Richard C. Dodel

Caspase-3-like proteases and 6-hydroxydopamine induced neuronal cell death

Neurotoxicity induced by 6-hydroxydopamine (6-OHDA) is believed to be due, in part, to the production of reactive oxygen species (ROS) and/or an inhibition of mitochondrial function. However, little is known about the ensuing intracellular events which ultimately result in cell death. Here we show that exposure to relatively low concentrations of 6-OHDA induces apoptosis of cerebellar granule neurons (CGN). 6-OHDA-induced apoptosis of CGN is associated with activation of a caspase-3-like protease. Western blots of cytosolic extracts from 6-OHDA-treated CGN reveal a translocation of cytochrome c from mitochondria to the cytosol, which precedes activation of the protease detected by Ac-DEVD-pNA. DNA laddering can be blocked by caspase inhibitors zVAD-FMK and Ac-DEVD-CHO, however cell death can only be attenuated for a short time period in the presence of these inhibitors. Our data suggest that 6-OHDA-induced apoptosis of CGN involves activation of a caspase-3-like protease. In contrast to the neurotoxicity induced by MPP+, however, the peptide inhibitors zVAD-FMK and Ac-DEVD-CHO can only attenuate early neuronal death induced by 6-OHDA. At later time points, neuronal death lacking DNA laddering occurs even in the presence of the peptide inhibitor zVAD-FMK or Ac-DEVD-CHO.

Peptide inhibitors of caspase-3-like proteases attenuate 1-methyl-4-phenylpyridinum-induced toxicity of cultured fetal rat mesencephalic dopamine neurons.

Abstract Multiple aspartate-specific cysteine proteases have been identified and specific members of this family have been implicated in the apoptotic death of many mammalian cell types. Caspase-3-like proteases seem to play a pivotal role in neuronal apoptosis since mice with germline inactivation of the caspase-3 gene manifest profound alterations in neurogenesis. Moreover, inhibitors of caspase-3-related proteases have been shown to inhibit neuronal apoptosis. Here we extend recent work from our laboratory on the mechanisms mediating the neurotoxic actions of 1-phenyl-4-methylpyridinium using ventral mesencephalon cultures containing dopamine neurons. We demonstrate that low concentrations of 1-phenyl-4-methylpyridinium induce apoptosis in dopamine neurons by morphological and biochemical criteria. Moreover, pretreatment of ventral mesencephalon cultures with the tetrapeptide inhibitors of the caspase-3-like proteases; zVAD-FMK or Ac-DEVD-CHO specifically inhibit death of dopamine neurons neurons induced by low concentrations of 1-phenyl-4-methylpyridinium, whereas the caspase-1-like inhibitor Ac-YVAD-CHO was without effect. Our data indicate that exposure of cultured ventral mesencephalondopamine neurons to low concentrations of 1-phenyl-4-methylpyridinium results in apoptotic death and that caspase-3-like proteases may mediate the neurotoxic apoptotic actions of 1-phenyl-4-methylpyridinium.

Caffeic acid phenethyl ester prevents cerebellar granule neurons (CGNs) against glutamate-induced neurotoxicity.

Caffeic acid phenethyl ester (CAPE) is an active component of propolis obtained from honeybee hives and is found to have the following properties: anti-mitogenic, anti-carcinogenic, anti-inflammatory, immunomodulatory, and antioxidant. Recent reports suggest that CAPE also has a neuronal protective property against ischemic injury. Since excitotoxicity may play an important role in ischemia, in this study, we investigated whether CAPE could directly protect neurons against excitotoxic insult. We treated cultured rat cerebellar granule neurons (CGNs) with excitotoxic concentrations of glutamate in the presence or absence of CAPE and found that CAPE markedly protected neurons against glutamate-induced neuronal death in a concentration-dependent fashion. Glutamate-induced CGNs death is associated with time-dependent activation of caspase-3 and phosphorylation of p38, both events of which can be blocked by CAPE. Treating CGNs with specific inhibitors of these two enzymes together exerts a synergistic neuroprotective effect, similar to the neuroprotective effect of CAPE exposure. These results suggest that CAPE is able to block glutamate-induced excitotoxicity by inhibiting phosphorylation of p38 and caspase-3 activation. This finding may further help understanding of the mechanism of glutamate-induced neuronal death and CAPE-induced neuroprotection against excitotoxicity.

Caffeic acid phenethyl ester prevents 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced neurodegeneration

Parkinsons disease is associated with the loss of dopaminergic neurons in the substantia nigra and decreased striatal dopamine levels. We now report that caffeic acid phenethyl ester (CAPE), an active component of propolis, attenuated dopaminergic neurodegeneration and dopamine loss in the MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) mouse model of Parkinsons disease. The neuroprotective effect of CAPE was associated with marked reductions in inducible nitric oxide synthase (iNOS) and caspase 1 expression. Additionally, CAPE inhibited MPP+-induced neurotoxicity in vitro and directly inhibited MPP+-induced release of cytochrome c and apoptosis inducing factor (AIF) from mitochondria. Thus, CAPE may have beneficial effects in slowing or preventing the progression of Parkinsons disease and other neurodegenerative disorders.

bcl-2 Protects SK-N-SH Cells From 6-Hydroxydopamine Induced Apoptosis by Inhibition of Cytochrome c Redistribution

Neurotoxicity induced by 6-hydroxydopamine (6-OHDA) is, in part, due to the production of reactive oxygen/nitrogen species (RNOS) and/or an inhibition of mitochondrial function. However, little is known about the ensuing intracellular events which ultimately result in cell death. Here we show that exposure to relatively low concentrations of 6-OHDA induce apoptosis of SK-N-SH cells. Western blots of cytosolic extracts from 6-OHDA treated cells reveal a translocation of cytochrome c from mitochondria into the cytosol. To further delineate the pathway by which 6-OHDA causes apoptosis, we investigated the effects on cell survival in bcl-2 overexpressing SK-N-SH cells and the relation to translocation of cytochrome c and free radical production. Overexpressing bcl-2 in SK-N-SH cells shifted the toxicity as well as the release of cytochrome c to higher concentrations of 6-OHDA. At concentrations of 6-OHDA in SK-N-SH-bcl-2 cells where a release of cytochrome c and subsequent cell death occurs, an increase of the oxidation of the fluorescent dye dihydrorhodamine-123 was detectable. This RNOS production paralleled the release of cytochrome c and was inhibited by bcl-2 as well. Prom these experiments, we hypothesize that cytochrome c might interact with 6-OHDA to promote an oxidative burst, which can be inhibited by bcl-2.