Involvement of Activated Caspase‐3‐Like Proteases in N‐Methyl‐D‐Aspartate‐Induced Apoptosis in Cerebrocortical Neurons

Abstract

Excessive activation of glutamate receptors mediates neuronal death in a number of neurodegenerative diseases. The intracellular signaling pathways that mediate this type of neuronal death are only partly understood. Following mild insults via NMDA receptor activation, central neurons undergo apoptosis, but with more fulminant insults, necrosis intervenes. Caspases are important in several forms of apoptosis in vivo and in vitro. Previously, we have demonstrated that caspases are important in excitotoxicity-mediated apoptosis of cerebrocortical neurons. To determine the possible activation of caspase-3 in NMDA-induced neuronal apoptosis, we used an affinity-labeling technique: Biotinylated N-acetyl-Asp-Glu-Val-Asp-aldehyde (DEVD.CHO) preferentially labels conformationally active caspase-3-like proteases, allowing us to visualize affinity-labeled caspases with streptavidin-fluorescein isothiocyanate under confocal microscopy. NMDA-induced apoptosis of cerebrocortical neurons was associated with a time-dependent increase in conformationally active caspase-3-like proteases. The activation of caspases was apparent within 20 min of NMDA stimulation and was localized primarily in the cytosol. However, following incubation of neurons for 18 -24 h, conformationally active caspase-3-like proteases were also detectable in nuclei. Double labeling with propidium iodide to detect chromatin condensation indicated that affinity-labeled caspase-3-like proteases were specifically expressed in apoptotic cells. To further confirm this, we used an antibody specific for the conformationally active fragment of caspase-3 and found largely concordant results. Moreover, preincubation with DEVD.CHO prevented NMDA-induced apoptosis. Our results suggest that caspase-3-like proteases play a major role in excitotoxin-induced neuronal apoptosis.

Mitochondrial-dependent manganese neurotoxicity in rat primary astrocyte cultures

“…In each of these conditions, the participation of executioner caspase activation and subsequent apoptosis in the neuronal cell death process has been established both in the adult and postnatal brain. Similarly, in vitro studies have also suggested that caspase-3 proteolytic activity plays a crucial role in excitotoxin-induced neuronal apoptosis (Allen et al, 1999;Du et al, 1997;Tenneti and Lipton, 2000). In addition, expression of caspase-3 has been described in oligodendrocytes (Beer et al, 2000;Nottingham and Springer, 2003) and astrocytes following CNS damage (Beer et al, 2000;Benjelloun et al, 2003;Mouser et al, 2006;Su et al, 2000).…”

Molecular aspects of glutamate dysregulation: implications for schizophrenia and its treatment

“…NMDA-mediated neurotoxicity activates caspases inside neurons (Fig. 5) (Du et al, 1997;Hirashima et al, 1999;Martin et al, 1998;Nath et al, 1998;Tenneti et al, 1998;Tenneti & Lipton, 2000). The damage to the mitochondria by Ca 2 + causes a release of cytochrome c (Luetjens et al, 2000); cytochrome c, in turn, activates caspase-3 .…”

Inhibitory effect of some tropical green leafy vegetables on key enzymes linked to Alzheimer’s disease and some pro-oxidant induced lipid peroxidation in rats’ brain

“…There are two main mechanisms by which QA has been implicated in Alzheimer's disease. The first is the activation of NMDA receptors by Quinolinic acid (Guillemin et al 2004), which is already known to mediate neuronal apoptosis with caspase-3 activation (Tenneti and Lipton 2000;Stone 2001). The second possibility, which represent another important aspect of Quinolinic acid toxicity, is lipid peroxidation (Rios and Santamaria 1991).…”