA-mediated silencing of endogenous AKAP121 drastically impaired synthesis and accumulation of mitochondrial ATP. These findings indicate that AKAP121, through its role in enhancing cAMP and tyrosine kinase signaling to distal organelles, is an important regulator in mitochondrial metabolism. INTRODUCTIONProtein kinase A (PKA) is an essential mediator in most cAMP-dependent signaling pathways. A family of proteins named A-kinase anchor proteins (AKAPs) has been identified that enhance cAMP-dependent PKA signaling pathways (Rubin, 1994;Gray et al., 1998;McKnight et al., 1998;Feliciello et al., 2001;Houslay and Adams, 2003;Tasken and Aandahl, 2004;Taylor et al., 2004;Wong and Scott, 2004). AKAP121 (also called D-AKAP1), AKAP149, and AKAP84 arise from a single gene by alternative pre-mRNA splicing (Lin et al., 1995;Trendelenburg et al., 1996;Chen et al., 1997;Huang et al., 1997Huang et al., , 1999Furusawa et al., 2002). AKAP121 and AKAP84 tether PKA to the mitochondrial outer surface. This localization is mediated by the interaction of AKAP121 and AKAP84 with ␤ tubulin, an integral component of mitochondrial outer membrane (Cardone et al., 2002). AKAP121 is widely expressed in several tissues and its accumulation is regulated at the transcriptional level by the cAMP/PKA pathway (Feliciello et al., 1998). Anchoring of PKA to mitochondria supports cAMP signaling and suppresses apoptosis (Harada et al., 1999;Affaitati et al., 2003). AKAP121, via a KH domain at its COOH-terminus, binds at least two mRNAs that encode mitochondrial proteins Ranganathan et al., 2005). This multicomponent system, reminiscent of other AKAP complexes at cell membranes, ensures efficient translation and import of nuclear-encoded mitochondrial proteins. It is suggested that PKA may phosphorylate some of these proteins cotranslationally, as well as acting on AKAP121 itself to regulate the stability of the RNA-AKAP121 complex Feliciello et al., 2005).In addition, AKAP121 and AKAP84 bind the central core of PTPD1, a classical nonreceptor protein tyrosine phosphatase (Moller et al., 1994). PTPD1 binds to and activates src, enhancing EGF-dependent mitogenic signaling (Cardone et al., 2004). By translocating PTPD1 to the outer membrane of mitochondria, AKAP121 inhibits PTPD1-dependent EGF signaling to the nucleus. These data suggest a model whereby AKAP121, by targeting PTPD1/src complex to mitochondria, may shift the focus of tyrosine kinase signaling from membrane to specific distal...

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Naϩ /Ca 2ϩ exchanger 3 (NCX3), one of the three isoforms of the NCX family, is highly expressed in the brain and is involved in the maintenance of intracellular Na ϩ and Ca 2ϩ homeostasis. Interestingly, whereas the function of NCX3 under physiological conditions has been determined, its role under anoxia is still unknown. To assess NCX3 role in cerebral ischemia, we exposed ncx3Ϫ/Ϫ mice to transient middle cerebral artery occlusion followed by reperfusion. In addition, to evaluate the effect of ncx3 ablation on neuronal survival, organotypic hippocampal cultures and primary cortical neurons from ncx3Ϫ/Ϫ mice were subjected to oxygen glucose deprivation (OGD) plus reoxygenation. Here we report that ncx3 gene suppression leads to a worsening of brain damage after focal ischemia and to a massive neuronal death in all the hippocampal fields of organotypic cultures as well as in cortical neurons from ncx3Ϫ/Ϫ mice exposed to OGD plus reoxygenation. In addition, in ncx3Ϫ/Ϫ cortical neurons exposed to hypoxia, NCX currents, recorded in the reverse mode of operation, were significantly lower than those detected in ncx3ϩ/ϩ. From these results, NCX3 protein emerges as a new molecular target that may have a potential therapeutic value in modulating cerebral ischemia.