The role of microRNAs in Schwann cell development and disease

描述(申请人提供)：因此，雪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变化驱动的。我们假设，microRNAs作为其他系统中重要的发育调节因子，将对SC的发育转变至关重要。为了评估microRNAs在雪旺细胞(SC)发育中的功能，我们有条件地去除了microRNA加工酶DICER。缺乏DICER的干细胞在分化过程中停滞不前，不会产生髓鞘，先天性髓鞘减少的表型。在特定的Aim1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被阻止在早幼粒-髓鞘形成过程中，维持Sox2的表达，并且缺乏SC主要调节基因Egr2(Krox20)。为了证明Egr2的缺失是髓鞘功能障碍的根本原因，我们将确定强制表达Egr2是否可以绕过缺乏DICER的SC的表型。最后，我们将生成DICER的马赛克删除，以验证在DICER CKO中观察到的变化是细胞自主的。我们将识别动态表达的关键microRNAs，然后通过生物信息确定它们的候选靶标。然后，我们将使用异源细胞中的荧光素酶报告分析、ArgAerte免疫共沉淀和SC-神经元共培养系统中的过度表达来验证microRNA与靶标的相互作用。最后，我们将确定一些关键的microRNAs本身是否被Egr2激活，验证Egr2在microRNAs的帮助下抑制SC中先前基因程序的假设。此外，由于SC分化的基础过程被认为是SC去分化的“镜像”，这些研究不仅将阐明发育转变，而且还将阐明发生在外周髓鞘疾病和神经损伤中的反向转变。 公共卫生相关性：这笔赠款侧重于MicroRNAs在雪旺细胞谱系中的作用。拟议的实验将影响我们对雪旺细胞在发育过程中的分化，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.