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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要大脑是迄今为止最复杂的器官，有数百甚至数千种不同的细胞类型混合在一起乍一看，在组织中似乎相当均匀，这给生化带来了问题。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其中纯化例如，在纹状体中，直接通路和间接通路的中型棘神经元。（dMSN 与 iMSN）表达不同的分子标记，细胞类型特异性激活或沉默产生在吸毒成瘾的情况下，沉默 iMSN 会增强自我动机。管理药物，同时沉默 dMSN 可以减少寻求药物的行为。纠正滥用药物引起的广泛分子改变是有前途的治疗策略。然而，考虑到 iMSN 与 dMSN 的相反行为输出，一种可以减少药物寻求的药物通过一种途径的行为可以通过另一种途径增强相同的行为细胞类型的识别。特定的目标会增加调节特定纹状体通路和行为的可能性，但我们的无法纯化 iMSN 与 dMSN 蛋白排除了此类探索性实验。开发一种基于常见免疫学技术磁细胞分选 (MACS) 的分选方法。将在 iMSN 或 dMSN 神经元的树突棘中表达一种可识别的惰性细胞外“TAG” 通过高度特异性的市售 MACS 分选试剂，可以使用细胞类型特异性来实现。病毒双重注射策略，其中将注射含有反向 TAG 构建体的 DIO/FLEX 载体进入纹状体，逆行 Cre 病毒将被注射到腹侧被盖区 (VTA) 或腹侧苍白球（VP）将分别标记直接或间接通路的神经元。使用金属偶联抗体和磁化柱完成，可以快速分离大分子在目标 1 中，我们在大鼠中开发并验证了 TAG 系统。在目标 2 中，我们标记了 iMSN。和 dMSNs 在大鼠中，并使用延长的间歇性访问 Herion 自我管理范例来产生表现出低水平和高水平的成瘾行为（以及控制食物奖励）的老鼠群体。然后将从表达高和低成瘾行为的大鼠中分离出 iMSN 和 dMSN，并使用定量质谱方法来鉴定 iMSN 中差异表达的蛋白质或大鼠中的 dMSNs 显示出高水平与低水平的成瘾样行为，后续工作超出了研究范围。赠款将探索在表达成瘾样物质的大鼠中调节这些蛋白质表达的潜力行为，目标是通过专门针对 iMSN 或 dMSN 的细胞来减少寻药行为更一般地说，我们的标记和分选方法可以广泛适用于细胞类型的研究。大脑中特定的蛋白质表达。