Evaluation of intramitochondrial zinc with ratiometric-pericam-mt

Strong evidences, in vitro and in vivo, supports the idea that Zn2+ is a potent promoter of neuronal loss in a variety of neurological conditions. Zn2+-mediated neuronal death can be triggered by the activation of mitochondrial and extramitochondrial pathways. Zn2+ enters mitochondria and favors the initiation of apoptotic and necrotic processes. The issue of measuring changes in intramitochondrial levels is therefore critical. Past studies have employed fluorescent Zn2+ indicators, like Rhod-2 and RhodZin-3, however, the use of these probes is impaired by their extramitochondrial sequestration. In this study, we have employed the ratiometric mitochondria-targeted pericam, RPmt, to detect changes of intramitochondrial free Zn2+ ([Zn2+]m) levels. Using RPmt in neuronal and non neuronal cell lines we demonstrate that mitochondria can take up the cation mobilized from the cytosolic pool of protein-bound Zn2+ and that mitochondrial Zn2+ sequestration is largely mediated by the activity of the Ca2+ uniporter. In the past decade our understanding of the complex biology of Zn2+ has increased dramatically. Nevertheless, a better blueprint of the intracellular systems controlling [Zn2+]i homeostasis and, in particular, of the role played by mitochondria in such processes is needed. Present results indicate that RPmt can be a valuable tool for detecting physiologically and pathophysiologically relevant changes in [Zn2+]m.