title: Spatial, temporal, and quantitative manipulation of intracellular hydrogen peroxide in cultured cells.
abstract: Hydrogen peroxide (H2O2) is produced endogenously in a number of cellular compartments, including the mitochondria, the endoplasmic reticulum, peroxisomes, and at the plasma membrane, and can play divergent roles as a second messenger or a pathological toxin. It is assumed that the tuned production of H2O2 within neuronal and nonneuronal cells regulates a discreet balance between survival and death. However, a major challenge in understanding the physiological versus pathological role of H2O2 in cells has been the lack of validated methods that can spatially, temporally, and quantitatively modulate H2O2 production. A promising means of regulating endogenous H2O2 is through the expression of peroxide-producing enzyme d-amino acid oxidase (DAAO from Rhodotorula gracilis lacking a peroxisomal targeting sequence). Using viral vectors to express DAAO in distinct cell types and using targeting sequences to target DAAO to distinct subcellular sites, we can manipulate H2O2 production by applying the substrate d-alanine or permeable analogs of d-alanine. In this chapter, we describe the use of DAAO to produce H2O2 in culture models and the real-time visual validation of this technique using two-photon microscopy and chemoselective fluorescent probes.
authors.affiliation: Department of Neurology and Neuroscience, The Burke Medical Research Institute, Weill Medical College of Cornell University, White Plains, New York, USA.
authors.forename: Ishraq
authors.initials: I
authors.lastname: Alim
authors.affiliation: Department of Neurology and Neuroscience, The Burke Medical Research Institute, Weill Medical College of Cornell University, White Plains, New York, USA; Department of Health and Natural Sciences, Mercy College, Dobbs Ferry, New York, USA.
authors.forename: Renee E
authors.initials: RE
authors.lastname: Haskew-Layton
authors.affiliation: Department of Neurology and Neuroscience, The Burke Medical Research Institute, Weill Medical College of Cornell University, White Plains, New York, USA; Fishberg Department of Neuroscience, Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, USA.
authors.forename: Hossein
authors.initials: H
authors.lastname: Aleyasin
authors.affiliation: Department of Neurology and Neuroscience, The Burke Medical Research Institute, Weill Medical College of Cornell University, White Plains, New York, USA; Fischell Department of Bioengineering, University of Maryland, College Park, Maryland, USA.
authors.forename: Hengchang
authors.initials: H
authors.lastname: Guo
authors.affiliation: Department of Neurology and Neuroscience, The Burke Medical Research Institute, Weill Medical College of Cornell University, White Plains, New York, USA. Electronic address: rrr2001@med.cornell.edu.
authors.forename: Rajiv R
authors.initials: RR
authors.lastname: Ratan
mesh_terms: D000409:Alanine; D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000378:metabolism; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D003605:D-Amino-Acid Oxidase / Q000235:genetics / Q000378:metabolism*; D004867:Equipment Design; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000032:analysis* / Q000378:metabolism*; D036641:Microscopy, Fluorescence, Multiphoton / Q000295:instrumentation / Q000379:methods*; D008928:Mitochondria / Q000378:metabolism; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D012248:Rhodotorula / Q000201:enzymology; D014161:Transduction, Genetic / Q000379:methods
chemical_list: D005456:Fluorescent Dyes; D005182:Flavin-Adenine Dinucleotide; D006861:Hydrogen Peroxide; D003605:D-Amino-Acid Oxidase; D000409:Alanine
keywords: Hydrogen Peroxide; Oxidative Stress; ROS; astrocytes; neurons