Differential effects of Bcl‐2 overexpression on fibre outgrowth and survival of embryonic dopaminergic neurons in intracerebral transplants
1999-09, Schierle, Gabriele S., Leist, Marcel, Martinou, Jean-Claude, Widner, H, Nicotera, Pierluigi, Brundin, Patrik
The causes of death of transplanted neurons are not known in detail, but apoptotic mechanisms involving caspase activation are likely to play a role. We examined whether overexpression of the anti‐apoptotic protein Bcl‐2 may enhance the survival of dopaminergic [tyrosine hydroxylase (TH)‐immunoreactive] grafted neurons. For this purpose, we prepared cells from embryonic day 13 ventral mesencephalon (VM) of mice overexpressing human Bcl‐2, or from their wild‐type littermates. The bcl‐2 transgene was strongly expressed in these cells, and resulted in protection of neuronal cultures from death triggered by serum deprivation or exposure to staurosporine. To model pretransplantation stress more closely in vitro, we stored dissociated embryonic mesencephalic cells for 8 h in the same type of medium used for intracerebral transplantation. This resulted in massive cell death as quantified by lactate dehydrogenase (LDH) release, and increased DNA fragmentation. Although this cell loss was strongly reduced by a caspase inhibitor, Bcl‐2 had no significant protective effect. Finally, mesencephalic cell suspensions were xenografted into the striatum of immunosuppressed hemiparkinsonian rats. Neither the survival of TH‐immunopositive transplanted neurons nor the functional recovery of the rats was improved by Bcl‐2, although the Bcl‐2 protein was strongly expressed in transgenic grafts 5 weeks after implantation, and dopaminergic fibre outgrowth from the grafts was significantly improved. These data suggest that cell death in neuronal transplants involves apoptotic mechanisms that can bypass negative regulation by Bcl‐2.
Cascade of Caspase Activation in Potassium-Deprived Cerebellar Granule Neurons : Targets for Treatment with Peptide and Protein Inhibitors of Apoptosis
2001-04, Gerhardt, Ellen, Kügler, Sebastian, Leist, Marcel, Beier, Christoph, Berliocchi, Laura, Volbracht, Christiane, Weller, Michael, Bähr, Mathias, Nicotera, Pierluigi, Schulz, Jörg B.
Cerebellar granule neurons (CGN) cultured in the presence of serum and depolarizing potassium concentrations undergo apoptosis when switched to serum-free medium containing physiological potassium concentrations. Here we show that processing of the key protease, caspase-3, depends on the activation of caspase-9, but not of caspase-8. Selective peptide inhibitors of caspase-9 block processing of caspase-3 and caspase-8 and inhibit apoptosis, whereas a selective inhibitor of caspase-8 blocks neither processing of caspase-3 nor cell death. The data obtained with peptide inhibitors were confirmed by adenovirally mediated ectopic expression of the cytokine response modifier A (crmA), the baculovirus protein p35, and the X chromosome-linked inhibitor of apoptosis (XIAP). Further, caspase-8-activating death receptors do not mediate apoptosis in CGN and potassium withdrawal-induced apoptosis evolves unaltered in gld or lpr mice, which harbor mutations in the CD95/CD95 ligand system. Thus, neuronal apoptosis triggered by potassium deprivation is death receptor-independent but involves the mitochondrial pathway of caspase activation.
Additive Effects of Caspase Inhibitor and Lazaroid on the Survival of Transplanted Rat and Human Embryonic Dopamine Neurons
2000-07, Hansson, Oskar, Castilho, Roger F., Kaminski Schierle, Gabriele S., Karlsson, Jenny, Nicotera, Pierluigi, Leist, Marcel, Brundin, Patrik
Major practical constraints on neural grafting in Parkinson's disease are the shortage of human donor tissue and the great loss of dopamine neurons during the grafting procedure. The vast majority of implanted embryonic dopamine neurons are believed to die within a few days of transplantation surgery, at least in part through apoptosis. We have previously found that survival of nigral grafts in rodents can be significantly augmented by pretreatment with the caspase inhibitor Ac-YVAD-cmk or by lazaroids (lipid peroxidation inhibitors). We now report that pretreatment with the caspase inhibitor Ac-DEVD-cmk, but not z-VAD-fmk, results in a significantly improved survival of transplanted dopamine neurons of similar magnitude to that achieved in this study using Ac-YVAD-cmk (both 220–230% of control). In addition, we found that treatment of the graft tissue with tirilazad mesylate (a lazaroid allowed for clinical use) almost doubled the survival of grafted dopamine neurons. When Ac-YVAD-cmk and tirilazad mesylate treatments were combined, the number of surviving dopamine neurons increased significantly further to 280% of control. Importantly, the same combination of neuroprotectants enhanced the survival of human dopamine neurons xenotransplanted to immunosuppressed rats (to 240% of control). In conclusion, these results suggest that combining treatments that counteract oxidative stress and caspase activation is a valuable strategy to enhance nigral graft survival that should be considered for clinical application.
Monocular deprivation (MD)-induced apoptosis in the lateral geniculate nucleus (LGN) of new-born mice involves poly(ADP)ribose polymerase (PARP)
2001, Nucci, Carlo, Piccirilli, Silvia, Nisticò, Robert, Cerulli, Luciano, Leist, Marcel, Nicotera, Pierluigi, Bagetta, Giacinto
Evidence that poly(ADP)ribose polymerase is involved in apoptosis induced by monocular deprivation (MD) in the lateral geniculate nucleus (LGN) of new-born mice
2000, Nucci, Carlo, Piccirilli, Silvia, Nisticò, Robert, Cerulli, Luciano, Leist, Marcel, Nicotera, Pierluigi, Bagetta, Giacinto
Execution of Apoptosis : Converging or Diverging Pathways?
1999-09, Nicotera, Pierluigi, Leist, Marcel, Single, Barbara, Volbracht, Christiane
There is increasing evidence that apoptosis and necrosis represent only two of several possible ways for cells to die. These two types of demise can occur simultaneously in tissues or cell cultures exposed to the same stimulus, and often local metabolic conditions and the intensity of the same initial insult decide the prevalence of either apoptosis or necrosis. Recent work has shown that execution of the apoptotic programme involves a relatively limited number of pathways. According to a general view, these would converge to activate the caspase family of proteases. However, there is increasing evidence that apoptotic-like features can be observed also in cells where caspases are inhibited by cell-permeable tripeptides, such as z-VaD-Ala-Asp-fluoromethyl ketone (z-VAD-fmk), or analogous compounds. This has posed the question as to whether apoptosis may or may not occur in a caspase independent way, and whether caspase inhibitors may be effective in the treatment of disease. Also relevant is the understanding that low intracellular energy levels during apoptosis can preclude caspase activation, and consequently decide the occurrence and mode of demise in damaged cells. In vivo, incomplete execution of damaged cells by apoptosis may have profound implications, as their persistence within a tissue, followed by delayed lysis, may elicit delayed pro-inflammatory reactions. In this minireview, we discuss some recent findings suggesting that cells may use diverging execution pathways, with different implications in pathology and therapy.