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Role of Trypanosoma cruzi mitochondrial Ca2+ in cell death

Patricia Laura Bustos

University of Georgia

Trypanosoma cruzi, the agent of Chagas disease, has a single mitochondrion and can phosphorylate ADP and take up cytosolic calcium ion (Ca2+) via a mechanism driven by an electrochemical proton potential generated by respiration. In our laboratory we have extensively studied Ca2+ as a key second messenger in protist, including T. cruzi. Ca2+ signaling appears to be important for several functions in T. cruzi, and among them, to be involved in cellular programmed cell death. The mitochondrial permeability transition pore (PTP) is apparently composed of an assembly of matrix, inner and outer membrane proteins such as the adenine nucleotide transporter, aspartate-glutamate and phosphate carriers, the voltage dependent anion channel, the CsA-binding protein cyclophilin D (CypD), hexokinase, and dimers of the ATP synthase. CypD is required for mediating Ca2+- and oxidative damage-induced cell death. There is a cyclophilin D gene ortholog in T. cruzi (TcCyp21). This protein localizes to the mitochondria. Stimulation of oxidative stress by incubating the cells in the presence of H2O2 or redox cycling agents resulted in alterations consistent with programmed cell death or apoptosis (decrease in mitochondrial membrane potential, cytochrome c translocation into the cytosol, phosphatidylserine exposure, and DNA degradation), and these effects were prevented with cyclosporin A, which is known to inhibit CyPD activity. We have also overexpressed the T. cruzi CypD in the parasites, and our aim is to test whether T. cruzi CypD is required for mediating Ca2+- and oxidative damage-mediated mitochondrial dysfunction and cell death. Our hypothesis is that the characterization of the pathway involving cell death in trypanosomes will lead to important insights into the biology of these parasites, the evolution of cell death pathways, and ultimately novel targets for anti-parasitic intervention. We will investigate the role of T. cruzi mitochondrial Ca2+ in cell death in a pioneer laboratory in the study of mitochondrial Ca2+ channels in trypanosomes. To accomplish this project, will explore whether overexpression of TcCypD (TcCypD-OE) leads to mitochondrial Ca2+ overload (measuring Ca2+ transport in permeabilized cells and isolated mitochondria using Calcium Green-5N and Ca2+ accumulation in whole cells using rhod2-AM loaded cells, loss of the mitochondrial membrane potential (using safranine O, ROS generation (measuring MitoSOX fluorescence increase, higher susceptibility to external oxidants, loss of oxidative phosphorylation (measuring changes in safranine O by ADP, ATP decrease (measure by a luciferin-luciferase assay), and release of cytochrome c and endoG. PCD will be measured with the TUNEL assay or by determining exposure of phosphatidylserine in the outer surface. We expect to detect mitochondrial Ca2+ overload and PCD in TcCypD-OE cells, suggesting a potential approach for drug targeting. We will also investigate whether TcCypD-OE cells are more susceptible to Ca2+ loading or to the presence of oxidants (t-butylhydroperoxide) and whether cyclosporin A inhibits these effects. We propose to investigate how cyclophilin D affects the mitochondrial membrane potential and Ca2+ transport and how deregulation of this pathway can lead to cell death. The study of an essential pathway in T. cruzi which also presents unique peculiarities making these studies significant not only from the disease control side but also for the advancement in our knowledge of the physiology of T. cruzi and for the understanding of Ca2+ transport pathways in general, and in addition, will fill gaps in the evolution of cell death pathways. We anticipate that our detailed analysis of the role of mitochondrial Ca2+ in T. cruzi cell death will lead to the discovery of new therapeutic targets, that could be exploited for development of new therapeutic approaches.

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