VX-765 alleviates motor and cognitive impairments via inhibiting PANoptosis activation in the neonatal rats after hypoxic–ischemic brain damage

Abstract

Neonatal hypoxia–ischemia (HI) is one of the main factors that cause neonatal severe neurologic impairment and death. Shown by a large number of studies, caspase-1 plays a significant effect in diseases such as hypoxic–ischemic brain damage (HIBD) and may be a key component of the protein complex that initiates PANoptosis. VX-765, an inhibitor of caspase-1, exerts a potential neuroprotective effect in traumatic brain injury. However, it is unknown whether the administration of VX-765 has neuroprotective effects on neonatal rats that suffered HIBD, and if so, the underlying mechanisms are also still unknown. In the present study, we found that treatment with VX-765 (50 mg/kg, i.p.) significantly ameliorated the impairment of locomotor coordination functions and myodynamia as well as the spatial learning and memory in neonatal rats subjected with HIBD. These behavior improvements were attributed to VX-765 reducing infarct volumes and neuronal loss in the CA1, CA3 region of hippocampus, and deeper layers of the cortex in HIBD rats. Moreover, the enzyme-linked immunosorbent assay showed that VX-765 obviously decreased the production of neuroinflammatory factors including TNF-α, IL-1β, and IL-6. Importantly, we identified HI promoted PANoptosis activation in vivo and in vitro, and VX-765 obviously suppressed PANoptosis activation. Finally, we demonstrated that VX-765 treatment reversed neuronal injury induced by oxygen–glucose deprivation (OGD). Taken together, these results suggest that VX-765 protects the neurons against damage by suppressing neuroinflammation and PANoptosis activation, thereby improving locomotor coordination and cognitive impairments in neonatal HIBD rats, indicating that VX-765 may be an underlying therapeutic drug for the clinical treatment of hypoxic–ischemic encephalopathy (HIE).

Keywords

caspase-1 / hypoxic-ischemic encephalopathy / neuroinflammation / PANoptosis / VX-765

References

[1] Kurinczuk JJ, White-Koning M, Badawi N. Epidemiology of neonatal encephalopathy and hypoxic-ischaemic encephalopathy. Early Hum Dev. 2010; 86(6): 329-338.

[2] Oza S, Lawn JE, Hogan DR, Mathers C, Cousens SN. Neonatal cause-of-death estimates for the early and late neonatal periods for 194 countries: 2000-2013. Bull World Health Organ. 2015; 93(1): 19-28.

[3] Liu L, Oza S, Hogan D, et al. Global, regional, and national causes of child mortality in 2000-13, with projections to inform post-2015 priorities: an updated systematic analysis. Lancet. 2015; 385(9966): 430-440.

[4] Douglas-Escobar M, Weiss MD. Hypoxic-ischemic encephalopathy a review for the clinician. JAMA Pediatr. 2015; 169(4): 397-403.

[5] Liu CL, Siesjö BK, Hu BR. Pathogenesis of hippocampal neuronal death after hypoxia-ischemia changes during brain development. Neuroscience. 2004; 127(1): 113-123.

[6] Hu BR, Liu CL, Ouyang Y, Blomgren K, Siesjö BK. Involvement of caspase-3 in cell death after hypoxia-ischemia declines during brain maturation. J Cerebr Blood Flow Metabol2000; 20(9): 1294-1300.

[7] Gluckman PD, Wyatt JS, Azzopardi D, et al. Selective head cooling with mild systemic hypothermia after neonatal encephalopathy: multicentre randomised trial. Lancet. 2005; 365(9460): 663-670.

[8] Man SM, Karki R, Kanneganti TD. Molecular mechanisms and functions of pyroptosis, inflammatory caspases and inflammasomes in infectious diseases. Immunol Rev. 2017; 277(1): 61-75.

[9] Vanden Berghe T, Linkermann A, Jouan-Lanhouet S, Walczak H, Vandenabeele P. Regulated necrosis: the expanding network of non-apoptotic cell death pathways. Nat Rev Mol Cell Biol. 2014; 15(2): 134-146.

[10] Man SM, Kanneganti TD. Converging roles of caspases in inflammasome activation, cell death and innate immunity. Nat Rev Immunol. 2016; 16(1): 7-21.

[11] Chan FKM, Luz NF, Moriwaki K. Programmed necrosis in the cross talk of cell death and inflammation. Annu Rev Immunol. 2015; 33(1): 79-106.

[12] Blander JM. A long-awaited merger of the pathways mediating host defence and programmed cell death. Nat Rev Immunol. 2014; 14(9): 601-618.

