title: In vitro and in vivo aggregation of a fragment of huntingtin protein directly causes free radical production.
abstract: Neurodegenerative diseases are characterized by intra- and/or extracellular protein aggregation and oxidative stress. Intense attention has been paid to whether protein aggregation itself contributes to abnormal production of free radicals and ensuing cellular oxidative damage. Although this question has been investigated in the context of extracellular protein aggregation, it remains unclear whether protein aggregation inside cells alters the redox homeostasis. To address this, we have used in vitro and in vivo (cellular) models of Huntington disease, one of nine polyglutamine (poly(Q)) disorders, and examined the causal relationship among intracellular protein aggregation, reactive oxygen species (ROS) production, and toxicity. Live imaging of cells expressing a fragment of huntingtin (httExon1) with a poly(Q) expansion shows increased ROS production preceding cell death. ROS production is poly(Q) length-dependent and not due to the httExon 1 flanking sequence. Aggregation inhibition by the MW7 intrabody and Pgl-135 treatment abolishes ROS production, showing that increased ROS is caused by poly(Q) aggregation itself. To examine this hypothesis further, we determined whether aggregation of poly(Q) peptides in vitro generated free radicals. Monitoring poly(Q) protein aggregation using atomic force microscopy and hydrogen peroxide (H(2)O(2)) production over time in parallel we show that oligomerization of httEx1Q53 results in early generation of H(2)O(2). Inhibition of poly(Q) oligomerization by the single chain antibody MW7 abrogates H(2)O(2) formation. These results demonstrate that intracellular protein aggregation directly causes free radical production, and targeting potentially toxic poly(Q) oligomers may constitute a therapeutic target to counteract oxidative stress in poly(Q) diseases.
authors: Southampton Neuroscience Group, School of Biological Sciences, University of Southampton, Southampton SO17 1BJ, United Kingdom.
authors: Sarah
authors: S
authors: Hands
authors: Mohammad U
authors: MU
authors: Sajjad
authors: Michael J
authors: MJ
authors: Newton
authors: Andreas
authors: A
authors: Wyttenbach
mesh_terms: D016923:Cell Death; D002460:Cell Line; D006801:Humans; D000071058:Huntingtin Protein; D006816:Huntington Disease / Q000235:genetics / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism*; D018625:Microscopy, Atomic Force; D008954:Models, Biological; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D057127:Single-Chain Antibodies / Q000737:chemistry
chemical_list: C086055:HTT protein, human; D000071058:Huntingtin Protein; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D010455:Peptides; D057127:Single-Chain Antibodies; C097188:polyglutamine; D006861:Hydrogen Peroxide