1Department of Neurobiology, School of Basic Medical Sciences and National Clinical Research Center for Aging and Medicine, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai, China 2Institute for Basic Research on Aging and Medicine, the State Key Laboratory of Medical Neurobiology, School of Basic Medical Sciences, Fudan University, Shanghai, China 3Shanghai Key Laboratory of Clinical Geriatric Medicine, Huadong Hospital, Fudan University, Shanghai, China
Pharmacological studies have indirectly shown that necroptosis participates in ischemic neuronal death. However, its mechanism has yet to be elucidated in the ischemic brain. TNFα-triggered RIPK1 kinase activation could initiate RIPK3/MLKL-mediated necroptosis under inhibition of caspase-8. In the present study, we performed middle cerebral artery occlusion (MCAO) to induce cerebral ischemia in rats and used immunoblotting and immunostaining combined with pharmacological analysis to study the mechanism of necroptosis in ischemic brains. In the ipsilateral hemisphere, we found that ischemia induced the increase of (i) RIPK1 phosphorylation at the Ser166 residue (p-RIPK1), representing active RIPK1 kinase and (ii) the number of cells that were double stained with P-RIPK1 (Ser166) (p-RIPK1+) and TUNEL, a label of DNA double-strand breaks, indicating cell death. Furthermore, ischemia induced activation of downstream signaling factors of RIPK1, RIPK3 and MLKL, as well as the formation of mature interleukin-1β (IL-1β). Treatment with necrostatin-1 (Nec-1), an inhibitor of necroptosis, significantly decreased ischemia-induced increase of p-RIPK1 expression and p-RIPK1+ neurons, which showed protection from brain damage. Meanwhile, Nec-1 reduced RIPK3, MLKL and p-MLKL expression levels and mature IL-1β formation in Nec-1 treated ischemic brains. Our results clearly demonstrated that phosphorylation of RIPK1 at the Ser166 residue was involved in the pathogenesis of necroptosis in the brains after ischemic injury. Nec-1 treatment protected brains against ischemic necroptosis by reducing the activation of RIPK1 and inhibiting its downstream signaling pathways. These results provide direct in vivo evidence that phosphorylated RIPK1 (Ser 166) plays an important role in the initiation of RIPK3/MLKL-dependent necroptosis in the pathogenesis of ischemic stroke in the rodent brain.
Xu-Xu Deng,Shan-Shan Li,Feng-Yan Sun. Necrostatin-1 Prevents Necroptosis in Brains after Ischemic Stroke via Inhibition of RIPK1-Mediated RIPK3/MLKL Signaling[J]. Aging and disease,
2019, 10(4): 807-817.
Figure 1. Expression and identification of phosphorylated RIPK1 in rat brains after ischemic injury
(A) Expression levels of RIPK1 in sham-operated (Sham) and ipsilateral (IPSI)/contralateral (CON) striatum at 24 h after MCAO. Expression levels of total RIPK1 (B) and low molecular weight RIPK1 (C) increased in the ipsilateral striatum compared with the contralateral striatum and sham-operated rats (n≥6). *p<0.05. (D) De-phosphorylation of RIPK1 in the ipsilateral striatum at 24 h after MCAO by calf intestinal alkaline phosphatase (CIP) treatment. (E) Expression levels of RIPK1 in the nuclear and cytoplasmic compartments of cells in the ipsilateral striatum, H3 was used as a specific nuclear marker and GAPDH as a specific cytoplasmic marker. (F) L-RIPK1 immuno-reacted with p-RIPK1 (Ser166) monoclonal antibody.
Figure 2. Distribution of phosphorylated RIPK1 (Ser166) in the ischemic rat brain
(A) Morphology of p-RIPK1 positive (p-RIPK1+) cells in the contralateral (CON) and the ipsilateral (IPSI) striatum at 24 h after MCAO. (B) p-RIPK1+ (red) signals co-localized with DAPI (blue) in the ipsilateral striatum of rats at 24 h after MCAO. (C) p-RIPK1 and NeuN double positive (p-RIPK1+-NeuN+) neurons in the ipsilateral striatum at 24 h after MCAO. (D) The number of p-RIPK1+-NeuN+ neurons increased in the ipsilateral (IPSI) compared with the contralateral (CON) striatum at 24 h after MCAO (n=5). *p<0.05. (E) p-RIPK1+ signals co-localized with TUNEL stain (green) in the ipsilateral striatum at 24 h after MCAO. Both black and white scale bars in all figures represent 50 μm.
