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© The Rockefeller University Press,
0021-9525/1997//1137 $5.00
The Journal of Cell Biology, Volume 136, Number 5,
, 1997 1137-1149
Article |
Neuroprotective Action of Cycloheximide Involves Induction of Bcl-2 and Antioxidant Pathways
Department of Physiology, Department of Anatomy and Neurobiology, University of Kentucky, Lexington, Kentucky 40536
The ability of the protein synthesis inhibitor cycloheximide (CHX) to prevent neuronal death in different paradigms has been interpreted to indicate that the cell death process requires synthesis of "killer" proteins. On the other hand, data indicate that neurotrophic factors protect neurons in the same death paradigms by inducing expression of neuroprotective gene products. We now provide evidence that in embryonic rat hippocampal cell cultures, CHX protects neurons against oxidative insults by a mechanism involving induction of neuroprotective gene products including the antiapoptotic gene bcl-2 and antioxidant enzymes. Neuronal survival after exposure to glutamate, FeSO4, and amyloid β-peptide was increased in cultures pretreated with CHX at concentrations of 50–500 nM; higher and lower concentrations were ineffective. Neuroprotective concentrations of CHX caused only a moderate (20–40%) reduction in overall protein synthesis, and induced an increase in c-fos, c-jun, and bcl-2 mRNAs and protein levels as determined by reverse transcription–PCR analysis and immunocytochemistry, respectively. At neuroprotective CHX concentrations, levels of c-fos heteronuclear RNA increased in parallel with c-fos mRNA, indicating that CHX acts by inducing transcription. Neuroprotective concentrations of CHX suppressed accumulation of H2O2 induced by FeSO4, suggesting activation of antioxidant pathways. Treatment of cultures with an antisense oligodeoxynucleotide directed against bcl-2 mRNA decreased Bcl-2 protein levels and significantly reduced the neuroprotective action of CHX, suggesting that induction of Bcl-2 expression was mechanistically involved in the neuroprotective actions of CHX. In addition, activity levels of the antioxidant enzymes Cu/ Zn-superoxide dismutase, Mn-superoxide dismutase, and catalase were significantly increased in cultures exposed to neuroprotective levels of CHX. Our data suggest that low concentrations of CHX can promote neuron survival by inducing increased levels of gene products that function in antioxidant pathways, a neuroprotective mechanism similar to that used by neurotrophic factors.
Abbreviations used in this paper: Aβ, amyloid β-peptide; BDNF, brainderived neurotrophic factor; bFGF, basic FGF; BSO, buthionine sulfoximine; CHX, cycloheximide; DCF, 2,7-dichlorofluorescein diacetate; hnRNA, heteronuclear RNA; NMDA, N-methyl-d-aspartate; ODN, oligodeoxynucleotide; RT, reverse transcription; SOD, superoxide dismutase.
Cycloheximide (CHX)1 is a protein synthesis inhibitor that has been widely used in studies of programmed cell death or apoptosis. Apoptosis and necrosis are two different forms of cell death whose distinguishing characteristics are based largely on morphological features (see Wyllie et al., 1980; for review see Steller, 1995). Cells dying by apoptosis undergo shrinkage, cell surface blebbing, and DNA condensation and fragmentation; their membranes remain intact as the cell dies. In contrast, cells dying by necrosis swell and lyse. Neuronal apoptosis has been most commonly studied in paradigms of natural developmental cell death in which withdrawal of trophic factor support initiates the cell death program (Deckwerth and Johnson, 1993). However, it is becoming increasingly recognized that apoptosis also occurs in both acute and chronic neurodegenerative conditions in the adult nervous system. For example, neurons may die by apoptosis in cerebral ischemia (MacManus et al., 1993; Linnik et al., 1993), epilepsy (Pollard et al., 1994), Huntington's disease (Portera-Calliau et al., 1995), and Alzheimer's disease (for review see Cotman and Anderson, 1995). CHX delays or prevents the death of neurons subjected to a range of insults. For example, CHX prevents apoptosis of cultured sympathetic neurons induced by withdrawal of NGF (Martin et al., 1988, 1992) and also prevents trophic factor deprivation–induced death of PC12 cells (Pittman et al., 1993). In addition, CHX protects: cultured retinal ganglion cells against excitotoxicity (Dreyer et al., 1995); PC12 cells against glutamate toxicity (Serghini et al., 1994); adult cortical neurons against ischemic injury in vivo (Goto et al., 1990; Linnik et al., 1993; Tortosa et al., 1994); adult septal neurons against NGF withdrawal in vivo (Svendsen et al., 1994); cultured striatal and cortical neurons against the toxicity of 3-nitropropionic acid (Behrens et al., 1995); cultured cortical neurons against oxidative stress-induced death (Ratan et al., 1994a); and cultured cortical neurons against amyloid β-peptide (Aβ) toxicity (Takashima et al., 1993). One widely accepted interpretation of the ability of CHX to protect neurons is that it prevents the synthesis of "killer" gene products (for review see Schwartz and Osborne, 1993). However, that mechanism of CHX action has not been firmly established and alternative explanations exist, including the quite opposite possibility that CHX induces the expression of cytoprotective gene products. Indeed, CHX is well known to induce increases in levels of an array of immediate early gene mRNAs (Carter, 1993; Oguchi et al., 1994), and recent findings suggest that CHX can activate kinases such as mitogen-activated protein kinase (Zinck et al., 1995) known to be involved in neurotrophic factor signaling cascades (Seger and Krebs, 1995).
