Ion Channels and Chemicals Controlling Synapse Stability

描述(申请人提供)：突触是我们大脑中信息传递的主要场所，也是许多疾病的目标，这些疾病可以从子宫中发育到死亡。因此，主要的研究工作是为了了解突触的形成和稳定的整个生命。有关突触的科学文献丰富了对神经肌肉接头(NMJ)研究的基本原理的发现。特别是，负责NMJ功能、形成和稳定性的蛋白质被相对较好地理解。然而，关于这些蛋白质之间的相互作用，基本的问题仍然存在。一个重要的实验模型表明，AChRs活性的异质性影响成年NMJ的稳定性。这一建议将该模型修改并扩展到发育中的NMJ，在NMJ成熟的关键阶段，未成熟的Gamma和成熟的epsilon AChRs的共同表达产生了终板活动的异质性。我们的模型认为，富含epsilon AChR的终板区介导了钙内流，激活了共定位的一氧化氮合酶(NNOS)。一氧化氮(NO)产生扩散到争夺运动终板的神经末梢。新的初步数据表明，NO增加了成年运动神经末梢的钙电流和递质释放。因此，发育中的神经末梢激活含有epsilon AChR的终板基因，可能通过不激活突触前鸟苷酸环化酶而在功能上得到增强和培育。相反，NO可能抑制激活epsilon AChR不良终板灶的竞争神经末梢的功能和稳定性。小鼠胸骨三角肌(TS)的制备方便了对我们模型的准确测试。我们的初步数据显示，从新生小鼠分离的TS制剂可以同时记录神经末梢电流和接受来自不同神经干终末的神经支配的终板上的突触后事件。这使得史无前例地研究哺乳动物神经末梢之间竞争突触后靶点的功能，并且没有中介的串扰。Epsilon亚单位和nNOS基因敲除小鼠的可用性，以及epsilon AChR选择性配体Waglerin-1的可用性进一步加强了为测试我们的模型而提出的实验。其他新的初步数据表明，神经元K-ATP通道的激活剂胰岛素抑制成年NMJ的Ach的量子释放。因此，这项提议的第二个目标是发现胰岛素和葡萄糖是否影响在发育中的NMJ竞争的神经末梢的功能和最终的稳定性。这将在I型糖尿病的非肥胖小鼠模型中进行探索。总体而言，这项研究具有临床意义，因为在杜氏肌营养不良症和中风动物模型中，信号级联信号没有显著改变。此外，epsilon AChR功能的改变是慢通道先天性肌无力综合征相关NMJ病理的原因。拟议的胰岛素效应评估是新颖的，将加强对成人和青少年糖尿病的神经学后果的理解。从这项研究中获得的知识将启发未来治疗折磨儿童和成人的病理学的分子方法。

DESCRIPTION (provided by applicant): Synapses are the major locus of information transfer within our brain as well as the target of numerous pathologies which can afflict humans from development in utero to death. Therefore, major research effort is given to understanding synapse formation and stabilization throughout life. The scientific literature concerning synapses is rich with discovery of fundamental principles derived from study of the neuromuscular junction (NMJ). In particular, proteins responsible for NMJ function, formation, and stability are relatively well understood. Nevertheless, fundamental questions remain concerning interactions between these proteins. An important experimental model suggests that heterogeneous activity of AChRs influences stability of the adult NMJ. This proposal modifies and extends that model to the developing NMJ where co-expression of immature gamma and mature epsilon AChRs during the critical phase of NMJ maturation produces heterogeneity of end-plate activity. Our model suggests that end-plate areas rich in epsilon AChR mediate Ca 2+ influx which activates co-localized nitric oxide synthase (nNOS). The nitric oxide (NO) produced diffuses to nerve terminals competing for the motor end-plate. New preliminary data suggest that NO enhances Ca2+ currents and transmitter release at adult motor nerve terminals. Thus, developing nerve terminals activating end-plate loci containing the epsilon AChR may be functionally enhanced and nurtured via NO activation of presynaptic guanylyl cyclase. In contrast, NO may repress function and stability of competing nerve terminals activating epsilon AChR poor end-plate foci. The mouse Triangularis sterni (TS) preparation facilitates exact testing of our model. Our preliminary data show that the TS preparation isolated from neonatal mice allows simultaneous recording of nerve terminal currents and post-synaptic events at end-plates receiving innervation from terminals originating in distinct nerve trunks. This allows unprecedented study of the function of, and NO-mediated cross talk between, mammalian nerve terminals competing for a postsynaptic target. The availability of epsilon subunit and nNOS knock out mice, as well as the epsilon AChR selective ligand Waglerin- 1 further strengthen experiments proposed to test our model. Additional novel preliminary data suggest that insulin, an activator of the neuronal K-ATP channel, suppresses quantal release of Ach at the adult NMJ. Therefore, a second goal of this proposal is to discover if insulin, as well as glucose, effects the function, and eventual stability, of nerve terminals competing at the developing NMJ. This will be explored in a non-obese mouse model of type I diabetes. Overall, this research is clinically relevant since NO signaling cascades are significantly altered in Duchenne muscular dystrophy as well as animal models of stroke. In addition, altered function of the epsilon AChR is responsible for NMJ pathology associated with slow channel congenital myasthenic syndrome. The proposed evaluation of insulin effects is novel and will enhance understanding of the neurologic consequence of adult and juvenile forms of diabetes. The knowledge gained from this research will enlighten future molecular approaches to treating pathologies which afflict children and adults.