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Mitochondrial dysfunction has been implicated in a wide variety of degenerative diseases, including age-related macular degeneration. Damage to mitochondria and mitochondrial DNA accumulates with age in the postmitotic retinal pigment epithelium (RPE), which could lead to RPE cell death and trigger disease. One possible mechanism for cells to avoid cell death is mitophagy, the targeted clearance of damaged mitochondria by autophagy. Here, we induced mitochondrial damage in human RPE cells (ARPE-19 and hRPE), using antimycin A, an inhibitor of complex III of the electron transport chain, and investigated cellular viability, mitochondrial structure and function, and autophagy activity. We observed that antimycin A evoked dose-dependent cell death, a rapid loss in mitochondrial membrane potential, and a collapse of oxidative phosphorylation. Mitochondria appeared swollen and there was clear damage to their cristae structure. At the same time, cells were undergoing active autophagy and were sensitive to autophagy inhibition by bafilomycin A1 or chloroquine. These results indicate that mitochondrial dysfunction can cause significant RPE damage and that autophagy is an important survival mechanism for cells suffering from mitochondrial damage.

RNA-binding protein dysregulation and altered expression of proteins involved in the autophagy/proteasome pathway play a role in many neurodegenerative disease onset/progression, including age-related macular degeneration (AMD). HuR/ELAVL1 is a master regulator of gene expression in human physiopathology. In ARPE-19 cells exposed to the proteasomal inhibitor MG132, HuR positively affects at posttranscriptional level p62 expression, a stress response gene involved in protein aggregate clearance with a role in AMD. Here, we studied the early effects of the proautophagy AICAR + MG132 cotreatment on the HuR-p62 pathway. We treated ARPE-19 cells with Erk1/2, AMPK, p38 MAPK , PKC, and JNK kinase inhibitors in the presence of AICAR + MG132 and evaluated HuR localization/phosphorylation and p62 expression. Two-hour AICAR + MG132 induces both HuR cytoplasmic translocation and threonine phosphorylation via the Erk1/2 pathway. In these conditions, p62 mRNA is loaded on polysomes and its translation in de novo protein is favored. Additionally, for the first time, we report that JNK can phosphorylate HuR, however, without modulating its localization. Our study supports HuR's role as an upstream regulator of p62 expression in ARPE-19 cells, helps to understand better the early events in response to a proautophagy stimulus, and suggests that modulation of the autophagy-regulating kinases as potential therapeutic targets for AMD may be relevant.

The aging process involves changes in immune regulation, i.e. adaptive immunity declines whereas innate immunity becomes activated. NF-kappaB signaling is the master regulator of the both immune systems. Two recent articles highlight the role of the NF-kappaB system in aging and immune responses. Adler et al showed that the NF-kappaB binding domain is the genetic regulatory motif which is most strongly associated with the aging process. Kwon et al studying HIV-1 infection and subsequent immune deficiency process demonstrated that HIV-1 Tat protein binds to SIRT1 protein, a well-known longevity factor, and inhibits the SIRT1-mediated deacetylation of the p65 component of the NF-kappaB complex. As a consequence, the transactivation efficiency of the NF-kappaB factor was greatly potentiated, leading to the activation of immune system and later to the decline of adaptive immunity. These observations support the scenario where immune responses and aging process can be enforced by the potentiation of NF-kappaB transactivation efficiency. Longevity factors, such as SIRT1 and its activators, might regulate the efficiency of the NF-kappaB signaling, the major outcome of which is inflamm-aging via proinflammatory responses.