The role of microRNAs in Schwann cell development and disease

描述（由申请人提供）：因此，许旺细胞分化是一个多阶段的过程，从一个阶段到下一个阶段的进展是由潜在遗传程序的动态改变驱动的。我们推测，microRNA，在其他系统中的重要发育调节因子，将是至关重要的SC发育过渡。为了评估微小RNA在雪旺细胞（SC）发育中的功能，我们有条件地去除了微小RNA加工酶，dicer。缺乏dicer的SC在分化过程中停滞，不产生髓鞘，形成先天性髓鞘形成不足的表型模型。在特定目标1中，我们将对这些缺乏dicer的SC进行详细的超微结构和分子表征。初步分析表明，这些SC在前髓鞘形成-髓鞘形成过渡期被阻止，维持Sox 2的表达，并且缺乏SC主调节基因Egr 2（Krox 20）。为了证明Egr 2的缺失是髓鞘形成障碍的根本原因，我们将确定Egr 2的强制表达是否可以规避SC缺乏dicer的表型。最后，我们将生成dicer的嵌合缺失，以验证在dicer cKO中观察到的变化是细胞自主的。在Aim Specific 2中，我们将在发育和病理情况下进行microRNA分析。我们将确定动态表达的关键microRNA，然后通过生物信息学确定其候选靶点。然后，我们将使用异源细胞中的荧光素酶报告基因测定、argonaute免疫共沉淀和SC神经元共培养系统中的过表达来验证microRNA与靶点的相互作用。最后，我们将确定一些关键的microRNA本身是否被Egr 2激活，验证Egr 2在microRNA的帮助下抑制SC中的先行基因程序的假设。总之，这些研究对于理解microRNA在发育中的作用非常重要。此外，由于SC分化的基础过程被认为是SC去分化的“镜像”，这些研究不仅揭示了发育转变，而且还揭示了外周髓鞘疾病和神经损伤中发生的匡威转变。 公共卫生相关性：该基金专注于microRNA在许旺细胞谱系中的作用。提出的实验将影响我们对发育过程中的雪旺细胞分化以及神经损伤和脱髓鞘疾病中的去分化的理解。

DESCRIPTION (provided by applicant): Schwann cell differentiation is thus a multistage process, the progression from one stage to the next being driven by dynamic alterations in underlying genetic programs. We hypothesize that microRNAs, being important developmental regulators in other systems, will be critical for SC developmental transitions. To evaluate the function of microRNAs in Schwann cell (SC) development we have conditionally ablated the microRNA processing enzyme, dicer. SC lacking dicer are stalled during differentiation and do not produce myelin, phenocopying models of congenital hypomyelination. Here in Specific Aim1, we will perform a detailed ultrastructural and molecular characterization of these SC lacking dicer. Preliminary analyses suggest that these SC are arrested at the promyelinating-myelinating transition, maintain the expression of Sox2, and lack the SC master regulator gene Egr2 (Krox20). To prove that loss of Egr2 is the underlying reason for dysmyelination, we will determine if forced expression of Egr2 can circumvent the phenotype of SC lacking dicer. Finally, we will generate a mosaic deletion of dicer, to verify that the changes observed in the dicer cKO are cell autonomous. In Specific Aim 2, we will perform microRNA profiling in developmental and pathological scenarios. We will identify key microRNAs that are dynamically expressed and then bioinformatically determine their candidate targets. We will then verify microRNA-target interactions using luciferase reporter assays in heterologous cells, argonaute co-immunoprecipitation, and overexpression in SC-neuron coculture systems. Finally we will determine whether some key microRNAs are themselves activated by Egr2, testing the hypothesis that Egr2 represses antecedent gene programs in SC with the help of microRNAs. Together these studies will be important in understanding the role of microRNAs in development. Further, since the processes underlying SC differentiation are considered a "mirror image" of SC dedifferentiation, these studies will not only shed light on developmental transitions but also on converse transitions that occur in peripheral myelin disorders and nerve injury. PUBLIC HEALTH RELEVANCE: This grant focuses on the role of microRNAs in the Schwann cell lineage. The experiments proposed will impact our understanding of both Schwann cell differentiation during development, as well as dedifferentiation in nerve injury and demyelinating diseases.