Molecular Dissection of Active Zone Functions in Neurotransmitter Release

描述（申请人提供）：突触传递的速度和精确调节对认知和学习等复杂的大脑功能至关重要。神经递质从突触前神经末梢的释放通常在神经疾病中受损，包括自闭症、精神分裂症、成瘾和神经变性。因此，神经递质释放的分子机制的确切知识对于理解脑部疾病至关重要。突触前神经末梢的活跃区是神经递质释放的部位。活性区由一个高度专业化的蛋白质网络组成，该网络组织突触囊泡以快速触发Ca2+释放，这是突触传输速度和精度的核心要求。我们的首要目标是了解活性区的蛋白质机制是如何运作的。我们通过剖析活性区组分的分子功能来实现这一目标。ELKS蛋白在活跃区高度富集，表明ELKS在活跃区神经元胞吐作用中起作用。在释放之前，活跃区停靠并启动突触囊泡，以便在突触前Ca2+通道附近进行胞吐。ELKS在这些过程中如何控制释放尚不清楚，部分原因可能是没有系统的遗传方法在脊椎动物中解决ELKS的功能。现在，我们已经培育出具有ELKS基因（ELKS1和ELKS2）的条件敲除小鼠。充足的初步数据导致我们的中心假设：ELKS蛋白通过控制突触前Ca2+内流增加释放概率，并且它们调节易释放囊泡池的大小。我们在三个具体目标中解决这个假设的单独组成部分，并剖析潜在的分子机制。在目的1中，我们假设ELKS1和ELKS2蛋白具有共享和不同的功能。我们通过系统地研究新生成的ELKS1和ELKS2条件单敲除小鼠以及ELKS1/2双敲除小鼠的突触前表型，确定每个ELKS基因如何促进神经递质释放活性区的功能。在初步实验中，我们发现ELKS蛋白增强突触前Ca2+内流，并且单个和双ELKS缺失对易释放囊泡池的影响不同。在目的2中，我们确定了ELKS控制突触前Ca2+内流的机制。在目标3中，我们提出了一个特定的假设，该假设统一了在ELKS突变体中观察到的对囊泡池的影响。我们检验这一假设，确定潜在的分子机制，并考虑许多替代的解释。我们的研究是创新的，因为它通过独特深度的遗传、生化和功能实验的结合来解决一个新的假设。最终，这种方法将导致对神经递质释放的分子控制的精确见解，这是在各种脑部疾病中失败的关键神经元过程。

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.