Marcel Leist

Four deaths and a funeral : from caspases to alternative mechanisms

A single family of proteases, the caspases, has long been considered the pivotal executioner of all programmed cell death. However, recent findings of evolutionarily conserved, caspase-independent controlled death mechanisms have opened new perspectives on the biology of cell demise, with particular implications for neurobiology, cancer research and immunological processes.

A roadmap for the development of alternative (non-animal) methods for systemic toxicity testing - t4 report

Systemic toxicity testing forms the cornerstone for the safety evaluation of substances. Pressures to move from traditional animal models to novel technologies arise from various concerns, including: the need to evaluate large numbers of previously untested chemicals and new products (such as nanoparticles or cell therapies), the limited predictivity of traditional tests for human health effects, duration and costs of current approaches, and animal welfare considerations. The latter holds especially true in the context of the scheduled 2013 marketing ban on cosmetic ingredients tested for systemic toxicity. Based on a major analysis of the status of alternative methods (Adler et al., 2011) and its independent review (Hartung et al., 2011), the present report proposes a roadmap for how to overcome the acknowledged scientific gaps for the full replacement of systemic toxicity testing using animals. Five whitepapers were commissioned addressing toxicokinetics, skin sensitization, repeated-dose toxicity, carcinogenicity, and reproductive toxicity testing. An expert workshop of 35 participants from Europe and the US discussed and refined these whitepapers, which were subsequently compiled to form the present report. By prioritizing the many options to move the field forward, the expert group hopes to advance regulatory science.

Lysosomes in cell death

For many years apoptosis research has focused on caspases and their putative role as sole executioners of programmed cell death. Accumulating information now suggests that lysosomal cathepsins are also pivotally involved in this process, especially in pathological conditions. In particular, the role of lysosomes and lysosomal enzymes in initiation and execution of the apoptotic program has become clear in several models, to the point that the existence of a ‘lysosomal pathway of apoptosis’ is now generally accepted. This pathway of apoptosis can be activated by death receptors, lipid mediators, and photodamage. Lysosomal proteases can be released from the lysosomes into the cytosol, where they contribute to the apoptotic cascade upstream of mitochondria. This review focuses on the players and the molecular mechanisms involved in the lysosomal pathway of apoptosis as well as on the importance of this pathway in development and pathology.

Concanavalin A - induced T-cell - mediated hepatic injury in mice : The role of tumor necrosis factor

Concanavalin A activates T lymphocytes in vitro and causes T‐cell‐dependent hepatic injury in mice. T lymphocytes were previously identified as effector cells of concanavalin A‐induced liver injury. Here we report that hepatic injury is characterized by apoptotic cell death. On concanavalin A challenge, the cytokines tumor necrosis factor‐α (TNF α), interleukin‐2, granulocyte macrophage‐colony stimulating factor, and interferon‐γ were detectable in the circulation of the mice. Pretreatment of mice with anti‐mouse TNF‐α antiserum protected them from concanavalin A‐induced liver injury. Nude mice failed to release TNF‐α or interleukin‐2 after concanavalin A challenge and were protected from liver injury. Lymph node cell transfer from responder mice to resistant nude mice resulted in susceptibility of the latter towards concanavalin A, i.e., to induction of cytokine release and hepatotoxicity. These experiments suggest that immunocompetent T cells play a pivotal role in concanavalin A‐stimulated TNF‐α release in vivo. After intravenous administration of fluorescein isothiocyanate‐labeled concanavalin A to mice, the most fluorescence was found within the liver. In vitro, concanavalin A stimulation of separate cultures of mouse lymph node cells or nonparenchymal liver cells induced the release of minute amounts of TNF, whereas stimulation of cocultures of these cells resulted in production of substantial amounts of TNF‐α. These findings may explain the hepatotropic effect of concanavalin A. In conclusion, T‐cell‐dependent concanavalin A‐induced apoptotic liver injury in mice is related to immunological and cytokinemediated disorders and possibly to autoreactive hepatic processes. (Hepatology 1995;21:190–198).

