Neurotoxicity induced by oxidative stress and aluminium in relation to parkinson's diseasea mitochondrial bioenergetic approach by high-resolution respirometry

Resumo

The study of mitochondria during the last years has revealed that this organelle plays an important role in the development and onset of neurodegenerative disorders such as Parkinson¿s disease. The main hallmarks found in post¿mortem studies remark the importance of brain free radicals levels, the alteration in brain metabolic parameters and the presence of xenobiotics like metals for the neurodegenerative processes. Also, any energetic deficit related with the metabolic impairment due to an exposition to toxics or an increase in oxidative stress might be a favorable condition for the onset of Parkinson¿s disease. Within this framework, and in order to gain insight in the aetiology of Parkinson¿s disease, this Thesis was developed as a bioenergetics study focused to oxidative stress, Parkinson¿s disease, and aluminium. In the first part of the study we set¿up an isolation protocol to guarantee the mitochondria structure and function for the further respiratory studies. Our study showed that the use of 3mM EDTA in the isolation medium for differential centrifugation results essential in order to obtain an enriched and well preserved mitochondrial pellet. In addition, we evaluated 6¿hydroxydopamine capacity to alter the mitochondrial bioenergetics in order to understand which reactive oxygen species involved on its neurotoxic mechanism. Our data showed that 6¿hydroxydopamine alters mitochondria bioenergetcis (IC50 200nM) by an interaction with the metabolic pathway linked to complex I and an inhibition of ATP synthase. This effect can be prevented by the addition of antioxidant enzymes such as catalase or superoxide dismutase. In order to gain insight in the neurotoxicity induced in cells, we evaluated the induced cellular death in human neuroblastoma cells (IC50 100¿M). The analysis of the antioxidant enzymes showed that the toxicity was promoted through oxidative stress that can be prevented by the presence of superoxide dismutase. However, the presence of catalase not prevents the death of human neuroblastoma cells, probably due to the transport of the neurotoxin inside the cell. The differential between cells and isolated mitochondria suggest that the reactive oxygen species formed in the catalase pathway are more toxic than those formed when superoxide dismutase is present. For this reason we purpose the anion radical superoxide and the hydroperoxyl radical as the main radical species involved in the neurotoxic mechanism of 6¿hydroxydopamine. Also, recent studies related the presence of metals at relatively high concentrations in the patient brain as a factor involved in the pathogenesis of neurodegenerative disorders. Indeed, in a previous study our group demonstrated that Al3+ promotes brain oxidative stress in an experimental model of PD, decreases the antioxidant activity of some enzymes, and triggers neuronal death (Sánchez Iglesias et al., 2009). In our study we evaluated aluminium ability to alter mitochondrial bioenergetics in order to understand the mechanisms underlying its neurotoxicity. Our data showed that aluminium alters the activity of complexes III and V in both models studied (in vitro and in vivo) in a dose¿dependent manner. Indeed, the complex II pathway was altered at the non¿couple respiration level, revealing a high capacity of the metal to alter electron transport system. Also, a remarkable mechanism of its neurotoxicity is the ability to modify the mitochondrial membrane properties, increasing the proton leak, and in last term the dissipation of the membrane potential. Consequently, aluminium exhibit the capacity to decrease the efficiency on the respiratory capacity and evidently, this effect might promote the rise of oxidative stress and the neurotoxicity.