Data from a variety of cell death paradigms ranging from withdrawal of trophic factor support in sympathetic neurons (Greenlund et al., 1995a), excitotoxicity (Bondy and LeBel, 1993; Mattson et al., 1995), metabolic impairment (Beal, 1995), and Aβ toxicity (Mattson et al., 1993a; Behl et al., 1994; Goodman and Mattson, 1994) suggest that free radicals and disruption of calcium homeostasis are convergence points in the cell death process in both apoptosis and necrosis. Further evidence supporting common mechanisms of apoptotic and necrotic neuronal death comes from studies showing that the same neurotrophic factor can protect neurons against death induced by the range of insults mentioned above including: withdrawal of trophic support (Deckwerth and Johnson, 1993); excitotoxicity (Mattson et al., 1989, 1995); metabolic insults (Cheng and Mattson, 1991; Lindvall et al., 1994); cerebral ischemia (Koketsu et al., 1994); and Aβ toxicity (Mattson et al., 1993a; Goodman and Mattson, 1994). General mechanisms of neuroprotection appear to involve induction of the expression of proteins involved in suppressing free radical accumulation (e.g., antioxidant enzymes) and stabilizing ion homeostasis (e.g., calcium-binding proteins) (for review see Mattson et al., 1993b). For example, NGF, basic FGF (bFGF), and brain-derived neurotrophic factor (BDNF) increased to varying degrees activities of one or more antioxidant enzymes (Cu/Zn–superoxide dismutase [SOD], catalase, glutathione peroxidase, and glutathione reductase) in hippocampal cell cultures (Mattson et al., 1995). Data suggest that BDNF can also induce expression of the antiapoptotic gene product bcl-2 (Allsopp et al., 1995), which is believed to function in antioxidant pathways (Hockenbery et al., 1993). bcl-2 can delay cell death when overexpressed in a variety of cell paradigms of apoptotic and necrotic cell injury (Hockenbery et al., 1990; Martinou et al., 1994; Bredesen, 1995; Chen et al., 1995; Linnik et al., 1995; Lawrence et al., 1996). Sympathetic neurons from mice deficient in bcl-2 die more rapidly after NGF withdrawal than do sympathetic neurons from wildtype mice (Greenlund et al., 1995b). These kinds of data led us to propose that cell death, whether apoptotic or necrotic, occurs when activation of cell life programs is not sufficient to overcome the level of stress (oxidative or ionic) imposed upon the neuron (Mattson and Barger, 1995). Because neurotrophic factors protect neurons against death in many of the same paradigms in which CHX is neuroprotective, we tested the hypothesis that CHX protects neurons by a mechanism similar to neurotrophic factors, namely, by inducing cytoprotective gene products.
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Materials and Methods |
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Quantification of Neuron Survival
These methods are detailed in our previous studies (Mattson et al., 1989, 1995). Briefly, viable neurons were counted in premarked microscope fields (x10 objective) before experimental treatment and 20–24 h after treatment. Most neurons that died during the exposure period to glutamate, FeSO4, or Aβ were absent at the 20-h time point, and viability of the remaining neurons was assessed by morphological criteria. Neurons with intact neurites of uniform diameter and a soma with a smooth appearance were considered viable. Neurons with fragmented neurites and a vacuolated and/or swollen soma were considered nonviable.
Protein Synthesis Assay
These methods were those described previously (Martin et al., 1992). Cultures (incubated in methionine- and cysteine-free medium) were pretreated with vehicle or CHX, and then [35S]methionine/cysteine (ICN Radiochemicals, Irvine, CA; sp act >1,000 Ci/mmol) was added to a final concentration of 25 µCi/ml (in the continued presence of CHX). 30 min later, cultures were washed once with medium, and 2 ml of a solution containing 0.5% SDS, 1 mM EDTA, 10 mM Tris (pH 7.5) was added. 40 µg of BSA was added to each sample, and protein was precipitated with 10% TCA on ice. Precipitated protein was collected by filtration onto 0.45-µm nitrocellulose filters. Filters were washed twice with cold 10% TCA and dried, and radioactivity was quantified in a liquid scintillation counter.
PCR Analyses
Analyses of gene expression were performed as described previously (Estus et al., 1994). Briefly, polyA+ RNA was isolated from cells after the indicated treatments by using an oligo-dT–cellulose-based purification kit (QuickPrep Micro kit; Pharmacia Fine Chemicals, Piscataway, NJ) and concentrated by coprecipitation with glycogen, all as directed by the manufacturer. Half of the mRNA was converted to cDNA by reverse transcription (RT) by using random hexamers (16 µM) to prime Moloney murine leukemia virus reverse transcriptase (Superscript; Life Technologies, Grand Island, NY). The 30-µl reaction contained 50 mM Tris (pH 8.3), 40 mM KCl, 6 mM MgCl2, 1 mM DTT, 500 µM each dATP, dTTP, dCTP, dGTP, and 20 U RNasin (Promega, Madison, WI). After 10 min at 20°C, the samples were incubated for 50 min at 42°C, and the reaction was then terminated by heating to 94°C for 5 min.
For PCR amplification of specific cDNAs, stock reactions (50 µl) were prepared on ice and contained 50 µM dCTP, 100 µM each dGTP, dATP, and dTTP, 15 µCi 
-[32P]dCTP (3,000 Ci/mmol), 1.5 mM MgCl2, 50 mM KCl, 10 mM Tris (pH 9.0), 0.1% Triton X-100, 1 µM each primer, 1 U of Taq polymerase, and 3% of the cDNA synthesized in the RT reaction. The sequences of the primers used in this study were: NFM sense primer: 5' ACG CTG GAC TCG CTG GGC AA 3', NFM antisense primer: 5' GCG AGC GCG CTG CGC TTG TA 3' (156-bp product); c-jun sense primer: 5' ACT CAG TTC TTG TGC CCC AA 3', c-jun antisense primer: 5' CGC ACG AAG CCT TCG GCG AA 3' (64-bp product); c-fos sense primer: 5' AAT AAG ATG GCT GCA GCC AA, c-fos antisense primer: 5' TTG GCA ATC TCG GTC TGC AA 3' (116-bp product); bcl-2 sense primer: 5'-CTGTACGGCCCCAGCATGCG-3', bcl-2 antisense primer: 5'-GCTTTGTTTCATGGTACATC-3' (210-bp product). The stock solutions were separated into three equal aliquots that were covered with a drop of mineral oil and subjected to various numbers of PCR cycles to determine the minimum number of cycles necessary to detect the PCR product. The typical reaction conditions were 1 min at 95°C, 1 min at 55°C, and 2 min at 72°C. The results depicted here represent 16–22 cycles of amplification. After amplification, the cDNAs were separated by electrophoresis on 12% polyacrylamide gels, visualized by autoradiography of the dried gels, and quantified with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The identity of the PCR product amplified by each primer pair was confirmed typically either by direct sequencing (fmole sequencing kit; Promega), or by subcloning the amplified cDNAs into pBluescript (Stratagene, La Jolla, CA) and then sequencing the inserts.
