Neuroprotective effects of bcl-2 overexpression in hippocampal cultures: interactions with pathways of oxidative damage

Neuroprotective effects of bcl-2 overexpression in hippocampal

cultures: interactions with pathways of oxidative damage

Sarah Howard,* Clement Bottino,* Sheila Brooke,* Elise Cheng,* Rona G. Giffard�,�and Robert Sapolsky*,�,§

*Department of Biological Sciences, Stanford University, Stanford, California, USA

Departments of �Anesthesiology, �Neurosurgery and §Neurology and Neurological Sciences, Stanford University School of Medicine,

Stanford, California, USA

Abstract

Overexpression of bcl-2 protects neurons from numerous

necrotic insults, both in vitro and in vivo. While the bulk of such

protection is thought to arise from Bcl-2 blocking cytochrome c

release from mitochondria, thereby blocking apoptosis, the

protein can target other steps in apoptosis, and can protect

against necrotic cell death as well. There is evidence that these

additional actions may be antioxidant in nature, in that Bcl-2

has been reported to protect against generators of reactive

oxygen species (ROS), to increase antioxidant defenses and to

decrease levels of ROS and of oxidative damage. Despite this,

there are also reports arguing against either the occurrence,

or the importance of these antioxidant actions. We have

examined these issues in neuron-enriched primary hippo-

campal cultures, with overexpression of bcl-2 driven by a her-

pes simplex virus amplicon: (i) Bcl-2 protected strongly against

glutamate, whose toxicity is at least partially ROS-dependent.

Such protection involved reduction in mitochondrially derived

superoxide. Despite that, Bcl-2 had no effect on levels of lipid

peroxidation, which is thought to be the primary locus of glu-

tamate-induced oxidative damage; (ii) Bcl-2 was also mildly

protective against the pro-oxidant adriamycin. However, it did

so without reducing levels of superoxide, hydrogen peroxide or

lipid peroxidation; (iii) Bcl-2 protected against permanent

anoxia, an insult likely to involve little to no ROS generation.

These findings suggest that Bcl-2 can have antioxidant actions

that may nonetheless not be central to its protective effects,

can protect against an ROS generator without targeting steps

specific to oxidative biochemistry, and can protect in the

absence of ROS generation. Thus, the antioxidant actions of

Bcl-2 are neither necessary nor sufficient to explain its pro-

tective actions against these insults in hippocampal neurons.

Keywords: apoptosis, bcl-2, necrosis, neurotoxicity, oxygen

radicals, reactive oxygen species.

J. Neurochem. (2002) 83, 914–923.

An extensive work demonstrates the capacity of Bcl-2 to block

cell death in numerous cell types, including both neurons and

glia (Green and Reed 1998). The protein is thought to

heterodimerize with pro-apoptotic proteins such as BAX,

thereby impeding the release of cytochrome c from mitochon-

dria. Release of cytochrome c is critical to the activation of

caspases and the execution of programmed cell death.

However, Bcl-2 has a variety of other actions within cells

and, as the most explicit example of this, Bcl-2 can also block

apoptosis following cytochrome c release (Rosse et al. 1998).

These other actions include Bcl-2 enhancing mitochondrial

calcium uptake (Murphy et al. 1996) and decreasing nuclear

calcium accumulation (Marin et al. 1996), forming ion

channels in mitochondria (Green and Reed 1998), and causing

the translocation of kinases to mitochondria (Wang et al.

1996). Moreover, Bcl-2 is capable of blocking instances of

necrotic, as well as apoptotic cell death (Kane et al. 1993, in

neural cell lines; Yang et al. 1998, in the substantia nigra;

Papadopoulos et al. 1998, in cortical astrocytes).

Thus, Bcl-2 appears to have salutary effects other than

preventing cytochrome c release. It has long been postulated

Resubmitted manuscript received August 13, 2002; accepted August 19,

2002.

Address correspondence and reprint requests to Robert Sapolsky,

Department of Biological Sciences, Gilbert Laboratory MC 5020,

Stanford University, Stanford, CA 94305–5020, USA.

E-mail: [email protected]

Abbreviations used: ABTS, 2,3-azino-bis(ethylbezothiazoline-6-sulf-

onic) acid; DCF, dichlorodihydrofluorescein diacetate; DMSO, dimethyl

sulphoxide; HE, dihydroethidium; MEM, a modified minimum essential

medium; MOI, multiplicity of infection; PBS, phosphate-buffered saline;

ROS, reactive oxygen species.

Journal of Neurochemistry, 2002, 83, 914–923

914 � 2002 International Society for Neurochemistry, Journal of Neurochemistry, 83, 914–923

that some of these additional effects occur in the oxidative

realm. Such protection could involve Bcl-2 acting as a

classical antioxidant [i.e. quenching reactive oxygen species

(ROS)]. However, there is little evidence for this (Lee et al.

