Clarissa A. Henry, Ph.D. - US grants

[unreadable] DESCRIPTION (provided by applicant): Muscle formation during early development is critical for normal muscle function. Many diseases disrupt muscle physiology. However, the cell and molecular networks that underlie the pathology of these diseases are not known. Because each step of muscle specification and differentiation translates to the progressive refinement of functional physiology, studying muscle development can significantly inform our understanding of muscle function and physiology. The long-term goal of our lab is to elucidate the signaling networks that lead to the carefully choreographed cell behaviors that generate functional muscle using the zebrafish model. The zebrafish is an excellent model system with which to integrate the genetic, molecular, and cell biological mechanisms that underlie muscle development. Zebrafish skeletal muscle is comprised of segmentally reiterated myotomes. These myotomes contain long muscle fibers that attach to myotome boundaries. The zebrafish myotome boundary is molecularly and functionally homologous to the mammalian tendon: it is comprised mainly of collagen and transmits muscle generated force to the skeletal system. Both muscle fiber and myotome boundary formation during development are critical for normal musculoskeletal function. Preliminary data elucidate the spatial complexity of the myotome boundary as well as the morphogenetic steps that underlie muscle fiber formation. We hypothesize that an extracellular basement membrane protein, laminin, is critical for multiple steps in muscle development. The aims of this proposal are to: 1) test the hypothesis that laminin is necessary for the initial elongation of muscle precursor cells into long muscle fibers and determine the mechanism by which laminin is required, 2) test the hypothesis that laminin is crucial for myotome boundary maintenance and determine the underlying mechanism for this requirement, and 3) test the hypothesis that laminin and Hedgehog signaling interact during boundary morphogenesis. Our use of the embryology and genetics of the zebrafish to elucidate novel aspects of muscle development may inform and benefit treatments of both muscle/tendon diseases and traumatic/overuse injuries. [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Muscle myopathies and dystrophies cause debilitating disease in children and are associated with premature death in nearly all patients affected. Many muscle disorders, such as Duchenne, Becker, and Merosin-deficient muscular dystrophies, are caused by mutations in adhesion complexes that anchor muscle cells to their surrounding basement membrane (BM). Despite great strides in identifying the genetic basis of many myopathies, mechanisms that mediate BM assembly and adhesion are not well understood. In particular, there is a need for small molecule therapeutics and discovery of new targets for drug development. We have shown that NAD+ biosynthesis is necessary for muscle development and sufficient to correct dystrophic phenotypes in zebrafish. Nicotinamide Riboside Kinase 2b (Nrk2b)-mediated NAD+ biosynthesis ameliorates muscle degeneration by increasing BM organization and promoting the subcellular localization of an intracellular Integrin-binding protein, Paxillin. Paxillin is an essential signaling nexus that regulates cell adhesion, morphology, and migration. Preliminary data indicate that Paxillin overexpression dramatically improves muscle structure and decreases degeneration in dystrophic zebrafish, but the mechanisms are not known. The rationale of the proposed research is that because mechanical failure in cell adhesion between muscle fibers and their surrounding BM underlies the etiology of many myopathies, the Nrk2b pathway may have therapeutic utility for multiple congenital muscular dystrophies (CMDs). The objectives of this application are to elucidate the molecular underpinnings of Paxillin function in this pathway. Our central hypothesis is that Nrk2b-mediated NAD+ biosynthesis regulates subcellular localization of Paxillin; resulting in downstream signaling events that increase organization and structure of the BM microenvironment. The contribution of the proposed research is expected to be the elucidation of molecular mechanisms underlying a novel pathway that restores muscle structure/function in dystrophic zebrafish. This contribution is significant because this is a new paradigm in the effort to eradicate myopathies. The approach is innovative because it represents a shift in thinking. Instead of focusing on canonical muscle adhesion proteins, the focus is on the compensatory response of adhesion proteins that are traditionally understudied in muscle. Thus, this unique approach focuses on improving muscle structure by strengthening existing machinery. The long-term goal of this work is to capitalize on elucidation of a novel pathway that ameliorates dystrophy to identify new drug targets for the treatment of muscle degenerative disorders.

