Functional role and therapeutic implications of neuronal caspase-1 and -3 in a mouse model of traumatic spinal cord injury.

Evidence indicates that both necrotic and apoptotic cell death contribute to tissue injury and neurological dysfunction following spinal cord injury. Caspases have been implicated as important mediators of apoptosis following acute central nervous system insults. We investigated whether caspase-1 and caspase-3 are involved in spinal cord injury-mediated cell death, and whether caspase inhibition may reduce tissue damage and improve outcome following spinal cord injury. We demonstrate a 17-fold increase in caspase-1 activity in traumatized spinal cord samples when compared with samples from sham-operated mice. Caspase-1 and caspase-3 activation were also detected by western blot following spinal cord injury, which was significantly inhibited by the broad caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. By immunofluorescence or in situ fluorogenic substrate assay, caspase-1 and caspase-3 expression were detected in neuronal and non-neuronal cells following spinal cord injury. N-Benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone treated mice, and transgenic mice expressing a caspase-1 dominant negative mutant, demonstrated a significant improvement of motor function and a reduction of lesion size compared with vehicle-treated mice. Our results demonstrate for the first time that both caspase-1 and caspase-3 are activated in neurons following spinal cord injury, and that caspase inhibition reduces post-traumatic lesion size and improves motor performance. Caspase inhibitors may be one of the agents to be used for the treatment of spinal cord injury.

Role of p38 mitogen-activated protein kinase in axotomy-induced apoptosis of rat retinal ganglion cells.

p38 is a member of the mitogen-activated protein (MAP) kinase superfamily and mediates intracellular signal transduction. Recent studies suggest that p38 is involved in apoptotic signaling in several cell types, including neurons. In the mammalian retina, approximately 50% of the retinal ganglion cells (RGCs) die by apoptosis during development. Additionally, transection of the optic nerve close to the eye bulb causes apoptotic cell death of RGCs in adulthood. We investigated the role of p38 in axotomy-induced apoptosis of RGCs. One day after axotomy, activated (phosphorylated) p38 was visualized by immunocytochemistry in the nuclei of RGCs, but not in control retinas. Phosphorylated p38 was first detected on immunoblots 12 hr after axotomy, reached a maximum at 1 d, and then decreased. To investigate possible roles of p38 in RGC death, a p38 MAP kinase inhibitor, SB203580, was administered intravitreally at the time of axotomy and repeated at 5 and 10 d. Assayed 14 d after axotomy, SB203580 increased the number of surviving RGCs in a dose-dependent manner (the minimum effective concentration was 1.6 micrometer). Furthermore, MK801, a selective inhibitor of NMDA receptors, not only showed protective effects against RGC apoptosis but also attenuated p38 MAP kinase activation in a dose-dependent manner. Our findings imply that p38 is in the signaling pathway to RGC apoptosis mediated by glutamate neurotoxicity through NMDA receptors after damage to the optic nerve. p38 inhibitors could be potentially useful for the treatment of optic nerve trauma and neurodegenerative diseases that affect RGCs, such as glaucoma.

Mitochondrial and extramitochondrial apoptotic signaling pathways in cerebrocortical neurons.

In cultured cerebrocortical neurons, mild excitotoxic insults or staurosporine result in apoptosis. We show here that N-methyl-d-aspartate (NMDA) receptor-mediated, but not staurosporine-mediated, apoptosis is preceded by depolarization of the mitochondrial membrane potential (Deltapsi(m)) and ATP loss. Both insults, however, release cytochrome c (Cyt c) into the cytoplasm. What prompts mitochondria to release Cyt c and the mechanism of release are as yet unknown. We examined the effect of inhibition of the adenine nucleotide translocator (ANT), a putative component of the mitochondrial permeability transition pore. Inhibition of the mitochondrial ANT with bongkrekic acid (BA) prevented NMDA receptor-mediated apoptosis of cerebrocortical neurons. Concomitantly, BA prevented Deltapsi(m) depolarization, promoted recovery of cellular ATP content, and blocked caspase-3 activation. However, in the presence of BA, Cyt c was still released. Because BA prevented NMDA-induced caspase-3 activation and apoptosis, the presence of Cyt c in the neuronal cytoplasm is not sufficient for the induction of caspase activity or apoptosis. In contrast to these findings, BA was ineffective in preventing staurosporine-induced activation of caspases or apoptosis. Additionally, staurosporine-induced, but not NMDA-induced, apoptosis was associated with activation of caspase-8. These results indicate that, in cerebrocortical cultures, excessive NMDA receptor activation precipitates neuronal apoptosis by means of mitochondrial dysfunction, whereas staurosporine utilizes a distinct pathway.

Involvement of activated caspase-3-like proteases in N-methyl-D-aspartate-induced apoptosis in cerebrocortical neurons.

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.

Molecular basis of NMDA receptor-coupled ion channel modulation by S-nitrosylation.

Several ion channels are thought to be directly modulated by nitric oxide (NO), but the molecular basis of this regulation is unclear. Here we show that the NMDA receptor (NMDAR)-associated ion channel was modulated not only by exogenous NO but also by endogenous NO. Site-directed mutagenesis identified a critical cysteine residue (Cys 399) on the NR2A subunit whose S-nitrosylation (NO+ transfer) under physiological conditions underlies this modulation. In cell systems expressing NMDARs with mutant NR2A subunits in which this single cysteine was replaced by an alanine, the effect of endogenous NO was lost. Thus endogenous S-nitrosylation can regulate ion channel activity.

Expression and trans-synaptic regulation of P2x4 and P2z receptors for extracellular ATP in parotid acinar cells. Effects of parasympathetic denervation.

