Optogenetic modeling of primary and secondary CNS proteinopathies in Drosophila

描述(由申请人提供)：最近的证据表明，多种疾病相关蛋白的异常积聚在几种神经退行性疾病中共存。例如，在一些阿尔茨海默病病例中可以发现Abeta、tau和TDP-43的病理，而在伴有认知障碍的ALS中，tau和TDP-43的异常是明显的。然而，人们对tau、Abeta和TDP-43在不同细胞类型和不同疾病背景下如何相互作用知之甚少。不幸的是，这些问题很难解决，因为当这些蛋白质在转基因动物中共表达时，会导致发育异常或早期死亡。因此，一种新的时空控制多个基因的策略将是应对这一挑战的关键。为此，我们开发了一种新的遗传编码的光可切换系统，该系统基于植物光敏色素B(PhyB)的快速、可逆光激活。光敏色素是一种感光感受器，它调节植物的生长以响应光信号，并需要一个共价连接的发色团才能正常发挥功能。作为对红光的响应，光敏色素-发色团复合体改变其构象并移位到细胞核以触发信号转导。在黑暗或远红光下，它的构象恢复到非活性状态，PhyB在胞质中积累。因此，我们将利用植物中这种依赖光的、构象特异的相互作用来开发一种新的光可切换的转基因动物基因表达系统。这个系统，我们称之为PhotoGal4，将编码在动物细胞中形成光敏色素-发色团复合体所需的所有元素，以及转录活动所需的蛋白质基序。我们将使用果蝇作为我们最初的动物模型来实现这个新的光遗传系统。在特定的目标1中，我们将表征成年果蝇PhotoGal4的光依赖激活和可逆性。在具体目标2中，我们将使用PhotoGal4来建立涉及Abeta、tau和TDP-43的原发和继发性中枢神经系统蛋白病的光遗传模型。我们的中心假设是，PhotoGal4将通过控制基因表达来应对光量、持续时间和方向，从而绕过致命性问题。这一提议具有重大的创新性和潜在的变革性，因为PhotoGal4的成功实施将提供一种工具，以前所未有的精度指导任何基因的表达。此外，它将提供一个独特的机会来定义事件序列，这些事件序列协调与Abeta、Tau和TDP43相互作用相关的伴随病理。阐明这三种蛋白在体内是如何相互作用的，将是确定一些最重要的原发和继发性中枢神经系统蛋白病变的潜在机制的重要步骤。

DESCRIPTION (provided by applicant): Recent evidence indicates that abnormal accumulation of multiple disease-associated proteins co-exists in several neurodegenerative disorders. For instance, Abeta, tau and TDP-43 pathology can be found in some Alzheimer's disease cases, while tau and TDP-43 abnormalities are evident in ALS with cognitive impairment. However, very little is known about how tau, Abeta and TDP-43 interact with each other in different cell types and in different disease context. Unfortunately, these questions are difficult to address because these proteins induce developmental abnormalities or early lethality when co-expressed in transgenic animals. Thus, a new strategy for the spatio-temporal control of multiple genes will be essential to address this challenge. To that end, we have developed a new genetically encoded light-switchable system based on the fast, reversible photoactivation of Phytochrome B (PhyB) from plants. Phytochromes are sensory photoreceptors that regulate plant growth in response to light signals and require a covalently linked chromophore for proper function. In response to red light, the phytochrome-chromophore complex changes its conformation and translocates to the nucleus to trigger signal transduction. In the dark or under far-red light, its conformation returns to the inactive state and PhyB accumulates in the cytosol. Thus, we will exploit this light-dependent, conformer-specific interaction in plants to develop a new light switchable gene expression system in transgenic animals. This system, which we have called PhotoGal4, will encode for all the elements required for the formation of the phytochrome-chromophore complexes in animal cells along with protein motifs required for transcriptional activity. We will use the fruit fly Drosophila melanogaster as our initial animal model to implement this new optogenetic system. In Specific Aim 1, we will characterize the light- dependent activation and reversibility of PhotoGal4 in adult flies. In Specific Aim 2, we wil use PhotoGal4 to generate optogenetic models of primary and secondary CNS proteinopathies involving Abeta, tau and TDP-43. Our central hypothesis is that PhotoGal4 will bypass lethality issues by controlling gene expression in response to light quantity, duration and direction. This proposal is significant, innovative and potentially transformative because a successful implementation of PhotoGal4 will provide a tool for directing expression of any gene with unprecedented precision. In addition, it will provide a unique opportunity to define the sequence of events that orchestrate concomitant pathology associated with Abeta, Tau and TDP43 interactions. The elucidation of how these three proteins interact in vivo will be a significant stp forward to define the mechanisms underlying some of the most important primary and secondary CNS proteinopathies.