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

项目总结/摘要 杜氏肌营养不良症（DMD）是一种退行性肌肉萎缩性疾病， dystrophin基因肌营养不良蛋白在肌肉中的缺失导致次级信号失调 其中许多途径仍然知之甚少。玛雅纳·扎茨博士的实验室已经确定了两个 来自一窝金毛猎犬肌营养不良症（GRMD）犬的肌营养不良蛋白缺陷犬， 影响和逃避早期致死和营养不良表型（肌肉萎缩），通常发生在 严重影响GRMD犬。我们对这些狗和其他GRMD和对照狗的微阵列分析， 揭示了Pitpna基因的强烈减少，Pitpna基因是已知的PTEN/AKT信号转导的调节因子。 通路在使用microRNA微阵列对来自患者的人类活检进行的平行研究中， 我们发现了一种microRNA，miR-486，它只在DMD肌肉中减少，并已被证明是DMD的。 我们和其他人改变PTEN/AKT信号。我们假设miR-486的过表达和/或 Pitpna的抑制导致PTEN水平降低，同时随后增加磷酸化（活化） AKT，这可能导致疾病进程的严重程度降低。磷酸化AKT的诱导（产生 来自miR-486和Pitpna调节），预计会影响该途径的几个下游靶基因 这将导致肌肉肥大，肌肉结构基因血管化基因， 增加神经肌肉接头分支点的数量，抑制凋亡基因，并验证 PTEN/AKT下游靶向肌肉基因（如Utrophin）。我们计划实现我们的长期目标， 了解这两种蛋白如何通过调节miR-486的表达来改变DMD的信号通路，和/或 根据以下三个具体目的在小鼠（mdx 5cv）模型中使用Pitpna： 486和Pitpna在体外对PTEN/AKT信号通路的调节。2)操作miR-486表达以 调节肌营养不良蛋白缺陷肌肉中的PTEN/AKT信号传导，用于未来治疗的建模。第三章 肌肉中Pitpna水平降低的小鼠的产生及其对PTEN/AKT的作用的表征 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.