Asialoerythropoietin is a nonerythropoietic cytokine with broad neuroprotective activity in vivo

Erythropoietin (EPO) is a tissue-protective cytokine preventing vascular spasm, apoptosis, and inflammatory responses. Although best known for its role in hematopoietic lineages, EPO also affects other tissues, including those of the nervous system. Enthusiasm for recombinant human erythropoietin (rhEPO) as a potential neuroprotective therapeutic must be tempered, however, by the knowledge it also enlarges circulating red cell mass and increases platelet aggregability. Here we examined whether erythropoietic and tissue-protective activities of rhEPO might be dissociated by a variation of the molecule. We demonstrate that asialoerythropoietin (asialoEPO), generated by total enzymatic desialylation of rhEPO, possesses a very short plasma half-life and is fully neuroprotective. In marked contrast with rhEPO, this molecule at doses and frequencies at which rhEPO exhibited erythropoiesis, did not increase the hematocrit of mice or rats. AsialoEPO appeared promptly within the cerebrospinal fluid after i.v. administration; intravenously administered radioiodine-labeled asialoEPO bound to neurons within the hippocampus and cortex in a pattern corresponding to the distribution of the EPO receptor. Most importantly, asialoEPO exhibits a broad spectrum of neuroprotective activities, as demonstrated in models of cerebral ischemia, spinal cord compression, and sciatic nerve crush. These data suggest that nonerythropoietic variants of rhEPO can cross the blood–brain barrier and provide neuroprotection.

Intracellular ATP, a switch in the decision between apoptosis and necrosis.

Regardless of whether apoptosis or necrosis are elicited by toxicants or by pathophysiological conditions they are considered conceptually distinct forms of cell death. Nevertheless, there is increasing evidence that classical apoptosis and necrosis represent only the extreme ends of a wide range of possible morphological and biochemical deaths. The two classical types of demise can occur simultaneously in tissues or cell cultures exposed to the same stimulus and often, the intensity of the same initial insult decides the prevalence of either apoptosis or necrosis. The execution of the death program seems to involve a relatively limited number of pathways. In many instances, their ordered execution results in characteristic morphological and biochemical changes termed apoptosis. However, some subroutines of the degradation program may not be active in all cases of cell death. Then, the morphological appearance of dying cells and some of their biochemical alterations differ from those of classical apoptosis. We have recently shown that intracellular energy levels and mitochondrial function are rapidly compromised in necrosis, but not in apoptosis of neuronal cells. Then we went on to show that pre-empting human T cells of ATP switches the type of demise caused by two classic apoptotic triggers (staurosporin and CD95 stimulation) from apoptosis to necrosis. Conditions of controlled intracellular ATP depletion, which was obtained by blocking mitochondrial and/or glycolytic ATP generation were used in combination with repletion of the cytosolic ATP pool with glucose to redirect the death program towards apoptosis or necrosis.

The Suitability of BV2 Cells as Alternative Model System for Primary Microglia Cultures or for Animal Experiments Examining Brain Inflammation

The role of microglia in neurodegeneration, toxicology and immunity is an expanding area of biomedical research requiring large numbers of animals. Use of a microglia-like cell line would accelerate many research programmes and reduce the necessity of continuous cell preparations and animal experimentation, provided that the cell line reproduces the in vivo situation or primary microglia (PM) with high fidelity. The immortalised murine microglial cell line BV-2 has been used frequently as a substitute for PM, but recently doubts were raised as to their suitability. Here, we re-evaluated strengths and potential short-comings of BV-2 cells. Their response to lipopolysaccharide was compared with the response of microglia in vitro and in vivo. Transcriptome (480 genes) and proteome analyses after stimulation with lipopolysaccharide indicated a reaction pattern of BV-2 with many similarities to that of PM, although the average upregulation of genes was less pronounced. The cells showed a normal regulation of NO production and a functional response to IFN-gamma, important parameters for appropriate interaction with T cells and neurons. BV-2 were also able to stimulate other glial cells. They triggered the translocation of NF-kappaB, and a subsequent production of IL-6 in astrocytes. Thus, BV-2 cells appear to be a valid substitute for PM in many experimental settings, incuding complex cell-cell interaction studies.

