Pyk2 inhibition attenuates hypoxic-ischemic brain injury in neonatal mice

Jie Zhu1, Shi-feng Chu1, Ye Peng1,2, Dan-dan Liu1, Chen Chen1, Wen-xuan Jian1,3, Hong-shuo Sun4, Zhong-ping Feng4, Zhao Zhang1, Nai-hong Chen1,2,3
1 State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica & Neuroscience Center, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
2 College of Pharmacy, Hunan University of Chinese Medicine, Changsha 410208, China
3 Institute of Clinical Pharmacology, Guangzhou University of Chinese Medicine, Guangzhou 510720, China
4 Department of Physiology, Faculty of Medicine, University of Toronto, Toronto, ON, Canada
Correspondence to: Zhao Zhang:, Nai-hong Chen:,
DOI: 10.1038/s41401-021-00694-5
Received: 3 January 2021
Accepted: 7 May 2021
Advance online: 5 July 2021


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.
Keywords: neonatal mice; hypoxic-ischemic brain injury; Pyk2; NMDA receptor; excitotoxicity; HIF-1α

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