Kelly R. Monk, Ph.D. - US grants

DESCRIPTION (provided by applicant): Myelin is a layer of insulation that covers neuronal projections called axons in the vertebrate nervous system. In the peripheral nervous system, specialized cells called Schwann cells spiral themselves around axons to form the myelin sheath. Myelin ensures that nerve impulses travel quickly and efficiently, ultimately allowing for the entire nervous system to function properly. Disruptions to the myelin sheath in disease (like multiple sclerosis or peripheral neuropathy) or after injury (like spinal cord trauma) lead to devastating symptoms, significant morbidity, and myelin loss can lead to permanent neuron loss, and ultimately, paralysis. Currently, no treatments exist to prevent demyelination or to hasten remyelination, and there is therefore a pressing need to develop therapies that address these issues. To this end, we must learn more about the mechanisms that govern myelination, myelin maintenance, and remyelination. We discovered that the orphan G protein-coupled receptor, Gpr126, is an essential component of the incompletely understood axon-Schwann cell signaling nexus that controls myelination. In Gpr126 mutant mice, Schwann cells associate with axons, but fail to spiral their membranes to generate the myelin sheath. G protein-coupled receptors are excellent drug targets, representing at least one-third of all approved drugs; thus, Gpr126 represents an extremely attractive potential target to stimulate remyelination in humans with myelin disease or injury. We therefore propose to dissect the mechanisms by which Gpr126 controls myelination and to determine if Gpr126 is required for myelin homeostasis or remyelination after injury. These studies encompass our broad goals to define the mechanisms that form, maintain, and regenerate myelinated axons in the vertebrate nervous system. In the first aim, we will define the signaling pathway downstream of Gpr126 activation by performing biochemical analyses on Gpr126 mutant tissue and cells. In the second aim, we will test the hypothesis that Gpr126 is required autonomously in Schwann cells for myelination. We will employ conditional mouse mutants to delete Gpr126 specifically in Schwann cells or in neurons, and we will perform immunohistochemical and ultrastructural analyses to determine the consequences of cell type specific loss. In this aim, we will also define the developmental window of Gpr126 requirement by temporally deleting Gpr126 in developing embryonic and perinatal transgenic mice. In the third aim, we will define the roles of Gpr126 in adult peripheral nerve. Specifically, we will determine if Gpr126 is required for myelin maintenance, demyelination, and/or remyelination after nerve injury by temporally deleting Gpr126 in mature nerve and in injured mature nerve. Together, these experiments will define the mechanisms by which Gpr126 controls myelination, will elucidate if Gpr126 is required for remyelination in adult nerve, and may lay the foundation for future therapeutics that stimulate myelin repair in humans. PUBLIC HEALTH RELEVANCE: Lack of robust remyelination represents one of the major barriers to recovery of neurological functions in disease or following injury in many disorders of the nervous system. Here, we propose to determine how Gpr126 controls myelination during development and to elucidate whether Gpr126 is required for myelin maintenance and remyelination in adult nerves. These studies will help to define new strategies to stimulate remyelination in the injured and diseased human nervous system.

DESCRIPTION (provided by applicant): In the peripheral nervous system, specialized glial cells called Schwann cells exist in intimate association with neuronal axons. Myelinating Schwann cells iteratively wrap their membrane around a single axonal segment to generate the myelin sheath, while Remak Schwann cells envelop multiple small caliber axons. Both types of Schwann cells arise from a common immature Schwann cell precursor, and although important questions remain unanswered, far more is known about the molecular mechanisms that govern the development of myelinating Schwann cells. The study of Remak Schwann cells has been significantly hindered by a paucity of tools - few specific markers are known, and most of these are also expressed by immature Schwann cells. Additionally, there are no transgenic mice that permit genetic deletion in Remak Schwann cells. The main goals of this proposal are to test a bacTRAP toolset for PNS glia, and to define the molecular signature of mature Remak Schwann cells. The secondary goals of this proposal, using data obtained from the bacTRAP analysis, are to generate test new reagents for reliably targeting transgenes to Remak Schwann cells, including Cre recombinase for conditional gene deletion.

