Ion Channels and Chemicals Controlling Synapse Stability

描述（由申请人提供）：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，这些疾病可以折磨人类从子宫发育到死亡。因此，主要的研究工作是了解突触在整个生命中的形成和稳定。关于突触的科学文献丰富，发现了神经肌肉连接（NMJ）研究的基本原理。特别是，对NMJ功能、形成和稳定性负责的蛋白质的了解相对较好。然而，关于这些蛋白质之间相互作用的基本问题仍然存在。一个重要的实验模型表明，achr的异质活性影响成人NMJ的稳定性。该研究对该模型进行了修改，并将其扩展到NMJ的发育过程中，在NMJ成熟的关键阶段，未成熟的γ和成熟的epsilon achr的共同表达产生了终板活性的异质性。我们的模型表明，富含epsilon AChR的终板区域介导ca2 +内流，激活共定位一氧化氮合酶（nNOS）。产生的一氧化氮（NO）扩散到神经末梢，竞争运动终板。新的初步数据表明，NO可增强成人运动神经末梢Ca2+电流和递质释放。因此，激活含有epsilon AChR的终板位点的发育中的神经末梢可能通过NO激活突触前冠酰环化酶而在功能上得到增强和培养。相反，NO可能会抑制激活epsilon AChR差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）制剂有助于我们模型的精确测试。我们的初步数据表明，从新生小鼠中分离的TS制备可以同时记录来自不同神经干末梢的神经支配的终板的神经末梢电流和突触后事件。这使得前所未有的研究功能，和no介导的串扰之间，哺乳动物神经末梢竞争突触后靶标。epsilon亚基和nNOS敲除小鼠，以及epsilon AChR选择性配体Waglerin- 1的可用性进一步加强了我们提出的测试我们模型的实验。另外，新的初步数据表明，作为神经元K-ATP通道的激活剂，胰岛素可抑制成人NMJ中乙酰胆碱的定量释放。因此，本研究的第二个目标是发现胰岛素和葡萄糖是否会影响NMJ发育过程中竞争的神经末梢的功能和最终稳定性。这将在非肥胖的1型糖尿病小鼠模型中进行探索。总的来说，这项研究具有临床意义，因为NO信号级联在杜氏肌营养不良症和中风动物模型中显著改变。此外，epsilon AChR功能的改变是与慢通道先天性肌无力综合征相关的NMJ病理的原因。提出的胰岛素效应评估是新颖的，将加强对成人和青少年形式糖尿病的神经系统后果的理解。从这项研究中获得的知识将启发未来治疗儿童和成人疾病的分子方法。

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.