THE ROLE OF A NOVEL GENE, NNAL, IN NEURONAL REGENERATION

描述(来自申请者的逐字摘要)：宏大的长期目标 是为了阐明成功的神经元背后的分子事件 再生。尽管人们对再生有很多了解，但许多分子 靶神经支配所需的信息仍不明确。PI已经克隆了一个 小鼠运动神经元表达上调的新基因NNAL 坐骨神经损伤。NNAL在肌肉重新神经支配时下调。 NNAL在分化的神经系统中也有高表达。数列 对全长4kb的cDNA进行分析，确定了一个核定位信号， 一个ATP/GTP结合基序和几个磷酸化位点。基于Motif 分析和表达模式，它被假设为NNAL作为 调节相关事件的磷酸化核ATP/GTP结合蛋白 有靶神经支配。具体目标将1)分析和关联 NNAL的表达模式，2)检测生化特性，3)评估NNAL的 对体外培养的神经突起生长的影响以及4)确定是否需要NNAL 在活体内成功再生神经元。研究设计使用了Northern、In 原位、免疫和免疫细胞化学分析mRNAs和蛋白质 表情。重组蛋白将用于评估ATP/GTP结合和 水解性。此外，将使用磷酸化分析来 体内检测NNAL的磷酸化状态及是否存在磷酸化 会影响核转位。最后，腺相关病毒(AAV)方法 将用于在体外和体内表达突变的NNAL。体外测定法 将检查对轴突生长的影响，而体内测试将评估 坐骨神经损伤后对神经元再生的影响。总而言之， 本申请描述了一种全新基因的作用， NNAL，在再生机制中。与健康相关的是要了解 成功再生所需的分子级联才能有效 可以设计治疗策略来治疗患有神经性疾病的患者 受伤。

DESCRIPTION (Verbatim from the Applicants Abstract): The broad long-term goals are to elucidate the molecular events underlying successful neuronal regeneration. Although much is known about regeneration, many molecules required for target innervation remain uncharacterized. The PI has cloned a novel gene, nnal, that is up-regulated in motor neurons following murine sciatic nerve injury. Nnal is down-regulated upon re-innervation of muscle. Nnal is also highly expressed in differentiating nervous system. Sequence analysis of the full length 4kb cDNA identified a nuclear localization signal, an ATP/GTP binding motif and several phosphorylation sites. Based upon motif analysis and expression patterns, it is hypothesized nnal acts as a phosphorylated nuclear ATP/GTP binding protein that regulates events associated with target innervation. The specific aims will 1) analyze and correlate expression patterns of nnal, 2) test biochemical properties, 3) assess nnal's effect on neurite outgrowth in vitro and 4) determine if nnal is required for successful neuronal regeneration in vivo. The research design uses northern, in situ, western and immunocytochemical analysis to map mRNA and protein expression. Recombinant protein will be used to assess ATP/GTP binding and hydrolysis characteristics. In addition, phosphorylation assays will be used to test the phosphorylation state of nnal in vivo and whether phosphorylation affects nuclear translocation. Finally, adeno-associated viral (AAV) approaches will be used to express mutant nnal in vitro and in vivo. The in vitro assays will examine effects on neurite outgrowth while in vivo assays will assess effects on neuronal regeneration following sciatic nerve injury. In summary, the present application characterizes the role of a completely novel gene, nnal, in regenerative mechanisms. The health relatedness is to understand the molecular cascades required for successful regeneration so that effective treatment strategies can be designed to treat patients suffering from neural injury.