Spotlight A New Look at Neurotoxicity Parkinson’s disease (PD) is a neurodegenerative disorder resulting from selective loss of dopaminergic neurons in the substantial nigra of the brain. Although the mechanism of the neuronal death is not known, a favored hypothesis is that a dysfunction of mitochondrial complex I (MCI) is involved in the process. In support of this hypothesis is the fact that exposure to known or suspected MCI inhibitors such as rotenone, paraquat, and MPP+ induces PD symptoms. To more fully explore the role of MCI in PD pathogenesis, Choi et al. [(2008) Proc. Natl. Acad. Sci. U.S.A. 105, 15136] have developed mesencephalic neuron cultures from the embryos of mice bearing a targeted deletion of the Ndufs4 gene that encodes a subunit of MCI. Neuronal cell cultures derived from embryonic Nudfs4-null mice were viable and morphologically
Lighting Up the ARE Activation of the antioxidant response element (ARE) is one mechanism by which cells protect themselves against exposure to reactive oxidants and electrophiles. The pathway is triggered when the reactive species chemically modify the protein, Keap1. Keap1 modification alters its interaction with the transcription factor, Nrf2, leading to Nrf2 activation and translocation to the nucleus. There, it binds to ARE-containing genes, leading to the transcription of a large number of protective proteins. Among the enzymes induced by the ARE pathway in human cell lines are the aldo-keto reductases AKR1C2 and AKR1C3. Now, Halim et al. [(2008) J. Am. Chem. Soc. 130, 14123] report on a new way to monitor ARE-dependent gene expression through a real-time fluorescence assay for the activity of these two enzymes. Their approach is based on the fact that AKR1C2 and AKR1C3 metabolize the nonfluorescent ketone, coumberone, to the corresponding highly fluorescent alcohol, coumberol. The activities of the two enzymes may be distinguished by the use of the highly selective inhibitors, ursodeoxycholic acid (>200-fold selectivity for AKR1C2) and indomethacin (>14-fold selectivity for AKR1C3). Halim et al. validated their approach by showing that tertbutylhydroquinone (t-BHQ) induces expression of AKR1C3 in IMR-32 neuroblastoma cells through the use of the coumberone fluorescence assay. They confirmed the fluorescence assay results by showing increased AKR1C3 protein levels by immunoblot and verifying ARE activation through increased expression of the ARE-dependent protein NQO1. Similarly, the coumberone assay demonstrated Published online 01/19/2009 • DOI: 10.1021/tx800451f © 2009 American Chemical Society
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indistinguishable from those from wild-type mice. Choi et al. confirmed that the Ndufs4-null neurons lacked MCI activity, but this did not affect the survival of the dopaminergic neurons in the cultures. The absence of MCI activity did not protect the dopaminergic cells from the toxic effects of rotenone, MPP+, or paraquat; in fact, rotenone toxicity was more severe in the cells lacking Ndufs4. Furthermore, there was no direct correlation between dopaminergic neuron death and MCI inhibition. These data raise serious doubts that the induction of PD by agents such as rotenone occurs primarily as a direct result of MCI inhibition and suggest that failure of MCI function in dopaminergic neurons is not a primary factor in the pathogenesis of spontaneous PD in human patients. • Carol Rouzer
induction of AKR1C2 in HepG2 hepatoma cells treated with t-BHQ, sulforaphane, or ethacrynic acid. Having verified the usefulness of the coumberone fluorescence assay, Halim et al. next used it to screen for new inducers of the ARE pathway. They discovered that panaxytriol, a component of red ginseng, caused a concentration-dependent induction of AKR1C3 in IMR-32 cells and AKR1C2 in HepG2 cells. The induction of AKR1C3 in IMR-32 cells was attenuated by selective kinase inhibitors LY294002 and Ro-31-8220, suggesting a role for phosphatidylinositol 3-kinase and protein kinase C, respectively, in the signaling pathway. These results are similar to those reported previously for t-BHQ in these cells.
Reproduced from Halim et al. [(2008) J. Am. Chem. Soc. 130, 14123]. Copyright 2008 American Chemical Society.
