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.

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We have identified the Pitpna gene (a regulator of PTEN/AKT signaling) as strongly decreased in expression in the muscle of a dog model of Duchenne Muscular Dystrophy (DMD) which is mildly affected and it's reduction in expression in dystrophin deficient sapje zebrafish increases their survival. In parallel studies, we identified a microRNA, miR-486, as being uniquely reduced in DMD human muscle biopsies. We have shown that miR- 486 is also a regulator of PTEN/AKT signaling through its direct inhibition of PTEN mRNA expression. Our studies aim to mechanistically characterize the functions of miR-486 and Pitpna as modulators of PTEN/AKT signaling (dysregulated in DMD) through a combinatorial approach utilizing our normal and DMD banked muscle cell lines along with transgenic mouse models that will test the hypothesis that the increased miR-486 expression and/or inhibition of Pitpna levels can ameliorate dystrophic symptoms, thus creating a novel therapeutic approach to help patients with this devastating disease.

National Institute of Health (NIH)
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
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Therapeutic Approaches to Genetic Diseases (TAG)
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Nuckolls, Glen H
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Children's Hospital Boston
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