Immunocytochemistry and Western Blot Analyses
These methods were similar to those described previously (Mattson et al., 1993c; Cheng et al., 1994). Briefly, cells were fixed in 4% paraformaldehyde, with membranes permeabilized by exposure for 5 min to 0.2% Triton X-100 in PBS, and placed in blocking serum (0.5% horse serum). Cells were then exposed to primary antibody overnight at 4°C, followed by incubation for 1 h with biotinylated anti–mouse IgG, a 30-min incubation in the presence of ABC reagent (Vector Labs, Inc., Burlingame, CA), and a 5-min exposure to DAB tetrahydrochloride. Parallel control cultures were processed identically except that the primary antibody was deleted from the procedure. The primary antibodies were affinity-purified rabbit anti– c-Fos (1:500) and anti–c-Jun (1:1,000) (Oncogene Science, Inc., Minecola, NY), and mouse monoclonal anti–Bcl-2 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA). Immunostained cultures were visualized under bright-field optics, and relative levels of immunoreactivity of neuronal cell bodies were scored according to the following scale: 0, no immunoreactivity; 1, weak; 2, moderate; 3, intense; 4, very intense. 100 neurons were scored per culture. All cultures were scored without knowledge of their treatment history. For Western blot analysis, solublized proteins were separated by SDS-PAGE (12% gel), transferred to a nitrocellulose sheet, and immunoreacted overnight with Bcl-2 antibody in the presence of blocking serum (1:500). The blot was then exposed for 1 h to HRP-conjugated secondary antibody (1:10,000; Jackson ImmunoResearch Laboratories Inc., West Grove, PA) and immunoreactive protein was visualized using a chemiluminescence-based detection kit according to the manufacturer's protocol (ECL kit; Amersham Corp., Arlington Heights, IL).
Measurement of Hydrogen Peroxide Levels
These methods were similar to those described previously (Goodman and Mattson, 1994). Briefly, cells were incubated for 50 min in the presence of 50 µM 2,7-dichlorofluorescin diacetate (DCF; Molecular Probes, Inc., Eugene, OR), and then were washed three times (2 ml per wash) in HBSS containing 10 mM Hepes and 10 mM glucose. Cells were imaged using a confocal laser scanning microscope (Sarastro 2000; Molecular Dynamics) coupled to an inverted microscope (Nikon, Inc., Garden City, NY). Cells were located under visible light and a single image was acquired by scanning with the laser (488-nm excitation and 510-nm barrier filter). The laser intensity and photodetector gain were held constant to allow quantitative comparisons of relative fluorescence intensity of cells between treatment groups. Values of relative DCF fluorescence (average pixel intensity per cell) were obtained using the Imagespace software supplied by the manufacturer (Molecular Dynamics).
Antioxidant Enzyme Activity Assays
Activities of Cu/Zn-SOD, Mn-SOD, and catalase in cultured cells were quantified by methods described previously (Mattson et al., 1995). Cells from 60-mm culture dishes were pelleted by low speed centrifugation and homogenized in 2.5 ml of a nitrogen-purged buffer consisting of 10 mM Hepes, 137 mM NaCl, 4.6 mM KCl, 1.1 mM KH2PO4, 0.6 mM MgSO4 (pH 7.4). The homogenate was centrifuged for 1 h at 100,000 g at 4°C, and the supernatant was used for enzyme assays. For the SOD activity assay, 200 µl of the supernatant was added to 1.48 ml of 68 mM NaH2PO4 containing 1.35 mM EDTA (pH 7.8), 100 µl of 4 mM xanthine, and 170 µl of 3.53 mM epinephrine (pH 11.5). After a 5-min incubation at 30°C, 50 µl of xanthine oxidase was added to the cuvette, and the absorbance was followed for 3 min at 320 nm. To control for variability among lots, xanthine oxidase was diluted until an assay mixture without SOD yielded 0.03 A change per min. Mn-SOD activity was taken as the SOD activity remaining after addition of potassium cyanide (200 mM). One unit of SOD activity was defined as the amount that reduced the absorbance change by 50%, and results were expressed as units per mg protein. For the catalase activity assay, cell homogenate supernatant (1.5 ml) was mixed with 18 µl absolute ethanol and incubated on ice for 8 min, and 7.8 µl of 10% Triton X-100 and 2.5 ml of 10 mM NaH2PO4 buffer (pH 7.4) were added. Aliquots (0.6 ml) of the solution were added to 20-ml test tubes along with 3.57 ml of cold 6 mM hydrogen peroxide and the tube was vortexed. After 3 min, 0.714 ml of 6 N H2SO4 was added and the mixture was vortexed. 5 ml of 0.01 N KMnO4 was added to the solution, the tube was vortexed, and the absorbance was measured at 480 nm within 60 s using a Spec 21 UV-Vis spectrophotometer (Shimadzu, Inc., Kyoto, Japan). Quantitation was based on the comparison of the tissue samples with a calibration curve of known peroxide concentrations. The activity of the enzyme was expressed as units per mg protein with 1 unit = 1 µmol of H2O2 catalyzed per min.
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1 µM were ineffective in protecting neurons, and concentrations >1 µM caused significant neuronal death during a 24-h exposure period (Fig. 1 D).
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60% of control levels within 1 h of exposure, recovered to 80% of control levels by 4 and 12 h, and then decreased somewhat at 24 h (Fig. 2 A). In contrast, levels of protein synthesis in cultures exposed to 10 µM CHX were reduced to 10% of control levels within 1 h of exposure and remained at this low level through 24 h (Fig. 2 A). In a separate experiment, the concentration dependence of inhibition of protein synthesis by CHX was examined in more detail. Levels of protein synthesis were reduced to 60–75% of control levels in cultures exposed to 10–300 nM CHX for 1 h. CHX concentrations of 1 µM caused a >90% inhibition of protein synthesis.
