Development of a non-invasive method to monitor expression and function of optogenetic tools in non-human primates

开发一种非侵入性方法来监测非人类灵长类动物中光遗传学工具的表达和功能

基本信息

批准号：
10451093
负责人：
Adriana Galvan
金额：
$ 27.3万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-06-15 至 2024-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10451093
关键词：
Aging Animals Applications Grants Area Autopsy Basal Ganglia Binding Brain Brain region Cells Clinic Clinical Communities Corpus striatum structure Data Development Electron Microscopy Electrophysiology (science)Estradiol Estrogen Receptor alpha FPS-FES Oncogene Failure Functional disorder Genes Genetic Glycols Goals Histologic Histology Human Image Injections Joints Knowledge Laboratories Lead Ligand Binding Domain Light Literature Macaca mulatta Mediating Methods Mission Monitor Monkeys Motor National Institute of Mental Health National Institute of Neurological Disorders and Stroke Nature Neurological Models Neurons Neurosciences Research Opsin Parkinson Disease Parkinsonian Disorders Pathologic Pathway interactions Pattern Peat Positron Positron-Emission Tomography Prevalence Primates Productivity Proteins Publishing Radiolabeled Research Research Personnel Research Project Grants Rodent Rodent Model Role Scanning Site Techniques Technology Time Transfection Translations Tweens Validation Viral Vector Virus analog base brain tissue brain volume cost extracellular in vivo innovation interest light microscopy method development nervous system disorder nonhuman primate novel optogenetics parkinsonian non-human primate putamen response tomography tool

项目摘要

PROJECT SUMMARY Optogenetic methods have been used extensively to expand our knowledge of the normal and pathological roles of the basal ganglia and related brain areas involved in Parkinson’s disease (PD). This revolutionary tech- nology has, so far, been restricted to studies in rodent models of parkinsonism, while technical constraints have largely limited the use of optogenetic experimentation in non-human primates (NHPs). The scarcity of optoge- netics studies in NHPs is not limited to PD-related research. A critical limitation to the expansion of optogenetics in primates is the lack of non-invasive methods to monitor opsin expression in vivo. Postmortem histology re- mains the only reliable method to confirm the correct targeting of brain regions and level of opsin expression in NHPs. Evidently, this method does not allow for in vivo corrections of experimental approaches. Expansion of the use of optogenetics in NHPs require the development of methods to monitor the temporal course and level of opsin expression, so that insufficient expression levels can be detected and corrected in vivo, thus avoiding costly experimental failures, and reducing the number of NHPs needed for research. We propose a non-invasive approach to monitor opsin expression repeatedly in vivo, based on positron imaging tomography (PET) imaging in NHPs. We will use an innovative version of the excitatory opsin channelrhodopsin-2 (ChR2), in which the opsin is fused to the ligand binding domain of the human estrogen receptor alpha, creating the novel opsin “ChRERa”. The radiolabeled fluorinated estradiol analog, [18F]-fluoroestradiol ([18F]-FES), binds strongly to ChRERa, and the brain distribution of [18F]-FES can be visualized with PET. We will use ChRERa and re- peated [18F]-FES PET scans to monitor opsin expression in Rhesus monkeys longitudinally. Using viral vectors, we will express ChRERa in the motor portion of the striatum, and then conduct several [18F]-FES PET scans over the next 12 months to describe the time course of PET detectable opsin expression. In parallel, we will examine if the PET-identified pattern of ChRERa expression corresponds to the presence of functional opsins, by conducting extracellular recordings during light activation. Finally, we will examine the correspondence be- tween the opsin expression pattern found in the [18F]-FES PET scans and the opsin distribution identified with post-mortem histology. These studies will establish a fundamental technology to evaluate in vivo the distribution of opsins in the NHP. In alignment with the purpose of this FOA PA-21-219 (“Joint NINDS/NIMH Exploratory Neuroscience Research Grant”), these studies have the long-term goal of using ChRERa and [18F]-FES PET scans in our NHP optogenetic studies of the pathophysiology of parkinsonism. More broadly, validation of a non-invasive in vivo method to monitor opsin expression could help accelerate reliable use of optogenetics for the study of NHP models of neurological disorders and could eventually facilitate the translation of optogenetic techniques to the clinic.

项目概要光遗传学方法已被广泛使用来扩展我们对正常和病理的认识这项革命性的技术涉及基底神经节和相关大脑区域在帕金森病 (PD) 中的作用。迄今为止，科学仅限于帕金森病啮齿动物模型的研究，而技术限制很大程度上限制了光遗传学实验在非人类灵长类动物（NHP）中的应用。 NHP 的遗传学研究不仅限于 PD 相关研究，这是光遗传学扩展的一个关键限制。灵长类动物的一个问题是缺乏非侵入性方法来监测体内视蛋白的表达。这是确认大脑区域正确定位和视蛋白表达水平的唯一可靠方法显然，这种方法不允许对实验方法进行体内校正。扩大光遗传学在 NHP 中的应用需要开发监测时间的方法视蛋白表达的过程和水平，以便可以在体内检测和纠正不足的表达水平，从而避免代价高昂的实验失败，并减少研究所需的 NHP 数量。基于正电子成像断层扫描的一种非侵入性体内重复监测视蛋白表达的方法我们将使用兴奋性视蛋白通道视紫红质 2 (ChR2) 的创新版本，其中视蛋白与人类雌激素受体α的配体结合域融合，创造了新的视蛋白“ChRERa”，放射性标记的氟化雌二醇类似物，[18F]-氟雌二醇 ([18F]-FES)，牢固结合。 ChRERa，[18F]-FES 的大脑分布可以用 PET 可视化，我们将使用 ChRERa 并重新进行。泥炭[18F]-FES PET扫描使用病毒载体纵向监测恒河猴中的视蛋白表达。我们将在纹状体的运动部分表达 ChRERa，然后进行多次 [18F]-FES PET 扫描在接下来的 12 个月内，我们将同时描述 PET 可检测视蛋白表达的时间过程。检查 PET 识别的 ChRERa 表达模式是否对应于功能性视蛋白的存在，通过在光激活期间进行细胞外记录最后，我们将检查对应关系 - [18F]-FES PET 扫描中发现的视蛋白表达模式与用以下方法识别的视蛋白分布之间的关系这些研究将建立评估体内分布的基础技术。 NHP 中的视蛋白。符合本 FOA PA-21-219 的目的（“NINDS/NIMH 联合探索性研究”）神经科学研究补助金”），这些研究的长期目标是使用 ChRERa 和 [18F]-FES PET 我们的 NHP 光遗传学研究对帕金森病的病理生理学进行了扫描，更广泛地验证了这一点。监测视蛋白表达的非侵入性体内方法有助于加速光遗传学的可靠应用对神经系统疾病 NHP 模型的研究，最终可能促进光遗传学的转化技术到临床。