Figure 3. Necrostatin-1 treatment protected brains against ischemic injury
(A) Experimental protocol of necrostatin-1 (Nec-1) treatment in MCAO rats. Nec-1/inactive necrostatin-1 (iNec) were stereotaxically injected to the right lateral ventricle 30 min before MCAO surgery. Rat brains were subjected to reperfusion at 30 min after occlusion of the MCA. Neurological evaluations were performed at 1 h before MCAO (pre-) and 12, 24, 72 h after MCAO. (B) Schematic of the brain shows the injection position of Nec-1/iNec (contralateral ventricle) and the areas of the infarct core (dotted line) after ischemic injury. (C-D) Nec-1 reduced infarct volume at 24 h after MCAO compared with iNec group (n=6-7). (E-F) Nec-1 reduced neurological impairments after cerebral ischemia. The graphs show neurological scores (E) and forelimb placing scores (F) of rats treated with Nec-1 (black bar) or iNec (white bar). **p<0.01.
Figure 4. Necrostatin-1 treatment prevented phosphorylation of RIPK1 at Ser166 in neurons after cerebral ischemia
(A-B) Nec-1 treatment significantly inhibited ischemia-induced increase of p-RIPK1 expression in the ipsilateral striatum (IPSI) at 24 h after MCAO (compared with iNec treatment, p<0.05, n≥10). (C-D) Nec-1 reduced the number of p-RIPK1-NeuN double positive (p-RIPK1+-NeuN+) neurons in the ipsilateral striatum (IPSI) at 24 h after MCAO (compared with iNec treatment, p<0.05, n=5). CON=contralateral striatum. Scale bars indicate 50 μm.
Figure 5. Necrostatin-1 treatment inhibited ischemia-induced expression of RIPK3/MLKL and formation of IL-1β in rat brains after MCAO
Expression levels of RIPK3, MLKL, p-MLKL and IL-1β (A) in the ipsilateral (IPSI) and contralateral (CON) striatum at 24 h after MCAO. Statistical analysis showed that Nec-1 treatment significantly suppressed RIPK3 (B), MLKL (C), p-MLKL (D) and mature IL-1β (E) expression levels in the ipsilateral striatum of MCAO rats compared with iNec-1 treatment (CON), n=4, *p<0.05.
Lipton P (1999). Ischemic cell death in brain neurons. Physiol Rev, 79: 1431-1568.
Kalogeris T, Baines CP, Krenz M, Korthuis RJ. 2012. CELL BIOLOGY OF ISCHEMIA/REPERFUSION INJURY. In International Review of Cell and Molecular Biology, Vol 298. Jeon KW, editor. San Diego: Elsevier Academic Press Inc. 229-317.
Puyal J, Ginet V, Clarke PGH (2013). Multiple interacting cell death mechanisms in the mediation of excitotoxicity and ischemic brain damage: A challenge for neuroprotection. Prog Neurobiol, 105: 24-48.
Holler N, Zaru R, Micheau O, Thome M, Attinger A, Valitutti S, et al. (2000). Fas triggers an alternative, caspase-8-independent cell death pathway using the kinase RIP as effector molecule. Nat Immunol, 1: 489-495.
Degterev A, Huang Z, Boyce M, Li Y, Jagtap P, Mizushima N, et al. (2005). Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury. Nat Chem Biol, 1: 112-119.
Degterev A, Hitomi J, Germscheid M, Ch'en IL, Korkina O, Teng X, et al. (2008). Identification of RIP1 kinase as a specific cellular target of necrostatins. Nat Chem Biol, 4: 313-321.
Xu XS, Chua KW, Chua CC, Liu CF, Hamdy RC, Chua BHL (2010). Synergistic protective effects of humanin and necrostatin-1 on hypoxia and ischemia/reperfusion injury. Brain Res, 1355: 189-194.
Wang YQ, Wang L, Zhang MY, Wang T, Bao HJ, Liu WL, et al. (2012). Necrostatin-1 Suppresses Autophagy and Apoptosis in Mice Traumatic Brain Injury Model. Neurochem Res, 37: 1849-1858.
You ZR, Savitz SI, Yang JS, Degterev A, Yuan JY, Cuny GD, et al. (2008). Necrostatin-1 reduces histopathology and improves functional outcome after controlled cortical impact in mice. J Cereb Blood Flow Metab, 28: 1564-1573.
Chavez-Valdez R, Martin LJ, Flock DL, Northington FJ (2012). Necrostatin-1 attenuates mitochondrial dysfunction in neurons and astrocytes following neonatal hypoxia-ischemia. Neuroscience, 219: 192-203.
Chavez-Valdez R, Martin LJ, Northington FJ (2012). Programmed Necrosis: A Prominent Mechanism of Cell Death following Neonatal Brain Injury. Neurol Res Int, 2012: 257563.
Northington FJ, Chavez-Valdez R, Graham EM, Razdan S, Gauda EB, Martin LJ (2011). Necrostatin decreases oxidative damage, inflammation, and injury after neonatal HI. J Cereb Blood Flow Metab, 31: 178-189.
Stanger BZ, Leder P, Lee T-H, Kim E, Seed B (1995). RIP: A novel protein containing a death domain that interacts with Fas/APO-1 (CD95) in yeast and causes cell death. Cell, 81: 513-523.
