Robert Hill, Ph.D. - US grants

DESCRIPTION (provided by applicant): Myelin is a fundamental component of mature neural networks that is affected in a large number of pathological conditions of the central nervous system (CNS). Critical for advancing knowledge about these conditions would be a better in vivo understanding of how oligodendrocytes and their respective myelin sheaths develop, are maintained throughout life and respond to injury. Growing evidence suggests neuronal activity and proper astrocyte function may play crucial roles in these processes. We have developed a new technique that allows high resolution label-free in vivo imaging of myelinated axons. This technique takes advantage of the high refractive index of lipid rich multilayered myelin and is based on spectral confocal reflectance (SCoRe) microscopy. Using SCoRe imaging I have obtained for the first time long-term images of the dynamics of cortical myelin on the cellular scale in a living animal. Preliminary data shows this technique as well as fluorescence imaging of oligodendrocytes, axons and astrocytes are a powerful set of tools that in combination provide a wealth of information about fine structural dynamics of these structures in vivo. I demonstrate the feasibility to track long- term changes in internode length in addition to documenting the temporal dynamics of demyelination after single oligodendrocyte and astrocyte ablation. I propose to use these powerful techniques to address three fundamental questions concerning the in vivo plasticity and regeneration of myelin and oligodendrocytes. First, I will determine the long term plasticity of myelin and oligodendrocytes in the mouse cortex. Next I will determine the effects of neuronal activity on myelin formation, plasticity and oligodendrocyte regeneration. Finally I will use single cell ablation techniques to determine if astrocytes are required for or alter the temporal dynamics of oligodendrocyte remyelination in vivo. Together these experiments will describe for the first time the longitudinal dynamics and regeneration capabilities of oligodendrocytes and myelin on the cellular scale. Furthermore these experiments will reveal how changes in axons and astrocytes influence these dynamics over weeks to months in the living brain.

Myelin formation and maintenance is vital for proper neuronal communication and its disruption is associated with numerous diseases of the central nervous system. Oligodendrocytes make myelin and are the only cells in the adult cerebral cortex that are continuously generated from a population of resident progenitors, called NG2 cells. Thus, protracted oligodendrocyte and myelin formation into adulthood constitutes a unique, understudied system for adult neuroplasticity, with broad implications for human cognition and disease. Understanding the process of oligodendrocyte generation is fundamental to dissect roles played by oligodendrocytes and myelination in nervous system function, plasticity, and disease. We have a rudimentary understanding of how new oligodendrocytes are generated in vivo. Reasons for this stem from inadequate tools for their dynamic investigation in the live brain. In light of these challenges, my long-term goals are to develop and apply optical and single cell molecular based approaches to dissect multicellular interactions in the intact developing and diseased nervous system, with a primary focus on the interface between the axon and oligodendrocyte. Realization of this goal has begun as we have now developed a range of novel complementary tools that allow unprecedented detailed investigation into the transformation of single progenitor cells into gap junction-coupled, mature myelinating oligodendrocytes in vivo. This proposal will implement and expand on these tools to ask several fundamental questions basic to our understanding of adult nervous system plasticity and response to injury. First, during the K99 phase, the in vivo dynamics of oligodendrocyte differentiation, gap junction coupling and internode assembly during initial myelin formation and after a demyelinating event will be determined. Next, a new method will be used to determine the developmental profile, longitudinal dynamics, and effects of demyelination on internode and Node of Ranvier assembly and distribution along extensive stretches of single axons. Finally, during the R00 phase, using a powerful combination of in vivo imaging and single cell molecular manipulation techniques learned during the K99 training period, the effects of myelin deposition on dynamic axonal structural plasticity will be tested. Overall the research portions of this proposal will uncover how functional internodes initially form, restructure throughout life, respond to oligodendrocyte death, and interact with the axon to influence its structural plasticity, all for the first time in the live brain. The aims set out in this proposal will provide the foundation for implementing these in vivo optical tools during the R00 phase. Furthermore this strategy will provide fundamental training in novel approaches for molecular design with unique intellectual, professional and academic guidance during the K99 phase under the mentorship of Dr. Jaime Grutzendler in collaboration with consultant Dr. Anthony Koleske and the vibrant neuroscience research environment at Yale University. The combination of learning a new set of molecular approaches and implementing our powerful in vivo imaging platform will ensure a unique skillset and perspective, critical components for a successful career as an independent investigator.

PROJECT SUMMARY Myelin has evolved to speed up, finely tune, and increase the metabolic efficiency of electrical signal transmission in the brain. In numerous human diseases however, myelin degenerates, ultimately resulting in devastating motor and cognitive impairment. Importantly, in order for tissue repair to proceed after myelin damage has occurred, the many layers of compacted cell membrane that constitute the myelin sheath must be rapidly and efficiently removed by resident phagocytic cells in the brain. Defective removal of these debris has been implicated in a number of degenerative conditions, including but not limited to, multiple sclerosis and aging, yet we know little about the cellular dynamics and molecular mechanisms governing these processes. In order to study these critical cellular events and answer questions centered on which cell populations are involved and what roles these different cell types play, we have developed advanced techniques for imaging and manipulating these discrete events in the live animal over a wide range of temporal scales from seconds to months. These techniques include intravital imaging of new combinations of fluorophore-based multicolor transgenic labels of distinct populations of neurons and glia together with label-free imaging modalities specific for compact myelin. In addition to these powerful labeling and optical imaging strategies, we have also developed a new technique for targeted induction of single-cell death, which we have recently established as a model of on-demand and titratable demyelination in the mouse cortical gray matter. Combining these techniques now allows dynamic investigation of demyelination and remyelination in the context of targeted genetic manipulations and animal models of human disease. Using these powerful tools this project will investigate three central aims. First, there is increasing evidence that in addition to microglia, the primary phagocytes of the brain, other resident glial cell types, namely astrocytes and NG2 glia, are also involved and play important roles in the phagocytosis and repair process. We will determine the precise contribution of each glial cell type in the dynamic detection and clearance of degenerating myelin debris. Next, we and others have shown the importance of phosphatidylserine receptors in the efficient detection and clearance of dying neurons and other cells in different organs. We will determine the role and consequences of both defective phagocytic receptors and debris digestion signaling on the dynamic response by NG2 glia to cortical demyelination and the resulting remyelination success and myelin patterning. Finally, there is evidence that neuronal activity and/or sensory experience can modify remyelination, but less is known about the roles of neuronal activity on phagocytic function in the context of demyelination. We will determine the consequences of bidirectional neuronal activity changes on the response by phagocytic cells to single-cell demyelination. Ultimately, these studies will reveal which cells are involved in myelin debris clearance, the role of major cell debris recognition pathways in successful clearance and repair, and how neuronal activity and sensory experience modify the response of phagocytic glia to a demyelinating event.