The specification and morphogenesis of skeletal muscle is critical for normal embryonic development. The critical role of muscle is highlighted by the fact that congenital muscular dystrophies cause debilitating disease in children and are associated with premature death in nearly all patients affected. Many congenital muscular dystrophies, such as Duchenne, Ulrich, and Merosin-deficient muscular dystrophies, are caused by mutations in adhesion complexes that anchor muscle cells to their surrounding extracellular matrix (the myomatrix). In addition to being required for muscle development, the myomatrix plays a fundamental role in muscle homeostasis because it bears much of the passive load. Despite the critical role that the myomatrix plays in muscle development and physiology, the mechanisms underlying myomatrix development are not known. In particular, mechanisms that mediate the dynamic changes in myomatrix composition are not well understood. Fibronectin (Fn) is a myomatrix protein that is necessary for muscle development and regeneration. Fn is transiently upregulated during muscle development and regeneration. The subsequent downregulation of Fn levels is important because excess Fn deposition leads to fibrosis. Fibrosis is the aberrant deposition of extracellular matrix proteins. In the context of skeletal muscle, the replacement of contractile muscle tissue with Fn-rich fibrotic material diminishes muscle function. Therefore, Fn levels have to be tightly controlled. The mechanisms that mediate Fn polymerization during muscle development are beginning to be elucidated, but nothing is known about how Fn is downregulated. Our central hypothesis is that the myomatrix protein laminin, along with Matrix Metalloproteinase 11 (Mmp11) and Tissue Inhibitor of Matrix Metalloproteinase 2a (Timp2a), comprise a regulatory network that controls Fn levels, muscle fiber type specification, and muscle morphogenesis. This hypothesis is based on our preliminary data showing that laminin polymerization mediates Mmp11 localization and that Mmp11 is necessary and sufficient for Fn downregulation during zebrafish development. Preliminary data also suggest that Timp2a is required for Fn regulation. Understanding Fn regulation during muscle development and regeneration is significant because transient Fn is necessary; but too much or sustained Fn is deleterious. These experiments are innovative because they are the first to address mechanisms underlying Fn downregulation during development, the first to address Mmp11 function during muscle development, and the first to analyze crosstalk between laminin and Fn. The contribution of this research will be identification of novel mechanisms regulating Fn levels and roles for Fn in muscle fiber type specification. Completion of this research will significantly advance our long-term goal, which is to understand how signaling between muscle cells and their myomatrix mediates muscle development, homeostasis, and regeneration.

ABSTRACT This proposal requests partial support for an international meeting on Myogenesis as part of the Gordon Research Conference (GRC) and Gordon Research Seminar (GRS) series to be held at the Renaissance Tuscany Il Ciocco in Lucca (Barga), Italy June 8-14, 2019. The international meeting on GRC Myogenesis has historically been viewed as one of the preeminent skeletal muscle meetings, attracting speakers and attendants from around the world. The major goal of the 2019 Conference is to broaden our current knowledge on the cellular and molecular mechanisms that control the formation, the maintenance and the regeneration of skeletal muscle. Leaders in the field will present recent unpublished data to educate conference attendees while stimulating interactive discussion amongst the group. GRC will convene invited speakers to the GRC to exchange new information on the underlying mechanisms of skeletal muscle development, homeostasis and regeneration. These topics will be communicated through nine scientific sessions focusing on: Building and maintaining skeletal muscle (I&II); Satellite cells and regeneration; The complexity of skeletal muscle architecture; Extrinsic regulation of the muscle microenvironment; Intrinsic signaling in muscle cells; Epigenetic, transcriptional, and post-transcriptional regulation of myogenesis; Cellular and molecular mechanisms of muscular diseases and a keynote lecture on Historical perspective on muscle development. In addition, four evening poster sessions will stimulate interactions between all attendees. GRS is a two-day conference program targeting graduate students and postdocs that takes place immediately preceding the GRC. The GRS will conduct four sessions of talks and two poster sessions by graduate students and postdoctoral fellows. Additional program events will ensure maximal level of interaction between established scientists and junior scientists/trainees. They include poster sessions, 2 sessions of 1-minute poster presentation that will provide a forum for junior scientists to present their work and receive feedback from other participants, meet the experts breakfast, specific social events and informal gatherings in the afternoons. These activities represent a unique opportunity for the next generation of scientists to present their research on myogenesis, interact with their peers, and form new collaborations. Thus, we expect the 2019 Myogenesis GRC will provide an ideal scenario to stimulate new breakthroughs in muscle biology, that will benefit attendees at every professional level and to place the field in a better position to accelerate the advancement of muscle biology and thus the development of new therapies for muscle.

