James J. Dowling, M.D., Ph.D. - US grants

DESCRIPTION (provided by applicant): Congenital myopathies are a heterogeneous group of muscle diseases that commonly present as weakness and hypotonia in infancy. Congenital myopathies are individually rare, but overall represent a significant cause of childhood morbidity and mortality. This application is centered on the development of an understanding of muscle formation, muscle maintenance and muscle disease as relates to congenital myopathies. The candidate's goal is to establish a research and clinical career in muscle biology and disease. His immediate goal is develop proficiency in the techniques utilized to study skeletal muscle in zebrafish, and to apply those techniques to the study of a question relevant to the pathogenesis of congenital myopathies. Specifically, he will master genetic and cell biologic manipulations in the developing zebrafish, with a focus on the examination of phosphoinositide signaling and membrane trafficking in skeletal muscle. He will apply these techniques to the study of myotubularin, a gene product hypothesized to be critical for endosomal dynamics and known to be the causative factor in the pathogenesis of myotubular myopathy. He will also study the relationship between myotubularin and the homologous gene MTMR14, which is mutated in another form of centronuclear myopathy. The goal of the research project is to establish the function of myotubularins in muscle development and homeostasis, and to determine the relationship between myotubularin dysfunction and muscle disease. In addition, the candidate will supplement the research project with a variety of non-laboratory experiences. These will include attendance at scientific courses in genetics and in pediatric muscle disease, participation in annual scientific meetings relevant to muscle biology, and advanced instruction in clinical neuromuscular disease. In all, this combination of laboratory and academic training in muscle biology will provide a strong foundation for the candidate's long term goal of establishing expertise in the field of pediatric muscle disease, and of utilizing his research to significantly advance understanding of the pathogenesis and treatment of congenital myopathies. RELEVANCE (See instructions): This application has high relevance to public health. In particular, the research is aimed at advancing the understanding of muscle disease in children. In addition, the training program is designed to provide the candidate with critical knowledge and expertise that will be applied to future studies aimed at understanding and treating childhood muscle disease.

DESCRIPTION (provided by applicant): Centronuclear myopathies are a group of childhood onset muscle diseases defined by shared muscle biopsy features and characterized by muscle weakness and severe motor disability. Currently there are 5 known genetic causes for CNM, and recent studies on these gene products have identified abnormal excitation- contraction coupling as a key aspect of disease pathogenesis. Despite these advancements, no treatments current exist for CNMs and much remains to be understood about these clinically severe conditions. Approximately 40% of cases of CNM are genetic unresolved. Determination of additional genetic causes is critical to advance the knowledge of and to develop treatments for this disease. We have used linkage analysis and whole exome sequencing to identify a novel gene mutation in the CCDC78 gene in a family with autosomal dominant CNM. CCDC78 encodes a previously uncharacterized gene product, and the gene mutation is predicted to result in production of a protein with an internal deletion. Our hypotheses are that (a) wild type CCDC78 is required for muscle development and in particular for stabilizing the excitation-contraction coupling machinery and that (b) mutant CCDC78 functions in a dominant negative manner to sequester ECC proteins and thereby impair motor function. These hypotheses will be tested in two aims. Aim 1 will examine the function(s) of wild type CCDC78 and Aim 2 will test the impact of the CCDC78 mutation on muscle development and function. Both aims will utilize a combinatorial approach that includes in vitro studies, biochemical and proteomic techniques, and in vivo experimentation in the zebrafish. In particular, the project will take advantage of the power in the zebrafish for manipulation of gene expression, used to create both loss of function and dominant negative models, and live image analysis, used to dynamically examine specific properties of muscle function. In all, this proposal will determine the function of CCDC78 in muscle development as well as the pathogenic mechanisms underlying its mutation in CNM. These data will be placed in the context of the existing knowledge of CNM, allowing for critical advancements in the understanding of muscle function and the pathogenesis of this devastating disease. PUBLIC HEALTH RELEVANCE: Centronuclear myopathies are a common childhood muscle disease, associated with significant morbidity and early mortality, for which no treatments currently exist. While recent studies have advanced the knowledge of this condition, much remains to be understood before therapies can be developed. The goal of this proposal is to provide this advancement in understanding of centronuclear myopathy by determining the function of CCDC78, mutations in which we have newly identified as one cause of this disorder.

