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: 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
• 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

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.

项目总结 光遗传学方法已被广泛用于扩展我们对正常和病理的知识。 基底节及相关脑区在帕金森氏病中的作用。这项革命性的技术- 到目前为止，精神病学仅限于对帕金森症啮齿动物模型的研究，而技术限制 在很大程度上限制了光遗传学实验在非人类灵长类动物(NHP)中的使用。眼镜片的稀缺- NHP中的嵌套研究并不局限于帕金森病相关的研究。光遗传学扩展的一个关键限制 在灵长类动物中，缺乏非侵入性的方法来监测体内视蛋白的表达。尸检组织学报告 Main是确认脑区正确靶向和视蛋白表达水平的唯一可靠方法 国家卫生保健计划。显然，这种方法不允许对实验方法进行活体校正。 扩大光遗传学在卫生保健计划中的应用需要开发监测颞叶的方法 视蛋白表达的进程和水平，从而可以在体内检测和纠正不足的表达水平， 从而避免了代价高昂的实验失败，并减少了研究所需的NHP数量。我们建议 一种基于正电子成像技术的体内重复检测视蛋白表达的非侵入性方法 (PET)在NHP中的成像。我们将使用一种创新版本的兴奋性视紫红质通道-2(ChR2)， 其中视蛋白被融合到人类雌激素受体α的配体结合区域，创造了新的 OPTING“CHRERA”。放射性标记的含氟雌二醇类似物[18F]-氟雌二醇([18F]-FeS)与强结合 ~(18)F]-FES的脑内分布可用正电子发射计算机断层扫描(PET)进行可视化。我们将使用ChRERa并重新- PEATED[18F]-FES PET扫描纵向监测恒河猴的视蛋白表达。利用病毒载体， 我们将在纹状体的运动部表达ChRERa，然后进行几次[18F]-FES PET扫描 在接下来的12个月中描述PET可检测到的视蛋白表达的时间进程。同时，我们将 检查经PET鉴定的ChRERa表达模式是否对应于功能视蛋白的存在， 通过在光激活过程中进行细胞外记录。最后，我们将审查这些通信是- 在[18F]-FeS PET扫描中发现的视蛋白表达模式与与 尸检组织学。这些研究将建立评估体内分布的基本技术 NHP中的Opsins。与本FOA PA-21-219(NINDS/NIMH联合探索性 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 扫描我们的NHP光遗传学研究帕金森病的病理生理学。更广泛地说，验证 非侵入性体内监测视蛋白表达的方法有助于加速光遗传学在 研究神经疾病的NHP模型，并最终可能促进光遗传学的翻译 把技术带到临床上。