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-glutamylcysteine synthase, an enzyme required for glutathione synthesis (Griffith and Meister, 1979). Hippocampal cultures were pretreated with vehicle or 100 nM CHX for 16 h, and then were exposed for 20 h to glutamate alone or in combination with 150 µM BSO. BSO significantly reduced the excitoprotective effect of CHX (Fig. 6 C). BSO alone was not toxic during a 20-h exposure period.
Bcl-2 Antisense Oligodeoxynucleotides Abrogate the Neuroprotective Actions of CHX
Previous studies used antisense approaches to demonstrate roles for Bcl-2 in prevention of cell death in several cell death paradigms (Kitada et al., 1994; Allsopp et al., 1995; Keith et al., 1995). In preliminary studies we showed that exposure of cultured hippocampal neurons to 10–50 µM of a Bcl-2 antisense ODN for 16 h resulted in a decrease in levels of Bcl-2 protein as determined by immunocytochemistry (Fig. 7 A) and Western blot analysis (Fig. 7 B). The ability of 100 nM CHX to protect neurons against glutamate toxicity in Bcl-2 antisense ODN-pretreated cultures and control (missense ODN- or vehicle-pretreated cultures) was then determined. Pretreatment with Bcl-2 antisense ODN significantly reduced the neuroprotective action of CHX, whereas the control ODN had no effect on the neuroprotective efficacy of CHX (Fig. 8). 20–35% of the neurons survived exposure to glutamate in the absence of CHX and in cultures pretreated with Bcl-2 antisense ODN plus CHX, whereas 50–65% of the neurons survived in cultures pretreated with CHX alone or CHX plus control ODN. Bcl-2 antisense and missense ODNs (Fig. 8), and sense and nonsense ODNs (data not shown), alone had no effect on neuronal survival during a 48-h exposure period.
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Discussion |
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It is increasingly recognized that apoptosis and necrosis often represent different manifestations of cell death that, nevertheless, share common underlying mechanisms. Indeed, the same initiator of cell death can kill the same cell type by either apoptosis or necrosis depending upon intensity/duration parameters of the insult (Ankarcrona et al., 1995). In the present study CHX protected hippocampal neurons against three different insults that are known to engender both apoptosis and necrosis. Our previous studies (Mattson et al., 1989; Mark et al., 1995) and unpublished data from studies of rat hippocampal cell cultures (M.P. Mattson and R.J. Mark) indicate that (at the concentrations used in the present study) glutamate and FeSO4 kill neurons mainly by necrosis, whereas Aβ induces mainly apoptotic death (see also Loo et al., 1993). The mechanisms of toxicities of these three insults have been well characterized in previous studies. Glutamate induces calcium influx through NMDA receptors and voltage- dependent channels, and the elevated calcium levels induce production of superoxide anion radical (Lafon-Cazal et al., 1993) and H2O2 (Mattson et al., 1995), probably by disrupting mitochondrial transmembrane potential (Mattson et al., 1993d). FeSO4 kills neurons by inducing hydroxyl radical production and lipid peroxidation; activation of NMDA receptors also contributes to FeSO4 neurotoxicity in hippocampal cell cultures (Zhang et al., 1993). Aβ induces membrane oxidation (Butterfield et al., 1994; Behl et al., 1994), which impairs function of ion- motive ATPases (Mark et al., 1995, 1997a) and glucose (Mark et al., 1997b) and glutamate (Keller et al., 1997) transporters, resulting in membrane depolarization and calcium influx; both free radicals and calcium appear to contribute to the neurotoxicity of Aβ (Mark et al., 1996). Our data indicate that the mechanism whereby CHX protects neurons against these different insults is by enhancing antioxidant defense systems. As evidence, we found that neuroprotective concentrations of CHX induce increased levels of Bcl-2. Moreover, exposure of hippocampal cultures to Bcl-2 antisense oligonucleotides reduced Bcl-2 levels and abrogated the neuroprotective effects of CHX. These findings suggest that the increase in Bcl-2 induced by neuroprotective concentrations of CHX was mechanistically linked to neuroprotection. Many different studies of both apoptotic and necrotic cell death paradigms have shown that Bcl-2 can protect cells against oxidative insults (Hockenbery et al., 1993; Fukunaga-Johnson et al., 1995; Kane et al., 1995). Measurements of levels of reactive oxygen species including H2O2 have shown that expression of Bcl-2 is correlated with reduced levels of oxidative stress in cells exposed to oxidative insults (Kane et al., 1993). Consistent with a role for Bcl-2 in the neuroprotective actions of CHX, we found that pretreatment of hippocampal neurons with neuroprotective concentrations of CHX resulted in a significant attenuation of FeSO4-induced accumulation of cellular H2O2. In addition, we found that activity levels of three different antioxidant enzymes were increased in CHX-treated cultures. It is known that oxidative stress itself can induce expression of immediate early gene products and antioxidant enzymes (Goldstone et al., 1995). However, we did not detect an increase in levels of H2O2 in cultured hippocampal neurons exposed to neuroprotective concentrations of CHX, suggesting that CHX was likely acting by a more direct effect on expression of the antioxidant enzymes. Another potential mechanism by which CHX might increase resistance to oxidative insults is suggested by the work of Ratan et al. (1994b) who showed that CHX can enhance cellular antioxidant pathways by shunting cysteine from protein synthesis to glutathione. Consistent with the latter mechanism, we found that BSO, which depletes cellular glutathione levels, significantly reduced the excitoprotective efficacy of CHX. The cumulative data therefore suggest that there may be several mechanisms, each involving enhancement of antioxidant pathways, whereby CHX protects neurons against excitotoxic and oxidative insults. Our data suggest that, in the cell death paradigms examined in the present study, the neuroprotective mechanism of CHX involves induction of cytoprotective gene products involved in antioxidant pathways rather than suppression of killer gene products. However, because the neuroprotective concentrations of CHX did cause a modest 20% decrease in overall protein synthesis, we cannot completely rule out the possibility that a selective inhibition of production of death genes occurred under those conditions.