PROJECT SUMMARY Myotubular myopathy is a fatal neuromuscular disease associated with severe morbidities (including wheelchair and ventilator dependence) and early mortality. There are critical barriers hindering treatment for this devastating disease. These include the need to uncover new therapeutic strategies, particularly ones with applicability for a broader spectrum of congenital myopathies, and to develop non-invasive biomarkers that correlate with treatment response and are thus suitable for use in clinical trials. Our overarching goal is to translate therapies for MTM. In this proposal, we will address this goal by establishing the first serum biomarker for MTM and identifying a novel therapeutic approach with broad treatment potential. MTM is an X-linked disorder caused by mutations in MTM1, a phosphatase that regulates endosomal sorting. We have shown that MTM1 mutation results in disruption of the triad, a muscle structure responsible for excitation-contraction coupling, and that triad disorganization is a critical driver of MTM pathogenesis. Several groups have demonstrated that myofiber hypotrophy is also an important contributor to MTM pathology. While the precise mechanisms underlying these changes remain to be elucidated, overexpression of DNM2 is a key driver of the triad defects, and unbalanced AKT-TOR pathway signaling is associated with the reduced myofiber size. In this study, we propose a treatment aimed at targeting and correcting these defects. MicroRNAs (miRNAs) are small, non-coding RNAs that modulate gene expression and serve as important regulators of myriad cellular processes. MiRNAs are emerging as both treatment effect biomarkers and potential therapeutics. In several disorders (including other muscle diseases), circulating miRNAs correlate with disease severity and treatment response, and are under consideration as biomarkers in clinical trials. The miRNAs miR-486 and miR-133a have direct relevance to MTM pathogenesis. We have shown that miR- 486 regulates myofiber size by rebalancing AKT signaling, and others have demonstrated that miR-133a directly down-regulates DNM2. These miRNAs are thus attractive as potential therapeutic targets, a concept validated by our demonstration of miR-486 overexpression as a treatment strategy for DMD. In preliminary data, we profiled miRNAs from MTM mouse muscle and found significant changes in 50 miRNAs including downregulation of miR-486 and miR-133a. Based on this, we hypothesize that changes in the levels of miRNAs will correlate with disease status and treatment response, and thus function as an ideal non-invasive biomarker for MTM. Further, we predict that restoring expression of miR-486 and miR-133a will increase myofiber size and reverse triad defects respectively, and thus rescue the MTM phenotype. We will test these hypotheses using rigorous methodology in the murine model of MTM. Successful completion of our study will identify the first biomarker of MTM suitable for clinical translation, and establish a novel treatment with applicability to muscle diseases that feature myofiber hypotrophy and/or triad defects.

