Original Contribution Increased oxidation, glycoxidation, and lipoxidation of brain proteins in prion disease Reinald Pamplona a, 1 , Alba Naudí a, 1 , Rosalina Gavín b , Miguel A. Pastrana c , Gustavo Sajnani c , Ekaterina V. Ilieva a , José Antonio del Río b , Manuel Portero-Otín a , Isidre Ferrer d , Jesús R. Requena c, ⁎ a Metabolic Pathophysiology Research Group, Departament de Medicina Experimental, Universitat de Lleida-IRBLLEIDA, Lleida 25008, Catalonia, Spain b Cellular and Molecular Basis of Regeneration and Neurorepair, Department of Cell Biology and Institute for Research in Biomedicine, University of Barcelona, Barcelona 08028, Catalonia, Spain c Prion Research Unit, Department of Medicine, School of Medicine, University of Santiago de Compostela, Santiago de Compostela 15782, Galiza, Spain d Institut de Neuropatologia, Servei de Anatomia Patològica, Hospital Universitari de Bellvitge, Hospitalet de Llobregat 08907, Catalonia, Spain abstract article info Article history: Received 12 March 2008 Revised 9 July 2008 Accepted 11 July 2008 Available online 23 July 2008 Keywords: Prion disease Protein oxidation Glutamic semialdehyde Aminoadipic semialdehyde Carboxymethyllysine Carboxyethyllysine Malondialdehydelysine Mass spectrometry The basic molecular underpinnings of the pathological changes that unfold in prion disease remain elusive. A key role of increased oxidative stress has been hypothesized. Given the transient nature of most intermediate molecules implicated, increased oxidative stress is better assessed by quantitating the damage it causes to macromolecules. We used mass spectrometry-based methods to measure specific products of protein oxidation, glycoxidation, and lipoxidation in brains from patients suffering from Creutzfeldt-Jakob disease and Syrian hamsters affected by scrapie. In both cases, increased amounts of glutamic and aminoadipic semialdehydes, products of metal-catalyzed oxidation, malondialdehydelysine (a product of lipoxidation), N- ɛ-carboxyethyllysine (a product of glycoxidation), and N-ɛ-carboxymethyllysine (generated by lipoxidation and glycoxidation) were measured. PrP Sc , the infectious isoform of the prion protein that accumulates in prion disease, was itself shown to be a target of increased oxidative modification. These changes were accompanied by alterations in fatty acid composition and increased phosphorylation of ERK 1/2 and p38, protein kinases known to respond to increased flows of ROS. These data support an important role of oxidative damage in the pathology of prion disease. © 2008 Elsevier Inc. All rights reserved. Introduction Prion diseases constitute a group of fatal neurodegenerative ailments that affect both humans and animals and are characterized by neuronal loss, astrocytic gliosis, microgliosis, spongiform change, and abnormal prion protein production and deposition [1–3]. The main prion diseases are scrapie and bovine spongiform encephalo- pathy (BSE) in animals, and Creutzfeldt-Jakob disease (CJD), fatal familial insomnia, kuru, and Gerstmann-Straussler-Scheinker disease in humans [1–3]. Prions are novel pathogenic entities that consist entirely of a misfolded protein with a self-propagating aberrant conformation [1–3]. According to the the prion paradigm, the normal (“cellular”) isoform of the prion protein, PrP C , is forced to the abnormal (“scrapie”) conformation, PrP Sc , by preexisting PrP Sc . The initial pool of PrP Sc molecules in a given brain might arise by infection, as a consequence of mutation-induced structural lability (genetic cases) or as the result of rare spontaneous misfolding events (sporadic cases). As PrP Sc propagates in the brain, neurodegeneration would ensue as a consequence of either a loss of function of PrP C or, more probably, a toxic gain of function associated with PrP Sc [1–3]; however, neither the function of PrP C nor the mechanisms by which a toxic effect of PrP Sc might be exerted are known. Among the data supporting a direct involvement of PrP Sc in the pathogenesis of prion disease are the temporal and anatomical correlations between neuropathology and PrP Sc deposition; however, this is not always the case, and exceptions have been described [4]. As with other “protein misfolding neurodegenerative diseases” [5] such as Alzhei- mer's, Parkinson's, and Huntington's diseases, PrP Sc oligomers might exert a direct pathogenic effect, or might induce more complex effects involving participation of other molecules and/or cells after recogni- tion by yet to be identified receptors [5,6]. One particularly relevant pathogenic mechanism that has been invoked in all these neurodegenerative diseases is increased Free Radical Biology & Medicine 45 (2008) 1159–1166 Abbreviations: AASA, aminoadipic semialdehyde; BSE, bovine spongiform encepha- lopathy; CEL, N-ɛ-carboxyethyllysine; CJD, Creutzfeldt-Jakob disease; CML, N-ɛ- carboxymethyllysine; DBI, double bond index; GSA, glutamic semialdehyde, 4-HNE, 4-hydroxynonenal; MCO, metal-catalyzed oxidation; MDAL, malondialdehydelysine; MUFA, monounsaturated fatty acids; PI, peroxidizability index; PrP, prion protein; PrP Sc , prion protein, “scrapie” isoform; PrP C , prion protein, “cellular” isoform; PUFA, polyunsaturated fatty acids; ROS, reactive oxygen species; SFA, saturated fatty acids, UFA, unsaturated fatty acids. ⁎ Corresponding author. Fax: +34 981 559904. E-mail address: jesus.requena@usc.es (J.R. Requena). 1 These authors contributed equally to this work. 0891-5849/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2008.07.009 Contents lists available at ScienceDirect Free Radical Biology & Medicine journal homepage: www.elsevier.com/locate/freeradbiomed