ABSTRACT Congenital muscular dystrophies (CMDs) are progressive debilitating diseases without cures. Many CMDs disrupt the adhesion of muscle cells to their surrounding extracellular matrix (ECM). Muscle-ECM adhesion is critical for muscle development, homeostasis, regeneration, and resilience to stress. Mutations in genes that modulate muscle-ECM adhesion frequently lead to CMDs. For example, Dystroglycan (DG) and Integrin alpha7 (Itga7) are transmembrane ECM receptors that, when mutated, result in CMDs. Whether and/or how these transmembrane receptors interact during muscle development/homeostasis is not known. In addition, the roles that post-translational modification of DG plays in modulating both the ECM proper and muscle-ECM adhesion are not known. We previously found that exogenous NAD+ potentiates ECM deposition and that NAD+ improves dystrophic phenotypes in zebrafish lacking either DG or Itga7. The basic cell biological mechanisms that underlie NAD+-mediated improvement in muscle-ECM adhesion are not well understood. Our long-term goal is to understand how signaling between muscle cells and their ECM mediates muscle health. Secondary Dystroglycanopathies are a subset of CMDs that result from mutations in genes that are necessary for glycosylation of DG, which is necessary for muscle-ECM adhesion. GDP-mannose, synthesized by GMPPB, is essential for glycosylation reactions. Mutations in GMPPB result in GMPPB-associated Dystroglycanopathy. Preliminary data show that muscle development, homeostasis, and regeneration are disrupted in gmppb mutants. In contrast to our previous data showing NAD+ improves ECM deposition in dg- deficient zebrafish, preliminary data show that NAD+ does not improve muscle structure in gmppb mutants. In this grant, we will compare and contrast the mechanisms underlying the effects of DG glycosylation and NAD+ on muscle development, homeostasis, and regeneration. Our central hypothesis is that both NAD+ and gmppb regulate muscle cell adhesion by altering sarcolemma architecture and ECM organization. In Aim 1 we will test the hypothesis that NAD+ increases cell adhesion in DG mutant zebrafish by increasing Itga7 clustering; and that hypoglycosylated DG disrupts sarcolemma architecture and prevents NAD+-mediated Itga7 clustering and increased cell adhesion. We will do this with a combination of longitudinal light sheet microscopy studies and super-resolution microscopy. In Aim 2 we will identify new muscle cell adhesion regulators through comparative studies of dysregulated muscle development in three zebrafish models of muscular dystrophy. We will take an unbiased approach to identify ECM regulatory nodes by using network modeling and network resilience analysis of co-expressed coding and non-coding genes. Completion of this grant will provide new insight into how cell-ECM adhesion mediates muscle development and homeostasis in vertebrate models of CMDs. These basic in vivo cell biological studies are crucial to provide a foundational understanding of the interplay between transmembrane receptors, ECM regulation, and cell adhesion.

ABSTRACT Dystroglycanopathies are neuromuscular diseases that result in progressive muscle wasting, which decreases quality of life and often leads to early death. Dystroglycanopathies result from mutations in genes that encode proteins that participate in dystroglycan (DG) glycosylation. DG is a transmembrane receptor for extracellular matrix (ECM) proteins and its glycosylation is necessary for cells to adhere to their surrounding ECM at both neuromuscular junctions (NMJs) and myotendinous junctions (MTJs). The contribution of disrupted cell-ECM adhesion to altered structure and function of the neuromuscular system in the context of dystroglycanopathies is poorly understood. This is partially because dystroglycanopathies caused by the same genetic mutation have variable clinical presentation, including severe congenital onset muscular dystrophy with eye/brain involvement, congenital myasthenic syndrome, and milder adult-onset limb girdle muscular dystrophies. There are multiple roadblocks to understanding the phenotypic variation of these incurable diseases. The neuromuscular system involves coordinated development of neural and muscle tissues to form NMJs, but mechanisms are not fully understood. The effects of disrupted primary motor neuron development on subsequently developing secondary motor neurons, muscle, and NMJ structure and function are not understood in the context of muscular dystrophies such as the dystroglycanopathies. A genetic model of dystroglycanopathies in a vertebrate model that allows longitudinal studies of neuromuscular development is needed to address these gaps. We generated a zebrafish model of gmppb-associated dystroglycanopathy. Our preliminary data suggest that gmppb is required for primary motoneuron, NMJ, and muscle development and/or homeostasis. Our central hypothesis is that gmppb is required for normal motor axon pathfinding; and that early disruption in motor axon pathfinding leads to defects in neuromuscular structure and homeostasis. We will test this hypothesis by conducting longitudinal studies that will test whether/how primary motoneuron development impacts muscle homeostasis. Elucidating cellular mechanisms is a crucial first step to understanding the molecular mechanisms of phenotypic variation in development and disease. This research is innovative because our preliminary data are the first to show early motoneuron axon pathfinding defects. No longitudinal studies of neuromuscular development in vertebrate models of dystroglycanopathies have been conducted. This study will have a significant impact on our understanding of roles for protein glycosylation in neuromuscular development. Thus, completion of this grant will provide new insight into how initial motor axon development affects neuromuscular development and homeostasis. This information is a critical foundation for understanding the basic biology underlying abnormal neuromuscular phenotypes in the dystroglycanopathies. Understanding these basic mechanisms is an important first step towards identifying future therapeutic targets. Taken together, this grant will significantly impact the field of neuromuscular development and homeostasis.