DESCRIPTION (provided by applicant): In the vertebrate nervous system, myelinating glia performs the spectacular feat of iteratively wrapping their membrane around axons to form the myelin sheath. Myelin allows for rapid nerve impulse propagation, and the glial cells that make myelin are also essential for neuronal health and survival. The importance of myelin is underscored in diseases in which it is disrupted, including multiple sclerosis, leukodystrophies, and numerous peripheral neuropathies. To date, no therapeutic strategies exist to halt demyelination or stimulate remyelination in disease or injury. As a vertebrate model organism that is amenable to both chemical and genetic screens, zebrafish represent the ideal system with which to dissect the molecular mechanisms that govern myelination. In a previous forward genetic screen, we discovered that the G protein-coupled receptor (GPCR) Gpr126 is essential for myelination in the peripheral nervous system, although the mechanisms by which Gpr126 functions are not completely understood. As a GPCR, Gpr126 represents an excellent potential therapeutic target to stimulate remyelination. To this end, we will define Gpr126-mediated pathways in glial development and myelination using both chemical and genetic screens in a hypomorphic gpr126 mutant, which generates reduced levels of myelin in the peripheral nervous system. (1) We will perform large-scale compound library screens to discover small molecules that can enhance or suppress the hypomorphic gpr126 mutant phenotype. (2) We will perform a forward genetic enhancer/suppressor screen of 2,000 genomes to define genetic modifiers of gpr126. Together, these experiments will define the mechanisms by which Gpr126 mediates myelination. Critically, these screens can also uncover new chemicals and genes that regulate glial cell development and myelination independently of Gpr126. These screens will enhance our understanding of the mechanisms that govern nervous system development and may point the way to novel therapeutics to promote nervous system repair in humans.

? DESCRIPTION (provided by applicant): Myelin is a layer of insulation that covers neuronal axon projections in the vertebrate nervous system. In the peripheral nervous system (PNS), Schwann cells (SCs) radially sort axons into a 1:1 relationship and then iteratively wrap axonal segments to form myelin. Myelin ensures that nerve impulses travel quickly and efficiently, ultimately allowing for the entire nervous system to function properly. Disruptions to the myelin sheath in disease (e.g., numerous peripheral neuropathies) or after injury lead to devastating symptoms, and significant morbidity. Moreover, myelin damage can lead to permanent neuron loss. Currently, no treatments exist to prevent demyelination or to hasten remyelination, and there is therefore a pressing need to develop therapies that address these issues. To this end, we must learn more about the mechanisms that govern SC development and myelination. We previously discovered that the adhesion G protein-coupled receptor (aGPCR) Gpr126 is essential for SC radial sorting and myelination, although the mechanisms by which Gpr126 controls these processes are only beginning to be understood. GPCRs are excellent drug targets, representing at least one-third of all approved drugs; thus, aGPCRs are extremely attractive therapeutic targets to stimulate remyelination in humans with myelin disease or injury. Interestingly, we have recently determined that Gpr56, an aGPCR related to Gpr126 is also required during SC radial sorting in development and myelin maintenance in adulthood. In addition to Gpr126 and Gpr56, we have determined that four other aGPCRs are expressed in SCs, though their functions are unknown. In this proposal, we seek to define the aGPCR-mediated SC developmental program. We will: (1) Determine how Gpr126 controls radial sorting; (2) Define the autonomy, downstream signaling, and ligands of Gpr56 in SCs; (3) Test if the four novel SC-expressed aGPCRs are required for PNS development, myelination, and/or myelin maintenance. These experiments will greatly strengthen our understanding of SC and aGPCR biology and may lay the foundation for future therapeutics that stimulate myelin repair in humans.

DESCRIPTION (provided by applicant): In the vertebrate nervous system, myelinating glia performs the spectacular feat of iteratively wrapping their membrane around axons to form the myelin sheath. Myelin allows for rapid nerve impulse propagation, and the glial cells that make myelin are also essential for neuronal health and survival. The importance of myelin is underscored in diseases in which it is disrupted, including multiple sclerosis, leukodystrophies, and numerous peripheral neuropathies. To date, no therapeutic strategies exist to halt demyelination or stimulate remyelination in disease or injury. As a vertebrate model organism that is amenable to both chemical and genetic screens, zebrafish represent the ideal system with which to dissect the molecular mechanisms that govern myelination. In a previous forward genetic screen, we discovered that the G protein-coupled receptor (GPCR) Gpr126 is essential for myelination in the peripheral nervous system, although the mechanisms by which Gpr126 functions are not completely understood. As a GPCR, Gpr126 represents an excellent potential therapeutic target to stimulate remyelination. To this end, we will define Gpr126-mediated pathways in glial development and myelination using both chemical and genetic screens in a hypomorphic gpr126 mutant, which generates reduced levels of myelin in the peripheral nervous system. (1) We will perform large-scale compound library screens to discover small molecules that can enhance or suppress the hypomorphic gpr126 mutant phenotype. (2) We will perform a forward genetic enhancer/suppressor screen of 2,000 genomes to define genetic modifiers of gpr126. Together, these experiments will define the mechanisms by which Gpr126 mediates myelination. Critically, these screens can also uncover new chemicals and genes that regulate glial cell development and myelination independently of Gpr126. These screens will enhance our understanding of the mechanisms that govern nervous system development and may point the way to novel therapeutics to promote nervous system repair in humans.

