Methods for studying synaptosomal copper release

Cu is thought to play an important role in the pathogenesis of several neurodegenerative diseases, such as Wilson's, Alzheimer's, and probably in prion protein diseases like Creutzfeld-Jakob's disease. Until now, no method existed to determine the concentration of this cation in vivo. Here, we present two possible approaches combined with a critical comparison of the results. The successful use of fluorescent ligands for the determination of Ca ²⁺-concentrations in recent years encouraged us to seek a fluorophore which specifically reacts to Cu²⁺ and to characterize it for our purposes. We found that the emission of TSPP (tetrakis-(4-sulfophenyl)porphine) at an emission wavelength of 645 nm is in vitro highly specific to Cu²⁺ (apparent dissociation constant K _d=0.43±0.07 μM at pH 7.4). It does not react with the most common divalent cations in the brain, Ca²⁺ and Mg²⁺, unlike most of the other dyes examined. In addition, Zn²⁺ quenches TSPP fluorescence at a different emission wavelength (605 nm) with a K _d of 50±2.5 μM (pH 7.0). With these findings, we applied the measurement of Cu with TSPP to a biological system, showing for the first time in vivo that there is release of copper by synaptosomes upon depolarisation. Our findings were validated with a completely independent analytical approach based on ICP-MS (inductively-coupled-plasma mass-spectrometry).