We have been investigating the pathogenesis of hypoxic-ischemic (HI) brain injury, as a model for neonatal stroke, with the long-term goals of developing therapeutic interventions specifically for neonatal stroke. Excitotoxicity mediated by NMDA-type glutamate receptors (NMDAR) is a major trigger of HI brain injury, and members of the Src family kinases (SFKs, mainly Fyn and Src) act as a molecular hub for the control of NMDAR. SFKs-mediated NMDAR phosphorylation is regulated by synaptic scaffolding proteins, such as postsynaptic density protein 95 (PSD-95) and receptor for activated C kinase 1 (RACK1). In adult rat models, HI differentially upregulates SFKs-mediated tyrosine phosphorylation of NMDAR subunits. However, there are no data regarding the interaction of NMDAR with SFKs in the immature mouse brain and how this interaction is regulated and contributes to ischemic neonatal brain injury. This project aims at exploring the role of Src family kinases, especially Fyn and Src, in the regulation of NMDAR function during the evolution of neonatal HI brain injury. We hypothesize that neonatal HI increases tyrosine phosphorylation of NR2A and NR2B mediated by SFKs (hypothesis I) and inhibition of SFKs activity or deletion of Fyn provides cerebral protection following ischemic neonatal brain injury (hypothesis II). These hypotheses will be tested in:
Aim1 To determine whether tyrosine phosphorylation of NMDAR is mediated by SFKs in response to HI in the immature mouse brain. We will measure tyrosine phosphorylation of NR2A and NR2B, as well as specific activation of Fyn or Src in the forebrain postsynaptic densities (PSDs) in both sham-operated and HI-injured C57b/6 mice at P7. The association of NR2A/2B with Fyn/Src, and RACK1 or PSD-95 will be determined by co-immunoprecipitation (Co-IP). The animals will be treated with PP2, a specific SFK inhibitor, to investigate whether NMDAR tyrosine phosphorylation is mediated by SFKs and whether inhibition of SFKs activity provides protection against neonatal HI. Histological protection of PP2 will be assessed 5 days and 8 weeks after HI. Long-term functional outcome will be measured by behavioral testing 2 months after HI injury.
Aim2 To determine the contribution of Fyn to HI damage in neonatal brain. Fyn deficient and overexpressing mice and their wildtype littermates will be subjected to HI injury at P7, brain damage will be scored 5 days later using cresyl violet and iron staining. Tyrosine phosphorylation of NR2A/2B and their interaction with associated proteins in Fyn deficient and overexpressing mice will be determined by Co-IP as above in Aim 1. Primary cortical neurons will be isolated from Fyn deficient and overexpressing mice and subjected to oxygen-glucose deprivation (OGD). Lactate dehydrogenase (LDH) activity and Live/Dead cytotoxicity assay will be used to assess neuronal cell death.
The long-term goal of our research is to develop safe and effective therapies specifically for neonatal stroke. This would have an enormous impact on the burden that our society bears to rehabilitate and care for severely brain-damaged children.
| Shao, Guo; Wang, Yongqiang; Guan, Shenheng et al. (2017) Proteomic Analysis of Mouse Cortex Postsynaptic Density following Neonatal Brain Hypoxia-Ischemia. Dev Neurosci 39:66-81 |
| Knox, Renatta; Jiang, Xiangning (2015) Fyn in Neurodevelopment and Ischemic Brain Injury. Dev Neurosci 37:311-20 |
| Knox, Renatta; Brennan-Minnella, Angela M; Lu, Fuxin et al. (2014) NR2B phosphorylation at tyrosine 1472 contributes to brain injury in a rodent model of neonatal hypoxia-ischemia. Stroke 45:3040-7 |
| Knox, Renatta; Zhao, Chong; Miguel-Perez, Dario et al. (2013) Enhanced NMDA receptor tyrosine phosphorylation and increased brain injury following neonatal hypoxia-ischemia in mice with neuronal Fyn overexpression. Neurobiol Dis 51:113-9 |
| Jiang, Xiangning; Knox, Renatta; Pathipati, Praneeti et al. (2011) Developmental localization of NMDA receptors, Src and MAP kinases in mouse brain. Neurosci Lett 503:215-9 |