Summary/Abstract This project would continue support for the Multidisciplinary Training Program in Neuroscience at the Oregon Health and Science University (OHSU) in Portland. The program, about to enter its 20th year, provides broad, early stage training for graduate students entering the Neuroscience Graduate Program (NGP). The program is based at the Vollum Institute but includes faculty and students in many centers, department and institutes on the OHSU campus. The program includes 55 students (ca. 10 per year). Our training faculty of 55 scientists offers thesis research opportunities that include all levels of modern neuroscience research from state-of-the-art cryoEM studies of membrane proteins to systems neuroscience to disease-oriented and translational neuroscience. The program has undergone a number of innovations and changes since the last renewal as fully outlined in the proposal, highlighted by the recruitment of new leaders and new faculty in the neuroscience community at OHSU. The new faculty members bring new areas of research strength that supplement the ongoing research strengths in the NGP. The innovations include a unique core curriculum structure that begins with a week-long Boot Camp of followed by a 12-week intensive course that provides a broad foundation in neuroscience for all students in the program. The curriculum format, now in its 3rd year, allows first year student to engage in fulltime laboratory rotations within 4 months of entry into graduate school. The core curriculum is supplemented by workshops and individual instruction in specific techniques as well as professional skills, ethics and career planning. Additional emphasis is placed on fostering skills in experimental design, programming, and quantitative approaches to address NIH mandates in rigor, reproducibility and transparency. The spirit of at OHSU among the large neuroscience community, numbering approximately 150 affiliated scientists, combined with the diversity of its many research institutes, and the close proximity of basic and clinical research facilities provide a unique opportunity for early stage pre-doctoral students to establish and benefit from cross-disciplinary collaborations. This foundation will foster the skills necessary for graduates to be successful in a variety of science-related careers needed to fully understand and treat complex neuropsychiatric diseases.

Summary/Abstract This project would continue support for the Multidisciplinary Training Program in Neuroscience at the Oregon Health and Science University (OHSU) in Portland. The program, about to enter its 20th year, provides broad, early stage training for graduate students entering the Neuroscience Graduate Program (NGP). The program is based at the Vollum Institute but includes faculty and students in many centers, department and institutes on the OHSU campus. The program includes 55 students (ca. 10 per year). Our training faculty of 55 scientists offers thesis research opportunities that include all levels of modern neuroscience research from state-of-the-art cryoEM studies of membrane proteins to systems neuroscience to disease-oriented and translational neuroscience. The program has undergone a number of innovations and changes since the last renewal as fully outlined in the proposal, highlighted by the recruitment of new leaders and new faculty in the neuroscience community at OHSU. The new faculty members bring new areas of research strength that supplement the ongoing research strengths in the NGP. The innovations include a unique core curriculum structure that begins with a week-long Boot Camp of followed by a 12-week intensive course that provides a broad foundation in neuroscience for all students in the program. The curriculum format, now in its 3rd year, allows first year student to engage in fulltime laboratory rotations within 4 months of entry into graduate school. The core curriculum is supplemented by workshops and individual instruction in specific techniques as well as professional skills, ethics and career planning. Additional emphasis is placed on fostering skills in experimental design, programming, and quantitative approaches to address NIH mandates in rigor, reproducibility and transparency. The spirit of at OHSU among the large neuroscience community, numbering approximately 150 affiliated scientists, combined with the diversity of its many research institutes, and the close proximity of basic and clinical research facilities provide a unique opportunity for early stage pre-doctoral students to establish and benefit from cross-disciplinary collaborations. This foundation will foster the skills necessary for graduates to be successful in a variety of science-related careers needed to fully understand and treat complex neuropsychiatric diseases. 7

