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

描述(申请人提供)：Duchenne肌营养不良症(DMD)是一种由肌营养不良蛋白基因突变引起的退行性肌肉萎缩疾病。肌营养不良蛋白在肌肉中的缺失导致二级信号通路的失调，其中许多信号通路仍然知之甚少。Mayana Zatz博士的实验室已经从一窝金毛猎犬肌肉营养不良症(GRMD)犬中发现了两只dystrophin缺陷型狗，它们受到的影响较轻，没有出现严重影响的GRMD犬通常会出现的早期死亡和营养不良表型(肌肉萎缩)。我们对这些狗以及其他GRMD和对照组狗进行的微阵列分析显示，作为PTEN/AKT信号通路调节因子的Pitpna基因显著减少。在对DMD患者的活检组织进行的使用microRNA微阵列进行的平行研究中，我们发现了一种microRNA miR-486，它只在DMD肌肉中减少，我们和其他人已经证明它可以改变PTEN/AKT信号。我们推测，miR-486的过表达和/或抑制Pitpna导致PTEN水平降低，而随后磷酸化(激活)的AKT增加，这可能导致疾病严重程度的降低。据预测，磷酸化的AKT(由miR-486和Pitpna调制引起)的诱导，将影响该途径的几个下游靶基因，从而导致肌肉肥大、肌肉结构基因和血管化基因的增加，神经肌肉分支点连接数量的增加，抑制凋亡基因，并验证PTEN/AKT下游靶肌肉基因(如Utroin)。我们计划通过调节mdx5Cv小鼠(Mdx5Cv)模型中miR-486和/或Pitpna的表达来达到了解这两个信号通路在DMD中如何改变的长期目标：1)体外研究miR-486的特性和Pitpna对PTEN/AKT信号通路的调节。2)操纵miR-486的表达来调节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)肌肉中垂体素水平降低的小鼠的建立及其对PTEN/AKT信号转导的影响。

DESCRIPTION (provided by applicant): 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.