* Sanders-Brown Research Center on Aging, 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 -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.
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 Data from a variety of cell death paradigms ranging
from withdrawal of trophic factor support in sympathetic
neurons (Greenlund et al., 1995a Hippocampal Cell Cultures and
Experimental Treatments
Dissociated cell cultures of fetal rat hippocampus were established from
18-d embryos and maintained as described previously (Mattson et al.,
1989 Quantification of Neuron Survival
These methods are detailed in our previous studies (Mattson et al., 1989 Protein Synthesis Assay
These methods were those described previously (Martin et al., 1992 PCR Analyses
Analyses of gene expression were performed as described previously (Estus et al., 1994 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 Immunocytochemistry and Western Blot Analyses
These methods were similar to those described previously (Mattson et al.,
1993c Measurement of Hydrogen Peroxide Levels
These methods were similar to those described previously (Goodman and
Mattson, 1994 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 CHX Protects Hippocampal Neurons
against Excitotoxic and Oxidative Injuries in a
Concentration-dependent Manner
Hippocampal neurons express NMDA and AMPA/kainate receptors and are vulnerable to glutamate-induced
excitotoxicity mediated by calcium influx (Choi, 1987
Neuroprotective Concentrations of CHX Do Not
Inhibit Protein Synthesis
Since the concentrations of CHX that were neuroprotective were below those expected to inhibit protein synthesis
maximally, we examined the time course and concentration dependence of CHX effects on incorporation of radiolabeled amino acids into TCA-precipitable protein (Fig. 2).
Parallel cultures were exposed for increasing times (1, 4, 12, and 24 h) to either 100 nM or 10 µM CHX. In cultures exposed to 100 nM CHX, levels of protein synthesis were
reduced to ~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
Neuroprotective Concentrations of CHX Induce
Expression of mRNA and Protein for bcl-2, c-fos,
and c-jun
A characteristic set of genes is rapidly induced when neurons are subjected to toxic insults. Perhaps the most widely
studied is the immediated early gene c-fos, which is induced at both the mRNA and protein levels in neural cells
subjected to ischemia (Aden et al., 1994
To provide insight into whether the increase of immediate early gene mRNA levels resulted from increased transcription or stabilization of mRNA, we examined levels of
c-fos heteronuclear RNA (hnRNA) in cultures exposed to
increasing concentrations of CHX. At the CHX concentrations that were most effective in protecting neurons
against excitotoxicity and oxidative insults (100-300 nM),
CHX induced a clear increase in the level of c-fos hnRNA (Fig. 4). Lower and higher concentrations of CHX had little or no effect on c-fos hnRNA levels. Because the concentrations of CHX effective in protecting neurons against
glutamate toxicity and oxidative insults were below concentrations that inhibit protein synthesis maximally, and
because c-fos and bcl-2 are induced by neurotrophic factors (Marsh et al., 1993
CHX Suppresses FeSO4-induced Accumulation
of Reactive Oxygen Species, and Depletion of an
Endogenous Antioxidant Reduces the Neuroprotective
Efficacy of CHX
Since neuroprotective concentrations of CHX induced increased levels of bcl-2 mRNA and protein, and since bcl-2
is known to have antioxidant functions (Hockenbery et al.,
1993
If the mechanism whereby CHX protected neurons involved enhancement of antioxidant pathways, then depletion of antioxidants should prevent protection by CHX.
Buthionine sulfoximine (BSO) is an agent that causes glutathione depletion by irreversibly inhibiting 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
CHX Increases Antioxidant Enzyme Activity Levels in
Hippocampal Cultures
Several different neurotrophic factors that protect neurons against the kinds of oxidative insults examined in the
present study have been shown to induce the expression of
one or more antioxidant enzymes in several types of neurons including hippocampal neurons (Jackson et al., 1994
The ability of CHX to protect cultured hippocampal neurons against the toxicities of glutamate, FeSO4, and A 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 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 While it is well documented that CHX can increase levels of immediate early gene mRNAs (Greenberg and Ziff,
1984 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; 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
-peptide (A
) 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
).
), excitotoxicity (Bondy
and LeBel, 1993
; Mattson et al., 1995
), metabolic impairment (Beal, 1995
), and A
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 A
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.
