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 biosensorexpressing 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.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Research Project (R01)
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Special Emphasis Panel (ZMH1-ERB-L (05))
Program Officer
Talley, Edmund M
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J. David Gladstone Institutes
San Francisco
United States
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Schachtrup, Christian; Ryu, Jae Kyu; Mammadzada, Könül et al. (2015) Nuclear pore complex remodeling by p75(NTR) cleavage controls TGF-? signaling and astrocyte functions. Nat Neurosci 18:1077-80
Davalos, Dimitrios; Baeten, Kim M; Whitney, Michael A et al. (2014) Early detection of thrombin activity in neuroinflammatory disease. Ann Neurol 75:303-8
Gyoneva, Stefka; Davalos, Dimitrios; Biswas, Dipankar et al. (2014) Systemic inflammation regulates microglial responses to tissue damage in vivo. Glia 62:1345-60
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Kwan, Wanda; Träger, Ulrike; Davalos, Dimitrios et al. (2012) Mutant huntingtin impairs immune cell migration in Huntington disease. J Clin Invest 122:4737-47
Davalos, Dimitrios; Ryu, Jae Kyu; Merlini, Mario et al. (2012) Fibrinogen-induced perivascular microglial clustering is required for the development of axonal damage in neuroinflammation. Nat Commun 3:1227
Davalos, Dimitrios; Akassoglou, Katerina (2012) Fibrinogen as a key regulator of inflammation in disease. Semin Immunopathol 34:43-62
Merlini, Mario; Davalos, Dimitrios; Akassoglou, Katerina (2012) In vivo imaging of the neurovascular unit in CNS disease. Intravital 1:87-94
Davalos, Dimitrios; Akassoglou, Katerina (2012) In vivo imaging of the mouse spinal cord using two-photon microscopy. J Vis Exp :e2760
Baeten, Kim M; Akassoglou, Katerina (2011) Extracellular matrix and matrix receptors in blood-brain barrier formation and stroke. Dev Neurobiol 71:1018-39

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