Tuning of Inhibitory Synaptic Transmission at the Presynaptic Active Zone

描述(申请人提供)：从突触前神经末梢释放神经递质启动突触传递，这是神经元之间信息传递的关键形式。突触前活动区是一个密集的蛋白质网络，是突触小泡融合的场所，与其他分泌途径相比，它是突触前神经末梢的独特特征。活动区协调突触小泡周期，赋予突触传输令人难以置信的速度和精度，这是大脑履行复杂功能所需的。ELKS是一种紧密定位于活动区的蛋白质。已发布的数据和我们的 初步结果表明，它在神经递质的释放中发挥了几个作用。人类遗传学研究发现ELKS1与言语的神经发育障碍有关，这表明ELKS在人类中具有相关功能。然而，哺乳动物ELKS的确切功能尚不清楚，其机制也不清楚。这项研究的目的是了解脊椎动物中由两个基因编码的ELKS如何在活动区发挥作用，从而促进其在释放中的作用。我们之前已经发现，ELKS2基因的缺失增加了抑制性突触上容易释放的突触小囊泡的数量。相反，我的初步数据表明，ELKS1可能在抑制传递中发挥相反的作用。这些数据导致了ELKS亚型不同地调节神经递质释放参数的假设。为了解决这一假设，我使用了全细胞膜片钳记录、分子生物学和小鼠遗传工具的组合。在第一个目标中，我将确定ELKS2抑制释放的机制。我将在ELKS2基因敲除中进行救援实验，以确定哪些序列是必要的，并且足以抑制释放。在目标2中，我会问ELKS1是否扮演与ELKS2类似的角色。我们的初步数据表明，ELKS1和ELKS2之间可能存在一些不同的角色，我将在新产生的ELKS1基因敲除小鼠中阐述这些角色。我的第三个目标是基于ELKS1和ELKS2之间错综复杂的相互作用释放微调的假设。我将测试两个具体的工作模型，它们可以解释我们的初步数据，我 将考虑其他结果。总而言之，我的工作将为活动区的组成如何控制突触传递，以及最终活动区的分子缺陷如何导致神经疾病提供新的见解。

DESCRIPTION (provided by applicant): Release of neurotransmitters from a presynaptic nerve terminal initiates synaptic transmission, a key form of information transfer between neurons. The presynaptic active zone, a dense protein network that serves as the site of synaptic vesicle fusion, is a unique feature of the presynaptic nerve terminal compared to other secretory pathways. The active zone orchestrates the synaptic vesicle cycle and endows synaptic transmission with incredible speed and precision required for the complex functions the brain fulfills. ELKS is a protein that localizes tightly to the active zone. Published data and our preliminary results suggest that it fulfills several roles in neurotransmitter release. Human genetic studies found ELKS1 to be associated with neurodevelopmental disorders of speech, indicating relevant functions of ELKS in humans. However, the exact functions of mammalian ELKS are unknown and its mechanisms are not understood. The goal of this study is to understand how ELKS, encoded by two genes in vertebrates, functions at the active zone to contribute to its roles in release. We have previously found that deletion of the ELKS2 gene in mice increases the pool of readily releasable synaptic vesicles at inhibitory synapses. In contrast, my preliminary data indicate that ELKS1 may serve opposing roles in inhibitory transmission. These data lead to the hypothesis that ELKS isoforms differentially tune the parameters of neurotransmitter release. To address this hypothesis, I am using a combination of whole-cell patch clamp recordings, molecular biology, and mouse genetic tools. In the first aim, I will determine the mechanisms by which ELKS2 suppresses release. I will pursue rescue experiments in an ELKS2 knock-out to determine which sequences are necessary and sufficient to suppress release. In aim 2, I will ask the question whether ELKS1 fulfills roles similar to ELKS2. Our preliminary data suggest that there may be some divergent roles between ELKS1 and ELKS2, and I will address these roles in newly generated ELKS1 knock-out mice. My third aim rests on the hypothesis that intricate interplay between ELKS1 and ELKS2 fine-tunes release. I will test two specific working models that could account for our preliminary data, and I will consider alternative outcomes. In summary, my work will provide novel insight into how the makeup of active zones controls synaptic transmission, and, ultimately, how molecular deficiencies at the active zone may contribute to neurological disease.