TRANSCRIPTIONAL DETERMINANTS OF THE FATE TRAJECTORIES OF SINGLE HUMAN GLIAL PROGENITOR CELLS IN RESPONSE TO DEMYELINATION IN VIVO

单个人胶质祖细胞响应体内脱髓鞘的命运轨迹的转录决定因素

• 批准号: 10251846
• 负责人: STEVEN Alan GOLDMAN
• 金额: $ 36.67万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10251846
• 关键词: Adult; Age; Aging; Astrocytes; Behavior; Biological; Biology; Brain; CSPG4 gene; Cell Aging; Cell Cycle; Cell Transplantation; Cells; Chimera organism; Competence; Cuprizone; Data; Demyelinating Diseases; Demyelinations; Diffuse; Epigenetic Process; Event; Experimental Models; Exposure to; Failure; Fibrous Astrocyte; Fluorescence-Activated Cell Sorting; Gene Expression Profile; Genes; Genetic Transcription; Goals; Heterogeneity; Human; Injury; Interruption; Lead; Length; Literature; Mediating; Messenger RNA; Methylation; Mitotic; Modeling; Multiple Sclerosis; Mus; Myelin; Neonatal; Neuroglia; Oligodendroglia; Pathway interactions; Pattern; Periodicity; Phenotype; Platelet-Derived Growth Factor alpha Receptor; Population; Process; RNA; RNA analysis; Recovery; Recurrence; Regulation; Rodent; Shivering; Terminology; Testing; Therapeutic; Time; Work; base; differential expression; exhaustion; experimental study; falls; human subject; in vivo; indexing; miRNA expression profiling; mutant; neonatal mice; novel; oligodendrocyte progenitor; progenitor; remyelination; response; self-renewal; senescence; single-cell RNA sequencing; stem cells; therapeutic target; transcriptome sequencing; white matter

Abstract In this application, we will test a longstanding but effectively untested hypothesis in myelin biology, that white matter recovery after sustained or recurrent demyelination might be constrained by the finite mitotic competence of the human glial progenitor cell pool. Such a mobilization-dependent depletion of mitotically- competent progenitors might lead to mitotic senescence, and hence to the eventual failure of remyelination noted in progressive multiple sclerosis. In addition, any such depletion of competent progenitor cells might also be expected to limit the utility of differentiation-based approaches towards induced remyelination. We thus propose to assess the responses of human glial progenitor cells (hGPCs) to demyelination in vivo, by defining the single cell RNA expression patterns of hGPCs, both at baseline and in response to sustained cuprizone demyelination in vivo. To this end, we will use mice neonatally chimerized with human GPCs, a novel model we have developed in which mouse oligodendrocytes and astrocytes are largely replaced by their human counterparts in vivo. Using these human glial chimeras, we will ask the following questions: 1) What is the phenotypic and transcriptional heterogeneity among single human glial progenitor cells in vivo, in the otherwise undisturbed adult glial chimeric brain? Are all GPCs multi-lineage competent? Are some more restricted than others to astrocytic or oligodendrocytic fate? Are some in cell cycle while others are more quiescent? How do these phenotypic distributions change with age? 2) How heterogeneous are the transcriptional responses of resident human GPCs to demyelination in vivo? In response to cuprizone-mediated demyelination, what differentially regulated pathways distinguish quiescent, initially mobilized, and actively remyelinating hGPCs? These experiments will combine the use of human glial chimeras engrafted with genetically tagged GFP+ hGPCs, with later single cell RNA-seq of both the post-demyelination white matter, and of pooled hGPC isolates after post-demyelination FACS, to define the transcriptional events associated with human GPC mobilization and remyelination in vivo. 3) Does the efficiency of remyelination by hGPCs fall with sustained demyelination? Are human GPCs capable of self-renewal during sustained demyelination, or is remyelination delimited by their mitotic senescence? These experiments will assess both methylation state and telomeric length of GPCs in vivo, both before and after sustained cuprizone exposure, so as to define the effects of sustained demyelination on these hallmarks of cellular aging. In addition, we will assess the transcriptional concomitants to methylation state-defined aging, by RNA-seq of the same cells as a function of time after demyelination. By this means, we intend to define both the transcriptional hallmarks of mitotic exhaustion by hGPCs, and the epigenetic correlates to that process, and by doing so to identify therapeutic targets by which to delay or control the demyelination-associated depletion of mitotically-competent hGPCs.

摘要 在这个应用程序中，我们将测试髓鞘生物学中一个长期存在但实际上未经检验的假说，即 持续性或复发性脱髓鞘后白质的恢复可能受到有丝分裂的限制。 人神经胶质前体细胞库的能力。这种依赖动员的有丝分裂的枯竭- 有能力的祖细胞可能导致有丝分裂衰老，从而最终导致重新髓鞘形成失败。 进行性多发性硬化症。此外，任何这种有能力的祖细胞的枯竭也可能是 预计将限制以分化为基础的方法诱导重新髓鞘形成的效用。因此，我们建议 目的：评价人神经胶质前体细胞(HGPC)对体内脱髓鞘的反应。 HGPC的细胞RNA表达模式，无论是在基础状态下还是在持续的铜比林脱髓鞘反应中 在活体内。为此，我们将使用新生小鼠与人类GPC嵌合，这是我们开发的一种新模型 在体内，小鼠的少突胶质细胞和星形胶质细胞在很大程度上被它们的人类同行所取代。vbl.使用 这些人类神经胶质嵌合体，我们将问以下问题：1)什么是表型和转录 体内单个人神经胶质前体细胞的异质性，在未受干扰的成人神经胶质嵌合体中 大脑？所有普通科医生都有多血统的能力吗？有些比其他的更局限于星形细胞或 少突胶质细胞的命运？有些处于细胞周期，而另一些则处于静止状态？这些表型是如何 分布随年龄变化？2)常驻人类GPC转录反应的异质性有多大 在活体内脱髓鞘？在铜酮介导的脱髓鞘反应中，哪些差异调节 途径区分静止的、最初动员的和活跃的再髓鞘hGPC？这些实验将 将植入有基因标记的GFP+hGPC的人神经胶质嵌合体与后来的单细胞相结合 脱髓鞘后白质和脱髓鞘后混合hGPC分离株的rna-seq FACS，以确定与体内人GPC动员和重新髓鞘形成相关的转录事件。 3)hGPC的再髓鞘形成效率是否随脱髓鞘时间的延长而下降？人类GPC有能力吗？ 在持续脱髓鞘过程中的自我更新，还是以有丝分裂衰老为界的重新髓鞘形成？这些 实验将评估体内GPC的甲基化状态和端粒长度，包括前后 持续暴露铜试剂，以确定持续脱髓鞘对这些特征的影响 细胞老化。此外，我们将通过以下方式评估甲基化状态定义的衰老的转录伴随 脱髓鞘后相同细胞的RNA-Seq随时间变化。通过这种方式，我们打算定义 HGPC有丝分裂衰竭的转录特征，以及与这一过程相关的表观遗传学，以及通过 这样做是为了确定治疗靶点，通过这些靶点来延缓或控制脱髓鞘相关的耗竭 有丝分裂能力的hGPC。