PROJECT SUMMARY/ABSTRACT Nemaline myopathy (NM) is a childhood-onset skeletal muscle disease that is characterized by severe disabilities, including (in many cases) wheelchair and feeding tube dependence. Mutations in more than a dozen genes can cause NM. Most of these genes encode components of the thin filament (a principle part of the sarcomere), and mutations associated in these genes alter the structure and/or function of the thin filament, resulting in impaired muscle contraction and generalized weakness. There are currently no treatments for NM. The overarching goal of this proposal is to develop therapies for this devastating disease. Recessive mutations in NEB are the most common cause of NM. NEB encodes the giant protein Nebulin, which functions to regulate the length of the thin filament. Many NEB mutations cause single exon skipping or single exon deletion. Such mutations do not alter the RNA reading frame of NEB; however, despite only removing a very small part of an otherwise giant protein, they unexpectedly result in significant reduction (or even complete loss) of the whole Nebulin protein. The reason for this is not known. We hypothesize that the reason for this surprising observation is that these mutations remove an incomplete portion of repeat elements within NEB, the consequence of which is to make the Nebulin protein out of register, thereby preventing it from incorporating into the thin filament. We will test this hypothesis in Aim 1. Aim 1: we will use cutting edge imaging and biochemical strategies to study the Nebulin protein in a series of in-frame nebulin mutants. To accomplish this, we will employ the zebrafish model system, which is ideal for this purpose because we can visualize the Nebulin protein in intact skeletal muscle in a living organism. Nebulin protein levels in patients with NEB mutations are correlated with disease severity (less protein = more severe disease). This fact means that a therapeutic strategy targeted at increasing Nebulin protein expression should be very effective. Based on our hypothesis above, we predict that we can accomplish this by removing more of the Nebulin RNA to take out complete repeats, the result of which should be to enable a shortened Nebulin to re-integrate into the thin filament and be stably maintained. We will test this idea, which we call ?domain skipping?, in Aims 2 and 3. Aim 2: Using either morpholino mediated multi exon skipping or CRISPR/Cas9 genomic deletion, we will establish the feasibility and efficacy of ?domain skipping? in zebrafish. Zebrafish allow us to rapidly and comprehensively examine this strategy across the entire nebulin gene. Aim 3: We will translate our findings from zebrafish to the mouse model and to patient cells, focusing specifically on two common Neb mutations (exon 55 deletion and a nonsense mutation in exon 61). The use of the mouse model will enable us to test efficacy in a mammalian system, and testing in human cells will provide vital proof of concept and reagent development necessary for clinical translation.

Mutations in the gene that encodes the skeletal muscle type I ryanodine receptor (RYR1) result in a wide range of muscle disorders that collectively comprise the most common cause of non-dystrophic myopathy. The most severe cases of RYR1-related myopathy (RYR1-RM) exhibit a recessive pattern of inheritance and present in infancy with muscle hypotrophy, weakness, respiratory insufficiency, short stature, and a marked reduction in RYR1 protein expression in muscle. Despite their severity, high prevalence and association with significant disability and early mortality, there are no treatments or disease-modifying therapies for RYR1-RM. A major barrier to therapy development has been the lack of an animal model that mirrors the early onset and clinical severity of recessive RYR1-RM. To overcome this barrier, we developed two mouse models of recessive RYR1-RM that pheno-copy key characteristics of the human disorder including myofiber hypotrophy, reduced muscle/body mass, muscle weakness, markedly reduced RYR1 expression, and premature death. The scientific premise of this proposal is that these new mouse models of RYR1-RM provide a unique opportunity to explore the underlying patho-mechanisms of RYR1-RM and test the therapeutic efficacy of mechanism-based interventions. The overall goal of the project is to elucidate the patho-mechanisms responsible for muscle dysfunction in recessive RYR1-RM and to develop and validate effective treatments. We hypothesize that reduced folding/stability of mutated RYR1 homotetramers results in increased RYR1 protein degradation that markedly reduces RYR1 expression, and that even a modest increase in either RYR1 expression or function will ameliorate the myopathy and prolong survival. Furthermore, we also hypothesize that reduced myofiber size in RYR1-RM is a key aspect of disease pathogenesis, that hypotrophy is due to epigenetic abnormalities, and that drugs that target the epigenome or promote muscle growth can ameliorate the disease phenotype. The validity of these hypotheses will rigorously evaluated in three specific aims. Aim 1 will characterize RYR1 expression, function and myopathy in two mouse models of severe, recessive RYR1-RM and assess the therapeutic potential of systemic treatment with ebselen, an FDA-approved drug and known RYR1 activator. Aim 2 will elucidate the mechanism(s) for reduced RYR1 expression in our mouse models of RYR1-RM mice and evaluate the therapeutic efficacy of systemic treatment with a chemical chaperone and ER stress inhibitor (4PBA). Aim 3 will determine the mechanisms leading to muscle hypotrophy in RYR1-RM mice and test the potential of treatment with either HDAC inhibitors or modulators of myofiber size. The results of these studies will provide novel insights into the patho-mechanisms responsible for reduced RYR1 expression and muscle fiber hypotrophy in recessive RYR1-RM and determine the therapeutic potential of several mechanism-based interventions designed to enhance RYR1 function, reduce RYR1 degradation, and limit muscle hypotrophy in pre-clinical models of recessive RYR1-RM.