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MicroRNA-29 overexpression by adeno-associated virus suppresses fibrosis and restores muscle function in combination with micro-dystrophin.

ABSTRACT: Duchenne muscular dystrophy (DMD) is caused by dystrophin deficiency resulting in progressive muscle weakness and fibrotic scarring. Muscle fibrosis impairs blood flow, hampering muscle repair and regeneration. Irrespective of the success of gene restoration, functional improvement is limited without reducing fibrosis. The levels of miR-29c, a known regulator of collagen, are reduced in DMD. Our goal is to develop translational, antifibrotic therapy by overexpressing miR-29c. We injected the gastrocnemius muscle with either self-complementary AAV.CMV.miR-29c or single-stranded AAV.MCK.micro-dystrophin alone or in combination in the mdx/utrn+/- mouse, a DMD mouse model. Treatment of 3-month-old mdx/utrn+/- mice with AAV.miR-29c showed a reduction in collagen and increased absolute and specific force compared with untreated animals, but neither parameter reached WT levels. Combinatorial gene delivery in 3-month-old mdx/utrn+/- mice further decreased fibrosis, and showed a reduction of transcript levels for Col1A, Col3A, fibronectin, and Tgfb1. In addition, absolute and specific force was normalized and equivalent to WT. However, protection against eccentric contraction fell short of WT levels at this time point. When this same mouse model was treated with miR-29c/micro-dystrophin combinatorial therapy at 1 month of age, there was complete normalization of specific and absolute force and protection against eccentric contraction-induced injury was comparable to WT. These findings highlight the potential for miR-29c as an important addition to the armamentarium for translational gene therapy, especially when used in combination with micro-dystrophin in DMD.

SUBMITTER: Heller KN

PROVIDER: S-EPMC5414552 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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MicroRNA-29 overexpression by adeno-associated virus suppresses fibrosis and restores muscle function in combination with micro-dystrophin.

Heller Kristin N KN Mendell Joshua T JT Mendell Jerry R JR Rodino-Klapac Louise R LR

JCI insight 20170504 9

Duchenne muscular dystrophy (DMD) is caused by dystrophin deficiency resulting in progressive muscle weakness and fibrotic scarring. Muscle fibrosis impairs blood flow, hampering muscle repair and regeneration. Irrespective of the success of gene restoration, functional improvement is limited without reducing fibrosis. The levels of miR-29c, a known regulator of collagen, are reduced in DMD. Our goal is to develop translational, antifibrotic therapy by overexpressing miR-29c. We injected the gas ...[more]

PMID: 28469083

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Project description:ObjectiveHypothalamic arcuate proopiomelanocortin (Arc-POMC) neurons are involved in different physiological processes such as the regulation of energy balance, glucose homeostasis, and stress-induced analgesia. Since these neurons heterogeneously express different biological markers and project to many hypothalamic and extrahypothalamic areas, it is proposed that Arc-POMC neurons could be classified into different subpopulations having diverse physiological roles. The aim of the present study was to characterize the contribution of the subpopulation of Arc-POMC neurons cosecreting gamma-aminobutyric acid (GABA) neurotransmitter in the control of energy balance.MethodsArc-Pomc expression restricted to GABAergic-POMC neurons was achieved by crossing a reversible Pomc-deficient mouse line (arcPomc-) with a tamoxifen-inducible Gad2-CreER transgenic line. Pomc expression was rescued in the compound arcPomc-/-:Gad2-CreER female and male mice by tamoxifen treatment at postnatal days 25 (P25) or 60 (P60), and body weight, daily food intake, fasting glycemia, and fasting-induced hyperphagia were measured. POMC recovery was quantified by immunohistochemistry and semiquantitative RT-PCR. Neuropeptide Y (NPY) and GABAergic neurons were identified by in situ hybridization. Arc-POMC neurons projecting to the dorsomedial hypothalamic nucleus (DMH) were studied by stereotactic intracerebral injection of fluorescent retrobeads into the DMH.ResultsTamoxifen treatment of arcPomc-/-:Gad2-CreER mice at P60 resulted in Pomc expression in ∼23-25% of Arc-POMC neurons and ∼15-23% of Pomc mRNA levels, compared to Gad2-CreER control mice. Pomc rescue in GABAergic-POMC neurons at P60 normalized food intake, glycemia, and fasting-induced hyperphagia, while significantly reducing body weight. Energy balance was also improved in arcPomc-/-:Gad2-CreER mice treated with tamoxifen at P25. Distribution analysis of rescued POMC immunoreactive fibers revealed that the DMH is a major target site of GABAergic-POMC neurons. Further, the expression of the orexigenic neuropeptide Y (NPY) in the DMH was increased in arcPomc-/- obese mice but was completely restored after Pomc rescue in arcPomc-/-:Gad2-CreER mice. Finally, we found that ∼75% of Arc-POMC neurons projecting to the DMH are GABAergic.ConclusionsIn the present study, we show that the expression of Pomc in the subpopulation of Arc-GABAergic-POMC neurons is sufficient to maintain normal food intake. In addition, we found that DMH-NPY expression is negatively correlated with Pomc expression in GABAergic-POMC neurons, suggesting that food intake may be regulated by an Arc-GABAergic-POMC → DMH-NPY pathway.

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MicroRNA-29 overexpression by adeno-associated virus suppresses fibrosis and restores muscle function in combination with micro-dystrophin.

Publications

MicroRNA-29 overexpression by adeno-associated virus suppresses fibrosis and restores muscle function in combination with micro-dystrophin.

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