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

• 批准号: 10633118
• 负责人: Adriana Galvan
• 金额: $ 22.75万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 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/10633118
• 关键词: Acceleration; 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; 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; Positron; Positron-Emission Tomography; Prevalence; Primates; Productivity; Proteins; Publishing; Radiolabeled; Research; Research Personnel; Research Project Grants; Rodent; Rodent Model; Role; Site; Techniques; Technology; Time; Transfection; Translations; Validation; Viral Vector; Virus; Visualization; analog; 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; wasting

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.

项目摘要 光遗传学方法已被广泛用于扩大我们的知识，正常和病理 基底神经节和相关脑区在帕金森病（PD）中的作用这项革命性的技术- 到目前为止，神经学仅限于对帕金森氏症啮齿动物模型的研究，而技术上的限制 这在很大程度上限制了光遗传学实验在非人灵长类动物（NHP）中的使用。光的稀缺性- NHP的遗传学研究不仅限于PD相关研究。光遗传学扩展的关键限制 缺乏非侵入性方法来监测体内视蛋白表达。死后组织学检查 主要是唯一可靠的方法来确认脑区域的正确靶向和视蛋白表达水平， NHP。显然，该方法不允许实验方法的体内校正。 光遗传学在NHP中的使用的扩展需要开发监测时间的方法。 视蛋白表达的过程和水平，使得可以在体内检测和校正不足的表达水平， 从而避免昂贵的实验失败，并减少研究所需的NHP数量。我们提出 一种基于正电子成像断层扫描的非侵入性方法，用于在体内重复监测视蛋白表达 (PET)NHP中的成像。我们将使用兴奋性视蛋白通道视紫红质-2（ChR 2）的创新版本， 其中视蛋白与人雌激素受体α的配体结合结构域融合， 视蛋白“ChRERa”。放射性标记的氟化雌二醇类似物，[18 F]-氟雌二醇（[18 F]-FES）， 至ChRERa，并且[18F]-FES的脑分布可用PET可视化。我们将使用ChRERA和重新- PEED [18F]-FES PET扫描以纵向监测恒河猴中的视蛋白表达。使用病毒载体， 我们将在纹状体的运动部分表达ChRERa，然后进行几次[18 F]-FES PET扫描， 在接下来的12个月中，描述PET可检测的视蛋白表达的时间过程。同时，我们将 检查PET鉴定的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，我们将检查通信是- 在[18F]-FES PET扫描中发现的视蛋白表达模式与用 尸体组织学这些研究将建立一个基本的技术，以评估在体内的分布 在NHP中。根据本FOA PA-21-219（“NINDS/NIMH联合探索性 神经科学研究基金”），这些研究的长期目标是使用ChRERa和[18F]-FES PET 我们在帕金森病病理生理学的NHP光遗传学研究中进行了扫描。更广泛地说， 监测视蛋白表达的非侵入性体内方法可以帮助加速光遗传学的可靠使用， 研究神经系统疾病的NHP模型，并最终促进光遗传学的翻译。 技术到诊所。