The University of Maine Graduate School of Biomedical Science and Engineering (GSBSE) is a unique biomedical science PhD program that spans the state of Maine. With partner institutions including the Jackson Laboratory, Mount Desert Island Biological Laboratory, Maine Medical Center Research Institute, and the University of New England, the GSBSE leverages the existing expertise and infrastructure of the state?s premier biomedical research programs. The GSBSE?s research spans the areas of computational biology, bioinformatics, mammalian genetics, cell and molecular biology, developmental biology, biomedical engineering, neuroscience, and general medical sciences. Since 2006, the GSBSE has successfully trained 47 PhD graduates, who have all continued in science- or medical-related careers. With the recognition that the future of biomedical research requires transdisciplinary collaboration and team science, a new T32 program is proposed to emphasize these training elements in the basic biomedical sciences. The broad objective of the Transdisciplinary Predoctoral Training Program in Biomedical Science and Engineering is to prepare trainees for exceptional careers in the biomedical workforce by focusing on transdisciplinary collaborations, training in team science, and supporting experiential professional career exploration. The GSBSE has built a strong foundation for this program, which includes: active participation of 50 funded faculty members who are committed to co-mentorship in transdisciplinary research, full support of partner institutes who provide resources and commitment to predoctoral trainees, a strong foundational curriculum in the biomedical sciences, and advisory committees to oversee and evaluate program outcomes. Layered on top of this foundation, this T32 will initiate new training strategies, including innovative, evidence-based training in team science, a co-mentorship model that supports transdisciplinary research, a unique experiential professional rotation in industry or other non-academic setting, a new professionalism and responsible conduct of research course, and a new strategy for increasing the diversity of the trainees. This program will be administered by the University of Maine and incorporate a multiple PI model. Dr. David Neivandt is a Professor at the University of Maine and the current Director of GSBSE, has scientific expertise in chemical and biomedical engineering, and has extensive mentorship and administrative leadership experience. Dr. Lucy Liaw is a GSBSE faculty member with a primary appointment at Maine Medical Center, has scientific expertise in cardiovascular biology and disease, and directs research training programs at her home institute. The two PIs complement each other scientifically and administratively, and have been working in partnership within the GSBSE since 2013. The two PIs also are geographically distributed in each in the two hubs of concentration of the partner institutions. This T32 proposes to provide the first two years of support for 4 new predoctoral students annually, to join the cohort of GSBSE students, all of whom will receive the enhanced training strategies described in this proposal.

ABSTRACT: Enhancing Wellness and Resilience Supplement PARENT AWARD: 1T32GM132006-01 (PI: Henry, Clarissa) The current T32 award supports an innovative, evidence-based training program that includes a co-mentorship framework and transdisciplinary research opportunities. This proposed supplement will endeavor to increase the resilience and wellness of our graduate students with particular focus on: (1) a multi-tiered strategy designed to enhance mentoring, wellness, and resilience, and (2) long-term sustainability and integration. The combination of social unrest and the pandemic has been difficult for everyone and especially so for graduate students. Dr. Henry, as GSBSE director (and PI of the Parent T32), along with Zhen Zhang, MBA, GSBSE Administrative Coordinator, meet annually with each of our graduate students every spring. In Spring 2020 it became very clear that our graduate students were severely affected by the pandemic ? especially the newer graduate students, who generally lack a robust social network. At that time, we took immediate action to try to mitigate some of the effects of the pandemic. In this supplement, we propose to augment our current efforts by implementing a series of activities within a three-tiered public health model with the first tier focused on prevention, the second tier focused on direct support, and the third tier focused on professional interventions. We do this by leveraging primary campus collaborators in the Graduate School and Counseling Center with support from faculty experts on curriculum development and evaluation as well as the Psychological Services Center. In the first tier of prevention, we propose (1) hosting the ?Becoming a Resilient Scientist? workshop and extending it with professional development modules from the UMaine Graduate School?s GRAD Initiative, (2) starting ?Career Communities? featuring working alumni of the program that will include in-person workshops, (3) offering wellness-oriented student clubs in line with the UMaine Graduate Student Government requirements, (4) expanding our Annual Meeting with a wellness and community-building day, and (5) running a ?Resilient Researcher? retreat in conjunction with the Counseling Center. In the second tier of direct support, we propose (1) starting a formal Peer Advising program that supports younger graduate students who will be advised by senior graduate students and (2) conducting regular individual check-ins with all PhD students. In the third tier of professional interventions, we are partnering with the UMaine Counseling Center and UMaine Psychological Services Center to educate students, faculty mentors, and staff on resources to increase access to and reduce stigma about mental health resources. The proposed enhancements to wellness and resiliency will focus on substantially improving and integrating the new efforts into the curriculum and co-curricular requirements of the GSBSE PhD programs with a major emphasis on assessment and sustainability.