Materials and Methods
, 1995). Cells were plated in 35-mm-diam plastic or glass-well dishes
on a polyethyleneimine substrate in 0.8 ml of medium consisting of MEM
with Earle's salts supplemented with heat-inactivated FBS (5%; vol/vol),
1 mM l-glutamine, 1 mM pyruvate, 20 mM KCl, and 26 mM sodium bicarbonate (pH 7.2). Cells were grown at a density of 80-120 cells per mm2
culture surface, and experiments were performed in 8-10-d-old cultures, a
time period during which neurons express both N-methyl-d-aspartate (NMDA) and non-NMDA glutamate receptors, and are vulnerable to excitotoxicity (Mattson et al., 1993c
), A
toxicity (Mattson et al., 1993a
), and
FeSO4 toxicity (Zhang et al., 1993
). Cycloheximide, glutamate, buthionine
sulfoximine, and FeSO4 were purchased from Sigma Chemical Co. (St.
Louis, MO) and prepared as 200-500 × stocks in saline (pH 7.2). A
25-35
(lot ZM500) was purchased from Bachem California (Torrance, CA) and
stored in lyophilized form, and 1 mM stocks were prepared by dissolving
the peptide in sterile distilled water 2-4 h before use. Oligodeoxynucleotides
(ODNs) were purchased from IDT Inc. (Coralville, IA). The sequence of the Bcl-2 antisense ODN was 5
-TCCCGGCTTGCGCCAT-3
. Three different control ODNs were used: sense ODN; missense ODN, 5
-TCGCGGCATGCCCCAT-3
; and nonsense ODN, 5
-CTGTCGCGCTCGACTC3
. Immediately before experimental treatment, the culture maintenance
medium was replaced with Locke's solution that had the following composition (mM): 154 NaCl; 5.6 KCl; 2.3 CaCl2; 1.0 MgCl2; 3.6 NaHCO3; 5 Hepes; 10 glucose.
,
1995). Briefly, viable neurons were counted in premarked microscope
fields (×10 objective) before experimental treatment and 20-24 h after
treatment. Most neurons that died during the exposure period to
glutamate, FeSO4, or A
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.
). 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.
). 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.
-[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.
; 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).
). 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).
). 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.
Results
;
Mattson et al., 1989
) and generation of reactive oxygen
species (Lafon-Cazal et al., 1993
; Dugan et al., 1995
; Mattson et al., 1995
). Cultured hippocampal neurons are also
vulnerable to killing by a variety of oxidative insults including exposure to FeSO4 (Zhang et al., 1993
) and to A
(see Yankner, 1996
; for review Mark et al., 1996
); A
-
induced death manifests as apoptosis (Loo et al., 1993
).
Exposure of cultures to 10 µM glutamate, 5 µM FeSO4, or
5 µM A
resulted in killing 60-70% of the neurons (Fig.
1). Pretreatment of cultures with increasing concentrations
of CHX for 16 h before exposure to glutamate, FeSO4, or
A
resulted in biphasic concentration-dependent increase in neuronal survival. CHX concentrations of 50-100 nM significantly increased neuronal survival such that 80-90% of
the neurons survived exposure to each of the three insults
(Fig. 1). Concentrations of CHX of
1 µM were ineffective in protecting neurons, and concentrations >1 µM
caused significant neuronal death during a 24-h exposure period (Fig. 1 D).
Fig. 1.
CHX protects cultured hippocampal neurons
against excitotoxic and oxidative insults in a concentration-dependent manner. Cultures were preincubated for
16 h with vehicle or the indicated concentrations of
CHX, and then were exposed
to 10 µM glutamate (A), 5 µM FeSO4 (B), 5 µM A
(C), or vehicle control (D).
Neuronal survival was quantified 20 h later. Values represent the mean and SEM
(n = 4 separate cultures). *P < 0.05, **P < 0.01 compared
with 0 CHX value. ANOVA
with Scheffe's post-hoc tests.
[View Larger Version of this Image (29K GIF file)]
1 µM
caused a >90% inhibition of protein synthesis.
Fig. 2.
(A) Time course of effects of neuroprotective (100 nM)
and high (10 µM) concentrations of CHX on protein synthesis.
Cultures were exposed to 100 nM or 10 µM CHX for 1, 4, 12, or
24 h, and incorporation of [35S]methionine into TCA-precipitable
protein was quantified. Values are the mean and SEM of determinations made in three separate cultures. For 100 nM CHX, the
1 h value was significantly less than the 0 h value (P < 0.05).
