Abstract
Newborns suffering from hypoxia-ischemia (HI) brain injury still lack effective treatment. Proline-rich tyrosine kinase 2 (Pyk2) is a non-receptor tyrosine kinase, which is highly correlated with transient ischemic brain injury in adult. In this study, we investigated the role of Pyk2 in neonatal HI brain injury. HI was induced in postnatal day 7 mouse pups by unilateral common carotid artery ligation followed by hypoxic exposure. Pyk2 interference lentivirus (LV-Pyk2 shRNA) was constructed and injected into unilateral cerebral ventricle of neonatal mice before HI. Infarct volume, pathological changes, and neurological behaviors were assessed on postnatal day 8–14. We showed that the phosphorylation level of Pyk2 was significantly increased in neonatal brain after HI, whereas LV-Pyk2 shRNA injection significantly attenuated acute HI brain damage and improved neurobehavioral outcomes. In oxygen-glucose deprivation-treated cultured cortical neurons, Pyk2 inhibition significantly alleviated NMDA receptor-mediated excitotoxicity; similar results were also observed in neonatal HI brain injury. We demonstrated that Pyk2 inhibition contributes to the long-term cerebrovascular recovery assessed by laser speckle contrast imaging, but cognitive function was not obviously improved as evaluated in Morris water maze and novel object recognition tests. Thus, we constructed lentiviral LV-HIF-Pyk2 shRNA, through which HIF-1α promoter-mediated interference of Pyk2 would occur during the anoxic environment. Intracerebroventricular injection of LV-HIF-Pyk2 shRNA significantly improved long-term recovery of cognitive function in HI-treated neonatal mice. In conclusion, this study demonstrates that Pyk2 interference protects neonatal brain from hypoxic-ischemic injury. HIF-1α promoter-mediated hypoxia conditional control is a useful tool to distinguish between hypoxic period and normal period. Pyk2 is a promising drug target for potential treatment of neonatal HI brain injury.
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References
Higgins RD. Hypoxic ischemic encephalopathy and hypothermia: a critical look. Obstet Gynecol. 2005;106:1385–7.
Douglas-Escobar M, Weiss MD. Hypoxic-ischemic encephalopathy: a review for the clinician. JAMA Pediatr. 2015;169:397–403.
Kurinczuk JJ, White-Koning M, Badawi N. Epidemiology of neonatal encephalopathy and hypoxic-ischaemic encephalopathy. Early Hum Dev. 2010;86:329–38.
Dixon BJ, Reis C, Ho WM, Tang J, Zhang JH. Neuroprotective strategies after neonatal hypoxic ischemic encephalopathy. Int J Mol Sci. 2015;16:22368–401.
Pregnolato S, Chakkarapani E, Isles AR, Luyt K. Glutamate transport and preterm brain injury. Front Physiol. 2019;10:417.
Davis SM, Lees KR, Albers GW, Diener HC, Markabi S, Karlsson G, et al. Selfotel in acute ischemic stroke: possible neurotoxic effects of an NMDA antagonist. Stroke. 2000;31:347–54.
Ikonomidou C, Bosch F, Miksa M, Bittigau P, Vockler J, Dikranian K, et al. Blockade of NMDA receptors and apoptotic neurodegeneration in the developing brain. Science. 1999;283:70–4.
Tian D, Litvak V, Lev S. Cerebral ischemia and seizures induce tyrosine phosphorylation of PYK2 in neurons and microglial cells. J Neurosci. 2000;20:6478–87.
Ma J, Zhang GY, Liu Y, Yan JZ, Hao ZB. Lithium suppressed Tyr-402 phosphorylation of proline-rich tyrosine kinase (Pyk2) and interactions of Pyk2 and PSD-95 with NR2A in rat hippocampus following cerebral ischemia. Neurosci Res. 2004;49:357–62.
Wenzel A, Fritschy JM, Mohler H, Benke D. NMDA receptor heterogeneity during postnatal development of the rat brain: differential expression of the NR2A, NR2B, and NR2C subunit proteins. J Neurochem. 1997;68:469–78.
Sans N, Petralia RS, Wang YX, Blahos J 2nd, Hell JW, Wenthold RJ. A developmental change in NMDA receptor-associated proteins at hippocampal synapses. J Neurosci. 2000;20:1260–71.
Kilkenny C, Browne W, Cuthill IC, Emerson M, Altman DG, Group NCRRGW. Animal research: reporting in vivo experiments: the ARRIVE guidelines. Br J Pharmacol. 2010;160:1577–9.
McGrath JC, Lilley E. Implementing guidelines on reporting research using animals (ARRIVE etc.): new requirements for publication in BJP. Br J Pharmacol. 2015;172:3189–93.
Hasselmann J, Coburn MA, England W, Figueroa Velez DX, Kiani Shabestari S, Tu CH, et al. Development of a chimeric model to study and manipulate human microglia in vivo. Neuron. 2019;103:1016–33.
Xu B, Xiao AJ, Chen W, Turlova E, Liu R, Barszczyk A, et al. Neuroprotective effects of a PSD-95 inhibitor in neonatal hypoxic-ischemic brain injury. Mol Neurobiol. 2016;53:5962–70.