2001). In addition, Bcl-2 may decrease ROS generation,

increase the level/activity of antioxidants and/or protecting

targets of oxidative damage. Overexpression of bcl-2

attenuates the cell death caused by numerous insults whose

toxicities depend heavily upon ROS generation (such as

adriamycin, paraquat, hydrogen peroxide, 6-OHDA, MPTP;

see below). Moreover, many of these instances of protection

are accompanied by a reduction in ROS accumulation (Kane

et al. 1993, insult of glutathione depletion in neural cell

lines; Lawrence et al. 1996, adriamycin in primary hippo-

campal cultures; Papadopoulos et al. 1998, aglycemia in

cortical astrocyte cultures). In addition, Bcl-2 decreases

oxidative damage (Kane et al. 1993, glutathione depletion in

neural cell lines; Myers et al. 1995, cyanide/aglycemia in

hypothalamic tumor lines; Bruce-Keller et al. 1998, hydro-

gen peroxide and amyloid b-peptide in PC12 cells; Giardinoet al. 1996, hyperglycemia in peripheral tissue; Lee et al.

2001, hydrogen peroxide in teratocarcinoma and neurobla-

stoma cell lines). Studies also report that Bcl-2 can increase

the activity or levels of antioxidants (Kane et al. 1993, in

neural cell line; Ellerby et al. 1996, in a hypothalamic cell

line; Papadopoulos et al. 1998, in cultured cortical astro-

cytes; Steinman 1995, in peripheral tissue; Voehringer et al.

1998, in peripheral cell lines; Lee et al. 2001, in a peripheral

and a neuroblastoma cell line). In one scenario revolving

around the putative antioxidant effects of Bcl-2, the protein is

thought to protect mitochondrial membranes from peroxida-

tive damage (Bruce-Keller et al. 1998). Another study

focuses on the capacity of Bcl-2 to maintain mitochondrial

potential and decrease mitochondrial ROS production (Green

and Reed 1998).

Thus, it is clearly that Bcl-2 can exert significant

antioxidant actions. However, there are findings which

suggest that this is not always the case:

1. In a number of cases in peripheral tissue, Bcl-2 prevents

cell death under circumstances which are highly unlikely to

involve ROS (for example, growth factor deprivation under

anaerobic conditions; (Jacobson and Raff 1995; Shimizu

et al. 1995).

2. In at least some reports, Bcl-2 does not prevent the cell

death induced by the pro-oxidant 6-OHDA (Oh et al. 1995,

1998, in a dopaminergic cell line; Yamada et al. 1999, in the

substantia nigra for example of protection against 6-OHDA).

3. In one instance where Bcl-2 reduced both the ROS

accumulation and cell death caused by an insult, the

reduction in ROS accumulation was shown to be irrelevant

to the sparing from death (Gardner et al. 1997, in fibro-

blasts), and could only account for part of the protection

observed (Papadopoulos et al. 1998).

4. Finally, there has been the suggestion that Bcl-2 can act

as a pro-oxidant. One report involving bcl-2 overexpression

in Escherichia coli, and in a B-cell line (Steinman 1995), and

one involving cultured cortical astrocytes (Papadopoulos

et al. 1998) showed that a primary effect of Bcl-2 was to

increase ROS levels, and the increase in antioxidant levels

could be viewed as a secondary compensation.

Relatively few of the reports in this confusing literature are

derived from studies of the nervous system, which is

particularly vulnerable to ROS and in which Bcl-2 can be

highly protective from various insults. Because of this, we

have examined these issues in primary hippocampal cultures

overexpressing bcl-2. We observe that: (i) Bcl-2 can reduce

the neurotoxicity of an excitotoxic insult while having

inconsistent effects upon indices of oxidative stress; (ii)

Bcl-2 can reduce the neurotoxicity of a pro-oxidant while

having no effect on indices of oxidative stress; (iii) Bcl-2 can

spare neurons from an insult whose toxicity is not likely to

involve the generation of ROS. Collectively, these findings

argue against the importance of an antioxidant role for Bcl-2 in

its neuroprotective actions in cultured hippocampal neurons.

Materials and methods

Hippocampal cell cultures

Tissue culture methods were described previously (Brooke et al.

1997) Briefly, hippocampus from 18-day-old fetal rats were

removed, dissociated with papain, filtered through an 80-lm cell

strainer, and resuspended in a modified minimum essential medium

(MEM; UCSF Tissue Culture Facility, San Francisco, CA, USA)

containing 25 mM glucose and 10% horse serum (Hyclone, Logan,

UT, USA). Cells were plated at a density of 30 000/cm2 on poly-D-

lysine-treated 96-well plates for the toxicity and superoxide studies,

on 24-well plates for the lipid peroxidation studies, and on 48-well

plates for the anoxia and anoxia/aglycemia studies. Cells were used

after 10–12 days. Cells used in glutamate experiments were treated

with 10 lM cytosine arabinoside on day 3 in culture, which

increased the percentage of neurons to approximately 70–80%.