The similarities between the neuroprotective actions of CHX demonstrated in the present study and the neuroprotective actions of neurotrophic factors documented in prior studies (for review see Mattson et al., 1993b) are striking. Pretreatment of hippocampal cell cultures with bFGF, NGF, and BDNF protected neurons against glutamate toxicity and suppressed accumulation of reactive oxygen species (Mattson et al., 1989; Cheng and Mattson, 1994; Mattson et al., 1995). bFGF and NGF also protected cultured hippocampal neurons against FeSO4 toxicity (Zhang et al., 1993). bFGF (Mattson et al., 1993b) and BDNF (Mattson, M.P., unpublished data) protected cultured hippocampal neurons against Aβ toxicity. Each of these neurotrophic factors has been shown to induce the expression of one or more gene products linked to resistance to oxidative insults. Examples include: bFGF induced Cu/Zn-SOD and glutathione reductase in hippocampal cell cultures and protected neurons against glutamate toxicity (Mattson et al., 1995); NGF induced catalase expression in PC12 cells and protected them against H2O2 toxicity (Jackson et al., 1994); BDNF induced glutathione peroxidase in striatal neurons and protected them against metabolic/excitotoxic insults (Spina et al., 1992); and PDGF induced a doubling of catalase and Cu/Zn-SOD activity levels in hippocampal cultures and protected neurons in those cultures against FeSO4- and glucose deprivation–induced injury (Cheng et al., 1995). None of the latter studies examined the effects of the trophic factors on Bcl-2 levels. However, recent data suggest that BDNF can induce bcl-2 expression in neurons that are dependent upon BDNF for survival, and that Bcl-2 may mediate the survival response to BDNF (Allsopp et al., 1995). Since we found that Bcl-2 antisense blocked the neuroprotective effects of CHX, the contribution of CHXinduced increases in antioxidant enzyme activities to the neuroprotective action of CHX remains to be established.
While it is well documented that CHX can increase levels of immediate early gene mRNAs (Greenberg and Ziff, 1984), our demonstrations of increased levels of immediate early gene proteins and antioxidant enzyme activities are novel findings. The mechanism whereby CHX induces increased levels of immediate early gene products and antioxidant enzymes may involve stimulation of transcription and/or stabilization of mRNAs. We found that neuroprotective concentrations of CHX induced an increase in c-fos hnRNA, indicating that, at least for this gene, CHX can induce transcription. Consistent with our findings are previous data showing that CHX can induce activation of transcription factors (Greenberg and Ziff, 1984; Subramaniam et al., 1989; Zinck et al., 1995). On the other hand, CHX has also been shown to reduce degradation of certain mRNAs (Edwards and Mahadevan, 1992) and could, in that way, increase mRNA levels. Such actions of CHX at concentrations below those that inhibit protein synthesis could, in theory, result in increased protein synthesis. Indeed, our data suggest that levels of c-Fos, c-Jun, and Bcl-2 were increased in cultures exposed to neuroprotective concentrations of CHX.
Taken together with previous findings, the present data suggest that CHX can suppress cell death by two quite different mechanisms, one involving suppression of production of presumptive death genes, and the other involving induction of expression of cytoprotective gene products. Some prior studies have provided strong evidence that CHX can suppress cell death by inhibiting protein synthesis. For example, in sympathetic neurons where CHX acts at higher concentrations, CHX may suppress cell death by inhibiting synthesis of killer gene products (Martin et al., 1988, 1992). However, in most studies that have used CHX to block cell death, the extent to which the cytoprotective concentrations of CHX suppressed protein synthesis was not determined. Moreover, in in vivo studies of cerebral ischemia and other insults that have documented neuroprotective actions of CHX, no information was obtained concerning levels of protein synthesis in the "saved" neurons (e.g., Goto et al., 1990; Linnik et al., 1993; Svendsen et al., 1994; Tortosa et al., 1994). The collective data from the present study demonstrate that CHX protects cortical neurons against insults relevant to the pathogenesis of ischemic and excitotoxic brain injury by inducing production of antioxidant gene products. Therefore, we suggest that the fact that CHX protects neurons against a particular insult does not justify the conclusion that death genes are involved in the cell death process. It will be necessary to rigorously evaluate the mechanism of neuroprotection by CHX in each paradigm and, ultimately, to identify specific life gene products that are induced, or death gene products that are suppressed.
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Acknowledgments |
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This work was supported by grants to M.P. Mattson from the National Institutes of Health (NIH) (NS29001 and NS30583), the Metropolitan Life Foundation, and the Kentucky Spinal Cord and Head Injury Trust, and by grants to S. Estus from the NIH and the Alzheimer's Association.
Submitted: 16 July 1996
Revised: 2 December 1996
Address all correspondence to Mark P. Mattson, Sanders-Brown Research Center on Aging, Department of Anatomy and Neurobiology, University of Kentucky, 211 Sanders-Brown Building, 800 South Limestone, Lexington, KY 40536-0230. Tel.: (606) 257-6040. Fax: (606) 3232866. email: MMattson{at}aging.coa.uky.edu
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References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Aden U, Bona E, Hagberg H & Fredholm BB. Changes in c-fos mRNA in the neonatal rat brain following hypoxic ischemia, Neurosci Lett, 1994, 180, 91–95.[Medline]
Allsopp TE, Kiselev S, Wyatt S & Davies AM. Role of Bcl-2 in the brain-derived neurotrophic factor survival response, Eur J Neurosci, 1995, 7, 1266–1272.[Medline]
Ankarcrona M, Dypbukt JM, Bonfoco E, Zhivotovsky B, Orrenius S, Lipton SA & Nicotera P. Glutamate-induced neuronal death: a succession of necrosis or apoptosis depending on mitochondrial function, Neuron, 1995, 15, 961–973.[Medline]
Beal MF. Aging, energy, and oxidative stress in neurodegenerative diseases, Ann Neurol, 1995, 38, 357–366.[Medline]
Behl C, Davis J, Lesley R & Schubert D. Hydrogen peroxide mediates amyloid β protein toxicity, Cell, 1994, 77, 817–827.[Medline]
Behrens MI, Koh J, Canzoniero LM, Sensi SL, Csernansky CA & Choi DW. 3-Nitropropionic acid induces apoptosis in cultured striatal and cortical neurons, Neuroreport, 1995, 6, 545–548.[Medline]
Bondy SC & LeBel CP. The relationship between excitotoxicity and oxidative stress in the central nervous system, Free Radical Biol Med, 1993, 14, 633–642.[Medline]
Bredesen DE. Neural apoptosis, Ann Neurol, 1995, 38, 839–851.[Medline]
Butterfield DA, Hensley K, Harris M, Mattson MP & Carney J. β-Amyloid peptide free radical fragments initiate synaptosomal lipoperoxidation in a sequence-specific fashion: implications to Alzheimer's disease, Biochem Biophys Res Commun, 1994, 200, 710–715.[Medline]
Carter DA. Up-regulation of β1-adrenoceptor messenger ribonucleic acid in the rat pineal gland: nocturnally, through a beta-adrenoceptor-linked mechanism, and in vitro, through a novel posttranscriptional mechanism activated by specific protein synthesis inhibitors, Endocrinology, 1993, 133, 2263–2268.
