Necroptosis is associated with Rab27‐independent expulsion of extracellular vesicles containing RIPK3 and MLKL

Abstract

Extracellular vesicle (EV) secretion is an important mechanism used by cells to release biomolecules. A common necroptosis effector—mixed lineage kinase domain like (MLKL)—was recently found to participate in the biogenesis of small and large EVs independent of its function in necroptosis. The objective of the current study is to gain mechanistic insights into EV biogenesis during necroptosis. Assessing EV number by nanoparticle tracking analysis revealed an increased number of EVs released during necroptosis. To evaluate the nature of such vesicles, we performed a newly adapted, highly sensitive mass spectrometry‐based proteomics on EVs released by healthy or necroptotic cells. Compared to EVs released by healthy cells, EVs released during necroptosis contained a markedly higher number of unique proteins. Receptor interacting protein kinase‐3 (RIPK3) and MLKL were among the proteins enriched in EVs released during necroptosis. Further, mouse embryonic fibroblasts (MEFs) derived from mice deficient of Rab27a and Rab27b showed diminished basal EV release but responded to necroptosis with enhanced EV biogenesis as the wildtype MEFs. In contrast, necroptosis‐associated EVs were sensitive to Ca2+ depletion or lysosomal disruption. Neither treatment affected the RIPK3‐mediated MLKL phosphorylation. An unbiased screen using RIPK3 immunoprecipitation‐mass spectrometry on necroptotic EVs led to the identification of Rab11b in RIPK3 immune‐complexes. Our data suggests that necroptosis switches EV biogenesis from a Rab27a/b dependent mechanism to a lysosomal mediated mechanism.

Systematic compositional analysis of exosomal extracellular vesicles produced by cells undergoing apoptosis, necroptosis and ferroptosis

“…In the present work, cdEVs released following different RCD modalities exhibit strong similarities in biophysical properties and protein composition, suggesting the involvement of a common pathway in their biogenesis and cargo loading. Here, we reported the presence of tetraspanin (CD63, CD81), lipid raft-associated proteins flotillin-1 and -2, and ESCRT-III components (CHMP2-A and -B), in agreement with previous reports on apoptotic (Park et al, 2018;Tucher et al, 2018), necroptotic (Gupta et al, 2022;Shlomovitz et al, 2021;Yoon et al, 2017;Zargarian et al, 2017) and ferroptotic EVs (Brown et al, 2019;Ito et al, 2021;Kajiwara et al, 2022). However, the amount of these classically used exosomal markers (CD63, CD81, CHMP2A, CHMP2B, CHMP4B, CHMP4C, TSG101, FLOT1, FLOT2, Alix, nSMase) was quite similar between the different cdEVs, and they were relatively underrepresented in cdEVs compared to ssEVs, as revealed by proteomics.…”

Necroptosis in CNS diseases: Focus on astrocytes

“…Unlike "apoptotic bodies, " "necroptotic bodies" do not appear to contain DNA. These necroptotic bodies can probably modulate the inflammatory response of surrounding cells (Edry-Botzer and Gerlic, 2017;Gupta et al, 2022). It remains to be investigated whether "necroptotic bodies" can undergo phagocytosis.…”

Ferroptosis: Underlying mechanism and the crosstalk with other modes of neuronal death after intracerebral hemorrhage

“…It is a regulated form of necrotic cell death mediated by receptor-interacting kinase 1 (RIPK1), receptor-interacting protein kinase 3 (RIPK3), and mixed lineage kinase domain-like (MLKL) ( Conrad et al, 2016 ; Cao et al, 2022 ). RIPK1 activates RIPK3 and thereby recruits MLKL at the cell membrane, which causes membrane rupture and eventually triggers necroptosis ( Samson et al, 2021 ; Gupta et al, 2022 ). Necroptosis is involved in cell death associated with ICH ( Galluzzi et al, 2018 ).…”

“…Neurovascular injury and related hemolysis of extravasated erythrocytes post-ICH producing hemoglobin degradation metabolites may trigger the neuroinflammatory response of surrounding astroglia resulting in activation of the necroptotic pathway. Meanwhile, Necrostatin-1, a specific RIPK1 inhibitor, has been shown to reduce cell death, hematoma volume, and neurobehavioral outcomes in a mouse model of ICH ( Gupta et al, 2022 ). Numerous reports have suggested that ferroptosis is always accompanied by necroptosis ( Lv et al, 2021 ).…”