Mitochondrial signaling dynamics in cocaine use disorder

可卡因使用障碍中的线粒体信号动力学

• 批准号: 10681667
• 负责人: Antony Daniel Abraham
• 金额: $ 65.28万
• 依托单位: RESEARCH TRIANGLE INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-15 至 2028-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10681667
• 关键词: Adenosine Triphosphate; Behavior; Calcium; Cardiolipins; Chemistry; Clustered Regularly Interspaced Short Palindromic Repeats; Cocaine; Cocaine use disorder; Complex; Drug Targeting; Enzymes; Event; Excision; Fiber; Future; Generations; Genes; Health; Hydrogen Peroxide; Knock-out; Lead; Measures; Mitochondria; Mitochondrial Matrix; Mitochondrial Proteins; Monitor; Mus; Neurons; Neurophysiology - biologic function; Nucleus Accumbens; Pharmaceutical Preparations; Pharmacology; Phospholipids; Photometry; Presynaptic Terminals; Production; Proteins; Reactive Oxygen Species; Research; Signal Transduction; Specificity; Stimulant overdose; Substance Use Disorder; Superoxide Dismutase; Superoxides; Synapses; System; Testing; Therapeutic; Viral; awake; calcium uniporter; cocaine exposure; dopaminergic neuron; drug of abuse; in vivo; knockout gene; novel; overdose death; pharmacologic; response; sensor; tool

PROJECT SUMMARY Cocaine use disorder continues to be a significant health burden, with psychostimulant overdose deaths rising significantly over the past few years. Cocaine exposure can alter aspects of mitochondrial function, but the tools to access and manipulate mitochondria in vivo are limited. Mitochondria contribute a significant amount of the energy required for events in the synapse in the form of adenosine triphosphate (ATP). Production of ATP in mitochondria near synapses is tightly regulated by the influx of calcium into mitochondria and leads to the generation of reactive oxygen species within the mitochondrial matrix. The studies in this proposal aim to measure and manipulate mitochondrial function, specifically calcium and hydrogen peroxide signaling in vivo, to determine how mitochondrial activity in dopamine neurons is altered in response to cocaine exposure. We will target three mitochondria-specific protein-encoding genes using a novel CRISPR viral strategy to knock out genes from dopamine neurons in DATCre mice in vivo. The target genes are involved in regulating calcium and hydrogen peroxide concentrations in mitochondria: Micu1, which is part of the mitochondrial calcium uniporter complex and allows mitochondrial calcium influx; Crls1, which encodes an enzyme necessary for the synthesis of cardiolipin, a mitochondrial phospholipid involved in integrating calcium and reactive oxygen species signaling; and Sod2, a mitochondria-specific superoxide dismutase that converts superoxide into hydrogen peroxide and enables removal of ROS from the mitochondrial matrix. Notably, Micu1 and Crls1 encode mitochondrial proteins that have been identified as targets of lead compounds, allowing for a relatively rapid determination of whether these systems can be modulated in vivo for substance use disorder treatments. To begin developing drugs for Sod2 and to refine drugs targeting Micu1 and Crls1, these studies will use mitochondrially-targeted fluorescent sensors for calcium and hydrogen peroxide in combination with fiber photometry in vivo. My preliminary evidence shows that HyPerRed, a hydrogen peroxide sensor, can be used in vivo in cortical neurons to record responses to drugs of abuse. The studies in this proposal will expand on these characterizations and further determine how cocaine may alter reactive oxygen species activity in presynaptic terminals of dopamine neurons in the nucleus accumbens core. I will also test mitochondrially- targeted GCaMP to develop a comprehensive understanding of how calcium and reactive oxygen species activity is coordinated within mitochondria. These sensors will be combined with the CRISPR viral knockout strategies and pharmacological tools targeting mitochondria to further characterize the specificity of these signals and the possible impact of these pharmacological approaches on neural function and behavior. Together, these studies will enable the exploration of a novel avenue of research by monitoring and manipulating mitochondrial function in vivo to develop treatments for cocaine use disorders.

项目摘要 可卡因使用障碍仍然是一个重大的健康负担，精神兴奋剂过量 死亡人数在过去几年中大幅上升。暴露于可卡因可以改变线粒体的某些方面， 功能，但在体内访问和操纵线粒体的工具是有限的。线粒体有助于 突触中事件所需的大量能量以三磷酸腺苷的形式存在 （ATP）中指定。突触附近线粒体中ATP的产生受到钙离子流入突触的严格调节。 线粒体内的活性氧物质，并导致线粒体基质内的活性氧物质的产生。的 该提案中的研究旨在测量和操纵线粒体功能，特别是钙和 体内过氧化氢信号传导，以确定多巴胺神经元中的线粒体活性如何改变， 可卡因暴露的反应。我们将使用一种新的靶向三个特定的蛋白质编码基因， CRISPR病毒策略敲除体内DATCre小鼠多巴胺神经元的基因靶基因是 参与调节线粒体中的钙和过氧化氢浓度：Micu 1，它是 线粒体钙单向转运体复合物，允许线粒体钙内流; Crls 1，编码一种 心磷脂合成所必需的酶，心磷脂是一种线粒体磷脂，参与钙的整合 和活性氧信号;和Sod 2，一种拟南芥特异性超氧化物歧化酶， 通过将超氧化物转化为过氧化氢，能够从线粒体基质中去除ROS。值得注意的是，Micu 1 和Crls 1编码线粒体蛋白，这些蛋白已被鉴定为先导化合物的靶点， 相对快速地确定这些系统是否可以在体内被调节用于物质使用障碍 治疗。为了开始开发针对Sod 2的药物并改进针对Micu 1和Crls 1的药物，这些研究 将使用钙和过氧化氢的靶向荧光传感器， 活体纤维光度法。我的初步证据表明，HyperRed，一种过氧化氢传感器，可以 在体内用于皮质神经元记录对滥用药物的反应。本提案中的研究将扩大 这些特征，并进一步确定可卡因如何可能改变活性氧物种的活动， 突触前末梢的多巴胺神经元在延髓核的核心。我也会做个心理测试- 有针对性的GCaMP，以全面了解钙和活性氧如何 活动在线粒体内协调。这些传感器将与CRISPR病毒敲除相结合 针对线粒体的策略和药理学工具，以进一步表征这些特异性， 信号以及这些药理学方法对神经功能和行为的可能影响。 总之，这些研究将使探索一个新的研究途径，监测和 操纵体内线粒体功能，以开发可卡因使用障碍的治疗方法。