Rice JE 3rd, Vannucci RC, Brierley JB. The influence of immaturity on hypoxic-ischemic brain damage in the rat. Ann Neurol. 1981;9:131–41.
Levine S. Anoxic-ischemic encephalopathy in rats. Am J Pathol. 1960;36:1–17.
Qi X, Hosoi T, Okuma Y, Kaneko M, Nomura Y. Sodium 4-phenylbutyrate protects against cerebral ischemic injury. Mol Pharmacol. 2004;66:899–908.
Chen C, Chu SF, Ai QD, Zhang Z, Guan FF, Wang SS, et al. CKLF1 aggravates focal cerebral ischemia injury at early stage partly by modulating microglia/macrophage toward m1 polarization through CCR4. Cell Mol Neurobiol. 2019;39:651–69.
Sun HS, Xu B, Chen W, Xiao A, Turlova E, Alibraham A, et al. Neuronal K(ATP) channels mediate hypoxic preconditioning and reduce subsequent neonatal hypoxic-ischemic brain injury. Exp Neurol. 2015;263:161–71.
Chen W, Xu B, Xiao A, Liu L, Fang X, Liu R, et al. TRPM7 inhibitor carvacrol protects brain from neonatal hypoxic-ischemic injury. Mol Brain. 2015;8:11.
Liu XH, Yan H, Xu M, Zhao YL, Li LM, Zhou XH, et al. Hyperbaric oxygenation reduces long-term brain injury and ameliorates behavioral function by suppression of apoptosis in a rat model of neonatal hypoxia-ischemia. Neurochem Int. 2013;62:922–30.
Morris R. Developments of a water-maze procedure for studying spatial learning in the rat. J Neurosci Methods. 1984;11:47–60.
Sun HS, Jackson MF, Martin LJ, Jansen K, Teves L, Cui H, et al. Suppression of hippocampal TRPM7 protein prevents delayed neuronal death in brain ischemia. Nat Neurosci. 2009;12:1300–7.
Huang YR, Xie XX, Ji M, Yu XL, Zhu J, Zhang LX, et al. Naturally occurring autoantibodies against alpha-synuclein rescues memory and motor deficits and attenuates alpha-synuclein pathology in mouse model of Parkinson’s disease. Neurobiol Dis. 2019;124:202–17.
Liu SY, Lu S, Yu XL, Yang SG, Liu W, Liu XM, et al. Fruitless wolfberry-sprout extract rescued cognitive deficits and attenuated neuropathology in Alzheimer’s disease transgenic mice. Curr Alzheimer Res. 2018;15:856–68.
Zhang Z, Chu SF, Wang SS, Jiang YN, Gao Y, Yang PF, et al. RTP801 is a critical factor in the neurodegeneration process of A53T alpha-synuclein in a mouse model of Parkinson’s disease under chronic restraint stress. Br J Pharmacol. 2018;175:590–605.
Kang N, Zhang J, Yu X, Ma Y. Radial extracorporeal shock wave therapy improves cerebral blood flow and neurological function in a rat model of cerebral ischemia. Am J Transl Res. 2017;9:2000–12.
Chu SF, Zhang Z, Zhou X, He WB, Chen C, Luo P, et al. Ginsenoside Rg1 protects against ischemic/reperfusion-induced neuronal injury through miR-144/Nrf2/ARE pathway. Acta Pharmacol Sin. 2019;40:13–25.
Sun XY, Dong QX, Zhu J, Sun X, Zhang LF, Qiu M, et al. Resveratrol rescues tau-induced cognitive deficits and neuropathology in a mouse model of tauopathy. Curr Alzheimer Res. 2019;16:710–22.
Liu SY, Yu XL, Zhu J, Liu XM, Zhang Y, Dong QX, et al. Intravenous immunoglobulin ameliorates motor and cognitive deficits and neuropathology in R6/2 mouse model of Huntington’s disease by decreasing mutant huntingtin protein level and normalizing NF-kappaB signaling pathway. Brain Res. 2018;1697:21–33.
Zha J, Liu XM, Zhu J, Liu SY, Lu S, Xu PX, et al. A scFv antibody targeting common oligomeric epitope has potential for treating several amyloidoses. Sci Rep-Uk. 2016;6:36631.
Giralt A, Brito V, Chevy Q, Simonnet C, Otsu Y, Cifuentes-Diaz C, et al. Pyk2 modulates hippocampal excitatory synapses and contributes to cognitive deficits in a Huntington’s disease model. Nat Commun. 2017;8:15592.
Besshoh S, Bawa D, Teves L, Wallace MC, Gurd JW. Increased phosphorylation and redistribution of NMDA receptors between synaptic lipid rafts and post-synaptic densities following transient global ischemia in the rat brain. J Neurochem. 2005;93:186–94.
Montalban E, Al-Massadi O, Sancho-Balsells A, Brito V, de Pins B, Alberch J, et al. Pyk2 in the amygdala modulates chronic stress sequelae via PSD-95-related micro-structural changes. Transl Psychiatry. 2019;9:3.
