Celia Shiau - US grants

DESCRIPTION (provided by applicant): Macrophages and microglia are phagocytic cells ofthe innate immune system that have important roles in the nervous system. Myelinating glial cells wrap axons with the myelin sheath and thereby allow for fast axonal conduction. Abnormal function of macrophages and microglia have been implicated in many neurodegenerative and myelin-related diseases, such as multiple sclerosis and peripheral neuropathies. Interestingly, aberrant activation of macrophages and myelin phagocytosis are prominent in demyelinating diseases, suggesting interactions between macrophages and myelinating glia. However, very little is known about the molecular and cellular mechanisms underlying the interactions between macrophages, microglia, and myelinated axons. The goal of the proposed research is to begin to dissect these relationships on a cellular and genetic level, using the zebrafish as the model system to address questions about the role of macrophages and microglia during development of the myelinated axons. Macrophages and microglia are present and functional in the vertebrate embryo from an early stage, but the roles of these cells in normal development have not been well characterized. This project will investigate the hypothesis that these immune cells play an essential role in development of myelinated axons. In light ofthe known role of macrophages in removing axonal and myelin debris after injury, the first aim is to test whether this same function occurs in the embryo even in undamaged nerves by analyzing the organization and ultrastructure of myelinated axons in zebrafish mutants lacking macrophages. This experiment will provide information on what role(s) macrophage and microglia may have during normal development of the myelinated axons. To identify genes involved in macrophage function and axonal myelination, a genetic screen will be conducted using the zebrafish model system. Mutants will be screened for defects in macrophage distribution, activation, and number in addition to myelination using known markers. Finally, a few mutated genes will be studied in depth. Phenotypic studies, including marker studies, cell transplantation, uitrastructural analysis, macrophage activation analyses, and time-lapse imaging if the effect involves abnormal cell migration, will define the function of the mutated genes at the cellular level. Genetic mapping and positional cloning will identify the genes and help define their functions at the biochemical level. These experiments will provide new insights into the mechanisms that dictate macrophage and microglia function and their relationship with myelination. This project will cast light on the potential causes of a wide array of neurological disorders derived from aberrant immune function, and may lead to new therapeutic approaches.

Project Summary Understanding how a cell is switched off and maintains its quiescence is fundamentally as important as how it is activated. The long-term goal of the proposed research is to determine how cell-intrinsic processes control and modulate activation states of macrophages in vivo. Because dysregulation and unprovoked activation of the immune system cause a host of human diseases associated with inappropriate inflammation, deciphering the molecular networks regulating immune activation normally is critical for addressing these health challenges. This will lead to new knowledge and technologies needed to harness the properties of macrophages for disease prevention and treatment. We are leveraging the unique advantages of the highly tractable vertebrate model system, Danio rerio, for exquisite genetic manipulations, high throughput screening, and in vivo imaging to dissect the complex relationship between intrinsic metabolic signaling and macrophage activation. The proposal encompasses a series of projects that collectively define essential negative regulators and their functions for keeping the innate immune system in check to maintain a normal equilibrium in macrophages. The starting basis of our projects stems from emerging evidence that metabolic and immune signaling pathways intersect to shape immune activation in macrophages, and a discovery of a null mutation in an intracellular NOD-like receptor (NLR) in zebrafish. A gene inactivation in this novel NLR, nlrc3l, causes unprovoked macrophage activation possibly due to metabolic dysregulation. The proposal seeks to define the network of molecular interactions of nlrc3l to understand this very important mechanism that keeps macrophages in check under normal biological conditions. We are taking a highly integrated approach at multiple levels-- using differential transcriptomics, proteomics, and metabolomics to inform candidate genes and pathways that constitute possible interactors and effectors of nlrc3l, and validating interactions using genetic mutants and biochemical studies. The proposal will also use the power of a forward genetic screen to discover additional genes akin to nlrc3l that prevent macrophage activation at steady state that act in the same or completely new pathways. We designed an innovative assay for the screen to assess macrophage activation using a live-cell reporter for an activation marker irg1. Finally, the proposal will examine the influence of lipid and glucose metabolic pathways on macrophage activation in zebrafish using genetic analyses and chemical screening. This work will benefit from collaboration with an expert group in extending our findings to mouse and human models. Taken together, these projects provide the important foundation for understanding the genetic and metabolic basis of how the innate immune system is kept in check, and will impact the direction of my lab far beyond the 5 years of MIRA funding.