Death of dopaminergic neurons in vitro and in nigral grafts: Reevaluating the role of caspase activation

Caspases are cysteine proteases involved in apoptotic cell death, and pharm acological caspase inhibition has been demonstrated to prevent neuronal cel l death in certain experimental paradigms. In this study, the role of caspa se-1 and -3 in the death of dopaminergic neurons derived from the E14 rat v entral mesencephalon (VM) has been examined in two model systems using pept ide caspase inhibitors. First, cell death was induced in vitro by withdrawi ng serum after 2 days. Different doses of caspase-1 (IL-1 beta converting e nzyme) and caspase-3 inhibitors (Ac-DEVD-cmk) were added to the medium at t he time of serum withdrawal, and the ability of the inhibitors to promote d opaminergic neuronal survival and prevent activation of caspase-3 was asses sed at 7 days. Immunostaining using tyrosine hydroxylase (TH) and cleaved c aspase-3 antibodies demonstrated that caspase-1 and -3 inhibitors reduce ca spase-3 activation as well as overall cell death. This did not, however, im prove the survival of TH-positive neurons, although it did appear to promot e their maturation. The second paradigm investigated the effects of these i nhibitors in the 6-hydroxydopamine rat model of PD, and similarly, addition of caspase-1 or -3 inhibitor during tissue preparation or immediately prio r to grafting of VM tissue did not promote dopaminergic neuronal survival. These results demonstrate that the reduction of apoptotic cell death by pha rmacological inhibition of caspase-1 and -3 does not increase dopaminergic neuronal survival in these paradigms and suggest either that caspase-3 acti vation is not the major determinant of dopaminergic neuronal death in vitro and in grafts or that the ability of caspase inhibitors to rescue cells de pends upon the degree of apoptotic stress. This implies that strategies to improve dopaminergic cell survival in clinical programmes of transplantatio n for PD will need to target other pathways of cell death. (C) 2001 Academi c Press.