Molecular Dissection of Active Zone Functions in Neurotransmitter Release

描述(由申请人提供)：突触传递的速度和精确调节对复杂的大脑功能如认知和学习至关重要。在包括自闭症、精神分裂症、成瘾和神经变性在内的神经疾病中，突触前神经末梢的神经递质释放经常受到损害。因此，准确了解神经递质释放的分子机制对于了解大脑疾病至关重要。突触前神经末梢的活动区是神经递质释放的部位。活动区由高度专业化的蛋白质网络组成，该网络组织突触小泡以快速触发钙释放，这是突触传递速度和精确度的核心要求。我们的首要目标是了解活动区的蛋白质机制是如何运作的。我们通过剖析活性区域组分的分子功能来实现这一目标。ELKS蛋白在活动区高度浓缩，表明ELKS在活动区参与神经元胞吐。在释放之前，活动区停靠在突触前钙通道附近，并启动突触小泡以进行胞吐。ELKS在这些过程中如何控制释放还不清楚，可能部分原因是脊椎动物还没有采取系统的遗传学方法来解决ELKS的功能。我们现在已经产生了哺乳动物ELKS基因ELKS1和ELKS2的条件性基因敲除小鼠。大量的初步数据导致了我们的中心假设：ELKS蛋白通过控制突触前钙离子的内流增加了释放的可能性，并且它们调节了容易释放的囊泡池的大小。我们在三个特定的目标中解决了这一假说的不同组成部分，并剖析了潜在的分子机制。在目标1中，我们假设ELKS1和ELKS2蛋白既有共同的功能，也有不同的功能。我们通过系统地研究新产生的ELKS1和ELKS2条件性单基因敲除小鼠以及ELKS1/2双基因敲除小鼠的突触前表型，确定了每个ELKS基因如何在神经递质释放的活动区功能中发挥作用。在初步实验中，我们发现ELKS蛋白促进突触前钙内流，并且ELKS单个和双个缺失不同地影响易释放的囊泡池。在目标2中，我们确定了ELKS控制突触前钙内流的机制。在目标3中，我们提出了一个特定的假设，统一了在ELKS突变体中观察到的对囊泡池的影响。我们检验了这一假设，确定了潜在的分子机制，并考虑了许多替代的解释。我们的研究是创新的，因为它结合了独特深度的遗传、生化和功能实验，提出了一个新的假设。最终，这种方法将导致对神经递质释放的分子控制的精确洞察，神经递质释放是在各种大脑疾病期间失败的关键神经过程。

DESCRIPTION (provided by applicant): Speed and precise regulation of synaptic transmission are critical for complex brain functions such as cognition and learning. Release of neurotransmitters from a presynaptic nerve terminal is often impaired in neurological disorders, including autism, schizophrenia, addiction and neurodegeneration. Exact knowledge of the molecular mechanisms for neurotransmitter release is thus critical for understanding brain disease. The active zone of a presynaptic nerve terminal is the site of neurotransmitter release. An active zone consists of a highly specialized network of proteins that organizes synaptic vesicles for fast Ca2+-triggering of release, a central requirement for speed and precision of synaptic transmission. It is our over-arching goal to understand how the protein machinery at the active zone operates. We approach this goal by dissecting the molecular functions of active zone components. ELKS proteins are highly enriched at active zones, indicating that ELKS functions in neuronal exocytosis at the active zone. Before release, active zones dock and prime synaptic vesicles for exocytosis close to presynaptic Ca2+-channels. How ELKS operates during these processes to control release is not understood, maybe in part because no systematic genetic approach has been taken in vertebrates to address ELKS function. We have now generated conditional knockout mice for both mammalian ELKS genes, ELKS1 and ELKS2. Ample preliminary data lead to our central hypothesis: ELKS proteins increase release probability though controlling presynaptic Ca2+-influx, and they modulate the size of the pool of readily releasable vesicles. We address separate components of this hypothesis in three specific aims, and we dissect the underlying molecular mechanisms. In aim 1, we hypothesize that ELKS1 and ELKS2 proteins have both shared and distinct functions. We determine how each ELKS gene contributes to the functions of active zones in neurotransmitter release by systematically studying presynaptic phenotypes in the newly generated conditional single knockout mice for ELKS1 and ELKS2, and in the ELKS1/2 double knockout mice. In preliminary experiments we find that ELKS proteins enhance presynaptic Ca2+-influx, and that individual and double ELKS deletions differentially affect the pool of readily releasable vesicles. In aim 2, we determine the mechanisms by which ELKS controls presynaptic Ca2+-influx. In aim 3, we propose a specific hypothesis that unifies effects on vesicle pools observed in ELKS mutants. We examine this hypothesis, determine the underlying molecular mechanisms and consider numerous alternative explanations. Our research is innovative because it addresses a novel hypothesis by a combination of genetic, biochemical and functional experiments of unique depth. Ultimately, this approach will lead to precise insights into the molecular control of neurotransmitter release, a key neuronal process that fails during various brain diseases.