Jiang Xiangning, Mu Dezhi, Biran Valerie, Faustino Joel, Chang Shengjun, Rincón Christina M, Sheldon R Ann, Ferriero Donna M
Department of Neurology, University of California San Francisco, San Francisco, CA 94143-0663, USA.
Ann Neurol. 2008 May;63(5):632-41. doi: 10.1002/ana.21365.
Neonatal stroke is associated with the N-methyl-D-aspartate receptor (NMDAR)-mediated excitotoxic brain injury. Src family kinases (SFKs) are considered to be the molecular hub for NMDAR regulation. We determined the relationship between SFKs activation and NMDAR tyrosine phosphorylation after neonatal hypoxia-ischemia (HI) and investigated the neuroprotective potential of a selective SFKs inhibitor, PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3, 4-d] pyramidine), against neonatal brain ischemic injury.
The Rice-Vannucci model was adapted for neonatal HI injury in postnatal day 7 CD1 mice. SFKs activity in the postsynaptic densities was measured by Western blot. NMDAR tyrosine phosphorylation and their association with SFKs were determined by coimmunoprecipitation. Brains from animals treated with PP2 or its inactive analog, PP3, were examined histologically with cresyl violet and iron stain to assess the degree of damage.
Neonatal HI resulted in a rapid and transient increase in tyrosine phosphorylation of NMDAR subunits NR2A and NR2B. This upregulation correlated with the enhanced association of Fyn and Src with NR2A and NR2B. SFKs were activated in the postsynaptic densities after HI. Inhibition of SFKs with PP2 attenuated brain injury after neonatal HI, whereas PP3 did not protect the brain from the HI insult.
SFKs may play an important role in NMDAR-mediated excitotoxicity and downstream events leading to neuronal death after neonatal HI. Inhibition of SFKs may provide protection against neonatal stroke. Rather than blockade of NMDAR after HI in the developing brain, it may be safer and more beneficial to manipulate components of the NMDAR signaling complex at the postsynaptic density.
新生儿脑卒中与N-甲基-D-天冬氨酸受体（NMDAR）介导的兴奋性毒性脑损伤有关。Src家族激酶（SFKs）被认为是NMDAR调节的分子枢纽。我们确定了新生儿缺氧缺血（HI）后SFKs激活与NMDAR酪氨酸磷酸化之间的关系，并研究了选择性SFKs抑制剂PP2（4-氨基-5-（4-氯苯基）-7-（叔丁基）吡唑并[3,4-d]嘧啶）对新生儿脑缺血损伤的神经保护潜力。
采用Rice-Vannucci模型对出生后第7天的CD1小鼠进行新生儿HI损伤。通过蛋白质印迹法测量突触后致密物中SFKs的活性。通过免疫共沉淀确定NMDAR酪氨酸磷酸化及其与SFKs的关联。用甲酚紫和铁染色对用PP2或其无活性类似物PP3处理的动物的大脑进行组织学检查，以评估损伤程度。
新生儿HI导致NMDAR亚基NR2A和NR2B的酪氨酸磷酸化迅速且短暂增加。这种上调与Fyn和Src与NR2A和NR2B的增强关联相关。HI后突触后致密物中的SFKs被激活。用PP2抑制SFKs可减轻新生儿HI后的脑损伤，而PP3不能保护大脑免受HI损伤。
SFKs可能在新生儿HI后NMDAR介导的兴奋性毒性和导致神经元死亡的下游事件中起重要作用。抑制SFKs可能为新生儿脑卒中提供保护。在发育中的大脑中，与其在HI后阻断NMDAR，在突触后致密物处操纵NMDAR信号复合物的成分可能更安全且更有益。
