Src Family Kinases - Mediated Ischemic Neonatal Brain Injury

描述（由申请人提供）：我们一直在研究缺氧缺血性（HI）脑损伤的发病机制，作为新生儿中风的模型，长期目标是开发针对新生儿中风的治疗干预措施。nmda型谷氨酸受体（NMDAR）介导的兴奋性毒性是HI脑损伤的主要触发因素，Src家族激酶（SFKs，主要是Fyn和Src）是控制NMDAR的分子中枢。sfks介导的NMDAR磷酸化受突触支架蛋白调控，如突触后密度蛋白95 （PSD-95）和活化C激酶1受体（RACK1）。在成年大鼠模型中，HI差异上调sfks介导的NMDAR亚基酪氨酸磷酸化。然而，关于NMDAR与未成熟小鼠大脑中SFKs的相互作用以及这种相互作用如何被调节并导致缺血性新生儿脑损伤，尚无数据。本项目旨在探讨Src家族激酶，尤其是Fyn和Src在新生儿HI脑损伤演化过程中对NMDAR功能的调控作用。我们假设新生儿HI增加了SFKs介导的NR2A和NR2B酪氨酸磷酸化（假设I），抑制SFKs活性或Fyn的缺失可在缺血性新生儿脑损伤后提供脑保护（假设II）。这些假设将在以下研究中得到验证：目的1确定在未成熟小鼠大脑中，NMDAR的酪氨酸磷酸化是否由SFKs介导，以响应HI。我们将在假手术和hi损伤的C57b/6小鼠P7时测量NR2A和NR2B酪氨酸磷酸化，以及Fyn或Src在前脑突触后密度（psd）中的特异性激活。NR2A/2B与Fyn/Src、RACK1或PSD-95的关联将通过共免疫沉淀（Co-IP）来确定。这些动物将接受PP2（一种特异性SFK抑制剂）治疗，以研究SFKs是否介导NMDAR酪氨酸磷酸化，以及SFKs活性的抑制是否对新生儿HI有保护作用。在HI后5天和8周评估PP2的组织学保护作用。HI损伤后2个月通过行为测试测量长期功能结果。目的探讨Fyn在新生儿脑HI损伤中的作用。Fyn缺失和过表达小鼠及其野生型仔鼠在P7时进行HI损伤，5天后用甲酚紫和铁染色对脑损伤进行评分。在Fyn缺陷和过表达小鼠中，酪氨酸磷酸化NR2A/2B及其与相关蛋白的相互作用将在Aim 1中通过Co-IP测定。从Fyn缺乏和过表达的小鼠中分离初级皮质神经元，并进行氧-葡萄糖剥夺（OGD）。乳酸脱氢酶（LDH）活性和活/死细胞毒性试验将用于评估神经元细胞死亡。公共卫生相关性：我们研究的长期目标是开发安全有效的新生儿中风治疗方法。这将对我们的社会承担的康复和照顾严重脑损伤儿童的负担产生巨大影响。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.