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: 9904385
• 负责人: STEVEN Alan GOLDMAN
• 金额: $ 36.21万
• 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/9904385
• 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; 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，我们将测试一个长期存在但未经有效验证的假设，在髓鞘生物学， 持续性或复发性脱髓鞘后白色物质的恢复可能受到有限的有丝分裂抑制， 人神经胶质祖细胞库的能力。这种依赖于细胞动员的有丝分裂- 有能力的祖细胞可能会导致有丝分裂衰老，从而导致髓鞘再生的最终失败 进行性多发性硬化症此外，任何此类有能力的祖细胞的耗竭也可能是不可避免的。 预期将限制基于分化的方法对诱导髓鞘再生的效用。因此，我们建议 为了评估人神经胶质祖细胞（hGPCs）对体内脱髓鞘的反应，通过定义单一的 hGPCs的细胞RNA表达模式，包括基线和持续铜腙脱髓鞘反应 in vivo.为此，我们将使用与人类GPCs嵌合的小鼠，这是我们开发的一种新模型 其中小鼠少突胶质细胞和星形胶质细胞在体内大部分被它们的人类对应物取代。使用 对于这些人类神经胶质嵌合体，我们将提出以下问题：1）什么是表型和转录 体内单个人胶质祖细胞之间的异质性，在其他未受干扰的成人胶质嵌合体中， 大脑？是否所有GPC都有多谱系能力？有些细胞比其他细胞更局限于星形胶质细胞或 少突胶质细胞的命运一些细胞处于细胞周期中，而另一些则处于静止状态？这些表型如何 分布随年龄变化？2)常驻人类GPCs的转录反应有多异质 体内脱髓鞘在对铜腙介导的脱髓鞘反应中， 途径区分静止，最初动员，并积极髓鞘再生hGPCs？这些实验将 联合收割机使用植入有遗传标记的GFP + hGPCs的人神经胶质嵌合体， 脱髓鞘后白色物质和脱髓鞘后合并的hGPC分离物的RNA-seq 流式细胞仪，以确定与人GPC动员和髓鞘再生在体内相关的转录事件。 3)hGPCs的髓鞘再生效率是否随持续脱髓鞘而下降？人类的GPC 在持续的脱髓鞘过程中自我更新，或者髓鞘再生是由有丝分裂衰老限定的？这些 实验将评估体内GPC的甲基化状态和端粒长度，无论是之前还是之后， 持续的cuprizone暴露，以确定持续脱髓鞘对这些标志的影响， 细胞老化此外，我们将评估甲基化状态定义的衰老的转录伴随物， 相同细胞的RNA-seq作为脱髓鞘后时间的函数。通过这种方式，我们打算定义两个 hGPCs有丝分裂耗竭的转录标志，以及与该过程相关的表观遗传学， 这样做是为了鉴定治疗靶点，通过该靶点来延迟或控制脱髓鞘相关的 有丝分裂能力的hGPCs。