In vivo imaging by two-photon microscopy has revolutionized our understanding of neurobiological and immunological processes and revealed unexpected information on the dynamic morphology of central nervous system structures and immune processes in health and disease. Currently, intracellular signaling cascades can only be studied in isolated cells or slices of brain tissue. The goal of this EUREKA proposal is to image signal transduction pathways in the living brain in mice, with the aim to study signaling events as they are regulated in real time by trauma, inflammation, and hypoxia. In this proposal we will (1) develop genetically modified biosensor expressing mice to image the activation of five major signal transduction pathways-redox changes, NFB, Rho, PKA, and Ras- in microglia and macrophages in vivo;(2) generate novel genetically encoded biosensors for the PKA, Rho, and Ras pathways, optimized for in vivo imaging by two-photon microscopy;(3) develop the first pharmacologic tools (biosensor-conjugated Quantum Dots) to deliver biosensors to microglia and macrophages in the brain;and (4) demonstrate dynamic signaling events in microglia in living mouse brain in real time during hypoxia, inflammation, or traumatic injury. The tools we propose to develop will fundamentally change the methods we use to study the dynamic activation of intracellular signaling pathways. Results from this proposal will elucidate inflammatory signal transduction pathways in an unprecedented way-by correlating molecular mechanisms of microglial activation with dynamic morphologic alterations in response to extracellular stimuli in the brain of a living animal. This proposal will generate tools to make possible imaging of signal transduction in vivo. It will make available to the scientific community novel biosensors, delivery methods, and animal models to study the temporal and spatial activation of five major signal transduction pathways in the living animal. In vivo imaging of signal transduction pathways in microglia and macrophages can be applied to a wide range of diseases with an inflammatory component, including cancer, diabetes, asthma, infection, and autoimmune disorders, and nervous system disorders characterized by microglia activation, such as multiple sclerosis, Alzheimer's disease, amyotrophic lateral sclerosis, pain, and spinal cord injury.

Public Health Relevance

The goal of this EUREKA proposal is to develop novel tools and image signal transduction pathways in the brain of the living animal with the aim to study signaling events as they are regulated in real time by trauma, inflammation, and hypoxia. This proposal will generate biosensor-expressing animal models and pharmacologic tools to enable imaging of five major signaling pathways-redox changes, NF-B, Rho, PKA, and Ras-in microglia and macrophages by two-photon microscopy. These tools will become available to the scientific community to enable studies that will unravel the sequence of signaling events and their causative relationships that lead to disease and provide information to design and test effective treatments.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS066361-04
Application #
8294755
Study Section
Special Emphasis Panel (ZMH1-ERB-L (05))
Program Officer
Talley, Edmund M
Project Start
2009-07-01
Project End
2014-06-30
Budget Start
2012-07-01
Budget End
2014-06-30
Support Year
4
Fiscal Year
2012
Total Cost
$382,000
Indirect Cost
$182,000
Name
J. David Gladstone Institutes
Department
Type
DUNS #
099992430
City
San Francisco
State
CA
Country
United States
Zip Code
94158
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