For 10 µM CHX, the 1, 4, 12, and 24 h values were each significantly
less than the 0 h value and each value for cultures exposed to 100 nM CHX (P < 0.001). (B) Effect of increasing concentrations of
CHX on protein synthesis. Cultures were exposed for 1 h to the
indicated concentrations of CHX, and incorporation of [35S]methionine into TCA-precipitable protein was quantified. Values are the mean and SEM of determinations made in three separate cultures. The values for cultures exposed to either 100 or 300 nM
CHX were different than the values for cultures exposed to 0 (P < 0.01), 10 (P < 0.05), 1,000 (P < 0.001), and 10,000 (P < 0.001) nM CHX. Statistical comparisons used ANOVA with
Scheffe's post-hoc tests.
[View Larger Version of this Image (18K GIF file)]
; Dragunow et al.,
1994
), excitotoxins (Ghosh et al., 1994
), oxidative insults
(Naveilhan et al., 1994
; Goldstone et al., 1995
), and trophic
factor withdrawal (Mesner et al., 1995
). We previously used
RT-PCR approaches to examine expression of immediate
early genes after NGF withdrawal from cultured sympathetic neurons (Estus et al., 1994
), and after exposure to
A
in cultured rat cortical neurons (Estus et al., 1995
). To
gain insight into the neuroprotective mechanism of CHX,
we determined the effects of CHX on levels of mRNA encoding c-Fos, c-Jun, and the antiapoptotic gene Bcl-2.
CHX induced a concentration-dependent increase in levels of bcl-2, c-fos, and c-jun mRNAs (Fig. 3). In the case of
bcl-2 mRNA, a twofold increase occurred in cells exposed
to 30 nM CHX, and the peak increase of approximately fivefold occurred in cultures exposed to 100 nM CHX. Higher
concentrations of CHX were ineffective in increasing bcl-2
mRNA levels. In the cases of c-fos and c-jun mRNAs, the
concentration-response curves were also biphasic and exhibited similar profiles with peak increases occurring in
cultures exposed to 100-300 nM CHX (Fig. 3). Similar results
were obtained in two additional concentration-response experiments using separate cultures. The time course of
changes in bcl-2, c-fos and c-jun mRNAs after exposure to
100 nM CHX is shown in Fig. 3 C. The mRNA levels for
each gene increased within 10 min of exposure to CHX,
peaked by 30 min, and remained elevated for at least 8 h.
Fig. 3.
Neuroprotective concentrations of CHX induce expression of c-fos, bcl-2, and c-jun mRNAs. Hippocampal cultures were
exposed for 1 h to the indicated concentrations of CHX (A and
B), or were exposed to 100 nM CHX for the indicated time periods (C). RNA was isolated, and c-fos, c-jun, bcl-2, and neurofilament (NFM) mRNAs were amplified by RT-PCR. Data in B and
C are representative of at least three independent experiments.
[View Larger Versions of these Images (24 + 21 + 53K GIF file)]
; Downen et al., 1993
) that can protect neurons against some of the same insults examined in
the present study (Mattson et al., 1993b
,c, 1995), we determined the effects of CHX on levels of Bcl-2, c-Fos, and c-Jun
proteins. Cultures were exposed for 4 h to increasing concentrations of CHX, and then were immunostained with antibodies to Bcl-2, c-Fos, and c-Jun. Relative levels of neuronal immunoreactivity were quantified (see Materials
and Methods). Photomicrographs of control and CHXtreated cells immunostained with bcl-2 and c-fos antibodies are shown in Fig. 5 A. CHX induced concentration-
dependent increases in levels of Bcl-2, c-Fos, and c-Jun,
with maximum increases occurring in neurons exposed to
100 nM CHX (Fig. 5 B). Higher concentrations of CHX
did not increase levels of any of the three proteins.
Fig. 4.
Neuroprotective concentrations of CHX induce c-fos
transcription. Cultures were exposed for 1 h to the indicated concentrations of CHX and RNA was isolated. (A) RT-PCR analysis
of RNA amplified with c-fos, c-jun, and NFM primers. Locations
of c-fos heteronuclear RNA (hnRNA) and mRNA are indicated
at the left. Levels of c-fos hnRNA were increased in cells exposed
to 100 and 300 nM CHX, but not in cells exposed to 1 µM CHX.
