Cellular calcium deficiency plays a role in neuronal death caused by proteasome inhibitors

Inhibition of the ubiquitin proteasome system (UPS) causes programmed cell death in many cell types, including neurons. We hypothesized that the neuronal death caused by proteasome inhibition might be associated with dysregulation of calcium (Ca2+) homeostasis, specifically with a reduction, rather than an increase, in neuronal Ca2+. Many studies have focused on increases in intracellular Ca2+ in disorders including stroke, epilepsy, trauma and neurodegenerative disorders (for recent reviews see Arundine and Tymianski 2004; Berliocchi et al. 2005). The role of reductions in intracellular Ca2+ in neuronal death has also been studied, primarily in developing neurons, and a “calcium set point” has been hypothesized to play a key role in neuronal survival (Johnson et al. 1992). We have previously reported that free cytosolic Ca2+ ([Ca2+]i) is reduced in cultured murine neocortical neurons after 1-4 hr treatment with proteasome inhibitors. We observed a similar reduction in [Ca2+]i in cultures treated with staurosporine, a protein kinase inhibitor that causes neuronal death associated with activation of programmed cell death pathways (Snider et al. 2002; Canzoniero et al. 2004). However, other investigators have reported that treatment with proteasome inhibitors increases [Ca2+]i (Landowski et al. 2005; Lee et al. 2005; Nawrocki et al. 2005b; Nawrocki et al. 2005a). These apparently conflicting results could represent differences in cell type, intensity of injury or timing of assessment of [Ca2+]i relative to injury. For example, there could be an early increase and subsequent decline in [Ca2+]i levels, or Ca2+ could be reduced in some cellular compartments and increased in others, as reported in cultured mesencephalic neurons treated with ceramide (Darios et al. 2005). Understanding the nature and timing of these derangements in Ca2+ homeostasis might have significant implications for designing rational treatment strategies for conditions where neuronal proteasome activity is impaired. We hypothesized that the neuronal death associated with impairment of the UPS was caused in part by transfer of Ca2+ between cytosol, ER and mitochondria and set out to clarify the timing of changes in [Ca2+]i relative to cell death and to identify mechanisms responsible for the alterations in [Ca2+]i and neuronal death under these conditions. A preliminary report of these studies has appeared in abstract form (Wu et al. 2006).