DROSOPHILA MODELS FOR STUDYING FUNCTION OF THE NF1 GENE

这项建议利用果蝇模型来研究肿瘤抑制基因神经纤维瘤病1(NF1)的机制和功能。我们特别感兴趣的是它在调节G蛋白依赖的cAMP途径激活中的作用。NF1蛋白含有一个与RAS特异性间隙催化区域同源的片段，它的突变会导致一种常见的神经遗传学疾病，这种疾病由神经纤维瘤和包括特定学习障碍在内的其他症状来鉴定。我们对果蝇NF1突变体的研究表明，NF1除了作为RAS-GAP外，还参与调节Rutabaga基因编码的腺苷酸环化酶(AC)的G蛋白依赖性激活。这种NF1调节的cAMP通路对于神经肌肉连接、身体大小发育以及学习和记忆中的神经肽反应至关重要。然而，目前尚不清楚NF1如何调节AC活动，NF1调节的AC通路如何促进学习和记忆，以及这种NF1调节机制是否也适用于脊椎动物。该提案旨在调查这些问题。它包括两个具体目标。1)在人类NF1患者中发现的18个点突变将被用来确定NF1的哪些部分对AC活性的调节以及RAS是否参与这种调节。然后，将使用小鼠NF1基因敲除突变体来测试NF1是否可以调节脊椎动物的AC活性。2)生化和电生理分析将被用于确定激活果蝇脑中NF1调节的AC活性的神经递质，以及激活NF1调节的AC通路以调节已识别的蘑菇体神经元的膜电流的神经递质。这些神经元被认为参与了被NF1突变破坏的联想学习过程。我们期待这项研究将对NF1的功能有新的认识，并为理解NF1的发病机制提供新的线索。

This proposal utilizes Drosophila models for investigating mechanisms and functions of the tumor suppressor gene neurofibromatosis 1 (NF1). We are particularly interested in its role in regulation of G-protein-dependent activation of the cAMP pathway. NF1 protein contains a fragment homologous to catalytic domain of the Ras-specific GAPS and its mutations cause a common neurogenetic disorder that is identified by neurofibromas and other symptoms including specific learning defects. Our study of Drosophila NF1 mutants has revealed that in addition to as a Ras-GAP, NF1 is involved in regulation of G-protein dependent activation of adenylyl cyclase (AC) encoded by rutabaga gene. This NF1-regulated cAMP pathway is crucial for mediating a neuropeptide response at neuromuscular junction, body size development, and learning and memory. However, it is unclear how NF1 regulates AC activity, how the NF1-regulated AC pathway contributes to learning and memory, and whether such NF1-regulated mechanism also works in vertebrates. The proposal is intended to investigate these problems. It includes two specific aims. 1) Eighteen point mutations identified in human NF1 patients will be used to define which parts of NF1 is important for regulation of AC activity and whether Ras is involved in such regulation. Then mouse Nf1 knockout mutants will be used to assay whether NF1 can regulate AC activity in vertebrates. 2) Biochemical and electrophysiological analyses will be directed to determine neurotransmitters that activate NF1-regulated AC activity in the fly brain and neurotransmitters that activate the NF1-regulated AC pathway to modulate membrane currents in identified mushroom body neurons. These neurons have been suggested to be involved in the associative learning process that are disrupted by NF1 mutations. We expect that the proposed research will lead to new insights into NF1 function and provide new clues for understanding pathogenesis of NF1.