Transcriptional and post-transcriptional regulation of Ambra1 in physiological and pathological conditions

(Macro)autophagy is the process of self-digestion of cellular compartments through lysosomal pathway. Ambra1 is one of the genes involved in this process which is regulated at both transcriptional and post-translational levels. Considering the involvement of Ambra1 in various disease conditions, it is of great significance to identify the factors involved in its regulation. In this study we analyzed Amba1 promoter and identified two fragments with inducer and repressor activities. We also analyzed the effect of different stressors such as starvation, DNA damage, and hypoxia on Ambra1. Starvation could upregulate Ambra1 both at RNA and protein levels. Induction of DNA damage by etoposide resulted in an increase in Ambra1 protein levels without affecting RNA levels. The stressor which was studied most extensively was hypoxia induced by hypoxia mimetic, CoCl2. Interestingly, while some autophagy genes were up-regulated upon treatment, a decrease in amount of Ambra1 protein levels was detected. In the meantime, qRT-PCR analysis showed only a slight increase in Amrba1 mRNA levels at the early stages of hypoxia, but no significant changes were observed at the later time points. Long-term stress such as starvation or staurosporin treatment leads to Ambra1 protein degradation through caspase and calpain cleavage, therefore inhibiting the pro-survival role of autophagic machinery. After CoCl2 treatment for 18 hours, caspase 3 was cleaved, this showing the activation of apoptotic machinery. Still, the inhibition of caspases along with hypoxia did not completely restore the levels of Ambra1 protein, indicating that other mechanisms are possibly involved. This led us to investigate the translation rate of Ambra1 mRNA. We observed translocation of Ambra1 mRNA from polysomes to non-translating fractions. Besides, we analyzed the mRNA localization in the cytoplasm. Interestingly, Ambra1 mRNA was present in distinct foci in the cell under normal conditions while CoCl2 treatment resulted in more intense and larger foci. Co-detection of mRNA and protein experiments revealed the re-localization of Ambra1 to cytoplasmic P-bodies which results in translational repression. This data suggests a possible new mechanism regulating autophagy. Still, further experiments such as RNA-IP and localization of the mRNA of other autophagy genes are required to confirm this hypothesis.