Calcium has been implicated in the control of neuronal activity, differentiation, survival, and death [1, 2, 3, 4]. Calcium-binding proteins (CaBPs) are expressed in many types of neurons and maintain calcium homeostasis [5, 6]. Calbindin-D28K belongs to the calbindin family of CaBPs that has a common structural calcium-binding domain designated the EF-hand [7]. Other members of the EF-hand family of CaBPs, such as calmodulin and calpains, have been extensively investigated in neuronal cells [8]. Though the neuroprotection by overexpressed calbindin has been attributed to its calcium-chelating activity, the exact role of calbindin in neuronal cell death remains largely unknown (for review, see [9]). Recently, we reported that calbindin prevents caspase-3 activation and apoptosis in neuronal death induced by staurosporine (STS) [10]. However, the activity of calbindin with respect to calpain, calcium-dependent protease, and necrotic (or late-apoptotic) cell death in STS-induced neuronal death is not clear.

Here, we report that calbindin prevented STS-induced elevation of intracellular levels of free calcium ([Ca²⁺]_i) and calpain activation. We also demonstrated that calbindin expression prevents STS-induced necrotic death, as well as apoptosis.

Previously, we demonstrated that STS induced the activation of caspase and calpain in dopaminergic neuron death [10, 13]. Overexpression of calbindin prevented caspase activation and apoptosis in STS-induced cell death [10]. In this study, we investigated the role of calbindin in calpain activation and necrosis in STS-induced cell death. After 30 min of STS treatment, [Ca²⁺]_i was elevated and peaked at 1 hr (Fig. 1). Cotreatment with BAPTA prevented STS-induced [Ca²⁺]_i surge (Fig. 2A and B). Overexpression of calbindin prevented [Ca²⁺]_i elevation after treatment with STS (Fig. 2B). Three individual clones of stable cell lines expressing calbindin were similarly resistant to STS-induced cell death (Fig. 3).

To further define the protective mechanism of calbindin, we tested the effect of calbindin on the cleavage of Bax, which is a substrate of calpain. As we previously reported, STS treatment induced calpain activation in MN9D/Neo cells, as indicated by calpain-mediated cleavage of an 18 kDa Bax fragment, at the late phase of cell death (Fig. 4A; 24-hr time point). Calbindin overexpression prevented Bax cleavage in STS-induced neuron death, suggesting that calbindin inhibited calpain activation (Fig. 4A).

After 20 hr of treatment with STS, both apoptotic (chromatin condensation and fragmentation) and necrotic (propidium-iodide staining) events occurred in MN9D/Neo cells; whereas, calbindin overexpression significantly prevented both types of events (Fig. 4B). Few MN9D/Calbindin cells showed chromatin condensation or fragmentation, and virtually no cells were stained with propidium iodide (Fig. 4B). These data suggest that calbindin may exert its protective functions by preventing calpain activation and necrotic cell death, as well as by effects on the caspase-mediated apoptotic pathway [10].

Previously, we reported that calbindin prevents STS-induced caspase activation [10]. However, the role of calbindin on calcium elevation and calcium-dependent calpain activation has not been investigated in STS-induced dopaminergic neuron death. In this study, we demonstrated that calbindin overexpression prevented elevation of [Ca²⁺]_i, calpain activation, and cleavage of Bax in STS-induced dopaminergic neuron death. Our previous results demonstrated that MPP+-induced Bax cleavage was prevented by the expression of calbindin. Taken together, our data suggest that the prevention of [Ca²⁺]_i elevation, calpain activation, and Bax cleavage might be a common mechanism underlying the protective role of calbindin in dopaminergic neuron death. In support of this hypothesis, calpain activation has been reported in post-mortem midbrain of patients with Parkinson's disease (PD). Furthermore, calpain regulates other molecules in dopaminergic neuron death [14]. Therefore, the calpain pathway may likely be a good research area for discovery of new PD treatments.

Interestingly, the protection rate by calbindin in STS-induced cell death is much higher than in MPP+-induced death (approximately 40% vs. 25%, respectively). This implies that the protective role of calbindin is accomplished through inhibition of both the calcium/calpain and caspase pathways in parallel in STS-induced dopaminergic cell death. Calbindin's protective effect is relatively smaller in MPP⁺-induced cell death, in which only the calcium-calpain pathway is inhibited [10].

Permeabilization of the plasma membrane is a typical characteristic of necrotic cell death [15]. In this study, we observed that a large portion of STS-treated MN9D cells lost the integrity of their cell membrane, although the morphology of the nucleoli was not apoptotic, suggesting that STS-induced cell death is necrotic in at least a part of the cell population. Our data suggested that calbindin prevents both the necrotic and apoptotic components of cell death in STS-induced dopaminergic cell death. Indeed, the cell death mechanisms are rather complicated in PD. In brain of PD patients, both caspase and calpain activation have been reported [16, 17, 18]. Therefore, a multifunctional molecule, e.g., calbindin, that can prevent involvement of both caspase and calpain pathways, might be needed to protect dopaminergic neurons in the brain.

Interestingly, it has been demonstrated that calbindin is located in the midbrain dopaminergic neurons, which are relatively invulnerable to degeneration as seen in animal PD models [19, 20]. In PD patients, dopaminergic neurons of the substantia nigra pars compacta that express calbindin appear to be relatively more resistant to cell death than those not expressing calbindin [21]. Therefore, this protein might be part of endogenous protective machinery, and further research may reveal a potential treatment based on it to prevent dopaminergic neuron death in PD.