Encoding Long Term Stability of Neuronal Function

描述(申请人提供)：在胚胎发育过程中，转录因子对于建立决定神经元前体和神经元命运的密码至关重要。然而，大多数负责神经元功能特性的蛋白质的半衰期从几分钟到几个小时不等；因此，在神经发育结束后很长一段时间内，为了维持神经元的功能，它们必须受到严格的调控。令人惊讶的是，人们对转录因子在这一过程中扮演的角色知之甚少。我们假设，最初被描述为早期神经发育基因的转录因子，是充分发育的运动神经元(MN)维持神经元功能所必需的。我们建议在完全发育的MNS中显著影响三种转录因子GSB、ISL和EVE的水平，并确定它们所控制的神经元功能。这项研究的长期目标是建立这些转录因子的分子靶点。果蝇神经肌肉接头(NMJ)为研究发育完全的MNS提供了一种很好的模型。MN已经达到了它们的肌肉目标，并在胚胎发育结束之前释放神经递质。我们可以在MN完全发育后4到5天，接近幼虫生命的末期，比平均蛋白质的寿命长很多倍，来测试MN的活性、突触结构和生理学。此外，转基因RNAi和条件表达的使用允许敲除完全发育的MN中的基因表达，从而绕过了胚胎对转录因子的要求。我们最近发现，GSB在发育成熟的晚期中枢神经系统的MN中普遍表达。利用免疫组织化学方法，我们将首先证明EVE和ISL在完全发育的MN的一个子集内表达。在第二个目标中，我们将结合经典遗传学和Gal4/UAS系统与Gal80TS的使用，在发育后期敲除或过度表达转录因子。然后，我们将使用免疫组织化学和电子显微镜来确定MN的活性或NMJ的结构是否因在完全发育的MN中敲除或过度表达Eve、ISL或GSB而受到影响。然后，我们将在NMJ进行细胞内电生理记录，以确定Eve、ISL或GSB是否控制充分发育的MN的突触释放和内稳态可塑性。这项研究将提供一个概念性模板，将功能归因于充分发育的神经元中的转录因子；这反过来可能导致对神经元老化和神经元变性的分子过程的深入了解。

DESCRIPTION (provided by applicant): During embryonic development, transcription factors are essential for establishing a code that will determine the fate of neuronal precursors and neurons. However, most proteins that are responsible for a neuron's functional properties have a half-life ranging from minutes to several hours; they therefore must be tightly regulated long after neural development is over in order to maintain neuronal function. Surprisingly, very little is known about the role that transcription factors play in this process. We hypothesize that transcription factors that were initially characterized as early neuro-developmental genes, are required in fully developed motorneurons (MNs) to maintain neuronal function. We propose to drastically affect the levels of three transcription factors gsb, isl and eve within fully developed MNs and determine the neuronal functions they control. The long term goal of this research is to establish the molecular targets of these transcription factors. The Drosophila neuromuscular junction (NMJ) provides a model that is well-suited to the study of fully developed MNs. MNs have reached their muscle targets and are releasing neurotransmitter before the end of embryogenesis. We can test MN viability, synaptic structure and physiology 4 to 5 days after they are fully developed, near the end of the larval life, many times longer than the lifetime of the average protein. Moreover, the use of transgenic RNAi and conditional expression allows for knockout of gene expression in fully developed MNs, therefore bypassing the embryonic requirement for transcription factors. We have recently shown that gsb is ubiquitously expressed in fully developed MNs of the late larval CNS. Using immunohistochemistry, we will first show that eve and isl are expressed within a subset of fully developed MNs. In a second aim we will combine classical genetics and the use of the Gal4/UAS system in conjunction with Gal80TS to knock down or over-express transcription factors late, after development. We will then use immunohistochemistry and electron microscopy to determine whether the viability of the MN or the structure of the NMJ is affected by knocking down or over-expressing eve, isl or gsb within fully developed MNs. We will then perform intracellular electrophysiological recordings at the NMJ to determine whether eve, isl or gsb control synaptic release and homeostatic plasticity within fully developed MNs. This study will provide a conceptual template attributing a function to transcription factors within fully developed neurons; this in turn could lead to great insights into the molecular processes of neuronal aging and neuronal degeneration.