Project Summary/Abstract Cells of the immune system, bacteria, and neurons are constantly interacting on every surface of the human body. Recent studies show that bacteria have evolved mechanisms to engage with the nervous system, often using the phagocytes that regulate inflammation. Neuroinflammation, or inflammation in neurological tissue, is thought to contribute to common neurodegenerative conditions, including Alzheimer's disease and Parkinson's disease, although the basic mechanisms of neuroinflammation remain incompletely understood. The long-term objective of this New Innovator Award proposal is to define the molecular mechanisms of neuroinflammation during infection. The innovation is our adaptation of the cutting-edge zebrafish molecular toolkit (optogenetics, biosensors, in vivo biotinylation) to host-pathogen interactions. With this system, we can genetically modify host and pathogen, then observe bacteria invading the transparent zebrafish brain in real-time. As pathogens, we use mycobacterial species that activate neuroinflammation in humans (TB meningitis, leprosy) and in zebrafish. We have also developed new zebrafish brain infection models, using human pathogens isolated from meningitis patients, in order to determine: Project 1: How do bacteria invade the brain from the blood? Approach: Define the endothelial mechanisms of brain invasion and vascular injury. Project 2: How do host versus pathogen factors drive catastrophic brain inflammation? Approach: Optogenetic (light-activated) control of phagocytes in vivo. Project 3: How do host versus pathogen factors injure neurons? Approach: Identifying infectious mechanisms of neuronal injury using biosensors. To answer these questions, this proposal combines immunobiology and neurobiology tools with brain infection models in zebrafish. Using live imaging of zebrafish brain infection, and genetic modification of fluorescent bacteria, neurons, and phagocytes, the earliest inflammatory and neurodegenerative events can be directly observed in unprecedented detail. Completion of this work fulfills two goals. First, it will provide needed mechanistic information on the host cells and molecules that mediate injury in understudied brain infections that disproportionately affect people living in poverty. This will support the goal of developing new therapies that limit brain inflammation. Beyond infection, the basic biological mechanisms of neuroinflammation, revealed by infection, will likely be relevant to the many non-infectious neurodegenerative conditions that are characterized by neuroinflammation.