Purification of cell-type specific synaptic material using virally-expressed tags

使用病毒表达标签纯化细胞类型特异性突触物质

• 批准号: 9980828
• 负责人: Stephen Edward Paucha Smith
• 金额: $ 23.42万
• 依托单位: SEATTLE CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9980828
• 关键词: AMPA Receptors; Affect; Antibodies; Area; Behavior; Behavioral; Biochemical; Biocompatible Materials; Biotechnology; Blood; Brain; Calcium; Cell Separation; Cells; Chronic Disease; Complex; Complex Mixtures; Control Groups; Corpus striatum structure; Cues; Dendritic Spines; Drug Addiction; Electron Microscopy; Event; Extracellular Protein; Face; Flow Cytometry; Food; Globus Pallidus; Goals; Grant; Heroin; Heroin Dependence; Immunohistochemistry; Immunologist; Immunology; Injections; Label; Lead; Lymphocyte; Magnetism; Mass Spectrum Analysis; Metals; Microscope; Mitochondria; Modernization; Molecular; Motivation; Neurobiology; Neurons; Organ; Output; Pathologic; Pathway interactions; Permeability; Pharmaceutical Preparations; Phenotype; Play; Population; Preparation; Proteins; Proteomics; Rat-1; Rattus; Reagent; Recombinant Proteins; Research Personnel; Rewards; Role; Self Administration; Severities; Sorting - Cell Movement; Specificity; Structure of subthalamic nucleus; Synapses; Synaptosomes; System; Techniques; Therapeutic; Tissues; Validation; Ventral Tegmental Area; Viral; Virus; Western Blotting; Work; addiction; adeno-associated viral vector; antibody conjugate; base; cell type; combinatorial; design; differential expression; drug of abuse; drug seeking behavior; empowered; experimental study; extracellular; follow-up; heroin use; in vivo; member; molecular marker; novel; protein biomarkers; protein expression; therapy development; treatment strategy; vector

PROJECT SUMMARY The brain is by far the most complex organ, with hundreds or even thousands of distinct cell types intermingled in a tissue that appears, at first glance, to be fairly homogeneous. This presents a problem for biochemical studies of specific cell types, since the protein content of a given cell type is difficult or impossible to purify from the surrounding tissue. For example, in the striatum, medium spiny neurons of the direct vs. indirect pathway (dMSNs vs. iMSNs) express different molecular markers, and cell-type-specific activation or silencing produces opposing behavioral outputs. In the context of drug addiction, silencing iMSNs enhances the motivation to self- administer drugs, while silencing dMSNs reduces drug-seeking behavior. Treatment approaches that could correct the extensive molecular alterations caused by drugs of abuse are promising therapeutic strategies. However, given the opposing behavioral outputs of iMSNs vs. dMSNs, a drug that reduces drug-seeking behavior via one pathway could enhance the same behavior via the other pathway. Identification of cell-type specific targets would increase the possibility of modulating specific striatal pathways and behaviors, but our inability to purify iMSN vs. dMSN protein precludes such exploratory experiments. Here, we propose to develop a sorting approach based on a common Immunology technique, Magnetic Cell Sorting (MACS). We will express, in the dendritic spines of iMSN or dMSN neurons, an inert, extracellular “TAG” that is recognized by highly specific, commercially available MACS sorting reagents. Cell-type-specificity will be achieved using a viral dual-injection strategy, in which a DIO/FLEX vector containing an inverted TAG construct will be injected into the striatum, and a retrograde Cre virus will be injected into the Ventral Tegmental Area (VTA) or the Ventral Pallidum (VP) to label neurons of the direct or indirect pathway, respectively. Sorting will be accomplished using metal-conjugated antibodies and a magnetized column, allowing rapid isolation of a large amount of biomaterial. In aim 1, we develop and validate the TAG system in the rat. In Aim 2, we label iMSNs and dMSNs in rats, and use an extended intermittent access herion self-administration paradigm to produce groups of rats that express low- and high-levels of addict-like behavior (as well as food reward for control). We will then isolate iMSNs and dMSNs from rats expressing high and low addict-like behavior, and use a quantitative mass spectrometry approach to identify proteins that are differentially expressed in iMSNs or dMSNs in rats showing high- vs. low-levels of addict-like behavior. Follow-up work beyond the scope of the grant will explore the potential of modulating the expression of these proteins in rats expressing addict-like behavior, with the goal of reducing drug-seeking behavior by specifically targeting cells of the iMSN or dMSN pathway. More generally, our tagging-and-sorting approach could be widely applicable to the study of cell-type- specific protein expression in the brain.

项目摘要 大脑是迄今为止最复杂的器官，数百甚至数千种不同的细胞类型混合在一起 在乍看之下出现的组织中，相当均匀。这给生化的问题带来了 对特定细胞类型的研究，因为给定细胞类型的蛋白质含量很难或不可能从中纯化 周围的组织。例如，在纹状体中，直接和间接途径的中刺神经元 （DMSN与IMSN）表达不同的分子标记，以及细胞类型的激活或沉默生产 相反的行为输出。在添加药物的背景下，沉默IMSN会增强自我的动力 施用药物，而沉默的DMSN则减少了寻求药物的行为。可以的治疗方法 纠正由滥用药物引起的广泛分子改变是有希望的治疗策略。 但是，鉴于IMSNS与DMSN的相对行为输出相反，该药物可减少寻求毒品 通过一个途径的行为可以通过另一个途径增强相同的行为。鉴定细胞类型 具体目标将增加调节特定纹状体途径和行为的可能性，但是我们的 无法纯化IMSN与DMSN蛋白无法进行此类探索实验。在这里，我们建议 基于常见的免疫学技术，磁细胞分类（MAC）开发分类方法。我们 将在IMSN或DMSN神经元的树突状刺中表达惰性的细胞外“标签” 通过高度特定的市售Mac分类试剂。使用A将实现细胞类型特异性 病毒双注射策略，其中包含倒置标记构建体的dio/flex矢量将被注入 进入纹状体，将逆行CRE病毒注射到腹侧对段区域（VTA）或 分别标记直接或间接途径的神经元的腹侧颗粒（VP）。排序将是 使用金属偶联抗体和磁化柱完成，可以快速隔离大型 生物材料的量。在AIM 1中，我们在大鼠中开发和验证标签系统。在AIM 2中，我们标记IMSNS 和大鼠的DMSN，并使用延长的间歇性访问HERION自我管理范式生产 表达低水平和高级成瘾者行为的大鼠组（以及对控制的食物奖励）。我们 然后，将从表达高和低成瘾者行为的大鼠中隔离IMSN和DMSN，并使用 定量质谱方法来鉴定在IMSN或 大鼠显示高空的DMSN。低水平的成瘾者行为。后续工作超出了 格兰特将探索调节这些蛋白质表达的潜力 行为，目的是通过专门针对IMSN或DMSN的细胞来减少寻求药物的行为 路径。更一般地，我们的标签和分类方法可以广泛适用于细胞类型的研究 大脑中的特定蛋白质表达。