[13] Christgen S, Zheng M, Kesavardhana S, et al. Identification of the PANoptosome: a molecular platform triggering pyroptosis, apoptosis, and necroptosis (PANoptosis). Front Cell Infect Microbiol. 2020; 10:237.

[14] Malireddi RKS, Gurung P, Kesavardhana S, et al. Innate immune priming in the absence of TAK1 drives RIPK1 kinase activity-independent pyroptosis, apoptosis, necroptosis, and inflammatory disease. J Exp Med. 2020; 217(3).

[15] Lawlor KE, Khan N, Mildenhall A, et al. RIPK3 promotes cell death and NLRP3 inflammasome activation in the absence of MLKL. Nat Commun. 2015; 6(1):6282.

[16] Kuriakose T, Man SM, Malireddi RKS, et al. ZBP1/DAI is an innate sensor of influenza virus triggering the NLRP3 inflammasome and programmed cell death pathways. Sci Immunol. 2016; 1(2):aag2045.

[17] Zheng M, Williams EP, Malireddi RKS, et al. Impaired NLRP3 inflammasome activation/pyroptosis leads to robust inflammatory cell death via caspase-8/RIPK3 during coronavirus infection. J Biol Chem. 2020; 295(41): 14040-14052.

[18] Tsuchiya K. Switching from apoptosis to pyroptosis: gasdermin-elicited inflammation and antitumor immunity. Int J Mol Sci. 2021; 22(1):426.

[19] Shi JJ, Zhao Y, Wang YP, et al. Inflammatory caspases are innate immune receptors for intracellular LPS. Nature. 2014; 514(7521): 187-192.

[20] Wang YQ, Kanneganti TD. From pyroptosis, apoptosis and necroptosis to PANoptosis: a mechanistic compendium of programmed cell death pathways. Comput Struct Biotec. 2021; 19: 4641-4657.

[21] Heilig R, Dilucca M, Boucher D, et al. Caspase-1 cleaves bid to release mitochondrial SMAC and drive secondary necrosis in the absence of GSDMD. Life Sci Alliance. 2020; 3(6):e202000735.

[22] Ye XD, Song GN, Huang SS, et al. Caspase-1: a promising target for preserving blood-brain barrier integrity in acute stroke. Front Mol Neurosci. 2022; 15:856372.

[23] Wen S, Deng F, Li LL, Xu L, Li X, Fan QL. VX-765 ameliorates renal injury and fibrosis in diabetes by regulating caspase-1-mediated pyroptosis and inflammation. J Diabetes Invest. 2022; 13(1): 22-33.

[24] Flores J, Noël A, Foveau B, Lynham J, Lecrux C, LeBlanc AC. Caspase-1 inhibition alleviates cognitive impairment and neuropathology in an Alzheimer's disease mouse model. Nat Commun. 2018; 9(1):3916.

[25] Hou XW, Yuan ZJ, Wang X, Cheng R, Zhou XG, Qiu J. Peptidome analysis of cerebrospinal fluid in neonates with hypoxic-ischemic brain damage. Mol Brain. 2020; 13(1):133.

[26] Chen G, Shaw MH, Kim YG, Nuñez G. NOD-like receptors: role in innate immunity and inflammatory disease. Annu Rev Pathol Mech. 2009; 4(1): 365-398.

[27] Martinon F, Burns K, Tschopp J. The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-β. Mol Cell2002; 10(2): 417-426.

[28] Chen YX, Li XH, Xiong Q, et al. Inhibiting NLRP3 inflammasome signaling pathway promotes neurological recovery following hypoxic-ischemic brain damage by increasing p97-mediated surface GluA1-containing AMPA receptors. J Transl Med. 2023; 21(1):567.

[29] Zhang WH, Wang X, Narayanan M, et al. Fundamental role of the Rip2/caspase-1 pathway in hypoxia and ischemia-induced neuronal cell death. Proc Natl Acad Sci USA. 2003; 100(26): 16012-16017.

[30] Zhang YT, Yao ZH, Xiao Y, Zhang XL, Liu JX. Downregulated XBP-1 rescues cerebral ischemia/reperfusion injury-induced pyroptosis via the NLRP3/caspase-1/GSDMD Axis. Mediat Inflamm. 2022; 2022:8007078.

[31] Bellut M, Papp L, Bieber M, Kraft P, Stoll G, Schuhmann MK. NLPR3 inflammasome inhibition alleviates hypoxic endothelial cell death in vitro and protects blood-brain barrier integrity in murine stroke. Cell Death Dis. 2022; 13(1):20.

