Monitoring cellular redox signaling and oxidant stress in vivo

Summary

Principal Investigator: Paul T Schumacker
Abstract: Healthy cells use low levels of reactive oxygen species (ROS) as second messengers in signal transduction pathways. High levels of ROS cause oxidative damage to proteins, lipids and DNA. Oxidant stress has been implicated in the cellular dysfunction associated with ischemia- reperfusion injury, vascular disease, stroke, diabetes, neurodegenerative diseases, liver disease, renal disease, inflammation, cancer, and other disorders. As the awareness of the role of redox stress in health and disease has grown, the demand for new tools to monitor oxidant stress in vivo has increased. Current methods to assess redox events are limited by their inability to provide quantitative data or spatial information on the subcellular sites of oxidant generation. Moreover, existing probes are generally unsuitable for in vivo studies. New methods to monitor intracellular oxidant stress in intact tissues could enhance our understanding of how cell-cell interactions and tissue microenvironments influence the generation of ROS. We propose to create a new system to detect redox status and oxidative stress in specific cells within intact tissues, using a novel combination of existing methods. In Aim 1 we will create transgenic mice with DNA encoding the redox-sensitive fluorescent protein, RoGFP, inserted at a LoxP-silenced ROSA26 genomic locus. Three lines will be generated, which target the RoGFP sensor to cytosol, mitochondrial matrix, or mitochondrial intermembrane space. In Aim 2 we will activate expression of the RoGFP genes in primary cells cultured from these mice, using Cre recombinase to delete the stop codon. We will confirm correct targeting of the expressed protein, and confirm its function in response to redox stress. In Aim 3 we will breed the RoGFP mice with smooth muscle-specific Cre recombinase mice, to elicit RoGFP expression in pulmonary artery smooth muscle cells in the lung. Using that model system to demonstrate efficacy, we will measure redox changes in smooth muscle cells in the intact lung during ventilation with different concentrations of oxygen. Two-photon microscopy will be used to assess the redox status of the subcellular targeted RoGFP proteins in vivo. These animals will therefore provide exciting new tools that will enable us, and other investigators, to monitor subcellular oxidative stress in intact tissue in diverse cell types and disease models. Public Health Relevance Statement: Healthy cells in the body use oxygen free radicals (Reactive Oxygen Species, or ROS) to regulate various cellular functions. Excessive levels of ROS disrupt cell function, and they contribute to cellular injury in a large number of diseases. To understand how ROS affect cells, it is essential to monitor their levels. However, current tools are limited in their ability to assess intracellular ROS. We propose to correct this problem by inserting a gene encoding an ROS-sensitive fluorescent protein into mice. When the gene is turned on, the cell will generate a protein that moves to a known intracellular compartment and signals a change in ROS levels by altering its fluorescence. We will turn this gene on in certain types of cells in the mouse, and measure the fluorescence changes using a form of microscopy that can "see" deeply into intact tissues. We will test the performance of this sensor in the lungs, where we will measure the ROS response to changes in the concentration of oxygen that the animal is breathing. However, many other investigators will be able to use the same mice where, by turning on the reporter gene in other cell types, it will be possible to monitor ROS in a wide range of different tissues. Hence, this mouse will provide useful information on ROS levels in a wide range of disease models. The successful outcome of this project is supported by extensive preliminary studies demonstrating the feasibility of each step in the process. The end result should significantly extend our ability to assess ROS in intact tissues, in animal models of disease.
Funding Period: ----------------2009 - ---------------2011-
more information: NIH RePORT

Top Publications

  1. pmc ROS-mediated PARP activity undermines mitochondrial function after permeability transition pore opening during myocardial ischemia-reperfusion
    Jacqueline M Schriewer
    Division of Neonatology, Department of Pediatrics, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA
    J Am Heart Assoc 2:e000159. 2013
  2. ncbi Developmental differences in hyperoxia-induced oxidative stress and cellular responses in the murine lung
    Sara K Berkelhamer
    Department of Pediatrics, Northwestern University, Chicago, IL 60611, USA Electronic address
    Free Radic Biol Med 61:51-60. 2013
  3. pmc Mitochondria are required for antigen-specific T cell activation through reactive oxygen species signaling
    Laura A Sena
    Department of Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA
    Immunity 38:225-36. 2013
  4. pmc Calcium, bioenergetics, and neuronal vulnerability in Parkinson's disease
    D James Surmeier
    Department of Physiology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA
    J Biol Chem 288:10736-41. 2013
  5. pmc Physiological phenotype and vulnerability in Parkinson's disease
    D James Surmeier
    Department of Physiology, Northwestern University, Chicago, Illinois, USA
    Cold Spring Harb Perspect Med 2:a009290. 2012
  6. pmc Lung cell hypoxia: role of mitochondrial reactive oxygen species signaling in triggering responses
    Paul T Schumacker
    Department of Pediatrics, Division of Neonatology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA
    Proc Am Thorac Soc 8:477-84. 2011
  7. pmc The role of calcium and mitochondrial oxidant stress in the loss of substantia nigra pars compacta dopaminergic neurons in Parkinson's disease
    D J Surmeier
    Department of Physiology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA
    Neuroscience 198:221-31. 2011
  8. ncbi Oxidant stress evoked by pacemaking in dopaminergic neurons is attenuated by DJ-1
    Jaime N Guzman
    Department of Physiology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA
    Nature 468:696-700. 2010
  9. pmc Menadione triggers cell death through ROS-dependent mechanisms involving PARP activation without requiring apoptosis
    Gabriel Loor
    Department of Surgery, University of Chicago, Chicago, IL 60637, USA
    Free Radic Biol Med 49:1925-36. 2010
  10. pmc Is enough oxygen too much?
    Paul T Schumacker
    Division of Neonatology, Department of Pediatrics, Northwestern University, Feinberg School of Medicine, 310 E, Superior St, Morton Bldg 4 685, Chicago, IL 60611, USA
    Crit Care 14:191. 2010