PROJECT SUMMARY This application is to request support for the 2020 Gordon Research Seminar (GRS; May 16-17) and Conference (GRC; May 17-22) on Myelin to be held in Barga, Italy. The goal of these conferences is to provide an active forum for exchange of results at the cutting edge of myelin development, physiology, pathology, and treatment. This meeting uniquely focuses on central and peripheral myelin and is timely given the growing interest in myelin biology and human disease and major advances since the last meeting in 2018. These advances include: a better understanding of myelinating glial cell heterogeneity, new insights into myelinating glial cell responses to injury, a growing recognition of the importance of glial-glial interactions in myelination (in addition to neuron-glial interactions), and paradigm shifting breakthroughs in our understanding of how remyelination may occur in multiple sclerosis. Moreover, it has become clearer over the past two years that myelinating glia are exquisitely sensitive to perturbation and may be causative for some pathologies in neurodegenerative disease like Alzheimer?s disease and amyotrophic lateral sclerosis. Together, the 2020 Myelin GRC ?Myelin: Translational Science of Myelin ? from Glial Biology to Repair? and the GRS ?Myelin: Dynamics of Myelin Formation, Function, and Repair? will: 1) assemble an international meeting of academic scientists, clinician/scientists, and industry scientists engaged in studies of myelination and myelin repair; 2) discuss new and exciting developments in the field by selecting presenters who will largely present unpublished data and by reserving presentation slots for talks selected from the abstracts; 3) promote collaborative interactions to accelerate the pace of discovery to treat diseases of myelin; 4) provide an opportunity for junior attendees of diverse backgrounds to present their work, interact with other scientists, and promote collaborative interactions among all participants.

Astrocytes are the major glial cell type in the human brain and are critical for central nervous system (CNS) development and function. Astrocyte dysfunction has been implicated in human neurodevelopmental and psychiatric diseases. Given the important roles astrocytes play in CNS development and health, it is surprising that little is known about the molecular and cellular mechanisms that govern astrocyte maturation and their functional interactions with neighboring cells. One caveat for currently available model systems is the inability to manipulate and observe astrocyte development and dynamic changes in living, intact animals. Transparent zebrafish larvae would be ideally suited for such studies, but bone fide astrocytes have not been described in this system to date. Here, we aim to exploit the optical transparency of zebrafish larvae together with molecular and genetic approaches to characterize a new glial cell type in zebrafish that features key hallmarks of mammalian astrocytes. We have identified a previously unreported cell type in zebrafish CNS with several defining characteristics of mammalian astrocytes, such as intricate bushy morphology, glutamate transporter expression, and spontaneous microdomain Ca2+ transients. In this proposal, we will: 1) fully characterize the development and function of astrocytes in zebrafish; 2) develop a cell-specific CRISPR/Cas9 method to study astrocyte biology in vivo. We expect our work will establish zebrafish as a new model system to explore astrocyte development and function, provide new insights into the molecular and cellular mechanisms regulating astrocyte development and growth, and lay the foundation to study astrocyte function in the context of the entire nervous system in an intact and behaving animal.

In the vertebrate peripheral nervous system (PNS), specialized glial cells called Schwann cells form the myelin sheath, which is required for fast action potential propagation as well as neuronal health and survival. The importance of myelin in normal nervous system function is perhaps best underscored by myelin loss and inefficient remyelination of axon tracts observed in diseases such as demyelinating peripheral neuropathies. Such disruptions of myelin can lead to permanent neuron loss, significant pain and morbidity, and ultimately paralysis. Currently, no treatments exist to prevent demyelination or to enhance remyelination, in part because of our incomplete understanding of the genetic and molecular control of myelination. To identify new regulators of myelinating glial cell development, we previously performed a large-scale forward genetic screen in zebrafish. Through this screen, we identified new mutants in dedicator of cytokinesis (dock1) and previously showed that these global mutants exhibit severe defects in radial sorting and reduced myelination in the PNS during development. Moreover, our preliminary analyses suggest a critical function for Dock1 in nerve repair following injury in adult zebrafish. Dock1 encodes a highly conserved atypical guanine nucleotide exchange factor that can activate the small Rho GTPase Rac1. To date, no role for Dock1 function in Schwann cells has been described, although Rac1 is a known regulator of Schwann cell development. Here, we propose to use zebrafish and mouse models to dissect the mechanisms by which Dock1 controls PNS development and repair. We aim to define the function of Dock1 in Schwann cells (Aim 1), uncover pathways up- and downstream of Dock1 function (Aim 2), and test if Dock1 is required for myelin maintenance or repair following nerve injury in the mammalian PNS (Aim 3). Together, these experiments will define fundamental mechanisms underlying axon-Schwann cell interactions in development, injury, and repair and can lay the foundation for new therapies to treat human neuropathies and peripheral nerve damage.