(B) Relative levels of c-fos mRNA and c-fos hnRNA in cultures
exposed for 1 h to the indicated concentrations of CHX. Similar
results were obtained in three independent experiments.
[View Larger Versions of these Images (28 + 18K GIF file)]
Fig. 5.
CHX induces a concentration-dependent increase in levels of
Bcl-2, c-Fos, and c-Jun protein in cultured hippocampal neurons. (A)
Cultures were exposed for 5 h to vehicle or 100 nM CHX. Cultures were
then immunostained with antibodies to c-Fos or Bcl-2. Note increased
nuclear c-Fos and Bcl-2 immunoreactivities in neurons exposed to CHX
compared with neurons in control cultures. (B) Relative levels of immunoreactivity of neurons with antibodies to c-Fos, Bcl-2, or c-Jun were
determined in cultures exposed for 5 h to the indicated concentrations
of CHX. Staining intensity was rated on a scale from 0 to 4 (0, no staining; 1, weak; 2, moderate; 3, strong; 4, very strong). Values are the mean
and SEM of determinations made in three separate cultures (100 neurons scored per culture). *P < 0.01 compared with corresponding value for cultures not exposed to CHX; ANOVA with Scheffe's post-hoc tests.
[View Larger Versions of these Images (52 + 21K GIF file)]
; Kane et al., 1993
), we determined whether pretreatment of hippocampal cultures with CHX would reduce
levels of oxidative stress in neurons exposed to FeSO4.
Cultures were pretreated with vehicle or 100 nM CHX, and then exposed to FeSO4 for 20 min. Levels of peroxides
were quantified using the DCF probe, and confocal laser
scanning microscopy (Goodman and Mattson, 1994
).
FeSO4 induced a large increase in peroxide levels (Fig. 6,
A and B). The FeSO4-induced increase of peroxide was attenuated in neurons pretreated with 100 nM CHX (Fig. 6
B), but not with higher or lower concentrations of CHX (10 nM and 1 µM; data not shown), demonstrating that a
neuroprotective concentration of CHX reduces oxidative
stress.
Fig. 6.
Neuroprotective concentrations of CHX suppress
H2O2 accumulation induced by
FeSO4. (A) Representative
confocal laser scanning microscope images of DCF fluorescence in neurons in a vehicletreated control culture (left), a
culture exposed to 10 µM
FeSO4 for 20 min (middle),
and a culture that had been
pretreated with 100 nM CHX
for 16 h and then exposed to 10 µM FeSO4 for 20 min (right). Fluorescence intensity is represented by the color scale at
the lower left. (B) Cultures
were preincubated for 16 h with
100 nM CHX or vehicle (Control and FeSO4), and then were loaded with 2,7-dichlorofluorescin diacetate and exposed to vehicle or 10 µM FeSO4 for 20 min. Average fluorescence in neuronal cell bodies was quantified (see Materials and Methods). Values are the mean and SEM of determinations made in a total of 30-48 neurons in three separate cultures. *P < 0.02; ANOVA with Scheffe's post-hoc test.
(C) 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 BSO as indicated. Values are the mean and SEM of determinations made in four separate cultures. *P < 0.05;
ANOVA with Scheffe's post-hoc test.
[View Larger Versions of these Images (31 + 49 + 55K GIF file)]
-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.
; 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.
Fig. 7.
Exposure of hippocampal cell cultures to Bcl-2
antisense oligodeoxynucleotide
decreases levels of Bcl-2. (A)
Parallel cultures were exposed
for 16 h to 50 µM control
(sense) ODN or 50 µM Bcl-2 antisense ODN. Cultures
were then fixed and immunostained in parallel using an
anti-Bcl-2 primary antibody.
Shown are phase-contrast (left) and bright-field (right)
micrographs. Note that levels of Bcl-2 immunoreactivity are
greater in neurons in the control culture than in the culture
exposed to Bcl-2 antisense
ODN (arrowheads point to
neuron cell bodies). (B) Cultures were exposed for 16 h to
vehicle (water), 50 µM Bcl-2
antisense ODN, or 50 µM
sense ODN. Solubilized proteins were electrophoretically
separated, transferred to a nitrocellulose sheet, and immunoreacted with a Bcl-2 antibody. Levels of Bcl-2 were
markedly decreased in the culture exposed to Bcl-2 antisense
ODN compared with the control cultures.
