Purpose: The pathogenesis of age-related macular degeneration involves impaired protein degradation in retinal pigment epithelial (RPE) cells. The ubiquitin-proteasome pathway and the lysosomal pathway including autophagy are the major proteolytic systems in eukaryotic cells. Recently, p62/sequestosome 1 (p62) has been shown to be a key player linking the proteasomal and lysosomal clearance systems. In the present study, the effects of AICAR (AICA ribonucleotide, 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside) and MG-132 (proteasome inhibitor) on autophagy regulation in ARPE-19 cells were evaluated. Methods: The AMP activated protein kinase (AMPK), p62 and ubiquitin protein levels were analyzed by western blotting. pDendra2-hLC3 construct was used to detect macroautophagy in confocal microscopy analysis. Transmission electron microscopy was used to detect protein aggregates and autophagosomes. Cellular permeability was measured by analyzing lactate dehydrogenase levels in culture medium. Results: MG-132 (5 microM) triggered the accumulation of perinuclear aggregates that strongly colocalized with p62 and ubiquitin. AICAR (2mM) induced autophagy clearance of p62 and ubiquitin positive protein aggregates without increasing cellular permeability. Cellular energy status regulator AMPK or p-AMPK levels were not significantly changed in response to AICAR treatment. Conclusion: Our findings open new avenues for understanding the mechanisms of proteolytic processes and indicate that AICAR could be useful in the acceleration of protein clearance in RPE cells.

AICAR INDUCES AUTOPHAGY IN ARPE-19 CELLS

AMADIO, MARIALAURA;MARCHESI, NICOLETTA;PASCALE, ALESSIA ANGELA;
2011

Abstract

Purpose: The pathogenesis of age-related macular degeneration involves impaired protein degradation in retinal pigment epithelial (RPE) cells. The ubiquitin-proteasome pathway and the lysosomal pathway including autophagy are the major proteolytic systems in eukaryotic cells. Recently, p62/sequestosome 1 (p62) has been shown to be a key player linking the proteasomal and lysosomal clearance systems. In the present study, the effects of AICAR (AICA ribonucleotide, 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside) and MG-132 (proteasome inhibitor) on autophagy regulation in ARPE-19 cells were evaluated. Methods: The AMP activated protein kinase (AMPK), p62 and ubiquitin protein levels were analyzed by western blotting. pDendra2-hLC3 construct was used to detect macroautophagy in confocal microscopy analysis. Transmission electron microscopy was used to detect protein aggregates and autophagosomes. Cellular permeability was measured by analyzing lactate dehydrogenase levels in culture medium. Results: MG-132 (5 microM) triggered the accumulation of perinuclear aggregates that strongly colocalized with p62 and ubiquitin. AICAR (2mM) induced autophagy clearance of p62 and ubiquitin positive protein aggregates without increasing cellular permeability. Cellular energy status regulator AMPK or p-AMPK levels were not significantly changed in response to AICAR treatment. Conclusion: Our findings open new avenues for understanding the mechanisms of proteolytic processes and indicate that AICAR could be useful in the acceleration of protein clearance in RPE cells.
File in questo prodotto:
Non ci sono file associati a questo prodotto.

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11571/443136
Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus 20
  • ???jsp.display-item.citation.isi??? ND
social impact