000136376 001__ 136376
000136376 005__ 20240731105613.0
000136376 0247_ $$2doi$$a10.1038/s41418-024-01313-6
000136376 0248_ $$2sideral$$a139157
000136376 037__ $$aART-2024-139157
000136376 041__ $$aeng
000136376 100__ $$aKelepouras, Konstantinos
000136376 245__ $$aThe importance of murine phospho-MLKL-S345 in situ detection for necroptosis assessment in vivo
000136376 260__ $$c2024
000136376 5060_ $$aAccess copy available to the general public$$fUnrestricted
000136376 5203_ $$aNecroptosis is a caspase-independent modality of cell death implicated in many inflammatory pathologies. The execution of this pathway requires the formation of a cytosolic platform that comprises RIPK1 and RIPK3 which, in turn, mediates the phosphorylation of the pseudokinase MLKL (S345 in mouse). The activation of this executioner is followed by its oligomerisation and accumulation at the plasma-membrane where it leads to cell death via plasma-membrane destabilisation and consequent permeabilisation. While the biochemical and cellular characterisation of these events have been amply investigated, the study of necroptosis involvement in vivo in animal models is currently limited to the use of Mlkl−/− or Ripk3−/− mice. Yet, even in many of the models in which the involvement of necroptosis in disease aetiology has been genetically demonstrated, the fundamental in vivo characterisation regarding the question as to which tissue(s) and specific cell type(s) therein is/are affected by the pathogenic necroptotic death are missing. Here, we describe and validate an immunohistochemistry and immunofluorescence-based method to reliably detect the phosphorylation of mouse MLKL at serine 345 (pMLKL-S345). We first validate the method using tissues derived from mice in which Caspase-8 (Casp8) or FADD are specifically deleted from keratinocytes, or intestinal epithelial cells, respectively. We next demonstrate the presence of necroptotic activation in the lungs of SARS-CoV-infected mice and in the skin and spleen of mice bearing a Sharpin inactivating mutation. Finally, we exclude necroptosis occurrence in the intestines of mice subjected to TNF-induced septic shock. Importantly, by directly comparing the staining of pMLKL-345 with that of cleaved Caspase-3 staining in some of these models, we identify spatio-temporal and functional differences between necroptosis and apoptosis supporting a role of RIPK3 in inflammation independently of MLKL versus the role of RIPK3 in activation of necroptosis.
000136376 536__ $$9info:eu-repo/grantAgreement/ES/AEI/PID2020-113963RB-I00$$9info:eu-repo/grantAgreement/ES/DGA/B29-23R$$9info:eu-repo/grantAgreement/ES/ISCIII/CB21-13-00087
000136376 540__ $$9info:eu-repo/semantics/openAccess$$aby$$uhttp://creativecommons.org/licenses/by/3.0/es/
000136376 655_4 $$ainfo:eu-repo/semantics/article$$vinfo:eu-repo/semantics/publishedVersion
000136376 700__ $$aSaggau, Julia
000136376 700__ $$aVaranda, Ana Beatriz
000136376 700__ $$aZrilic, Matea
000136376 700__ $$aKiefer, Christine
000136376 700__ $$aRakhsh-Khorshid, Hassan
000136376 700__ $$aLisewski, Ina
000136376 700__ $$aUranga-Murillo, Iratxe
000136376 700__ $$0(orcid)0000-0002-9730-2210$$aArias, Maykel
000136376 700__ $$aPardo, Julian
000136376 700__ $$aTonnus, Wulf
000136376 700__ $$aLinkermann, Andreas
000136376 700__ $$aAnnibaldi, Alessandro
000136376 700__ $$aWalczak, Henning
000136376 700__ $$aLiccardi, Gianmaria
000136376 773__ $$g31, 7 (2024), 897-909$$pCell death differ.$$tCell Death and Differentiation$$x1350-9047
000136376 8564_ $$s8550271$$uhttps://zaguan.unizar.es/record/136376/files/texto_completo.pdf$$yVersión publicada
000136376 8564_ $$s3007800$$uhttps://zaguan.unizar.es/record/136376/files/texto_completo.jpg?subformat=icon$$xicon$$yVersión publicada
000136376 909CO $$ooai:zaguan.unizar.es:136376$$particulos$$pdriver
000136376 951__ $$a2024-07-31-09:24:00
000136376 980__ $$aARTICLE