The cross-regulation of autophagy and necroptosis

Autophagy, apoptosis and necroptosis are three well studied intracellular signaling pathways to modulate cell fate. Among them, apoptosis and necroptosis predominantly regulate cell death and extrinsic apoptosis shares the same upstream signal transduction with necroptosis. Apoptotic cell death is mediated by the activation of a family of cysteine proteases termed caspases. However, when the activity of caspase 8 is inhibited, apoptotic cell death can switch to necroptotic cell death that is controlled by the RIPK1-RIPK3-MLKL protein complex. Although under certain conditions autophagy can also cause cell death via degrading pro-survival proteins, in general autophagy represents a pro-survival pathway via degrading misfolded proteins, damaged organelles and invaded pathogens to maintain intracellular homeostasis. While the cross-regulation of apoptosis and autophagy is under investigation for many years, the crosstalk of necroptosis and autophagy is a new field and so far poorly understood due to limited research. Therefore, my thesis focused on the cross-regulation of necroptosis and autophagy to explore the underlying mechanisms, specifically on the two protein kinase complexes of RIPK1-RIPK3 and AMPK-ULK1 that represent the signaling nodes of necroptosis and autophagy, respectively. In the first part of this thesis, it was shown that autophagy induced by amino acid starvation blocks necroptosis induction upon TNFα treatment in L929 cells. Since the Ser/Thr kinases ULK1 and RIPK1 represent the core of autophagy and necroptosis, respectively, and they are required for autophagy and necroptosis initiation, this part of my study focused on the ULK1-RIPK1 axis. The knockdown of ULK1 can enhance necroptotic cell death in L929 cells and necroptotic/apoptotic cell death in MEFs. Furthermore, ULK1 interacts with RIPK1 and restricts RIPK1 activity by direct phosphorylation at Ser 357. Moreover, ULK1-mediated phosphorylation of RIPK1 blocks the formation of complex II (RIPK1, FADD, caspase8 and/or RIPK3) and thereby cell death induced by TNFα. In the second part of this thesis, it was demonstrated that necroptosis also cross-regulates autophagy in L929 cells. This cross-regulation is mediated by the AMPK-RIPK3 axis. In L929 cells, TNFα-induced necroptosis inhibited LC3-II degradation by lysosomes. Meanwhile, TNFα-induced necroptosis also promotes autophagy via activating early pro-autophagic proteins, e.g., the phosphorylation of ULK1 at Ser 555, Beclin1 at Ser 91 and AMPK at Thr 172/Thr 183, or the formation of ATG14 puncta. This work identified necroptosis-related RIPK3 as a new regulator of autophagy. RIPK3 interacts with AMPK and directly phosphorylates it at Thr 172/Thr 183 to control AMPK activity and hence regulate autophagy. In summary, autophagy plays a pro-survival role in cell death mediated by TNFα since autophagy deficiency enhances TNFα-induced cell death. More importantly, this work provides new evidence for the cross-regulation between autophagy and necroptosis/apoptosis induced by TNFα and identifies mechanistic details, which might potentially provide novel therapeutic targets in the future. A scheme of the cross-regulatory mechanisms between autophagy and necroptosis/apoptosis identified in this thesis is shown in figure 1. Since my study mainly focuses on necroptosis and autophagy, a short introduction to these pathways is provided. In order to understand necroptosis, apoptosis will also be introduced briefly.