[View Larger Versions of these Images (142 + 26K GIF file)]
Fig. 8.
Blockade of the neuroprotective actions of CHX in cultures treated with bcl-2 antisense oligodeoxynucleotide. Cultures
were pretreated for 12 h with 10 or 50 µM bcl-2 antisense ODN
(bcl-2 AS) or control missense ODN (MS). Cultures were then
exposed to vehicle (saline) or 100 nM CHX for 16 h, followed by
a 20-h exposure to vehicle (saline) or 10 µM (A) or 50 µM (B)
glutamate (Glut) in the continued presence of ODN. Neuronal
survival was quantified and values (mean and SEM; n = 3 cultures) are expressed as a percentage of the initial number of neurons. *P < 0.05; *P < 0.01.
[View Larger Version of this Image (56K GIF file)]
;
Mattson et al., 1995
). Since CHX suppressed accumulation
of H2O2 and toxicity of oxidative insults, we quantified activities of the antioxidant enzymes catalase, Cu/Zn-SOD,
and Mn-SOD in control hippocampal cultures and in cultures pretreated for 24 h with increasing concentrations of
CHX. Activities of all three antioxidant enzymes were significantly increased in cultures treated with 100-500 nM
CHX (Fig. 9). Lower or higher concentrations of CHX did
not affect antioxidant enzyme activities.
Fig. 9.
CHX induces increases in antioxidant enzyme activities
in cultured hippocampal cells. Cultures were exposed to the indicated concentrations of CHX for 24 h. Activity levels of catalase,
Cu/Zn-SOD, and Mn-SOD in cell homogenates were then quantified. Values are expressed in units of enzyme activity per mg
protein and represent the mean and SEM of determinations
made in three separate cultures. *P < 0.01 compared with corresponding cultures not exposed to CHX.
[View Larger Version of this Image (41K GIF file)]
Discussion
is
in general agreement with previous reports in various cell
culture models. CHX protected cultured retinal ganglion
cells against NMDA toxicity (Dreyer et al., 1995
), protected PC12 cells against glutamate toxicity (Serghini et al.,
1994
), and protected cultured cortical neurons against oxidative injury (Ratan et al., 1994b
) and A
toxicity (Takashima et al., 1993
). In such prior studies, a single concentration of CHX was usually used and concentration-effect analyses were not performed. Our data clearly show that
the neuroprotective actions of CHX in hippocampal cultures are concentration dependent with significant effects
being observed within the range of 50-500 nM CHX. It
was reported that only intermediate doses of CHX protect neurons against ischemic injury in adult rats in vivo (Tortosa et al., 1994
), a finding consistent with our cell culture
data. Moreover, Oguchi et al. (1994)
directly demonstrated
that low concentrations of CHX that have a negligible effect on protein synthesis can induce a large increase in inducible nitric oxide synthase mRNA levels in cultured
macrophages. Several lines of evidence in the present
study suggest that the neuroprotective actions of CHX
were not the result of inhibition of protein synthesis. First,
the most effective concentrations of CHX (50-100 nM)
were considerably lower than the concentrations required
to inhibit protein synthesis maximally in most cell types
and, in our cultures, caused only a 40% reduction in incorporation of radiolabeled amino acids into TCA-precipitable protein. Second, higher micromolar concentrations of
CHX that inhibited protein synthesis maximally were ineffective in protecting neurons against the excitotoxic and
oxidative insults. Third, neuroprotective concentrations of CHX induced increases in levels of Bcl-2, c-Fos, and c-Jun
protein (and their encoding mRNAs); increased production of these proteins argues against a general inhibition of
protein synthesis at those CHX concentrations. Finally,
neuroprotective concentrations of CHX induced increased
activity levels of the antioxidant enzymes catalase, Cu/ZnSOD, and Mn-SOD.
). 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 A
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
). A
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 A
(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.
) 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 A
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.
), 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.
, 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.
Received for publication 16 July 1996 and in revised form 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.eduWe thank J. Begley, S. Bose, and R. Pelphrey for technical assistance.
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.
A, amyloid
-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.