[32] Stack JH, Beaumont K, Larsen PD, et al. IL-converting enzyme/caspase-I inhibitor VX-765 blocks the hypersensitive response to an inflammatory stimulus in monocytes from familial cold autoinflammatory syndrome patients. J Immunol2005; 175(4): 2630-2634.

[33] Wannamaker W, Davies R, Namchuk M, et al. (S)-1-((S)-2-{[1-(4-amino-3-chloro-phenyl)-methanoyl]-amino}-3,3-dimethyl-butanoyl)-pyrrolidine-2-carboxylic acid ((2R,3S)-2-ethoxy-5-oxo-tetrahydro-furan-3-yl)-amide (VX-765), an orally available selective interleukin (IL)-converting enzyme/caspase-1 inhibitor, exhibits potent anti-inflammatory activities by inhibiting the release of IL-1β and IL-18. J Pharmacol Exp Therapeut. 2007; 321(2): 509-516.

[34] Liu X, Zhang ZB, Ruan JB, et al. Inflammasome-activated gasdermin D causes pyroptosis by forming membrane pores. Nature. 2016; 535(7610): 153-158.

[35] Mula M. Emerging drugs for focal epilepsy. Expet Opin Emerg Drugs. 2013; 18(1): 87-95.

[36] Li J, Hao JH, Yao D, et al. Caspase-1 inhibition prevents neuronal death by targeting the canonical inflammasome pathway of pyroptosis in a murine model of cerebral ischemia. Cns Neurosci Ther. 2020; 26(9): 925-939.

[37] Qin ZJ, Song JQ, Lin AL, et al. GPR120 modulates epileptic seizure and neuroinflammation mediated by NLRP3 inflammasome. J Neuroinflammation. 2022; 19(1):121.

[38] Liang YB, Song PP, Chen W, et al. Inhibition of caspase-1 ameliorates ischemia-associated blood-brain barrier dysfunction and integrity by suppressing pyroptosis activation. Front Cell Neurosci. 2021; 14:540669.

[39] Dai CF, Wu B, Chen YX, et al. Aagab acts as a novel regulator of NEDD4-1-mediated PTEN nuclear translocation to promote neurological recovery following hypoxic-ischemic brain damage. Cell Death Differ. 2021; 28(8): 2367-2384.

[40] Zhang S, Taghibiglou C, Girling K, et al. Critical role of increased PTEN nuclear translocation in excitotoxic and ischemic neuronal injuries. J Neurosci. 2013; 33(18): 7997-8008.

[41] Yang DX, Qiu J, Zhou HH, et al. Dihydroartemisinin alleviates oxidative stress in bleomycin-induced pulmonary fibrosis. Life Sci. 2018; 205: 176-183.

[42] Tang M, Wang RY, Feng PP, et al. Dihydroartemisinin attenuates pulmonary hypertension through inhibition of pulmonary vascular remodeling in rats. J Cardiovasc Pharmacol. 2020; 76(3): 337-348.

[43] Yang DX, Yuan WD, Lv CJ, et al. Dihydroartemisinin supresses inflammation and fibrosis in bleomycine-induced pulmonary fibrosis in rats. Int J Clin Exp Pathol. 2015; 8(2): 1270-1281.

[44] Vorhees CV, Williams MT. Morris water maze: procedures for assessing spatial and related forms of learning and memory. Nat Protoc. 2006; 1(2): 848-858.

[45] Benedek A, Móricz K, Jurányi Z, et al. Use of TTC staining for the evaluation of tissue injury in the early phases of reperfusion after focal cerebral ischemia in rats. Brain Res. 2006; 1116(1): 159-165.

[46] Dai CF, Liu YM, Dong ZF. Tanshinone I alleviates motor and cognitive impairments via suppressing oxidative stress in the neonatal rats after hypoxic-ischemic brain damage. Mol Brain. 2017; 10(1):52.

[47] Shi XY, Lim YS, Myers AK, et al. PIK3R2/Pik3r2 activating mutations result in brain overgrowth and EEG changes. Ann Neurol. 2020; 88(6): 1077-1094.

[48] van Schie PEM, Schijns J, Becher JG, Barkhof F, van Weissenbruch MM, Vermeulen RJ. Long-term motor and behavioral outcome after perinatal hypoxic-ischemic encephalopathy. Eur J Paediatr Neurol. 2015; 19(3): 354-359.

