During ischemic brain injury, glutamate accumulation leads to overstimulation of postsynaptic glutamate receptors with intracellular Ca2+ overload and neuronal cell death. Here we show that glutamate can induce either early necrosis or delayed apoptosis in cultures of cerebellar granule cells. During and shortly after exposure to glutamate, a subpopulation of neurons died by necrosis. In these cells, mitochondrial membrane potential collapsed, nuclei swelled, and intracellular debris were scattered in the incubation medium. Neurons surviving the early necrotic phase recovered mitochondrial potential and energy levels. Later, they underwent apoptosis, as shown by the formation of apoptotic nuclei and by chromatin degradation into high and low molecular weight fragments. These results suggest that mitochondrial function is a critical factor that determines the mode of neuronal death in excitotoxicity.

N-Methyl-D-aspartate (NMDA) receptor-mediated neurotoxicity may depend, in part, on the generation of nitric oxide (NO.) and superoxide anion (O2.-), which react to form peroxynitrite (OONO-). This form of neurotoxicity is thought to contribute to a final common pathway of injury in a wide variety of acute and chronic neurologic disorders, including focal ischemia, trauma, epilepsy, Huntington disease, Alzheimer disease, amyotrophic lateral scelerosis, AIDS dementia, and other neurodegenerative diseases. Here, we report that exposure of cortical neurons to relatively short durations or low concentrations of NMDA, S-nitrosocysteine, or 3-morpholinosydnonimine, which generate low levels of peroxynitrite, induces a delayed form of neurotoxicity predominated by apoptotic features. Pretreatment with superoxide dismutase and catalase to scavenge O2.- partially prevents the apoptotic process triggered by S-nitrosocysteine or 3-morpholinosydnonimine. In contrast, intense exposure to high concentrations of NMDA or peroxynitrite induces necrotic cell damage characterized by acute swelling and lysis, which cannot be ameliorated by superoxide dismutase and catalase. Thus, depending on the intensity of the initial insult, NMDA or nitric oxide/superoxide can result in either apoptotic or necrotic neuronal cell damage.

No abstract

Fas (CD95) and Fas ligand (FasL) play major roles in staphylococcal enterotoxin B (SEB)-induced peripheral deletion of Vbeta8+ T cells. We found that peripheral deletion was defective in radiation chimeras with non-functional tissue FasL, regardless of the FasL status of the bone marrow-derived cells. SEB induced a dramatic upregulation of FasL expression and function in nonlymphoid cells of liver and small intestine. This effect was resistant to inhibition by cyclosporin A, which also failed to inhibit peripheral deletion. In SCID animals nonlymphoid tissues did not express FasL in response to SEB unless transplanted lymphocytes were present. Thus, some immune responses induce FasL in nonlymphoid tissues, which in turn kills activated lymphocytes, leading to peripheral T cell deletion.

No abstract

In vitro studies have demonstrated that intestinal intraepithelial lymphocytes (IEL) are constitutively cytotoxic; however, the mechanism and target of their cytotoxicity are unknown. Apoptosis of intestinal epithelial cells (IEC) and an increase in IEL numbers are classical signs of intestinal graft-versus-host disease (GVHD), although whether IEL can mediate IEC apoptosis directly in GVHD is unclear. Recent evidence suggests that target epithelial organ injury observed in GVHD is predominantly Fas-mediated; therefore, we investigated the possibility that IEL induce apoptosis of IEC through a Fas-mediated mechanism. Here, we demonstrate that the IEL isolated from normal mice readily display potent Fas ligand (FasL)-mediated killing activity after CD3 stimulation, and that IEC express Fas, suggesting that IEC are potential targets for FasL-mediated killing by IEL. In vitro, IEL isolated from GVHD mice have markedly increased FasL-mediated killing potential and are spontaneously cytolytic toward host-derived tumor cells predominantly through a Fas-mediated pathway. In vivo transfer of IEL isolated from GVHD mice induced significantly more IEC apoptosis in F1 wild-type mice than in Fas-defective F1lpr mice. Thus, these results demonstrate that FasL-mediated death of IEC by IEL is a major mechanism of IEC apoptosis seen in GVHD.

Apoptosis of smooth muscle cells (SMC) in atherosclerotic vessels can destabilize the atheromatus plaque and result in rupture, thrombosis, and sudden death. In efforts to understand the molecular processes regulating apoptosis in this cell type, we have defined a novel mechanism involving the ubiquitously expressed transcription factor Sp1. Subtypes of SMC expressing abundant levels of Sp1 produce the death agonist, Fas ligand (FasL) and undergo greater spontaneous apoptosis. Sp1 activates the FasL promoter via a distinct nucleotide recognition element whose integrity is crucial for inducible expression. Inducible FasL promoter activation is also inhibited by a dominant-negative form of Sp1. Increased SMC apoptosis is preceded by Sp1 phosphorylation, increased FasL transcription, and the autocrine/ paracrine engagement of FasL with its cell-surface receptor, Fas. Inducible FasL transcription and apoptosis are blocked by dominant-negative protein kinase C-, whose wild-type counterpart phosphorylates Sp1. Thus, Sp1 phosphorylation is a proapoptotic transcriptional event in vascular SMC and, given the wide distribution of this housekeeping transcription factor, may be a common regulatory theme in apoptotic signal transduction.

We have recently demonstrated that nitric oxide (NO) donors can trigger either apoptosis or necrosis of neurons as a function of the intensity of the exposure. Here, we show that the apoptosis induced by the NO donors S-nitrosocysteine (SNOC) or S-nitroso-N-acetylpenicillamine (SNAP) in cultured cerebellar granule cells (CGCs) depends on NMDA receptor (NMDA-R) activation leading to intracellular Ca'+ overload. Early dissolution of actin filaments followed by breakdown of microtubUles and nuclear lam ins preceded the appearance of Taken together, these data strongly suggest that Ca" influx through NMDA-R-gated ion channels is a critical event in CGC apoptosis induced by NO donors.