Iron-Induced Oxidative Stress and Rotenone Modify Alphasynuclein Phosphorylation in a Cellular Model of Parkinson's Disease

Abstract

Event Abstract Back to Event Iron-Induced Oxidative Stress and Rotenone Modify Alphasynuclein Phosphorylation in a Cellular Model of Parkinson's Disease Rita Perfeito1*, Catarina R. Oliveira1, 2 and Cristina A. Rego1, 2 1 University of Coimbra, Center for Neuroscience and Cell Biology, Portugal 2 University of Coimbra, Institute of Biochemistry, Faculty of Medicine, Portugal Accumulation of alpha-synuclein (alpha-syn) as intracellular filamentous aggregates is a pathological feature of Parkinson´s disease (PD). These aggregates localize in neuronal cytoplasm as Lewy bodies (LBs), mainly composed of alpha-syn, ubiquitin and synphilin-1, and are associated with degeneration of dopaminergic neurons in both sporadic and familial forms of PD. Furthermore, changes in alpha-syn post-translational modifications, as well as mitochondrial dysfunction (through inhibition of complex I) and oxidative stress constitute key pathogenic events of this disorder. Indeed, brains of PD patients contain more iron in the substantia nigra than normal controls, and LBs from surviving neurons display oxidative damage. Since mitochondrial dysfunction and oxidative stress have been linked to PD, and alpha-syn includes several sites susceptible to undergo post-translational modifications, the aim of this work was to analyse the role of FeSO4 and rotenone (complex I inhibitor) on alpha-syn phosphorylation and aggregation, and the formation of reactive oxygen species. We used a human neuroblastoma cell line (SH-SY5Y) transiently transfected with wild type (WT) and mutant A53T alpha-syn, linked to eGFP, as a PD model. Results showed that prolonged exposure (4 days) to FeSO4 increased phosphorylation at Ser129, particularly of mutant A53T alpha-syn. Similar results were observed in the presence of rotenone, whereas phosphorylation at Tyr125 was not affected. Analysis of cell death with propidium iodide indicated that the chosen concentrations of iron caused approximately 1% necrotic cell death, whereas rotenone caused very little cell death. Aggregate formation was also examined by confocal microscopy. Prolonged iron and rotenone exposure did not cause the formation of Ub-labelled alpha-syn aggregates in SH-SY5Y cells, suggesting that alpha-syn phosphorylation does not correlate with protein aggregation under these experimental conditions. However, in cells expressing WT or A53T alpha-synGFP, and further exposed to iron or rotenone, we observed a decrease in MitoTracker Red fluorescence, suggesting mitochondrial depolarization. Preliminary data using the fluorescent probe dichlorodihydrofluorescein-diacetate (DCFH2-DA) showed that short incubations (up to 2h) with FeSO4 increased the formation of endogenous hydroperoxides in SH-SY5Y cells expressing WT alpha-synGFP and this effect was more pronounced in cells expressing A53T alpha-synGFP. These data suggest that early ROS formation may be important for defining subsequently alpha-syn phosphorylation, which precede alphasyn aggregation and prominent cell death in PD.Supported by: SFRH/BD/25515/2005 FCT Conference: 11th Meeting of the Portuguese Society for Neuroscience, Braga, Portugal, 4 Jun - 6 Jun, 2009. Presentation Type: Poster Presentation Topic: Neurodegenerative Disorders Citation: Perfeito R, Oliveira CR and Rego CA (2009). Iron-Induced Oxidative Stress and Rotenone Modify Alphasynuclein Phosphorylation in a Cellular Model of Parkinson's Disease. Front. Neurosci. Conference Abstract: 11th Meeting of the Portuguese Society for Neuroscience. doi: 10.3389/conf.neuro.01.2009.11.061 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 07 Aug 2009; Published Online: 07 Aug 2009. * Correspondence: Rita Perfeito, University of Coimbra, Center for Neuroscience and Cell Biology, Alicante, Portugal, rita_perfeito@hotmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Rita Perfeito Catarina R Oliveira Cristina A Rego Google Rita Perfeito Catarina R Oliveira Cristina A Rego Google Scholar Rita Perfeito Catarina R Oliveira Cristina A Rego PubMed Rita Perfeito Catarina R Oliveira Cristina A Rego Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.