The presence of aggregated alpha-synuclein molecules is a common denominator in a variety of neurodegenerative disorders. Here, we show that alpha-synuclein (alpha-syn) is an outstanding substrate for the protein tyrosine kinase p72syk (Syk), which phosphorylates three tyrosyl residues in its C-terminal domain (Y-125, Y-133, and Y-136), as revealed from experiments with mutants where these residues have been individually or multiply replaced by phenylalanine. In contrast, only Y-125 is phosphorylated by Lyn and c-Fgr. Eosin-induced multimerization is observed with wild-type alpha-syn, either phosphorylated or not by Lyn, and with all its Tyr to Phe mutants but not with the protein previously phosphorylated by Syk. Syk-mediated phosphorylation also counteracts alpha-syn assembly into filaments as judged from the disappearance of alpha-syn precipitated upon centrifugation at 100,000 x g. We also show that Syk and alpha-syn colocalize in the brain, and upon cotransfection in Chinese hamster ovary cells, alpha-syn becomes Tyr-phosphorylated by Syk. Moreover, Syk and alpha-syn interact with each other as judged from the mammalian two-hybrid system approach. These data suggest that Syk or tyrosine kinase(s) with similar specificity may play an antineurodegenerative role by phosphorylating a-syn, thereby preventing its aggregation.

Silver(I) and gold(I)-N-heterocyclic carbene (NHC) complexes bearing a fluorescent anthracenyl ligand were examined for cytotoxicity in normal and tumor cells. The silver(I) complex exhibits greater cytotoxicity in tumor cells compared with normal cells. Notably, in cell extracts, this complex determines a more pronounced inhibition of thioredoxin reductase (TrxR), but it is ineffective towards glutathione reductase (GR). Both gold and silver complexes lead to oxidation of the thioredoxin system, the silver(I) derivative being particularly effective. In addition, the dimerization of peroxiredoxin 3 (Prx3) was also observed, demonstrating the ability of these compounds to reach the mitochondrial target. The fluorescence microscopy visualization of the subcellular distribution of the complexes shows a larger diffusion of these molecules in tumor cells with respect to normal cells.

] occurring in the mitochondrial matrix of mdx myotubes are significantly larger than in controls upon KCl-induced depolarization or caffeine application. The augmented response of mitochondria precedes the alterations in the Ca 2؉ responses of the cytosol and of the cytoplasmic region beneath the membrane, which become significant only at a later stage of myotube differentiation. Taking into account the key role played by mitochondria Ca 2؉ handling in the control of cell death, our data suggest that mitochondria are potential targets of impaired Ca 2؉ homeostasis in muscular dystrophy.Although it is well established that the lack of dystrophin expression is the primary genetic defect in Duchenne's muscular dystrophy (DMD), 1 the mechanism leading to progressive muscle damage is still largely unknown (1). It has been suggested that an elevation of cytosolic Ca 2ϩ concentration ([Ca 2ϩ ] c ), under resting conditions, and a concurrent activation of Ca 2ϩ -dependent proteases may represent the mechanistic link between the genetic defect and the DMD phenotype (2). The differences in [Ca 2ϩ ] c between normal and dystrophic muscles have been found also in myotubes and in the classical animal model of the disease, the mdx mouse. Several groups (3, 4), however, have been unable to confirm these data, and the question remains controversial.Evidence has been accumulated over the last few years indicating that a key aspect of the Ca 2ϩ signaling pathway is represented by its spatial and temporal complexity. Localized changes in the cytosol, much larger than those occurring in the bulk cytosol, are known to occur close to the mouth of Ca 2ϩ channels, and these localized events are pivotal in triggering important cellular events such as secretion, gene expression, and metabolic activation. In this respect, mitochondria represent a privileged sensor of local [Ca 2ϩ ] increases. Not only their Ca 2ϩ accumulation depends on microdomains of high Ca 2ϩ generated in their vicinity, but their capacity to take up Ca 2ϩ is essential to shape the kinetics of cytoplasmic Ca 2ϩ changes (5). Last, but not least, mitochondrial Ca 2ϩ accumulation results in activation of ATP production under physiological conditions (6) but leads to initiation of apoptotic signaling when excess Ca 2ϩ is taken up by the organelles (7). Taking into account the key role played by mitochondria Ca 2ϩ handling in the control of cell death, our data suggest that mitochondria are potential targets of impaired Ca 2ϩ homeostasis in muscular dystrophy.In this study, we tested the hypothesis that the differences in cytoplasmic [Ca 2ϩ ] in muscles lacking dystrophin might be amplified in specific cellular regions, in particular within the mitochondrial matrix. We addressed this issue directly by using the strategy of targeted aequorin that we previously developed and used to analyze Ca 2ϩ handling in different types of cells ranging from cell lines to primary cultures of neurons or rat skeletal myotubes (8 -11). To evaluate the importance of mechanical...

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