Sarah C. Kucenas - US grants

DESCRIPTION (provided by applicant): P2X receptors in mammals have been found on peripheral nociceptive sensory neurons and are implicated in sensation. In the fish, a subset of P2X receptors has been found to be localized to a primary sensory neuron called the Rohon-Beard neuron. This cell type is the primary mediator of the touch-evoked escape response in the embryonic zebrafish. By utilizing this dynamic animal model to study a similar phenomenon to sensation in mammals, more direct evidence implicating P2X receptors in sensory pathways can be achieved. To elucidate if P2X receptors mediate the mechanosensory mechanism that underlies this behavior in the fish, studies directly addressing P2X receptor function will be conducted. Through the use of transgenic fish, morpholino technology and calcium indicators, we will demonstrate that P2X receptors are an absolutely necessary component to the mechanosensory mechanism that underlies the escape behavior in the fish. By directly implicating P2X receptors in sensation in this animal model, a better understanding of sensation in the mammal can be gleaned. Ultimately, in human conditions like Causalgia and Rheumatoid Arthritis, direct evidence pointing to P2X receptors will aid in drug discovery.

[unreadable] DESCRIPTION (provided by applicant): In developmental and disease studies of vertebral motor nerve roots, motor axons and Schwann cells have been extensively studied while the perineurium has been mostly ignored. Because of its juxtaposition to these cell populations, the perineurium is poised to potentially play an integral role in nerve root organogenesis and disease. We have an unprecedented opportunity to elucidate the cell-cell interactions that occur among motor axons, Schwann cells and perineurium by using the zebrafish model system and its unique and powerful combination of in vivo time-lapse imaging and genetics. The overall goal of this application is to uncover the origin of the perineurium and subsequently elucidate the interactions that occur between this cell population and Schwann cells. Through the use of transgenic reporters, morpholino oligonucleotide (MO) technology, pharmacological inhibitors and mutants, we will demonstrate that the perineurium is an essential and integral part of the motor nerve root and plays a role in nerve root organogenesis. Ultimately, by directly implicating the perineurium in nerve root development, research aimed at understanding the cause and progression of many peripheral neuropathies, including Charcot- Marie-Tooth disease, can be expanded to include this structure and lead to a deeper understanding of these disorders. [unreadable] [unreadable]

? DESCRIPTION (provided by applicant): Motor nerves play the critical role of shunting information out of the central nervous system to targets in the periphery. Their formation requires the coordinated development of distinct cellular components, including motor axons, the glial cells that ensheath them and surrounding muscle. During nervous system construction, these cells must migrate long distances and coordinate their differentiation, ensuring the efficient propagation of electrical information. In this project, we will investigate the role of perineurial glia in early spinal motor nerve development as well as the role of muscle-nerve interactions during peripheral nerve development and disease. Although spinal motor axons exit the spinal cord in stereotyped positions along the anterior-posterior axis of the vertebrate neural tube and neural crest streams to these locations, the mechanisms that prefigure these exit points are poorly understood. In Aim 1 of this project, we will investigate the hypothesis that perineurial glia and their precursors prefigure motor exit points (MEP) and direct motor axon outgrowth and neural crest migration. In Aim 2, we will investigate the role of dystrophin in early nervous system development in a zebrafish model of Duchenne Muscular Dystrophy (DMD). Coupling genetics and in vivo imaging in the zebrafish, we can: 1) distinctly tease apart facets of motor nerve development, 2) elucidate how they are regulated, 3) lay the groundwork for a more fundamental understanding of the rules that form a functional nervous system and 4) shed light on mechanisms that could be perturbed in disease.

? DESCRIPTION (provided by applicant): Development of a functional and efficient nervous system requires the orchestrated migration and differentiation of axons and their associated glia. Motor axons connect the central nervous system (CNS) with targets in the periphery, including muscle. These axons interact with myelinating glial cells both in the CNS and peripheral nervous system (PNS). Ultimately, the differentiation of these two distinct glial populations forms a specialized structure known as the transition zone (TZ), which exists at every boundary between the spinal cord and periphery. Interestingly, at motor exit point (MEP) TZs, oligodendrocytes and peripheral myelinating glia normally stay restricted to their respective half of the nervous system, while other glia, including perineurial glia and a newly described population of cells, MEP glia, freely migrate from the spinal cord out into the periphery. How MEP TZs are selectively permeable, restricting myelinating cells from mixing, while allowing the passage of other populations, is unknown. In this project, we will characterize the development and function of a novel population of glia, motor exit point (MEP) glia, that we demonstrate are essential for restricting oligodendrocyte progenitor cells (OPC) to the spinal cord (Aim 1). In Aim 2, using both a candidate and unbiased approach, we will investigate the molecular mechanism that mediates MEP glia-OPC interactions during development. Defects in the development or maintenance of myelin along axons are the cause of many disorders collectively known as myelinopathies, one such example being Charcot-Marie-Tooth Disease (CMT). Some of the most severe types of this disease lead to demyelination, neurodegeneration and subsequent muscle atrophy in young children. Utilizing an in vivo system, zebrafish, to directly investigate the glial-glial interactions that establish MEP TZs, we will provide important insights into how functional nervous systems are assembled, maintained and behave during disease.