Bcl-2 overexpression

A modified herpes simplex virus was used to deliver plasmids to

hippocampal neurons in culture. A bipromoter plasmid, pa22bgala4bcl-2, containing bcl-2 and the lacZ reporter gene under a4and a22 promoters, respectively, was used to overexpress bcl-2. Thecontrol plasmid, pa4bgal, contained the a4 promoter and lac Z

reporter gene. Both plasmids also contained the oriS and a

sequences required for replication and packaging. Construction of

plasmids was described previously (Lawrence et al. 1996).

Vectors were generated by transfection of plasmids into E5 cells

using lipofectamine (Gibco-BRL, Gaithersburg, MD, USA) and

superinfecting 24 h later with the helper virus d120 at a multiplicity

of infection (MOI) of 0.03 (Ho 1994). E5 cells stably transformed

with the a4 gene allowed d120, which lacks a4, propagation(DeLuca et al. 1985). Amplicons and d120 helper virus were titered

Neuroprotective effects of bcl-2 overexpression 915

� 2002 International Society for Neurochemistry, Journal of Neurochemistry, 83, 914–923

on Vero and E5 cells, respectively, and both were in the range of

0.5–3 · 107 infectious particles/mL. In all experiments, hippocam-pal cultures were infected approximately 12 h prior to experimental

treatment. Under these conditions, 63% of neurons are infected, as

are 11% of glia.

Solutions

For all experiments, working solutions were prepared in MEM

Eagle media (UCSF Tissue Culture Facility). A 1-M glutamate stock

solution in water was prepared prior to each experiment and further

diluted in MEM Eagle media. A 2-mM adriamycin (also known as

doxorubicin hydrochloride; Sigma, St Louis, MO, USA) stock

solution was kept at 4�C and diluted in MEM Eagle media for use.

EDTA (Sigma) was diluted from a 100-mM stock for use.

Toxicity studies

Glutamate or adriamycin was added directly to tissue culture wells

to the final concentrations of 5, 10, 15, 20 lM and 10, 30, 40, 50 and60 lM, respectively. After 24 h, cells were fixed with cold methanoland analyzed according to a published method (Brooke et al. 1997).

Briefly, cells were blocked with 5% milk in phosphate-buffered

saline (PBS), followed by immunocytochemistry with a neuron

specific primary antibody against MAP-2 (Sigma) at a dilution of

1 : 1000 in 5% milk in PBS. Following incubation with a rat-

adsorbed biotinylated secondary anti-IgG antibody (Vector, Burlin-

game, CA, USA), cells were treated with avidin-bound horseradish

peroxidase (ABC reagent, Vector). Finally, 2,3-azino-bis(ethylbezo-

thiazoline-6-sulfonic) acid (ABTS) was added according to manu-

facturer’s instructions (Vector), producing a color change in

proportion to amount of MAP-2 present. Absorbance at 405 nM

wavelength was read on a plate reader. During analysis, blanks

(wells treated with cold media to kill neurons), were subtracted from

all values and data were expressed as percentage survival according

to comparison with control wells that received no insult.

Hydroethidine studies

Generation of intracellular superoxide was determined according to

fluorescence of ethidium as a result of oxidation of hydroethidine

[also known as dihydroethidium (HE); Molecular Probes, Eugene,

OR, USA; Lagrange et al. 1994]. HE, 10 lg/lL in dimethyl

sulphoxide (DMSO), was stored under nitrogen at )80�C. Experi-mental treatments were added in 10–20 lL aliquots directly to tissueculture wells. Final concentrations of treatments were 40 lMadriamycin with and without 1.5 mM EDTA, and 10 and 20 lMglutamate. HE (16 lM) or DMSO vehicle was added at the same

time. Thirty minutes later, media were aspirated and replaced with

PBS containing 1% Triton-X. Fluorescence with excitation 480 nm

and emission 590 nm was read on a fluorescence plate reader.

Blanks without HE were subtracted from readings. A Pierce assay

was done in order to standardize for the amount of protein. Data

were expressed as percentage of control on each plate. For the

rotenone experiments, cells were pretreated with a final concentra-

tion of 10 lM rotenone (Sigma) in MEM, 40 min prior to

experimental treatment.

DCF studies

Dichlorodihydrofluorescein diacetate (DCF; Molecular Probes)

fluoresces upon oxidation by hydrogen peroxide (Lebel et al.

