Muscular dystrophies are a heterogeneous group of myopathic disorders characterized by the progressive degeneration of skeletal and cardiac muscle. One type of dystrophy that leads to muscle weakness and a loss of muscle strength is called Limb-Girdle Muscular Dystrophy type 2H (LGMD2H). LGMD2H is caused by a mutation in the E3 ubiquitin ligase protein TRIM32. Diverse TRIM32 substrates have been identified in cell cycle regulation, neuronal differentiation, muscle physiology, and tumorigenesis. The generation of a Trim32-/- knockout mouse was a key advance in studying LGMD2H muscle degeneration. However, the ubiquitous expression of TRIM32 and pleiotropic phenotypes present in these mutant mice did not clarify the role of TRIM32 in LGMD2H pathogenesis. For these reasons, it is clear that alternative models are needed to fully understand the muscle-intrinsic role of TRIM32. We were the first group to publish a Drosophila model for LGMD2H. Mutations in thin (tn), which encodes for Drosophila TRIM32, exhibit a degenerative muscle phenotype and are defective in locomotor ability. These features recapitulate the histological and mobility defects present in LGMD2H patients. TRIM32 is characterized by an N-terminal RING domain and C-terminal NHL (Ncl-1, HT2A, Lin-41) repeats. Pathogenic alleles that cause LGMD2H are located within the NHL region, which is predicted to mediate protein interactions. It has already been reported that the NHL domain prevents muscle degeneration in our Drosophila LGMD2H model. Herein we determined the structure of the Drosophila NHL region and compared this to the mammalian NHL domain. The superimposition of these two structures demonstrate that the NHL region of Drosophila TRIM32 is a faithful model to understand NHL function. Using a proteomics approach to identify proteins that physically interact with the NHL domain, we find that TRIM32-NHL binds to glycolytic proteins. Moreover, loss of Drosophila TRIM32 alters the subcellular localization of these enzymes. The overall objective of the proposed research is to use our Drosophila LGMD2H model to determine how and why TRIM32 is required for the metabolic maintenance of glycolytic muscles. We will pursue this goal by completing two specific aims. First, we will biochemically and genetically characterize the TRIM32-glycolytic complex in muscle tissue.
The second aim will assess the in vitro and in vivo consequences of muscle-specific expression of pathogenic TRIM32 mutations on glycolytic protein levels, sarcomeric localization, and metabolism. Our simpler Drosophila model that is devoid of satellite cells or adaptive immunity eliminates complications associated with muscle regeneration and the infiltration of immune cells that drive disease progression in mammalian models. Collectively, completion of these aims will be a major step forward in understanding how metabolism is regulated to maintain healthy muscle tissue.

Public Health Relevance

There are currently no cures for muscular dystrophies, characterized by progressive muscle weakness, loss of mobility, and possibly early morbidity. We have developed a Drosophila muscular dystrophy model to characterize the cellular and molecular changes that contribute to the initiation and progression of muscle degeneration. Our overall goal is to understand how metabolic alterations influence muscle homeostasis to eventually develop successful therapeutic strategies that prevent muscle degeneration.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21AR073373-01A1
Application #
9669324
Study Section
Skeletal Muscle and Exercise Physiology Study Section (SMEP)
Program Officer
Cheever, Thomas
Project Start
2018-09-13
Project End
2020-07-31
Budget Start
2018-09-13
Budget End
2019-07-31
Support Year
1
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Kansas State University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
929773554
City
Manhattan
State
KS
Country
United States
Zip Code
66506