Chen J, Graham SH, Chan PH, Lan J, Zhou RL & Simon RP. bcl-2 is expressed in neurons that survive focal ischemia in the rat, Neuroreport, 1995, 6, 394–398.[Medline]
Cheng B & Mattson MP. NGF and bFGF protect rat and human central neurons against hypoglycemic damage by stabilizing calcium homeostasis, Neuron, 1991, 7, 1031–1041.[Medline]
Cheng B & Mattson MP. NT-3 and BDNF protect hippocampal, septal, and cortical neurons against metabolic compromise, Brain Res, 1994, 640, 56–67.[Medline]
Cheng B, Christakos S & Mattson M P. Tumor necrosis factors protect neurons against excitotoxic/metabolic insults and promote maintenance of calcium homeostasis, Neuron, 1994, 12, 139–153.[Medline]
Choi DW. Ionic dependence of glutamate neurotoxicity, J Neurosci, 1987, 7, 369–379.[Abstract]
Collazo D, Takahashi H & McKay RDG. Cellular targets and trophic functions of neurotrophin-3 in the developing rat hippocampus, Neuron, 1992, 9, 643–656.[Medline]
Cotman CW & Anderson AJ. A potential role for apoptosis in neurodegeneration and Alzheimer's disease, Mol Neurobiol, 1995, 10, 19–45.[Medline]
Deckwerth TL & Johnson EM Jr. Temporal analysis of events associated with programmed cell death (apoptosis) of sympathetic neurons deprived of nerve growth factor, J Cell Biol, 1993, 123, 1207–1222.
Downen M, Mudd L, Roback JD, Palfrey HC & Wainer BH. Early nerve growth factor-induced events in developing rat septal neurons, Dev Brain Res, 1993, 74, 1–13.[Medline]
Dragunow M, Beilharz E, Sirimanne E, Lawlor P, Williams C, Bravo R & Gluckman P. Immediate-early gene protein expression in neurons undergoing delayed death, but not necrosis, following hypoxic-ischaemic injury to the young rat brain, Mol Brain Res, 1994, 25, 19–33.[Medline]
Dreyer EB, Zhang D & Lipton SA. Transcriptional or translational inhibition blocks low dose NMDA-mediated cell death, Neuroreport, 1995, 6, 942–944.[Medline]
Dugan LL, Sensi SL, Canzoniero LMT, Handran SD, Rothman SM, Lin T-S, Goldberg MP & Choi DW. Mitochondrial production of reactive oxygen species in cortical neurons following exposure to N-methyl-daspartate, J Neurosci, 1995, 15, 6377–6388.
Edwards DR & Mahadevan LC. Protein synthesis inhibitors differentially superinduce c-fos and c-junby three distinct mechanisms: lack of evidence for labile repressors, EMBO (Eur Mol Biol Organ) J, 1992, 11, 2415–2424.[Medline]
Estus S, Zaks WJ, Freeman R S, Gruda M, Bravo R & Johnson EM Jr. Altered gene expression in neurons during programmed cell death: identification of c-junas necessary for neuronal apoptosis, J Cell Biol, 1994, 127, 1717–1727.
Estus S, van Rooyen C, Mattson MP, Brigham EF & Rydel RE. Comparison of altered gene expression during neuronal death induced by NGF withdrawl, amyloid β-protein treatment, or glucose deprivation, Soc Neurosci Abstr, 1995, 21, 1721.
Fukunaga-Johnson N, Ryan JJ, Wicha M, Nunez G & Clarke MF. Bcl-2 protects murine erythroleukemia cells from p53-dependent and -independent radiation-induced cell death, Carcinogenesis, 1995, 16, 1761–1767.
Ghosh A, Ginty DD, Bading H & Greenberg ME. Calcium regulation of gene expression in neuronal cells, J Neurobiol, 1994, 25, 294–303.[Medline]
Goldstone SD, Fragonas JC, Jeitner TM & Hunt NH. Transcription factors as targets for oxidative signalling during lymphocyte activation, Biochim Biophys Acta, 1995, 1263, 114–122.[Medline]
Goodman Y & Mattson MP. Secreted forms of β-amyloid precursor protein protect hippocampal neurons against amyloid β-peptide-induced oxidative injury, Exp Neurol, 1994, 128, 1–12.[Medline]
Goto K, Ishige A, Sekiguchi K, Iizuka S, Sugimoto A & Yuzurihara M. Effects of cycloheximide on delayed neuronal death in rat hippocampus, Brain Res, 1990, 534, 299–302.[Medline]
Greenberg ME & Ziff EB. Stimulation of 3T3 cells induces transcription of the c-fosproto-oncogene, Nature (Lond), 1984, 311, 433–438.[Medline]
Greenlund LJS, Deckwerth TL & Johnson EM Jr. Superoxide dismutase delays neuronal apoptosis: a role for reactive oxygen species in programmed neuronal death, Neuron, 1995a, 14, 303–315.[Medline]
Greenlund L J, Korsmeyer SJ & Johnson EM Jr. Role of BCL-2 in the survival and function of developing and mature sympathetic neurons, Neuron, 1995b, 15, 649–661.[Medline]
Griffith OW & Meister A. Potent and specific inhibition of glutathione synthesis by buthionine sulfoximine (S-n-butyl homocysteine sulfoximine), J Biol Chem, 1979, 254, 7558–7560.
