Cerebellar Granule Cell Death Induced by Aluminum

Abstract

Using flow cytometry of acutely isolated cerebellar granule cell neurons, we have determined the effects of Al³⁺ on viability, membrane potential, intracellular calcium concentration and generation of reactive oxygen species (ROS). Al³⁺ killed granule cells in a time- and concentration-dependent fashion when monitored by use of the DNA-binding dye, propidium iodide. The threshold concentration was about 50 µM, and cell death at 100 µM was apparent after 30 min exposure and increased over time. Cell death was accompanied by cell swelling and a decrease in membrane potential, and was not dependent on external calcium concentration. While exposure to Al³⁺ was accompanied by an increase in ROS and an elevation of intracellular calcium concentration, calcium chelators and ROS scavengers did not reduce cell death. The action of Al³⁺ was not accompanied by activation of caspase-3 or an increase in annexin-V binding, both indicators of apoptosis. In the presence of intracellular O,O’-bis(2-aminophenyl)ethyleneglycol-N,N,N’,N’-tet-raacetic acid (BAPTA) and absence of extracellular calcium there was still a fluo-3 signal, which likely reflects an accumulation of intracellular Al³⁺. These observations suggest that the cell death is subsequent to intracellular accumulation of Al³⁺ and subsequent perturbation of cellular metabolism.