权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Src Family Kinases - Mediated Ischemic Neonatal Brain Injury

Src 家族激酶 - 介导的缺血性新生儿脑损伤

基本信息

批准号：
7849015
负责人：
Xiangning Jiang
金额：
$ 19.31万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-06-01 至 2012-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7849015
关键词：
Adult Animals Apoptotic Binding Biological Assay Brain Brain Injuries Brain Ischemia Caring Cell Death Cerebrum Child Childhood Co-Immunoprecipitations Complex Data Elderly Evolution Family Glucose Glutamate Receptor Goals Hand Hippocampus (Brain)Hypoxia Incidence Injury Iron Ischemia Ischemic-Hypoxic Encephalopathy Lactate Dehydrogenase Life Live Birth Measures Mediating Modeling Molecular Mus N-Methylaspartate Neonatal Neonatal Brain Injury Nerve Degeneration Neurons Newborn Infant Oxidative Stress Oxygen Pathogenesis Pathway interactions Phosphorylation Prevention Prosencephalon Proteins Rattus Receptor Protein-Tyrosine Kinases Regulation Reporting Research Rodent Role Scaffolding Protein Serine Signal Transduction Slice Societies Staining method Stains Stroke Synapses Testing Therapeutic Intervention Threonine Tyrosine Kinase Inhibitor Tyrosine Phosphorylation Up-Regulation Ursidae Family behavior test cresyl violet cytotoxicity density deprivation effective therapy excitotoxicity functional outcomes injured member mortality neonatal hypoxic-ischemic brain injury neonate neuron loss overexpression postnatal postsynaptic postsynaptic density protein presynaptic density protein 95 public health relevance receptor function receptors for activated C kinase response src-Family Kinases

项目摘要

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.

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，作为新生儿卒中的模型，长期目标是开发专门针对新生儿卒中的治疗干预措施。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，并且Src家族激酶（SFK，主要是Fyn和Src）的成员充当控制NMDAR的分子枢纽。SFK介导的NMDAR磷酸化受突触支架蛋白如突触后密度蛋白95（PSD-95）和活化C激酶1受体（RACK 1）的调节。在成年大鼠模型中，HI差异上调SFK介导的NMDAR亚基酪氨酸磷酸化。然而，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调节和促成缺血性新生儿脑损伤的数据。本项目旨在探讨Src家族激酶，尤其是Fyn和Src在新生儿HI脑损伤演变过程中NMDAR功能调节中的作用。我们假设新生儿HI增加了由SFKs介导的NR 2A和NR 2B的酪氨酸磷酸化（假设I），并且SFKs活性的抑制或Fyn的缺失提供了缺血性新生儿脑损伤后的脑保护（假设II）。这些假设将在以下方面进行检验：目的1确定在未成熟小鼠脑中，NMDAR的酪氨酸磷酸化是否由SFKs介导以响应HI。我们将测量NR 2A和NR 2B的酪氨酸磷酸化，以及假手术和HI损伤的C57 b/6小鼠在P7前脑突触后密度（PSD）中Fyn或Src的特异性激活。通过免疫共沉淀（Co-IP）测定NR 2A/2B与Fyn/Src和RACK 1或PSD-95的结合。将用PP 2（一种特异性SFK抑制剂）处理动物，以研究NMDAR酪氨酸磷酸化是否由SFK介导以及SFK活性的抑制是否提供针对新生儿HI的保护。将在HI后5天和8周评估PP 2的组织学保护。在HI损伤后2个月通过行为测试测量长期功能结果。目的2探讨Fyn在新生儿脑HI损伤中的作用。Fyn缺陷和过表达小鼠及其野生型同窝仔将在P7时经受HI损伤，5天后使用甲酚紫和铁染色对脑损伤进行评分。如上文目的1所述，通过Co-IP测定NR 2A/2B的酪氨酸磷酸化及其与Fyn缺陷和过表达小鼠中相关蛋白的相互作用。原代皮质神经元将从Fyn缺陷和过表达小鼠中分离，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专门针对新生儿卒中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。