Caspase inhibition reduces apoptosis and increases survival of nigral transplants

Transplantation of embryonic nigral tissue ameliorates functional deficiencies in Parkinson disease. The main practical constraints of neural grafting are the shortage of human donor tissue and the poor survival of dopaminergic neurons grafted into patients, which is estimated at 5–10% (refs. 3,4). The required amount of human tissue could be considerably reduced if the neuronal survival was augmented. Studies in rats indicate that most implanted embryonic neurons die within 1 week of transplantation, and that most of this cell death is apoptotic. Modified peptides, such as acetyl–tyrosinyl–valyl–alanyl–aspartyl–chloro–methylketone (Ac–YVAD–cmk), that specifically inhibit proteases of the caspase family effectively block apoptosis in a plethora of experimental paradigms, such as growth factor withdrawal, excitotoxicity, axotomy, cerebral ischemia and brain trauma. Here we examined the effects of caspase inhibition by Ac–YVAD–cmk on cell death immediately after donor tissue preparation and on long–term graft survival. Treatment of the embryonic nigral cell suspension with Ac–YVAD–cmk mitigated DNA fragmentation and reduced apoptosis in transplants. It also increased survival of dopaminergic neurons grafted to hemiparkinsonian rats, and thereby substantially improved functional recovery.

Neuronal cell death: a demise with different shapes

It is not surprising that initially simple death programmes, developed early during phylogeny, undergo complex modifications in mammalian cells. A further consequence of the increased complexity could be that an increasing number of feedback loops gives rise to many possibilities of initiation, control and execution (Fig. 1a, Fig. 2).Multiple pathways probably cooperate to ensure the removal of injured cells, and positive feedback loops amplify death signals to prevent survival of ‘undead’ cells. In complex cellular systems such as neuronal networks, one can even speculate that execution of the death programme is predominantly apoptotic in certain subcellular regions, whereas other subroutines prevail in other cellular domains. The main implication of this standpoint is the exclusion of a single, predominant and molecularly defined commitment step. It seems likely that accumulation of damage incompatible with cell survival would require disruption of several vital functions. Once such a threshold is trespassed, other positive feedback loops would ensure the progression of the death programme and the safe disposal of the injured cell. Also, it is apparent that the morphological appearance of cell death (apoptosis or necrosis) is not linked to a putative commitment point, but it is rather the result of a more-or-less complete execution of subroutines of the death programme. Overall, the arguments presented here support the view that in neuronal death, individual, intricately interconnected pathways self-amplify or delete each other in an inflationary process, which results in the many shapes of cell death.

Progressive Degeneration of Human Mesencephalic Neuron-Derived Cells Triggered by Dopamine-Dependent Oxidative Stress Is Dependent on the Mixed-Lineage Kinase Pathway

Models of Parkinsons disease (PD) based on selective neuronal death have been used to study pathogenic mechanisms underlying nigral cell death and in some instances to develop symptomatic therapies. For validation of putative neuroprotectants, a model is desirable in which the events leading to neurodegeneration replicate those occurring in the disease. We developed a human in vitro model of PD based on the assumption that dysregulated cytoplasmic dopamine levels trigger cell loss in this disorder. Differentiated human mesencephalic neuron-derived cells were exposed to methamphetamine (METH) to promote cytoplasmic dopamine accumulation. In the presence of elevated iron concentrations, as observed in PD, increased cytosolic dopamine led to oxidative stress, c-Jun N-terminal kinase (JNK) pathway activation, neurite degeneration, and eventually apoptosis. We examined the role of the mixed-lineage kinases (MLKs) in this complex degenerative cascade by using the potent inhibitor 3,9-bis[(ethylthio)methyl]-K-252a (CEP1347). Inhibition of MLKs not only prevented FeCl2+/METH-induced JNK activation and apoptosis but also early events such as neurite degeneration and oxidative stress. This broad neuroprotective action of CEP1347 was associated with increased expression of an oxidative stress-response modulator, activating transcription factor 4. As a functional consequence, transcription of the cystine/glutamate and glycine transporters, cellular cystine uptake and intracellular levels of the redox buffer glutathione were augmented. In conclusion, this new human model of parkinsonian neurodegeneration has the potential to yield new insights into neurorestorative therapeutics and suggests that enhancement of cytoprotective mechanisms, in addition to blockade of apoptosis, may be essential for disease modulation.