Trans-synaptic regulation of muscarinic, peptidergic, and purinergic responses after denervation has been reported previously in rat parotid acinar cells (McMillian, M. K., Soltoff, S. P., Cantley, L. C., Rudel, R., and Talamo, B. R. (1993) Br. J. Pharmacol. 108, 453-461). Characteristics of the ATP-mediated responses and the effects of parasympathetic denervation were further analyzed through assay of Ca2+ influx, using fluorescence ratio imaging methods, and by analysis of P2x receptor expression. ATP activates both a high affinity and a low affinity response with properties corresponding to the recently described P2x4 and the P2z (P2x7)-type purinoceptors, respectively. Reverse transcription-polymerase chain reaction analysis reveals mRNA for P2x4 as well as P2x7 subtypes but not P2x1, P2x2, P2x3, P2x5, or P2x6. P2x4 protein also is detected by Western blotting. Distribution of the two types of ATP receptor responses on individual cells was stochastic, with both high and low affinity responses on some cells, and only a single type of response on others. Sensitivity to P2x4-type activation also varied even among cells responsive to low concentrations of ATP. Parasympathetic denervation greatly enhanced responses, tripling the proportion of acinar cells with a P2x4-type response and increasing the fraction of highly sensitive cells by 7-fold. Moreover, P2x4 mRNA is significantly increased following parasympathetic denervation. These data indicate that sensitivity to ATP is modulated by neurotransmission at parasympathetic synapses, at least in part through increased expression of P2x4 mRNA, and suggest that similar regulation may occur at other sites in the nervous system where P2x4 receptors are widely expressed.

Role of caspases in N-methyl-D-aspartate-induced apoptosis in cerebrocortical neurons.

Overactivation of glutamate receptors mediates neuronal death in several acute and chronic neurodegenerative diseases. The intracellular processes underlying this form of death, however, remain poorly understood. Depending on the severity of insult, N-methyl-D-aspartate (NMDA) receptor activation induces either apoptosis or necrosis. Cysteine proteases related to interleukin-1beta-converting enzyme (ICE), recently termed caspases, appear necessary for neuronal apoptosis in vivo and in vitro. To determine whether caspases play a role in NMDA-induced apoptosis, we used two functionally distinct approaches to decrease substrate cleavage by caspases. One is a novel peptide (V-ICEinh) that contains the caspase catalytic site and acts as a pseudoenzyme that binds caspase substrates and prevents their cleavage. The other is a pseudosubstrate peptide (Z-VAD x fmk) that inhibits caspase activity. Pretreatment with either V-ICEinh or Z-VAD-fmk protects cerebrocortical neurons from NMDA-induced apoptosis, suggesting a role for caspases in NMDA-induced apoptosis. To explore the signaling pathways involved, we looked at the effects of NMDA receptor activation on Ca2+ influx, production of reactive oxygen species (ROS), mitochondrial membrane potential, and lipid peroxidation. Neither NMDA-induced Ca2+ influx nor the initial collapse of mitochondrial membrane potential could be prevented by pretreatment with V-ICEinh or Z-VAD x fmk. In contrast, ROS formation and lipid peroxidation were completely blocked by both V-ICEinh and Z-VAD x fmk. Taken together, our results suggest that Ca2+ influx and mitochondrial depolarization occur upstream from caspase activation, whereas ROS formation and lipid peroxidation may be downstream events in the cascade leading to cortical neuronal apoptosis.

Suppression of neuronal apoptosis by S-nitrosylation of caspases.

S-Nitrosylation (reaction of nitric oxide (NO) species with a critical cysteine sulfhydryl) can regulate the physiological activity of proteins, including enzymes, ion channels, G-proteins, and transcription factors. Caspases are a family of interleukin-1beta-converting enzyme-like proteases involved in the signaling pathway to apoptotic cell death, and each member of this enzyme family contains a critical cysteine residue in its active site. Here we show that S-nitrosylation of caspases in human embryonic kidney (HEK)-293 cells and primary cerebrocortical neurons decreases enzyme activity and is associated with protection from apoptosis.

Modulation of extracellular ATP-induced Ca2+ responses: role of protein kinases.

Evidence for the modulation of the P2z-purinoceptor for extracellular ATP in dissociated rat parotid cells is presented in studies using compounds that inhibit protein kinases. Preincubation of acinar cells with the protein kinase catalytic-site inhibitors K-252a and staurosporine, as well as with the regulatory-domain inhibitor sphingosine, specifically potentiates the elevation in cytosolic Ca2+ concentration ([Ca2+]i) mediated by extracellular ATP, but has no effect on the [Ca2+]i elevation mediated by muscarinic receptors through phospholipase C activation. Phorbol dibutyrate (PDBu), which activates protein kinase C (PKC), has no modulatory effect on ATP-mediated [Ca2+]i elevation. Further, pretreatment with PDBu does not reverse or block the effects of K-252a or sphinogosine, arguing against the involvement of PKC. Other pharmacological manipulations indicate that neither calmodulin-dependent nor cyclic-AMP-dependent kinases are involved. Neither the peak intracellular Ca2+ mobilization nor the sustained Ca2+ entry in response to carbachol or to a Ca2+ ionophore (4-bromo-A23187) is altered by the kinase inhibitors that potentiate the [Ca2+]i response to ATP, indicating that effects on the ATP response are not due to non-specific permeability changes, nor to decreased Ca2+ removal from the cytosol. ATP-mediated influx of Mn2+ as well as ATP-induced membrane depolarization are potentiated in cells preincubated with K-252a, directly demonstrating that cation influx is enhanced through a P2z-specific route. These results show that P2z responses (or purinoceptors) can be modulated and suggest that phosphorylation events are involved.