Hockenbery D, Nunez G, Milliman C, Schreiber RD & Korsmeyer SJ. Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death, Nature (Lond), 1990, 348, 334–336.[Medline]
Hockenbery D, Oltvai ZN, Yin XM, Milliman CL & Korsmeyer SH. Bcl-2 functions in an antioxidant pathway to prevent apoptosis, Cell, 1993, 75, 241–251.[Medline]
Jackson GR, Werrbach-Perez K, Pan Z, Sampath D & Perez-Polo JR. Neurotrophin regulation of energy homeostasis in the central nervous system, Dev Neurosci, 1994, 16, 285–290.[Medline]
Kane DJ, Sarafian TA, Anton R, Hahn H, Gralla EB, Valentine JS, Ord T & Bredesen DE. Bcl-2 inhibition of neural death: decreased generation of reactive oxygen species, Science (Wash DC), 1993, 262, 1274–1277.
Kane DJ, Ord T, Anton R & Bredesen DE. Expression of bcl-2 inhibits necrotic neural cell death, J Neurosci Res, 1995, 40, 269–275.[Medline]
Keller, J.N., R.J. Mark, A.J. Bruce, E.M. Blanc, J.D. Rothstein, K. Uchida, and M.P. Mattson. 1997. 4-hydroxynonenal, an aldehydic product of membrane lipid peroxidation, impairs glutamate transport and mitochondrial function in synaptosomes. Neurosci. In press.
Keith FJ, Bradbury DA, Zhu YM & Russell NH. Inhibition of bcl2 with antisense oligonucleotides induces apoptosis and increases the sensitivity of AML blasts to Ara-C, Leukemia, 1995, 9, 131–138.[Medline]
Kitada S, Takayama S, De Riel K, Tanaka S & Reed JC. Reversal of chemoresistance of lymphoma cells by antisense-mediated reduction of bcl-2 gene expression, Antisense Res Dev, 1994, 4, 71–79.[Medline]
Koketsu N, Berlove DJ, Moskowitz MA, Kowall NW, Caday CG & Finklestein SP. Pretreatment with intraventricular basic fibroblast growth factor decreases infarct size following focal cerebral ischemia in rats, Ann Neurol, 1994, 35, 451–457.[Medline]
Lafon-Cazal M, Pietri S, Culcasi M & Bockaert J. NMDA-dependent superoxide production and neurotoxicity, Nature (Lond), 1993, 364, 535–537.[Medline]
Lawrence MS, Ho DY, Sun GH, Steinberg GK & Sapolsky RM. Overexpression of Bcl-2 with herpes simplex virus vectors protects CNS neurons against neurological insults in vitro and in vivo, J Neurosci, 1996, 16, 486–496.
Lindvall O, Kokaia Z, Bengzon J, Elmer E & Kokaia M. Neurotrophins and brain insults, Trends Neurosci, 1994, 17, 490–496.[Medline]
Linnik MD, Zobrist RH & Hatfield MD. Evidence supporting a role for programmed cell death in focal cerebral ischemia in rats, Stroke, 1993, 24, 2002–2008.
Linnik MD, Zahos P, Geschwind MD & Federoff HJ. Expression of bcl-2 from a defective herpes simplex virus-1 vector limits neuronal death in focal cerebral ischemia, Stroke, 1995, 26, 1670–1674.
Loo DT, Copani A, Pike CJ, Whittemore ER, Walencewicz AJ & Cotman CW. Apoptosis is induced by β-amyloid in cultured central nervous system neurons, Proc Natl Acad Sci USA, 1993, 90, 7951–7955.
MacManus JP, Buchan AM, Hill IE, Rasquinha I & Preston E. Global ischemia can cause DNA fragmentation indicative of apoptosis in rat brain, Neurosci Lett, 1993, 164, 89–92.[Medline]
Mark RJ, Hensley K, Butterfield DA & Mattson MP. Amyloid β-peptide impairs ion-motive ATPase activities: evidence for a role in loss of neuronal Ca2+homeostasis and cell death, J Neurosci, 1995, 15, 6239–6249.[Abstract]
Mark RJ, Blanc EM & Mattson MP. Amyloid β-peptide and oxidative cell injury in Alzheimer's disease, Mol Neurobiol, 1996, 12, 211–224.[Medline]
Mark RJ, Lovell MA, Markesbery WR, Uchida K & Mattson MP. A role for 4-hydroxynonenal in disruption of ion homeostasis and neuronal death induced by amyloid β-peptide, J Neurochem, 1997a, 68, 255–264.[Medline]
Mark RJ, Pang Z, Geddes JW & Mattson MP. Amyloid β-peptide impairs glucose uptake in hippocampal and cortical neurons: involvement of membrane lipid peroxidation, J Neurosci, 1997b, 17, 1046–1054.
Marsh HN, Scholz WK, Lamballe F, Klein R, Nanduri V, Barbacid M & Palfrey HC. Signal transduction events mediated by the BDNF receptor gp 145trkB in primary hippocampal pyramidal cell culture, J Neurosci, 1993, 13, 4281–4292.[Abstract]
Martin DP, Schmidt RE, DiStefano PS, Lowry OH, Carter JG & Johnson EM Jr. Inhibitors of protein synthesis and RNA synthesis prevent neuronal death caused by nerve growth factor deprivation, J Cell Biol, 1988, 106, 829–844.
Martin DP, Ito A, Horigome K, Lampe PA & Johnson EM Jr. Biochemical characterization of programmed cell death in NGF-deprived sympathetic neurons, J Neurobiol, 1992, 23, 1205–1220.[Medline]
Martinou JC, Dubois-Dauphin M, Staple JK, Rodriguez I, Frankowski H, Missotten M, Albertini P, Talabot D, Catsicas S & Pietra C. Overexpression of Bcl-2 in transgenic mice protects neurons from naturally occurring cell death and experimental ischemia, Neuron, 1994, 13, 1017–1030.[Medline]
Mattson, M.P., and S.W. Barger. 1995. Programmed cell life: neuroprotective signal transduction and ischemic brain injury. In Cerebrovascular Diseases: Nineteenth Princeton Stroke Conference. M.A. Moskowitz and L.R. Caplan, editors. Butterworth, Stoneham, MA. 271–290.
