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

描述（由申请人提供）：杜氏肌营养不良症（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。肌肉中肌营养不良蛋白的缺乏导致次级信号通路失调，其中许多仍然知之甚少。Mayana Zatz博士的实验室从一窝肌肉萎缩症（GRMD）金毛猎犬中发现了两只营养不良蛋白缺乏的狗，它们受到轻度影响，避免了早期致命性和营养不良表型（肌肉萎缩），而严重影响GRMD的狗通常会出现这种情况。我们对这些狗以及其他GRMD和对照狗的微阵列分析显示，Pitpna基因（PTEN/AKT信号通路的已知调节因子）明显减少。在使用microRNA微阵列对DMD患者的人体活检进行的平行研究中，我们发现了一种microRNA miR-486，它在DMD肌肉中完全减少，并且已经被我们和其他人证明可以改变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，同时随后磷酸化（活化）AKT增加，这可能导致病程严重程度降低。预计磷酸化AKT的诱导（由miR-486和Pitpna调节引起）会影响该通路的几个下游靶基因，导致肌肉肥大、肌肉结构基因、血管化基因、神经肌肉连接分支点数量的增加、凋亡基因的抑制，并验证PTEN/AKT下游靶肌肉基因（如Utrophin）。我们计划根据以下三个具体目标来实现我们的长期目标，即通过在小鼠（mdx5cv）模型中调节miR-486和/或Pitpna的表达来了解这两者如何改变DMD的信号通路：1)miR-486的表征和Pitpna对PTEN/AKT信号通路的调控。2)通过miR-486表达调控肌营养不良蛋白缺陷肌中PTEN/AKT信号传导，为未来的治疗方法建模。3)肌肉中Pitpna水平降低小鼠的产生及其对PTEN/AKT信号传导的影响。

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.