Sang Qian, Kim Mary H, Kumar Sharad, Bye Nicole, Morganti-Kossman Maria C, Gunnersen Jenny, Fuller Stephanie, Howitt Jason, Hyde Lavinia, Beissbarth Tim, Scott Hamish S, Silke John, Tan Seong-Seng
Howard Florey Institute, The University of Melbourne, Parkville, Victoria 3010, Australia.
J Neurosci. 2006 Jul 5;26(27):7234-44. doi: 10.1523/JNEUROSCI.1398-06.2006.
Understanding the transcriptional response to neuronal injury after trauma is a necessary prelude to formulation of therapeutic strategies. We used Serial Analysis of Gene Expression (SAGE) to identify 50,000 sequence tags representing 18,000 expressed genes in the cortex 2 h after traumatic brain injury (TBI). A similar tag library was obtained from sham-operated cortex. The SAGE data were validated on biological replicates using quantitative real-time-PCR on multiple samples at 2, 6, 12, and 24 h after TBI. This analysis revealed that the vast majority of genes showed a downward trend in their pattern of expression over 24 h. This was confirmed for a subset of genes using in situ hybridization and immunocytochemistry on brain sections. Of the overexpressed genes in the trauma library, Nedd4-WW (neural precursor cell expressed, developmentally downregulated) domain-binding protein 5 (N4WBP5) (also known as Ndfip1) is strongly expressed in surviving neurons around the site of injury. Overexpression of N4WBP5 in cultured cortical neurons increased the number of surviving neurons after gene transfection and growth factor starvation compared with control transfections. These results identify N4WBP5 as a neuroprotective protein and, based on its known interaction with the ubiquitin ligase Nedd4, would suggest protein ubiquitination as a possible survival strategy in neuronal injury.
了解创伤后神经元损伤的转录反应是制定治疗策略的必要前奏。我们使用基因表达序列分析（SAGE）来识别创伤性脑损伤（TBI）后2小时皮质中代表18,000个表达基因的50,000个序列标签。从假手术皮质获得了类似的标签文库。在TBI后2、6、12和24小时，使用多个样本的定量实时PCR对生物重复样本验证了SAGE数据。该分析表明，绝大多数基因在24小时内表达模式呈下降趋势。使用脑切片的原位杂交和免疫细胞化学对一部分基因进行了验证。在创伤文库中过表达的基因中，Nedd4-WW（神经前体细胞表达，发育下调）结构域结合蛋白5（N4WBP5）（也称为Ndfip1）在损伤部位周围存活的神经元中强烈表达。与对照转染相比，在培养的皮质神经元中过表达N4WBP5可增加基因转染和生长因子饥饿后存活神经元的数量。这些结果确定N4WBP5为一种神经保护蛋白，基于其与泛素连接酶Nedd4的已知相互作用，提示蛋白质泛素化可能是神经元损伤中的一种存活策略。
