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

项目摘要 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 在一个组织中，乍一看，似乎是相当均匀的。这给生物化学带来了问题， 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 周围组织。例如，在纹状体中，直接与间接通路的中型多刺神经元 （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不同的分子标记。 相反的行为输出。在药物成瘾的背景下，沉默iMSN增强了自我- 管理药物，而沉默dMSN减少了药物寻求行为。治疗方法可以 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相反的行为输出，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。细胞类型鉴定 特定的靶点会增加调节特定纹状体通路和行为的可能性，但我们的研究表明， 不能纯化iMSN与dMSN蛋白质排除了这种探索性实验。在此，我们建议 开发基于常见免疫学技术的分选方法，即磁性细胞分选（MACS）。我们 将在iMSN或dMSN神经元的树突棘中表达一种惰性的细胞外"TAG"， 通过高度特异性的市售MACS分选试剂。细胞类型特异性将使用 病毒双注射策略，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病毒将被注射到大脑皮层区（VTA）或纹状体。 神经元特异性荧光素（VP）分别标记直接或间接通路的神经元。排序将是 使用金属结合抗体和磁化柱完成，允许快速分离大的 量的生物材料。在目标1中，我们在大鼠中开发和验证TAG系统。在目标2中，我们将iMSN标记为 和dMSNs在大鼠中，并使用延长的间歇性访问herion自我管理范式，以产生 表达低水平和高水平成瘾样行为（以及食物奖励对照）的大鼠组。我们 然后将从表达高和低成瘾样行为的大鼠中分离iMSN和dMSN，并使用 定量质谱法鉴定iMSN中差异表达的蛋白质，或 大鼠中的dMSN显示出高水平与低水平的成瘾样行为。范围以外的后续工作 格兰特将探索在表达成瘾样蛋白的大鼠中调节这些蛋白表达的潜力。 行为，目的是通过特异性靶向iMSN或dMSN的细胞来减少药物寻求行为 通路更一般地说，我们的标记和分选方法可以广泛应用于细胞类型的研究， 大脑中的特定蛋白质表达。

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.