MANIPULATION OF PTEN/AKT SIGNALING IN DUCHENNE MUSCULAR DYSTROPHY AS A MEANS OF T

项目摘要/摘要 Duchenne肌营养不良症(DMD)是一种退行性肌肉萎缩疾病，由基因突变引起 抗肌营养不良蛋白基因。肌营养不良蛋白在肌肉中的缺失导致二级信号转导失调 路径，其中许多仍然鲜为人知。Mayana Zatz博士的实验室已经确定了两个 肌营养不良症(GRMD)犬中的营养不良蛋白缺乏症犬 影响并逃脱了通常发生在 严重感染GRMD犬。我们对这些狗和其他GRMD和对照狗的微阵列分析， 发现了作为PTEN/AKT信号调节因子的Pitpna基因的强烈减少 路径。使用微RNA微阵列对来自慢性支气管炎患者的活检组织进行平行研究 对于DMD，我们发现了一种microRNA miR-486，它只在DMD肌肉中还原，并已被证明 美国和其他国家改变PTEN/AKT信号。我们假设miR-486和/或 抑制甲状旁腺激素导致PTEN水平降低，而随后增加磷酸化(激活) AKT，可能导致病程严重程度降低。磷酸化AKT的诱导(结果 从miR-486和Pitpna调制)，预计会影响该途径的几个下游靶基因 这将导致肌肉肥大，肌肉结构基因，血管形成基因， 增加神经肌肉接头的分支点数量，抑制细胞凋亡基因，并验证 PTEN/AKT下游靶肌肉基因(如Utroin)。我们计划实现我们的长期目标 了解这两种基因如何通过调节miR-486和/或的表达来改变DMD的信号通路 Mdx5Cv小鼠(Mdx5Cv)模型根据以下三个特定目的：1)miR- 486和Pitpna对体外PTEN/AKT信号通路的调节作用。2)操纵miR-486表达式以 调制肌营养不良蛋白缺乏症肌肉中的PTEN/AKT信号，为未来的治疗建模。3) 肌肉垂体素水平降低小鼠的建立及其对PTEN/AKT影响的研究 发信号。

PROJECT SUMMARY/ABSTRACT Duchenne Muscular Dystrophy (DMD) is a degenerative muscle wasting disease caused by mutations in the dystrophin gene. The absence of dystrophin protein in muscle results in dysregulated secondary signaling pathways, many of which remain poorly understood. The laboratory of Dr. Mayana Zatz has identified two dystrophin-deficient dogs from a litter of Golden Retriever Muscular Dystrophy (GRMD) dogs that are mildly affected and escape the early lethality and dystrophic phenotype (muscle wasting) that normally occur in severely affected GRMD dogs. Our microarray analysis of these dogs and other GRMD and control dogs, revealed a strong reduction of the Pitpna gene, which is a known regulator of the PTEN/AKT signaling pathway. In parallel studies using microRNA microarrays performed on human biopsies from patients with DMD, we identified a microRNA, miR-486, that is exclusively reduced in DMD muscle and has been shown by us and others to alter PTEN/AKT signaling. We hypothesize that either overexpression of miR-486 and/or inhibition of Pitpna results in decreased PTEN levels while subsequently increasing phosphorylated (activated) AKT, which might lead to reduced severity of disease course. The induction of phosphorylated-AKT (resulting from miR-486 and Pitpna modulation), are predicted to affect several downstream target genes of this pathway which will result in an increase in muscle hypertrophy, muscle structural genes vascularization genes, the increase in the neuromuscular junction number of branch points, inhibition of apoptosis genes, and validated PTEN/AKT downstream target muscle genes (such as Utrophin). We plan to approach our long-term goal of understanding how these two alter signaling pathways in DMD by modulating expression of miR-486 and/or Pitpna in the mouse (mdx5cv) model according to the following three specific aims: 1) Characterization of miR- 486 and Pitpna regulation of PTEN/AKT signaling pathway in vitro. 2) Manipulation of miR-486 expression to modulate PTEN/AKT signaling in dystrophin-deficient muscle for modeling of future therapeutics. 3) Generation of mice with reduced Pitpna levels in muscle and characterization of its effects on PTEN/AKT signaling.