Development of a functional and efficient nervous system requires the orchestrated specification, migration and differentiation of glia. During gliogenesis in both the central and peripheral nervous systems (CNS and PNS, respectively), large populations of glia are specified that must migrate and differentiate into not only functional glial cells, but also coordinate their development so that they occupy discrete, non-overlapping territories with neighboring glia. This phenomenon of glial spacing, or tiling, can occur between glia found in the CNS, PNS, or between glial cells where one cell resides in the CNS and the other resides in the PNS. Although we know that these tiling events occur, we don?t know the molecular nature of these interactions or whether they are used by all tiling glia. Additionally, how local glial-glial interactions play a role in global glial tiling is unknown. In this proposal, using zebrafish as a model organism, we will investigate the cellular and molecular mechanisms that mediate pre-myelinating glial tiling at motor exit point (MEP) transition zones (TZ) and in the developing spinal cord. In Aim 1 of this project, we will use a combination of expression analysis, multi-color, in vivo, time-lapse imaging, electron microscopy, pharmacological and genetic manipulation (CRISPR) of newly identified mediators to elucidate the cellular and molecular mechanisms that govern myelinating glial tiling at MEP TZs. In Aim 2, we will determine if the same mechanisms we characterize in Aim 1 also mediate tiling between oligodendrocyte progenitor cells (OPC) in the developing spinal cord. To better understand, diagnose and treat the many degenerative disorders of the CNS and PNS, we need to comprehend the cellular and molecular mechanisms that mediate glial-glial interactions and tiling. Zebrafish provide a unique opportunity to directly observe and manipulate cell populations to gain insight into how glial cells interact under normal physiological conditions, and if those interactions that ultimately result in glial tiling are perturbed in disease.

PROJECT SUMMARY The Brain Immunology ang Glia Training Program (BIGTP) at the University of Virginia (UVA) will build on existing strengths in neuroimmunology and glia research and the programming that has been in place since the establishment of the center from Brain Immmunolgy and Glia (BIG) in 2012. The BIGTP brings together 20 mentors from eight different departments with the goal of providing interdisciplinary training that sparks discoveries and prepares a generation of researchers that are uniquely equipped to tackle research questions that arise at the interface of neuroscience and immunology. Funds are requested to support two predoctoral trainees each year for a duration of two years, typically in years three and four of graduate training. The 20 mentors are well-funded by a total of nearly $11 million in annual direct costs. The average mentor has trained more than nine pre- and postdoctoral trainees in the past ten years. Assistant Professors are included as Junior Mentors that will require senior BIGTP faculty to serve as co-mentors for trainees. Formal mentor training is required of all BIGTP mentors. Trainees will partake in the dynamic BIG research in progress seminars that are held weekly. Trainees will also have access to leaders in the field through the BIG Neuro seminar series. Annual retreats will focus on trainee development, review of the program, and will allow trainees to interact with members of the BIGTP External Advisory Board that are invited as keynote speakers. The quantitative literacy of trainees will be developed and supported through the Quantitative Literacy Series designed specifically for trainees that addresses topics in experimental design, reproducibility, power analysis, statistical analyses, transparency in reporting, etc. Importantly, Dr. Marieke Jones directs required courses in programming and statistical analyses and will be directly supported by and involved in BIGTP events, providing continuity in instruction and support for trainees for the duration of their predoctoral studies. The BIGTP will be evaluated by current and past trainees, program mentors, and members of a highly-qualified External Advisory Board. Predoctoral trainees trained in BIGTP mentor laboratories have completed their degrees in approximately 5.25 years and contributed to an average of 4.9 publications and 1.7 first-author publications in journals that include: Nature, Cell, Nature Neuroscience, Science Translational Medicine, Immunity, Neuron, PNAS, Science Advances, and Nature Communications. Current and past trainees have been supported by individual fellowships sponsored by NIH, NSF, and UVA. Importantly, 97% of trainees remain in science-related fields and 74% go on to complete further training. The BIGTP leadership and mentors understand the importance of diversity, which is essential for the strength and growth of the proposed Training Program. The BIGTP?s commitment to diversity is demonstrated by the inclusivity within the Program?s administration and programmatic events. The implementation of an NIH- sponsored T32 training program will allow existing strengths and programming at UVA to strengthen and expand to provide unparalleled training for predoctoral trainees in a burgeoning research area.

Peripheral nerve assembly requires the coordinated development of many components, including axons and the glial cells that ensheath them. Currently, we have a broad perspective of the structure of peripheral nerves and the events that govern nerve assembly. However, we still lack a complete picture of all of the cellular and molecular mechanisms that mediate these events, and whether perturbations to these processes underlie disease. This is in part due to the fact that we still don?t have a full appreciation of the diversity of glial populations that make up these structures and there are currently no specific markers for any peripheral glial population. Additionally, although peripheral nerves are often grouped together as a single, uniform structure, there are in fact many types, including cranial, trunk, sensory, and motor, as well as spatial organization along the proximal-distal axis. However, whether there is any glial diversity between or along these structures, and whether this diversity is what contributes to the differences observed between these nerves with respect to function and susceptibility to disease, has yet to be determined. Using single-nuclei RNA-sequencing coupled with the powerful system, zebrafish, we will create a molecular taxonomy of peripheral glial subpopulations and decipher their roles in nervous system development.