The role of microRNAs in Schwann cell development and disease

描述（由申请人提供）：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。我们假设在其他系统中作为重要的发育调节因子的microrna对SC的发育转变至关重要。为了评估microRNA在雪旺细胞（SC）发育中的功能，我们有条件地消融了microRNA加工酶dicer。缺乏dicer的SC在分化过程中停滞不前，不产生髓磷脂，这是先天性髓鞘化低的表型模型。在Specific Aim1中，我们将对这些SC缺乏dicer进行详细的超微结构和分子表征。初步分析表明，这些SC在早髓鞘-髓鞘转变过程中受阻，维持Sox2的表达，缺乏SC主调控基因Egr2 （Krox20）。为了证明Egr2的缺失是髓鞘发育异常的潜在原因，我们将确定Egr2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的马赛克缺失，以验证在dicer cKO中观察到的变化是细胞自主的。在Specific Aim 2中，我们将在发育和病理情况下进行microRNA分析。我们将识别动态表达的关键microrna，然后从生物信息学上确定它们的候选靶标。然后，我们将在异种细胞、argonaute共免疫沉淀和sc -神经元共培养系统中的过表达中使用荧光素酶报告基因检测来验证microrna与靶标的相互作用。最后，我们将确定一些关键的microrna本身是否被Egr2激活，从而验证Egr2在microrna的帮助下抑制SC中先前基因程序的假设。总之，这些研究对于理解microrna在发育中的作用将是重要的。此外，由于SC分化背后的过程被认为是SC去分化的“镜像”，这些研究不仅将阐明发育转变，还将阐明发生在外周髓鞘疾病和神经损伤中的反向转变。

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.