Aarts M, Liu Y, Liu L, Besshoh S, Arundine M, Gurd JW, et al. Treatment of ischemic brain damage by perturbing NMDA receptor-PSD-95 protein interactions. Science. 2002;298:846–50.
Sun HS, Doucette TA, Liu Y, Fang Y, Teves L, Aarts M, et al. Effectiveness of PSD95 inhibitors in permanent and transient focal ischemia in the rat. Stroke. 2008;39:2544–53.
Seabold GK, Burette A, Lim IA, Weinberg RJ, Hell JW. Interaction of the tyrosine kinase Pyk2 with the N-methyl-D-aspartate receptor complex via the Src homology 3 domains of PSD-95 and SAP102. J Biol Chem. 2003;278:15040–8.
Liu Y, Zhang GY, Hou XY, Xu TL. Two types of calcium channels regulating activation of proline-rich tyrosine kinase 2 induced by transient brain ischemia in rat hippocampus. Neurosci Lett. 2003;348:127–30.
Liu Y, Zhang G, Gao C, Hou X. NMDA receptor activation results in tyrosine phosphorylation of NMDA receptor subunit 2A(NR2A) and interaction of Pyk2 and Src with NR2A after transient cerebral ischemia and reperfusion. Brain Res. 2001;909:51–8.
Liu Y, Zhang GY, Yan JZ, Xu TL. Suppression of Pyk2 attenuated the increased tyrosine phosphorylation of NMDA receptor subunit 2A after brain ischemia in rat hippocampus. Neurosci Lett. 2005;379:55–8.
Clancy B, Darlington RB, Finlay BL. Translating developmental time across mammalian species. Neuroscience. 2001;105:7–17.
Rumajogee P, Bregman T, Miller SP, Yager JY, Fehlings MG. Rodent hypoxia-ischemia models for cerebral palsy research: a systematic review. Front Neurol. 2016;7:57.
Scott DB, Blanpied TA, Swanson GT, Zhang C, Ehlers MD. An NMDA receptor ER retention signal regulated by phosphorylation and alternative splicing. J Neurosci. 2001;21:3063–72.
Knox R, Brennan-Minnella AM, Lu F, Yang D, Nakazawa T, Yamamoto T, et al. NR2B phosphorylation at tyrosine 1472 contributes to brain injury in a rodent model of neonatal hypoxia-ischemia. Stroke. 2014;45:3040–7.
Northington FJ, Zelaya ME, O’Riordan DP, Blomgren K, Flock DL, Hagberg H, et al. Failure to complete apoptosis following neonatal hypoxia-ischemia manifests as “continuum” phenotype of cell death and occurs with multiple manifestations of mitochondrial dysfunction in rodent forebrain. Neuroscience. 2007;149:822–33.
Li B, Concepcion K, Meng X, Zhang L. Brain-immune interactions in perinatal hypoxic-ischemic brain injury. Prog Neurobiol. 2017;159:50–68.
Ziemka-Nalecz M, Jaworska J, Sypecka J, Polowy R, Filipkowski RK, Zalewska T. Sodium butyrate, a histone deacetylase inhibitor, exhibits neuroprotective/neurogenic effects in a rat model of neonatal hypoxia-ischemia. Mol Neurobiol. 2017;54:5300–18.
Hurn PD, Vannucci SJ, Hagberg H. Adult or perinatal brain injury: does sex matter? Stroke. 2005;36:193–5.
Johnston MV, Hagberg H. Sex and the pathogenesis of cerebral palsy. Dev Med Child Neurol. 2007;49:74–8.
Salazar SV, Cox TO, Lee S, Brody AH, Chyung AS, Haas LT, et al. Alzheimer’s disease risk factor Pyk2 mediates amyloid-beta-induced synaptic dysfunction and loss. J Neurosci. 2019;39:758–72.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (81730096, 81873026, 81973499), the CAMS Innovation Fund for Medical Sciences (CIFMS) (2016-I2M-1-004), the Drug Innovation Major Project (2018ZX09711001-003-005, 2018ZX09711001-002-007), the Beijing Key Laboratory of New Drug Mechanisms and Pharmacological Evaluation Study (BZ0150), the opening Program of Shanxi Key Laboratory of Chinese Medicine Encephalopathy (CME-OP-2017001), and the High-End Foreign Experts introduction program (G20200001485).
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NHC and ZZ designed research. JZ performed the animal models and all the cell and WB experiments; YP, DDL, and WXJ assisted the experiment, ZZ and CC analyze the data; SFC reviewed the data; JZ and ZZ drafted the manuscript; HSS and ZPF guided the animal experiments; HSS, ZPF, and NHC revised the manuscript.
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Zhu, J., Chu, Sf., Peng, Y. et al. Pyk2 inhibition attenuates hypoxic-ischemic brain injury in neonatal mice. Acta Pharmacol Sin 43, 797–810 (2022). https://doi.org/10.1038/s41401-021-00694-5
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DOI: https://doi.org/10.1038/s41401-021-00694-5
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