Lysosomal damage after spinal cord injury causes accumulation of RIPK1 and RIPK3 proteins and potentiation of necroptosis

Abstract

Necroptosis, a regulated necrosis pathway mediated by the receptor-interacting protein kinases 1 and 3 (RIPK1 and RIPK3), is induced following spinal cord injury (SCI) and thought to contribute to neuronal and glial cell death. However, mechanisms leading to activation of necroptosis after SCI remain unclear. We have previously shown that autophagy, a catabolic pathway facilitating degradation of cytoplasmic proteins and organelles in a lysosome-dependent manner, is inhibited following SCI in rats. Our current data confirm that inhibition of autophagy also occurs after thoracic contusive SCI in the mouse model, as indicated by accumulation of both the autophagosome marker, LC3-II and autophagy cargo protein, p62/SQSTM1. This was most pronounced in the ventral horn neurons and was caused by rapid inhibition of lysosomal function after SCI. Interestingly, RIPK1, RIPK3, and the necroptosis effector protein MLKL also rapidly accumulated after SCI and localized to neurons with disrupted autophagy, suggesting that these events may be related. To determine if lysosomal dysfunction could contribute to induction of necroptosis, we treated PC12 cells and primary rat cortical neurons with lysosomal inhibitors. This led to rapid accumulation of RIPK1 and RIPK3, confirming that they are normally degraded by the lysosomal pathway. In PC12 cells lysosomal inhibition also sensitized cells to necroptosis induced by tumor necrosis factor α (TNFα) and caspase inhibitor. Imaging studies confirmed that RIPK1 partially localized to lysosomes in both untreated and lysosomal inhibitor treated cells. Similarly, we detected presence of RIPK1, RIPK3 and MLKL in both cytosol and at lysosomes after SCI in vivo. Furthermore, stimulation of autophagy and lysosomal function with rapamycin treatment led to decreased accumulation of RIPK1 and attenuated cell death after SCI. These data suggest that lysosomal dysfunction after SCI may contribute to both inhibition of autophagy and sensitize cells to necroptosis by promoting RIPK1 and RIPK3 accumulation.

Rotenone: from modelling to implication in Parkinson’s disease

“…In addition to apoptotic and necrotic cell death, rotenone was reported to lead to necroptosis in primary rat dopaminergic neurons as measured by the upregulation of RIPK3 after 24 h of exposure [6]. Upregulation of RIPK3 is an indicator of necroptotic cell death [47].…”

Metformin mediates cardioprotection against aging‐induced ischemic necroptosis

“…p62 accumulation is believed to cause organ dysfunction, such as in the liver and brain (Hara et al, 2016;Takamura et al, 2011). Recent data have also shown that p62 can serve as a scaffold to modulate the mode of programmed cell death in cancer cells (Goodall et al, 2016) or neurons (Liu et al, 2018). However, in aged hearts, definitive in vivo evidence that p62 regulates stress-induced cardiomyocyte death is less well established.…”

Distinct photooxidation-induced cell death pathways lead to selective killing of human breast cancer cells

“…It has been previously shown by others that RIPK1 and RIPK3 can be degraded in lysosomes and that inhibition of lysosomal function, with LMP, leads to this kinases accumulation and necroptosis induction 59,60 , strengthening the possibility of a link between LMP and necroptosis. Indeed, we have shown that, in breast cancer cells submitted to MB-PDT, lysosomal cathepsin inhibition was able to suppress cell death and MLKL phosphorylation, providing a clear evidence of the existence of cross-talks between LDCD and necroptosis, triggered by MB-PDT.…”