Src Family Kinases - Mediated Ischemic Neonatal Brain Injury

描述(申请人提供)：我们一直在研究缺氧缺血性脑损伤的发病机制，作为新生儿中风的模型，长期目标是开发专门针对新生儿中风的治疗干预措施。由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，而Src家族成员(SFKs，主要是Fyn和Src)是NMDAR调控的分子枢纽。SFKs介导的NMDAR磷酸化受突触支架蛋白的调节，如突触后密度蛋白95(PSD-95)和活化C激酶受体1(RACK1)。在成年大鼠模型中，HI差异地上调SFKs介导的NMDAR亚单位的酪氨酸磷酸化。然而，目前还没有关于NMDAR与SFKs在未成熟小鼠脑中的相互作用以及这种相互作用如何受到调控并导致新生儿缺血性脑损伤的数据。本课题旨在探讨在新生儿缺氧缺血性脑损伤发生发展过程中，Src家族蛋白，尤其是Fyn和Src在NMDAR功能调节中的作用。我们假设新生儿缺氧缺血增加了SFKs介导的NR2A和NR2B的酪氨酸磷酸化(假说I)，抑制SFKs的活性或Fyn的缺失提供了新生儿缺血性脑损伤后的脑保护(假说II)。这些假说将在：AIM1中进行验证，以确定在未成熟的小鼠大脑中，NMDAR的酪氨酸磷酸化是否由SFKs介导，以响应HI。我们将在P7检测假手术和HI损伤的C57b/6小鼠前脑突触后密度(PSD)中NR2A和NR2B的酪氨酸磷酸化以及Fyn或Src的特异性激活。NR2A/2B与Fyn/Src以及RACK1或PSD-95的结合将通过免疫共沉淀(Co-IP)来确定。动物将用一种特异性的SFK抑制剂PP2处理，以研究NMDAR酪氨酸磷酸化是否由SFKs介导，以及抑制SFKs的活性是否对新生儿HI起到保护作用。在HI后5天和8周评估PP2的组织保护作用。在HI损伤后2个月通过行为学测试来评估远期功能结果。目的探讨Fyn在新生儿缺氧缺血性脑损伤中的作用。Fyn基因缺陷和过度表达的小鼠及其野生型仔鼠在出生后7天将受到HI损伤，5天后用甲酚紫和铁染色对脑损伤进行评分。在目标1中，将用Co-IP法检测Fyn缺乏和过度表达的小鼠NR2A/2B的酪氨酸磷酸化及其与相关蛋白的相互作用。从Fyn缺乏和过度表达的小鼠中分离原代皮质神经元，并接受缺氧-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细胞死亡。公共卫生相关性：我们研究的长期目标是开发专门针对新生儿中风的安全有效的治疗方法。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.