Imaging Intracellular and Intra-Mitochondrial Free Zinc Ion Concentrations Following Hypoxia/Hypoglycemia with an Expressible Fluorescence Biosensor

Abstract

Zinc is a “trace” metal necessary for cellular function, but excess free zinc ion is toxic to most cells (1,2,9) We and others have observed increased intra- and extra-cellular free zinc concentrations following cerebral ischemia (3,4). Substantial evidence indicates that mitochondrial dysfunction plays a significant role in neuronal death following ischemia (5), and both mitochondrial dysfunction and increased intracellular zinc have been associated with increased production of reactive oxygen species (ROS) and ultimately apoptosis (6,7). Zinc, potently, inhibits mitochondrial enzymes involved in energy production and destruction of ROS, potentially promoting mitochondrial dysfunction (8). We targeted our expressible fluorescent zinc biosensor (9) to mitochondria of PC12 cells, to ratiometrically image the intra-mitochondrial zinc concentration at resting (pM) levels. We imaged cytoplasmic and mitochondrial zinc levels in cells deprived of oxygen and glucose (OGD), a widely used model of ischemia. Our data indicate that both intra-mitochondrial and cytoplasmic zinc concentrations increase substantially following OGD. Further experiments indicate that the zinc is released from intracellular sites rather than entering the cell from external sources. Supported by NIH EB03924. 1. Canzoniero, L.M., et al. (1999) Journal of Neuroscience 19:RC31, 1–6 2. Zodl, B, et al. (2003) Journal of Inorganic Biochemistry 97, 324–330 3. Tonder, N., et al. (1990) Neuroscience Letters 109, 247–252 4. Frederickson, C.J., et al (2006) Experimental Neurology 198, 285–293 5. Fiskum G., et al. (2008) Mitochondria and Oxidative Stress in Neurodegenerative Disorders: Annals of the New York Academy of Science. 1147, 129–138 6. Weiss, J.H., et al. (2000) Trends in Pharmacological Science 21, 395–4017. Jiang, D., et al. (2001) Journal of Biological Chemistry 276, 47524–47529 8. Gazaryan, I.G., et al. (2007) Journal of Biological Chemistry 282, 24373–24380 9. Bozym, R.A., et al. (2006) ACS Chemical Biology 1, 103–111.