Optogenetic modeling of primary and secondary CNS proteinopathies in Drosophila

果蝇原发性和继发性中枢神经系统蛋白病的光遗传学模型

• 批准号: 8771014
• 负责人: Diego E Rincon-Limas
• 金额: $ 22.5万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8771014
• 关键词: Address; Adult; Alzheimer' s Disease; Amino Acid Motifs; Amyloid beta-Protein; Animal Model; Animals; Binding; Biochemical; Bypass; Cell Nucleus; Cells; Chemicals; Complex; Coupled; Cyanobacterium; Cytoplasm; Cytosol; DNA Binding Domain; Data; Development; Developmental Process; Devices; Disease; Drosophila genus; Drosophila melanogaster; Elements; Engineering; Enzymes; Epitopes; Event; Experimental Models; Figs - dietary; Gene Expression; Genes; Goals; Growth and Development function; Head; Heme Group; Human; Impaired cognition; Light; Lighting; Link; Modeling; Molecular Conformation; Nerve Degeneration; Neurodegenerative Disorders; Nuclear; Nuclear Localization Signal; Pathology; Photoreceptors; Physiologic pulse; Phytochrome; Plants; Proteins; Public Health; Reporter Genes; Research; Resolution; Sensory; Signal Transduction; System; Technology; Time; Transactivation; Transgenic Animals; Transgenic Organisms; Viral Proteins; Work; base; beta-site APP cleaving enzyme 1; cell type; chromophore; conformer; design; drug discovery; flexibility; fly; gene discovery; in vivo; innovation; nervous system disorder; neurotoxic; novel; optogenetics; photoactivation; phyB phytochrome; plant growth/development; protein TDP-43; protein expression; prototype; public health relevance; research study; response; spatiotemporal; tau Proteins; tool; transcription factor; transgene expression

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.

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共存于多种神经退行性疾病中。例如，Abeta，tau和TDP-43病理可以在一些阿尔茨海默病病例中发现，而tau和TDP-43异常在具有认知障碍的ALS中是明显的。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，这些问题是难以解决的，因为这些蛋白质诱导发育异常或早期致死时，在转基因动物共表达。因此，一个新的战略，多个基因的时空控制将是必不可少的，以应对这一挑战。为此，我们开发了一种新的基因编码的光开关系统的基础上快速，可逆的光敏色素B（PhyB）从植物的光活化。光敏色素是一种感受性光感受器，它响应光信号调节植物生长，并且需要共价连接的发色团才能发挥适当的功能。响应于红光，光敏色素-发色团复合物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，其构象恢复到非活性状态，PhyB在胞质溶胶中积累。因此，我们将利用植物中这种光依赖的、适形基因特异性的相互作用，在转基因动物中开发一种新的光开关基因表达系统。这个系统，我们称之为PhotoGal 4，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件，沿着转录活性所需的蛋白基序。我们将使用果蝇Drosophila melanogaster作为我们的初始动物模型来实现这种新的光遗传学系统。在具体目标1中，我们将表征成年果蝇中PhotoGal 4的光依赖性激活和可逆性。在具体目标2中，我们将使用PhotoGal 4来产生涉及Abeta、tau和TDP-43的原发性和继发性CNS蛋白病的光遗传学模型。我们的中心假设是，PhotoGal 4将通过控制基因表达来响应光量，持续时间和方向，从而绕过致死问题。这一提议意义重大，具有创新性和潜在的变革性，因为PhotoGal 4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的顺序，这些事件协调与Abeta，Tau和TDP 43相互作用相关的伴随病理学。阐明这三种蛋白在体内如何相互作用将是一个重要的stp前进，以确定一些最重要的原发性和继发性CNS蛋白病的机制。