The myelin sheath is a multilayered membrane generated by specialized glial cells called oligodendrocytes (OLs) that iteratively spiral their plasma membranes around axon segments in the vertebrate central nervous system (CNS). OLs derive from OL precursor cells (OPCs), and functional interactions between neurons and OLs as well as between neurons and OPCs are critical for CNS function and health. A specialized but very poorly understood interaction between neurons and OPCs occurs at the neuron-OPC synapse: nearly all OPCs form synapses postsynaptically to neurons. OPCs differ from mature neurons in many ways: they migrate, frequently remodel processes, and are capable of transforming their processes into myelin sheaths. These unique cellular features raise questions as to whether neuron-OPC synapses adapt to an OPC's unique biology and employ distinct mechanisms for synapse development. Despite previous EM and electrophysiological characterizations, almost nothing is known about synapse development in OPCs, the molecular mechanisms that govern neuron-OPC synapse formation, and how signaling via neuron-OPC synapses influences myelination. Here we propose to use zebrafish to investigate neuron-OPC synapse development, the relationship of these synapses to myelination, and to probe the underlying molecular mechanisms regulating these processes. Zebrafish provide unparalleled optical clarity for in vivo imaging and powerful tools for rapid genetic manipulations. We have identified the presence of two postsynaptic scaffolds, PSD-95 and gephyrin, at neuron-OPC synapses and generated new tools to label synapses containing these scaffolds in OPCs. Our preliminary results suggest unique synapse assembly and disassembly mechanisms in OPCs and highlight potential roles for synapses in OPC development and myelination. In this application, we will determine if and how neuron-OPC synapses are correlated with OPC differentiation and subsequent myelination (Aim 1). We will also employ cell-specific knockdown approaches to identify genes that are critical for synapse development and assess their roles in OPC biology (Aim 2). Together, our work can define previously unknown functions for neuron-OPC synapses and reveal important mechanisms that mediate neuron-glial interactions in the vertebrate CNS.

SUMMARY Astrocytes are the most abundant glial cell type in the human brain and are critical for central nervous system (CNS) development and function. Mature astrocytes are unusually elaborate cells, with an intricate and ramified morphology. Their numerous fine cellular processes interact closely with synapses, neuronal cell bodies, axons, blood vessels, and other glial cells throughout the CNS. Through these interactions, astrocytes fulfil diverse functions to support and enhance neuronal activity, maintain CNS homeostasis, and modulate circuits. Underscoring the importance of proper astrocyte development, defects in astrocyte growth or loss of astrocyte complexity are implicated in many neurological diseases, including Alexander's disease, autism, and epilepsy. However, it remains poorly understood how astrocytes develop their intricate morphological associations and regulate neural circuit function. Our long-terms goals are to understand how astrocyte acquire their remarkable morphology, target their processes to synapses, and use these cell-cell contacts to modulate brain function. We recently performed a genetic screen in Drosophila to identify new regulators of astrocyte development, and uncovered a novel gene, Trapped in endoderm 1 (Tre1), as required for astrocyte morphogenesis. We find that loss of Tre1 leads to severely reduced astrocyte complexity in vivo, resulting in decreased infiltration of the synaptic neuropil. Tre1 encodes a G protein-coupled receptor (GPCR) with no known function in the CNS. This proposal will use a synergistic combination of molecular-genetic tools available in Drosophila and zebrafish along with new tools we have generated and in vivo imaging to: determine how Tre1 regulates astrocyte morphogenesis, function, and animal behavior in Drosophila (Aim 1); elucidate signaling pathways upstream and downstream of Tre1 activation (Aims 1+2); and define the evolutionary conservation of Tre1 in vertebrates (Aim 3). Our work will provide exciting new insights into the mechanisms regulating astrocyte development and function in vivo and lay the foundation for understanding astrocyte growth and dysfunction in human disease.