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累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，而在认知障碍的ALS患者中，tau和TDP-43的异常是明显的。然而，对于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，因为这些蛋白在转基因动物中共表达时会诱导发育异常或早期致死。因此，一种新的多基因时空控制策略对于解决这一挑战至关重要。为此，我们开发了一种基于植物光敏色素B （Phytochrome B, PhyB）快速可逆光激活的新型遗传编码光开关系统。光敏色素是一种感觉光感受器，它根据光信号调节植物的生长，并需要共价连接的发色团才能正常发挥作用。在红光的作用下，光敏色素-发色团复合物改变其构象并转运到细胞核中触发信号转导。在暗光或远红光下，其构象恢复到无活性状态，PhyB在细胞质中积累。因此，我们将在植物中利用这种光依赖性、共构体特异性相互作用，在转基因动物中开发一种新的光可切换基因表达系统。这个系统，我们称之为PhotoGal4，将编码动物细胞中光敏色素-发色团复合物形成所需的所有元素，以及转录活性所需的蛋白质基元。我们将使用果蝇作为我们的初始动物模型来实现这个新的光遗传系统。在Specific Aim 1中，我们将描述成年果蝇中PhotoGal4的光依赖性激活和可逆性。在Specific Aim 2中，我们将使用PhotoGal4生成涉及β、tau和TDP-43的原发性和继发性中枢神经系统蛋白病变的光遗传学模型。我们的中心假设是，PhotoGal4将通过控制基因表达来响应光量、持续时间和方向，从而绕过致死性问题。这一建议意义重大，具有创新性，具有潜在的变革性，因为PhotoGal4的成功实施将为指导任何基因的表达提供前所未有的精确工具。此外，它将提供一个独特的机会来定义与Abeta， Tau和TDP43相互作用相关的伴随病理的事件序列。阐明这三种蛋白在体内如何相互作用，将对确定一些最重要的原发性和继发性中枢神经系统蛋白病变的机制具有重要意义。

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.