[49] Erdi-Krausz G, Rocha R, Brown A, et al. Neonatal hypoxic-ischaemic encephalopathy: motor impairment beyond cerebral palsy. Eur J Paediatr Neurol. 2021; 35: 74-81.

[50] Weinstein JR, Koerner IP, Moller T. Microglia in ischemic brain injury. Future Neurol. 2010; 5(2): 227-246.

[51] Iadecola C, Anrather J. The immunology of stroke: from mechanisms to translation. Nat Med. 2011; 17(7): 796-808.

[52] Ravizza T, Noé F, Zardoni D, Vaghi V, Sifringer M, Vezzani A. Interleukin converting enzyme inhibition impairs kindling epileptogenesis in rats by blocking astrocytic IL-1β production. Neurobiol Dis. 2008; 31(3): 327-333.

[53] Yuan JY, Amin P, Ofengeim D. Necroptosis and RIPK1-mediated neuroinflammation in CNS diseases. Nat Rev Neurosci. 2019; 20(1): 19-33.

[54] McKinstry RC, Miller JH, Snyder AZ, et al. A prospective, longitudinal diffusion tensor imaging study of brain injury in newborns. Neurology. 2002; 59(6): 824-833.

[55] Yang JP, Zhao YY, Zhang L, et al. RIPK3/MLKL-Mediated neuronal necroptosis modulates the M1/M2 polarization of microglia/macrophages in the ischemic cortex. Cerebr Cortex. 2018; 28(7): 2622-2635.

[56] Shan B, Pan HL, Najafov A, Yuan JY. Necroptosis in development and diseases. Gene Dev. 2018; 32(5-6): 327-340.

[57] Gong Z, Pan JR, Shen QY, Li M, Peng Y. Mitochondrial dysfunction induces NLRP3 inflammasome activation during cerebral ischemia/reperfusion injury. J Neuroinflammation. 2018; 15(1):242.

[58] Chen J, Chen YQ, Shi YJ, et al. VX-765 reduces neuroinflammation after spinal cord injury in mice. Neural Regen Res. 2021; 16(9): 1836-1847.

[59] Zhang XH, Li ML, Wang B, Guo MX, Zhu RM. Caspase-1 inhibition alleviates acute renal injury in rats with severe acute pancreatitis. World J Gastroenterol. 2014; 20(30): 10457-10463.

[60] Samir P, Malireddi RKS, Kanneganti TD. The PANoptosome: a deadly protein complex driving pyroptosis, apoptosis, and necroptosis (PANoptosis). Front Cell Infect Microbiol. 2020; 10:238.

[61] Zheng M, Kanneganti TD. The regulation of the ZBP1-NLRP3 inflammasome and its implications in pyroptosis, apoptosis, and necroptosis (PANoptosis). Immunol Rev. 2020; 297(1): 26-38.

[62] Gurung P, Burton A, Kanneganti TD. NLRP3 inflammasome plays a redundant role with caspase 8 to promote IL-1β-mediated osteomyelitis. Proc Natl Acad Sci USA. 2016; 113(16): 4452-4457.

[63] Lukens JR, Gurung P, Vogel P, et al. Dietary modulation of the microbiome affects autoinflammatory disease. Nature. 2014; 516(7530): 246-249.

[64] Tsuchiya K, Nakajima S, Hosojima S, et al. Caspase-1 initiates apoptosis in the absence of gasdermin D. Nat Commun. 2019; 10(1):2091.

[65] Sedgwick JD, Riminton DS, Cyster JG, Körner H. Tumor necrosis factor: a master-regulator of leukocyte movement. Immunol Today2000; 21(3): 110-113.

[66] Sriram K, Matheson JM, Benkovic SA, Miller DB, Luster MI, O'Callaghan JP. Deficiency of TNF receptors suppresses microglial activation and alters the susceptibility of brain regions to MPTP-induced neurotoxicity: role of TNF-α. FASEB J. 2006; 20(6): 670-682.

[67] Rothwell N. Interleukin-1 and neuronal injury: mechanisms, modification, and therapeutic potential. Brain Behav Immun. 2003; 17(3): 152-157.

[68] Li J, Zhang JY, Zhang YS, et al. TRAF2 protects against cerebral ischemia-induced brain injury by suppressing necroptosis. Cell Death Dis. 2019; 10(5):328.

RIGHTS & PERMISSIONS

2024 The Authors. Pediatric Discovery published by John Wiley & Sons Australia, Ltd on behalf of Children's Hospital of Chongqing Medical University.