The data presented by Halim et al. affirm the coumberone fluorescence assay as a potentially valuable method for monitoring ARE-dependent transcription and screening for new activators of the ARE pathway. They also identify panaxytriol as an ARE inducer, a finding that may explain its previously reported antitumor and neurotrophic activities. • Carol A. Rouzer Vol. 22,
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Spotlight Ceramide and Apoptosis Understanding the process of programmed cell death, apoptosis, is key to dissecting the mechanism by which many toxic substances kill cells. Deng et al. [(2008) Science 322, 110]. advance our knowledge of this complex process through their studies of the role of ceramide in radiationinduced apoptosis in germ line cells of C. elegans. These cells, which divide throughout the organism’s adult life, are highly sensitive to radiation, engaging an apoptotic pathway that involves the p53 homologue CEP-1, and proteins making up the apoptotic machinery, including the caspase CED-3, the apoptotic protease activating factor1-like protein CED-4, the Bcl-2 protein CED-9, and the BH3-domain protein EGL-1. The process is antagonized upstream by the ABL-1 tyrosine kinase. In C. elegans, there are three genes coding for ceramide synthase enzymes, hyl-1, hyl-2, and lagr-1. Deng et al. found that deletion mutants of hyl-1 and lagr-1, but not hyl-2, were resistant to germ line radiation-induced apoptosis. Intragonadal injection of C16-ceramide reversed the protection observed in hyl-1 and lagr-1 mutants, and higher doses induced apoptosis in wild-type organisms in the absence of radiation. Radiation-induced expression of EGL-1 protein was not affected by lagr-1 or hyl-1 deletion, and exogenous C16-ceramide did not induce apoptosis in loss-of-function mutants of egl-1, ced-4, or ced-3, or a gain-of-function mutant of ced-9. These results suggested that ceramide promotes apoptosis via a mechanism that is parallel to the function of the CEP-1-triggered apoptotic machinery and requires that machinery be operational. Fluorescence microscopy indicated that radiation-induced increases in ceramide levels were localized to the mitochondria of germ line cells and that these increases were eliminated in hyl-1;lagr-1 double mutants. Radiation exposure elicited a translocation of CED-4 from the mitochondria to the nucleus that was eliminated by hyl-1;lagr-1 double deletion. Deng et al. note that ceramide has a capacity to self-associate within biological membranes, producing ceramide-rich macrodomains that can alter protein association and function. The increase in mitochondrial ceramide in radiation-treated germ line cells may create such domains, thereby promoting the EGL-1-driven displacement of CED-4 from its resting-state association with CED-9 in mitochondrial membranes. This displacement is a critical first step allowing CED-4 to translocate to nuclear membranes and initiate the apoptotic response. Ceramide has been proposed to play a role in apoptosis in other cells, including human tumor cells. Perhaps these studies in C. elegans will shed light on apoptotic mechanisms in other species as well. • Carol A. Rouzer
MetsThe Natural Antioxidant Evolutionary biologists have been intrigued by the fact that the codon AUA specifies Ile for all nuclear-coded genes but Met for mitochondrial-coded genes of some, but not all, species. A number of hypotheses have been offered to explain this selective codon switch, but now, Bender et al. 16
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[(2008) Proc. Natl. Acad. Sci. U.S.A. 105, 16496] propose a new idea linking mitochondrial protein Met levels to protection against oxidative stress. Bender et al. discovered that Met constitutes approximately 2-3% of all amino acids in nuclear-encoded proteins and that the percentage is similar for mitochondrialencoded proteins in species in which AUA encodes for Ile. For mitochondrial-encoded proteins in which AUA codes for Met, however, levels of this amino acid are much higher, averaging approximately 6% and reaching as high as 10%. A comparison of mitochondrial-encoded proteins from the echinoderm F. serratissima (AUA ) Ile) and the insect M. bicolor (AUA ) Met) revealed that the “extra” Met residues in the M. bicolor proteins were localized primarily to transmembrane and surface domains.
Reproduced with permission from Bender et al. [(2008) Proc. Natl. Acad. Sci. U.S.A. 105, 16496]. Copyright 2008 National Academy of Sciences.
Bender et al. argue that the location of the added Met residues would be expected to minimize the structural effect of their substitution for Ile, while at the same time placing them at the point of maximal exposure to oxidants in the mitochondrial environment. The switch of the AUA codon from Ile to Met occurs primarily in species with high rates of oxidative metabolism and correlates with the appearance of genes for Met sulfoxide reductases, which repair oxidative damage to Met residues. These findings suggest that the mitochondrial Met residues may serve as oxidant scavengers, which are then enzymatically repaired. Support for this hypothesis was obtained in experiments in which membrane-soluble Met mimics were shown to provide protection against oxidative stress in SH-SY5Y neuroblastoma cells. Thus, it is quite conceivable that a switch from Ile to Met, which probably had minimal effect on three-dimensional structure, provided a strong adaptive advantage to protect organisms with a high rate of oxidative metabolism against oxidant damage. • Carol A. Rouzer TX800451F Published online 01/19/2009 •
DOI: 10.1021/tx800451f $40.75 © 2009 American Chemical Society
