Characterization of WNK function in Axonal Maintenance

描述(申请人提供)：正常的大脑功能需要建立和维持有效的神经回路。神经回路的发育需要轴突的引导、突触靶标的选择、突触的形成以及突触的生长和重塑，以响应发育和环境提示。突触前和突触后细胞中的大型蛋白质复合体起到协调突触成熟的作用，这涉及突触前神经递质释放机制与神经递质受体和信号蛋白的聚集和排列。有证据表明，突触一旦形成，就会在有机体的整个生命周期中保持不变。虽然轴突引导和突触发育的机制已经得到了很好的研究，但对涉及轴突和突触维持的机制知之甚少。我们的实验室正在使用果蝇的遗传屏幕来揭示形成和维持突触完整性的分子机制。我们在上述类型的筛查中发现的最有趣的突变之一是没有赖氨酸[K](WNK)，这是一种保守的丝氨酸-苏氨酸激酶，与许多人类疾病有关，包括高血压、遗传性神经病变和癌症。尽管WNK在神经系统中高度表达，但其在神经系统中的功能尚未被描述。我们发现WNK突变果蝇在突触素的轴突运输方面存在缺陷，突触素是一种参与钙依赖的囊泡释放的蛋白质。有趣的是，WNK突变体也表现出显著的突触回缩，这表明突触稳定的机制受到了损害。此外，我们还发现了WNK和Rab3-Global之间的一种新的相互作用，Rab3-Global是Rab3 GTP酶的激活剂，参与突触处的囊泡和蛋白质运输。我们的目标是以果蝇为模型，确定WNK控制轴突和突触维持的机制。我们将确定在突触上对WNK功能至关重要的结构域，并确定WNK的亚细胞定位。我们还将定义我们发现的WNK和Rab3-Global之间的新相互作用，并确定WNK信号通路中的蛋白质。此外，我们的目标是通过使用小鼠背根神经节(DRG)培养来研究在没有WNK的情况下轴突的完整性和突触的形成，从而将我们的发现扩展到哺乳动物的感觉神经元。这些研究的结果将导致对WNK如何塑造突触的发展和维持的新见解。此外，由于WNK的哺乳动物同源基因已经与遗传性感觉和自主神经病变II型(HSANII)有关，因此对WNK信号通路的研究也可能对这种疾病和其他神经性疾病产生新的见解，包括确定治疗候选药物。 与公共健康相关：轴突和突触的发育对建立和维持神经元电路和大脑功能至关重要。因此，确定控制这些过程的分子机制对于我们理解或正常的大脑功能以及功能障碍是如何发生的至关重要。我们计划表征丝氨酸/苏氨酸激酶WNK的作用，并描述其在调节轴突和突触发育和维持中的作用。这些实验的结果不仅将为调控突触维持的机制提供新的和基本的见解，而且还将导致可能的人类疾病的治疗靶点，包括遗传性感觉和自主神经病变II型(HSANII)等神经疾病。

DESCRIPTION (provided by applicant): Proper brain function requires the establishment and maintenance of effective neural circuits. Neural circuit development entails axon guidance, synaptic target selection, synapse formation, and synaptic growth and remodeling in response to developmental and environmental cues. Large protein complexes in both the pre- and post-synaptic cells act to coordinate synapse maturation, which involves clustering and aligning of the pre-synaptic neurotransmitter-releasing machinery with neurotransmitter receptors and signaling proteins. Evidence suggests that once formed, synapses are maintained over the lifetime of an organism. While mechanisms of axon guidance and synapse development have been well studied, little is known about the mechanisms involved in axon and synapse maintenance. Our lab is using genetic screens in Drosophila to uncover the molecular mechanisms that form and maintain synapse integrity. One of the most interesting mutations we've uncovered in the type of screen mentioned above is in With No Lysine [K] (wnk), a conserved serine-threonine kinase that has been implicated in a number of human diseases including hypertension, hereditary neuropathy and cancer. Although Wnk is highly expressed in the nervous system, its function there has not been described. We found that wnk mutant flies have defects in axon transport of synaptotagmin, a protein involved in calcium-dependent vesicle release. Interestingly, Wnk mutants also show significant synaptic retraction, indicating that mechanisms of synapse stability are compromised. In addition, we uncovered a novel interaction between Wnk and the Rab3-GEF, an activator of the Rab3 GTPase involved in vesicle and protein trafficking at the synapse. We aim to define the mechanism by which WNK controls axon and synapse maintenance using Drosophila as a model. We will identify the domains that are critical for WNK function at the synapse and determine the sub-cellular localization of WNK. We will also define the novel interaction between WNK and Rab3-GEF that we've discovered and identify proteins in the WNK signaling pathway. In addition, we aim to extend our findings to mammalian sensory neurons by using mouse dorsal root ganglion (DRG) cultures to study axon integrity and synapse formation in the absence of WNK. Results from these studies will lead to novel insights into how WNK shapes the development and maintenance of the synapse. In addition, because the mammalian orthologs of WNK have been implicated in hereditary sensory and autonomic neuropathy type II (HSANII), study of the WNK signaling pathway may also yield novel insights into this and other neuropathic disease, including identification of therapeutic candidates. PUBLIC HEALTH RELEVANCE: Axon and synapse development are crucial for establishing and maintaining neuronal circuits and brain function throughout life. Defining the molecular mechanisms that control these processes is therefore critical to our understanding or normal brain function as well as how dysfunction occurs. We plan to characterize the role of the serine/threonine kinase Wnk and delineate its role in regulating axon and synapse development and maintenance. The results of these experiments will not only provide novel and fundamental insights into mechanisms regulating the control of synaptic maintenance, but also lead to possible therapeutic targets for human diseases, including neuropathies such as hereditary sensory and autonomic neuropathy type II (HSANII).