Abstract
The aim of this study was to determine whether autophagy and AMPK contribute to premature senescence in auditory cells. Incubating HEI-OC1 auditory cells with 5 mM H2O2 for 1 h induced senescence, as demonstrated by senescence-associated β-galactosidase (SA-β-gal) staining. H2O2 treatment significantly delayed population-doubling time, leaving cell viability unchanged. Furthermore, the proportion of SA-β-gal-positive cells significantly increased. Autophagy-related protein expression increased, with Atg7 and LC3-II peaking 6 h and Lamp2 peaking 24 h after H2O2 treatment. The expression of these proteins decreased 48 h after treatment. Transmission electron microscopy revealed lipofuscin and aggregates within autolysosomes, which accumulated markedly in the cytoplasm of HEI-OC1 cells 48 h after treatment. Akt and P70S6 phosphorylation markedly decreased after H2O2 treatment, but 4EBP1 phosphorylation significantly increased 48 h after treatment. After RNAi-mediated knockdown (KD) of Atg7 and AMPK, H2O2-treated cells displayed dense SA-β-gal staining. Also, premature senescence was significantly induced. These suggest that a negative feedback loop may exist between autophagy and AMPK signaling pathways in HEI-OC1 cells. In our model, oxidative stress-induced premature senescence occurred due to impaired autophagy function through 4EBP1 phosphorylation. Our results also indicate that AMPK may regulate premature senescence in auditory cells in an autophagy-dependent and independent manner.
Highlights
Aging is a physiological phenomenon that occurs without fail in all eukaryotes
To determine whether oxidative stress plays a role in cellular senescence in auditory cells, we treated cultured House Ear Institute-Organ of Corti 1 (HEI-OC1) cells with different concentrations of H2O2 (2 mM or 5 mM for 1 h), washed out the H2O2 with normal culture medium, and incubated the cells under permissive conditions [26]
We examined SA-β-gal staining in cultured HEI-OC1 cells treated briefly with H2O2, as described in the Methods (Cell Viability Assay section)
Summary
Aging is a physiological phenomenon that occurs without fail in all eukaryotes. Cellular senescence manifests as stable cell cycle arrest with active metabolism. The study of cellular senescence began in 1961 when Hayflick and Moorhead discovered that human fibroblasts could only divide a finite number of times in culture [2]. This phenomenon, called replicative senescence, was subsequently determined to result from telomere shortening due to repeated chromosome replication and division [3]. SIPS occurs irrespective of telomere shortening status This shows that cellular senescence does not necessarily need to unfold after extended time passage
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