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Novel optogenetic tool for noninvasive neuronal inhibition

用于非侵入性神经元抑制的新型光遗传学工具

基本信息

批准号：
10353090
负责人：
Mark Gomelsky
金额：
$ 18.06万
依托单位：
UNIVERSITY OF WYOMING
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：
2022-09-01 至 2024-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10353090
关键词：
Acids Adenylate Cyclase Amino Acids Anxiety Biochemical Biological Assay Biomedical Engineering Brain Brain region Carbon Dioxide Carboxy-Lyases Cell Therapy Cyclic AMP Cyclic GMP Deinococcus radiodurans Dependovirus Dopamine Engineering Enzymes Epilepsy Future GAD67 enzyme Glutamate Decarboxylase Goals Guanylate Cyclase Histamine Hour Hyperactivity In Vitro Interneurons Length Light Location Mammals Membrane Mental disorders Methodology Modeling Molecular Conformation Monitor Mus Mutagenesis Neurons Neurosciences Neurotransmitters Performance Pharmaceutical Preparations Photoreceptors Protein Engineering Protein Tyrosine Kinase Proteins Research Saccharomyces cerevisiae Schizophrenia Seizures Serotonin Signal Transduction Site Slice Somatosensory Cortex Source Surface Synapses Synaptic Vesicles Testing The Sun Time Variant Virus Visible Radiation Work Yeasts autism spectrum disorder brain tissue cranium design design-build-test designer receptors exclusively activated by designer drugs enzyme activity gamma-Aminobutyric Acid gene therapy high risk high throughput screening in vivo innovation monomer nervous system disorder neuronal excitability novel optogenetics phosphoric diester hydrolase preservation promoter prototype quantum side effect spatiotemporal synthetic biology tool vector

项目摘要

PROJECT SUMMARY/ ABSTRACT This exploratory bioengineering project is aimed at designing, building and testing a light-activated glutamate decarboxylase (GAD) as a novel optogenetic tool for noninvasive inhibition of neuronal activity. GAD produces gamma-aminoburyric acid, GABA, a central neurotransmitter involved in reducing neuronal excitability. Enzy- matic activity of the engineered enzyme will be controlled by light in the near-infrared window (NIRW) of the spectrum (670-900 nm) that penetrates through the skull and brain tissue much better than visible light. We expect the NIRW light-activated GAD (NIRW-GAD) expressed in specific brain regions to be activated via ex- tracorporeal light (e.g., transcranially). NIRW-GAD will represent a unique optogenetic research tool for nonin- vasive, spatiotemporally controlled long-term inhibition of neuronal activity in deep or surface brain regions. In the future, it may be developed into a gene therapy for neurological and psychiatric disorders involving hyper- active brain, such as epilepsy, schizophrenia, anxiety and autism spectrum disorder. The NIRW-GAD protein will be engineered using the homodimeric bacteriophytochrome engineering approach, where bacteriophyto- chromes are a class of photoreceptors that sense light within the NIRW spectrum. In Aim 1, the NIRW-GAD prototypes will be designed and screened for light-inducible GAD activity in yeast. The dynamic range and back- ground activity of the enzyme will be optimized using mutagenesis. In Aim 2, we will examine the NIRW-GAD expression and the effects of NIRW treatment in the somatosensory cortex neurons of mice. We will also quantify the effect of NIRW-GAD on GABAergic synapses and GABA quantal content in cortical neurons in vitro. Upon completion of this project, we expect to have engineered and tested a unique optogenetic tool for noninvasive control of the major inhibitory neurotransmitter in silencing of neuronal activity in mammals. 0

项目总结/摘要这个探索性的生物工程项目旨在设计、构建和测试一种光激活的谷氨酸盐脱羧酶（GAD）作为一种新型的光遗传学工具用于非侵入性抑制神经元活性。GAD产生 γ-氨基丁酸，GABA，一种参与降低神经元兴奋性的中枢神经递质。恩齐工程化酶的活性将由光控制，在近红外窗口（NIRW）的这是一种比可见光更好地穿透颅骨和脑组织的光谱（670-900 nm）。我们预期在特定脑区表达的近红外线光激活GAD（NIRW-GAD）通过前跟踪灯（例如，经颅）。NIRW-GAD将代表一种独特的非蛋白质光遗传学研究工具，侵入性的、时空控制的对深部或表层脑区神经元活动的长期抑制。在在未来，它可能会发展成为一种基因疗法，用于治疗神经和精神疾病，活跃的大脑，如癫痫，精神分裂症，焦虑和自闭症谱系障碍。NIRW GAD蛋白将使用同源二聚体细菌光敏色素工程方法进行工程改造，其中细菌光敏色素- 色素是一类感光器，其感测NIRW光谱内的光。在目标1中，将设计原型并筛选酵母中的光诱导GAD活性。动态范围和背面- 酶的基础活性将使用诱变进行优化。在目标2中，我们将研究NIRW-GAD 表达和NIRW治疗的影响。我们还将量化 NIRW-GAD对体外培养皮层神经元GABA能突触和GABA量子含量的影响。后完成这个项目后，我们预计将设计和测试一种独特的光遗传学工具，在哺乳动物神经元活动沉默中控制主要抑制性神经递质。 0