Scientific Experts

  • Paul Schumacker
  • DALTON SURMEIER
  • Gregory B Waypa
  • Jacqueline M Schriewer
  • Sara K Berkelhamer
  • Laura A Sena
  • Navdeep S Chandel
  • Gabriel Loor
  • Jyothisri Kondapalli
  • Jaime N Guzman
  • Amit Jairaman
  • Gina A Kim
  • Clara Bien Peek
  • Robin H Steinhorn
  • Teresa Ezponda
  • Lyubov Czech
  • Harris Perlman
  • Sha Li
  • Jonathan D Licht
  • Paul J Bryce
  • Murali Prakriya
  • Josiah E Radder
  • Stephen Wedgwood
  • Chyung Ru Wang
  • Joseph Bass
  • David A Hildeman
  • Javier Sanchez-Padilla
  • Ema Ilijic
  • David Wokosin
  • Terry L Vanden Hoek

Detail Information

Publications11

  1. pmc ROS-mediated PARP activity undermines mitochondrial function after permeability transition pore opening during myocardial ischemia-reperfusion
    Jacqueline M Schriewer
    Division of Neonatology, Department of Pediatrics, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA
    J Am Heart Assoc 2:e000159. 2013
    ..We therefore wanted to determine whether oxidant stress, mPTP opening, and PARP activity contribute to the same death pathway after myocardial I/R...
  2. ncbi Developmental differences in hyperoxia-induced oxidative stress and cellular responses in the murine lung
    Sara K Berkelhamer
    Department of Pediatrics, Northwestern University, Chicago, IL 60611, USA Electronic address
    Free Radic Biol Med 61:51-60. 2013
    ..Therapies for neonatal and adult oxidative lung injury should therefore consider and address developmental differences in oxidative stress responses. ..
  3. pmc Mitochondria are required for antigen-specific T cell activation through reactive oxygen species signaling
    Laura A Sena
    Department of Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA
    Immunity 38:225-36. 2013
    ..Thus, mitochondrial metabolism is a critical component of T cell activation through the production of complex III ROS...
  4. pmc Calcium, bioenergetics, and neuronal vulnerability in Parkinson's disease
    D James Surmeier
    Department of Physiology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA
    J Biol Chem 288:10736-41. 2013
    ....
  5. pmc Physiological phenotype and vulnerability in Parkinson's disease
    D James Surmeier
    Department of Physiology, Northwestern University, Chicago, Illinois, USA
    Cold Spring Harb Perspect Med 2:a009290. 2012
    ....
  6. pmc Lung cell hypoxia: role of mitochondrial reactive oxygen species signaling in triggering responses
    Paul T Schumacker
    Department of Pediatrics, Division of Neonatology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA
    Proc Am Thorac Soc 8:477-84. 2011
    ..Although the specific targets of reactive oxygen species signals are not fully understood, this signaling pathway is critical for development and for normal lung responses in the newborn and the mature lung...
  7. pmc The role of calcium and mitochondrial oxidant stress in the loss of substantia nigra pars compacta dopaminergic neurons in Parkinson's disease
    D J Surmeier
    Department of Physiology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA
    Neuroscience 198:221-31. 2011
    ....
  8. ncbi Oxidant stress evoked by pacemaking in dopaminergic neurons is attenuated by DJ-1
    Jaime N Guzman
    Department of Physiology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA
    Nature 468:696-700. 2010
    ..Because drugs approved for human use can antagonize calcium entry through L-type channels, these results point to a novel neuroprotective strategy for both idiopathic and familial forms of Parkinson's disease...
  9. pmc Menadione triggers cell death through ROS-dependent mechanisms involving PARP activation without requiring apoptosis
    Gabriel Loor
    Department of Surgery, University of Chicago, Chicago, IL 60637, USA
    Free Radic Biol Med 49:1925-36. 2010
    ..These studies suggest that multiple redundant cell death pathways are activated by menadione, but that PARP plays an essential role in mediating each of them...
  10. pmc Is enough oxygen too much?
    Paul T Schumacker
    Division of Neonatology, Department of Pediatrics, Northwestern University, Feinberg School of Medicine, 310 E, Superior St, Morton Bldg 4 685, Chicago, IL 60611, USA
    Crit Care 14:191. 2010
    ..They suggest that 'permissive hypoxia' represents a therapeutic possibility. But before we turn down the inspired O2 levels we should consider the broader effects of hypoxia on tissue repair in critical illness...
  11. pmc Hypoxia-induced changes in pulmonary and systemic vascular resistance: where is the O2 sensor?
    Gregory B Waypa
    Department of Pediatrics, Division of Neonatology, Northwestern University, Morton Building 4 685, 310 East Superior St, Chicago, IL 60611, USA
    Respir Physiol Neurobiol 174:201-11. 2010
    ....