Sun X, Yin J, Starovasnik MA, Fairbrother WJ, Dixit VM (2002). Identification of a novel homotypic interaction motif required for the phosphorylation of receptor-interacting protein (RIP) by RIP3. J Biol Chem, 277: 9505-9511.
Cho YS, Challa S, Moquin D, Genga R, Ray TD, Guildford M, et al. (2009). Phosphorylation-driven assembly of the RIP1-RIP3 complex regulates programmed necrosis and virus-induced inflammation. Cell, 137: 1112-1123.
Sun L, Wang H, Wang Z, He S, Chen S, Liao D, et al. (2012). Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase. Cell, 148: 213-227.
Ofengeim D, Yuan J (2013). Regulation of RIP1 kinase signalling at the crossroads of inflammation and cell death. Nat Rev Mol Cell Biol, 14: 727-736.
Meng H, Liu Z, Li X, Wang H, Jin T, Wu G, et al. (2018). Death-domain dimerization-mediated activation of RIPK1 controls necroptosis and RIPK1-dependent apoptosis. Proc Natl Acad Sci U S A, 115(9): E2001-E2009.
Ofengeim D, Ito Y, Najafov A, Zhang Y, Shan B, DeWitt JP, et al. (2015). Activation of Necroptosis in Multiple Sclerosis. Cell Rep. 10(11): 1836-49.
Yang ZJ, Bao WL, Qiu MH, Zhang LM, Lu SD, Huang YL, et al. (2002). Role of vascular endothelial growth factor in neuronal DNA damage and repair in rat brain following a transient cerebral ischemia. J Neurosci Res, 70: 140-149.
Longa EZ, Weinstein PR, Carlson S, Cummins R (1989). Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke, 20: 84-91.
Schallert T, Fleming SM, Leasure JL, Tillerson JL, Bland ST (2000). CNS plasticity and assessment of forelimb sensorimotor outcome in unilateral rat models of stroke, cortical ablation, parkinsonism and spinal cord injury. Neuropharmacology, 39: 777-787.
Li J, McQuade T, Siemer AB, Napetschnig J, Moriwaki K, Hsiao YS, et al. (2012). The RIP1/RIP3 necrosome forms a functional amyloid signaling complex required for programmed necrosis. Cell, 150: 339-350.
Chen X, Li W, Ren J, Huang D, He WT, Song Y, et al. (2014). Translocation of mixed lineage kinase domain-like protein to plasma membrane leads to necrotic cell death. Cell Res, 24: 105-121.
Cai Z, Jitkaew S, Zhao J, Chiang HC, Choksi S, Liu J, et al. (2014). Plasma membrane translocation of trimerized MLKL protein is required for TNF-induced necroptosis. Nat Cell Biol, 16: 55-65.
Laster SM, Wood JG, Gooding LR (1988). Tumor necrosis factor can induce both apoptic and necrotic forms of cell lysis. J Immunol, 141: 2629-2634.
Vandenabeele P, Galluzzi L, Vanden Berghe T, Kroemer G (2010). Molecular mechanisms of necroptosis: an ordered cellular explosion. Nat Rev Mol Cell Biol, 11: 700-714.
Xie T, Peng W, Liu Y, Yan C, Maki J, Degterev A, et al. (2013). Structural Basis of RIP1 Inhibition by Necrostatins. Structure, 21: 493-499.
Degterev A, Maki JL, Yuan J (2013). Activity and specificity of necrostatin-1, small-molecule inhibitor of RIP1 kinase. Cell Death Differ, 20: 366.
Takahashi N, Duprez L, Grootjans S, Cauwels A, Nerinckx W, DuHadaway JB, et al. (2012). Necrostatin-1 analogues: critical issues on the specificity, activity and in vivo use in experimental disease models. Cell Death Dis, 3: e437.
Dondelinger Y, Jouan-Lanhouet S, Divert T, Theatre E, Bertin J, Gough PJ, et al. (2015). NF-kappaB-Independent Role of IKKalpha/IKKbeta in Preventing RIPK1 Kinase-Dependent Apoptotic and Necroptotic Cell Death during TNF Signaling. Mol Cell, 60: 63-76.
Lambertsen KL, Biber K, Finsen B (2012). Inflammatory cytokines in experimental and human stroke. J Cereb Blood Flow Metab, 32: 1677-1698.
Rickard JA, O'Donnell JA, Evans JM, Lalaoui N, Poh AR, Rogers T, et al. (2014). RIPK1 regulates RIPK3-MLKL-driven systemic inflammation and emergency hematopoiesis. Cell, 157: 1175-1188.
Suda J, Dara L (2016). Knockdown of RIPK1 Markedly Exacerbates Murine Immune-Mediated Liver Injury through Massive Apoptosis of Hepatocytes, Independent of Necroptosis and Inhibition of NF-kappaB. J Immunol, 197: 3120-3129.