Mattson MP, Murrain M, Guthrie PB & Kater SB. Fibroblast growth factor and glutamate: opposing actions in the generation and degeneration of hippocampal neuroarchitecture, J Neurosci, 1989, 9, 3728–3740.[Abstract]
Mattson MP, Barger SW, Cheng B, Lieberburg I, Smith-Swintosky VL & Rydel RE. β-Amyloid precursor protein metabolites and loss of neuronal calcium homeostasis in Alzheimer's disease, Trends Neurosci, 1993a, 16, 409–415.[Medline]
Mattson MP, Cheng B & Smith-Swintosky VL. Growth factor- mediated protection from excitotoxicity and disturbances in calcium and free radical metabolism, Semin Neurosci, 1993b, 5, 295–307.
Mattson MP, Kumar K, Cheng B, Wang H & Michaelis EK. Basic FGF regulates the expression of a functional 71 kDa NMDA receptor protein that mediates calcium influx and neurotoxicity in cultured hippocampal neurons, J Neurosci, 1993c, 13, 4575–4588.[Abstract]
Mattson MP, Zhang Y & Bose S. Growth factors prevent mitochondrial dysfunction, loss of calcium homeostasis and cell injury, but not ATP depletion in hippocampal neurons deprived of glucose, Exp Neurol, 1993d, 121, 1–13.[Medline]
Mattson MP, Lovell MA, Furukawa K & Markesbery WR. Neurotrophic factors attenuate glutamate-induced accumulation of peroxides, elevation of [Ca2+]iand neurotoxicity, and increase antioxidant enzyme activities in hippocampal neurons, J Neurochem, 1995, 65, 1740–1751.[Medline]
Mesner PW, Epting CL, Hegarty JL & Green SH. A timetable of events during programmed cell death induced by trophic factor withdrawal from neuronal PC12 cells, J Neurosci, 1995, 15, 7357–7366.[Abstract]
Naveilhan P, Neveu I, Jehan F, Baudet C, Wion D & Brachet P. Reactive oxygen species influence nerve growth factor synthesis in primary rat astrocytes, J Neurochem, 1994, 62, 2178–2186.[Medline]
Oguchi S, Weisz A & Esumi H. Enhancement of inducible-type NO synthase gene transcription by protein synthesis inhibitors. Activation of an intracellular signal transduction pathway by low concentrations of cycloheximide, FEBS Lett, 1994, 338, 326–330.[Medline]
Pittman RN, Wang S, DiBenedetto AJ & Mills JC. A system for characterizing cellular and molecular events in programmed neuronal cell death, J Neurosci, 1993, 13, 3669–3680.[Abstract]
Pollard H, Cantagrel S, Charriaut-Marlangue C, Moreau J & Ben-Ari Y. Apoptosis-associated DNA fragmentation in epileptic brain damage, Neuroreport, 1994, 5, 1053–1055.[Medline]
Portera-Calliau C, Hedreen JC, Price DL & Koliatsos VE. Evidence for apoptotic cell death in Huntington disease and excitotoxic animal models, J Neurosci, 1995, 15, 3775–3787.[Abstract]
Ratan RR, Murphy TH & Baraban JM. Oxidative stress induces apoptosis in embryonic cortical neurons, J Neurochem, 1994a, 62, 376–379.[Medline]
Ratan RR, Murphy TH & Baraban JM. Macromolecular synthesis inhibitors prevent oxidative stress-induced apoptosis in embryonic cortical neurons by shunting cysteine from protein synthesis to glutathione, J Neurosci, 1994b, 14, 4385–4392.[Abstract]
Schwartz LM & Osborne BA. Programmed cell death, apoptosis and killer genes, Immunol Today, 1993, 14, 582–590.[Medline]
Seger R & Krebs EG. The MAPK signaling cascade, FASEB (Fed Am Soc Exp Biol) J, 1995, 9, 726–735.[Abstract]
Serghini R, Froissard P, Sola B & Duval D. Cycloheximide and actinomycin D block the toxic effect of glutamic acid on PC12 cells, Neuroreport, 1994, 6, 29–32.[Medline]
Spina MB, Squinto SP, Miller J, Lindsay RM & Hyman C. Brainderived neurotrophic factor protects dopamine neurons against 6-hydroxydopamine and N-methyl-4-phenylpyridinium ion toxicity: involvement of glutathione system, J Neurochem, 1992, 59, 99–106.[Medline]
Steller H. Mechanisms and genes of cellular suicide, Science (Wash DC), 1995, 267, 1445–1449.
Subramaniam M, Schmidt LJ, Crutchfield CE III & Getz MJ. Negative regulation of serum-responsive enhancer elements, Nature (Lond), 1989, 340, 64–66.[Medline]
Svendsen CN, Kew JN, Staley K & Sofroniew MV. Death of developing septal cholinergic neurons following NGF withdrawal in vitro: protection by protein synthesis inhibition, J Neurosci, 1994, 14, 75–87.[Abstract]
Takashima A, Noguchi K, Sato K, Hoshino T & Imahori K. Tau protein kinase I is essential for amyloid β-protein-induced neurotoxicity, Proc Natl Acad Sci USA, 1993, 90, 7789–7793.
Tortosa A, Rivera R & Ferrer I. Dose-related effects of cycloheximide on delayed neuronal death in the gerbil hippocampus after bilateral transitory forebrain ischemia, J Neurol Sci, 1994, 121, 10–17.[Medline]
Wyllie AH, Kerr JF & Currie AR. Cell death: the significance of apoptosis, Int Rev Cytol, 1980, 68, 251–306.[Medline]
Yankner BA. Mechanisms of neuronal degeneration in Alzheimer's disease, Neuron, 1996, 16, 921–932.[Medline]
Zhang Y, Tatsuno T, Carney J & Mattson MP. Basic FGF, NGF, and IGFs protect hippocampal neurons against iron-induced degeneration, J Cereb Blood Flow Metab, 1993, 13, 378–388.[Medline]
Zinck R, Cahill MA, Kracht M, Sachsenmaier C, Hipskind RA & Nordheim A. Protein synthesis inhibitors reveal differential regulation of mitogen-activated protein kinase and stress-activated protein kinase pathways that converge on Elk-1, Mol Cell Biol, 1995, 15, 4930–4938.[Abstract]
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