1990). DCF was stored at 4 mM in DMSO at – 80�C. Experimentaltreatments were added in 10–20 lL aliquots with final concentra-

tions of 40 lM adriamycin with and without 1.5 mM EDTA. DCF

(20 lM) or DMSO vehicle was added at the same time. Fifteen

minutes later, media were aspirated and replaced with PBS

containing 1% Triton-X. Fluorescence was read on a plate reader

with excitation 480 nm and emission 520 nm. Blanks, without DCF,

were subtracted from readings. A Pierce assay was used to

determine amounts of protein for standardization. Data were

expressed as a percentage of control on each plate. Because DCF

is pH-sensitive and glutamate decreases intracellular pH, attempts to

detect glutamate-induced increases in hydrogen peroxide were not

useful.

Lipid peroxidation studies

Lipid peroxidation was determined by measuring the loss of

fluorescence due to peroxidation of the naturally fluorescent fatty

acid, cis-parinaric acid (Molecular Probes; Kuypers et al. 1987).

Hippocampal cultures were incubated with 10 lM cis-parinaric

acid or 90% EtOH vehicle for 1 h prior to addition of 10 lLinsult or vehicle. Final conditions were 10 lM glutamate or 40 lMadriamycin with and without 1.5 mM EDTA. Two hours later,

cells were scraped from tissue culture wells and suspended in

PBS bubbled with nitrogen. Fluorescence was measured on a

Perkin-Elmer LS50B spectrometer (Perkin-Elmer, Foster City,

CA, USA) with excitation 312 nm and emission 414 nm. Blanks

containing no cis-parinaric acid were subtracted from readings

and data were expressed as percent of control. Because cis-

parinaric acid is light sensitive, all manipulations were performed

in the dark.

Anoxia studies

Experimental and control cells received two media changes with a

balanced salt solution. The experimental solution contained NaCl

116 mM, CaCl2 1.8 mM, MgSO4 0.8 mM, KCl 5.4 mM, NaH2PO41 mM, glucose 5.5 mM, NaHCO3 14.7 mM, and HEPES 10 mM. The

control solution was the same except it lacked HEPES and had a

higher concentration of NaHCO3 (27 mM). Experimental cells were

transferred to an anoxia chamber (Sheldon Manufacturing, Cornel-

ius, OR, USA) in an atmosphere of 5% CO2, 5% H2 and 90% N2,

where the media was changed twice to the deoxygenated salt

solution lacking glucose. Control plates received the same treatment

with solution incubated in the 37�C incubator with 5% CO2.

Experimental plates were incubated at 37�C in the anoxia chamber(with N2 gas). After 5–6 h, survival was assessed by the ABTS

assay described.

Data analysis and statistics

For all studies, data were expressed as a percentage increase above

Bcl-2 or bgal control after comparison by t-test to determine that

Bcl-2 and bgal controls were not significantly different from each

other. For toxicity studies, two-way ANOVA followed by Tukey post-

hoc test was used to determine difference between bgal and Bcl-2groups. For cPnA, HE and DCF studies, t-test was used to compare

insult group with Bcl-2 versus insult group with bgal. In the HEstudy with two concentrations of glutamate, a 2-way ANOVA

followed by Tukey’s post-hoc test was used to compare the bgalgroup versus the Bcl-2 group. For the anoxia and anoxia/aglycemia

916 S. Howard et al.


studies, percentage survival with bgal was compared with

percentage survival with Bcl-2 by t-test. For all statistics, signifi-

cance was set at p < 0.05, and data are presented as mean ± SE.

Results

As would be expected, glutamate was neurotoxic to neuron-

enriched hippocampal cultures in a dose-dependent manner

[Fig. 1; the extent of toxicity did not differ from mock-

infected cultures exposed to equivalent amounts of glutamate

(data not shown)]. In support of the picture of excitotoxin-

induced neuron death involving ROS accumulation and

damage, glutamate also caused a significant accumulation of

superoxide (Fig. 2) and a significant amount of lipid

peroxidation (Fig. 3). Such superoxide appeared to be

derived from mitochondria. As evidence, treatment of

cultures with rotenone, which blocks mitochondrial super-

oxide production (Sensi et al. 1999; Saybasili et al. 2001),

blocked glutamate-induced superoxide accumulation (super-

oxide accumulation above baseline induced by 10 lMglutamate: 35% ± 9; p < 0.01 by t-test, compared with

0 glutamate; superoxide accumulation above baseline

induced by 10 lM glutamate plus 10 lM rotenone:

15% ± 6; n.s. as compared with 0 glutamate plus rotenone).

In agreement with a prior report (Lawrence et al. 1996),

overexpression of bcl-2 decreased the neurotoxicity of

glutamate, causing an approximate doubling of the LD50(Fig. 1). We then explored whether bcl-2 overexpression

attenuated the ROS-related effects of glutamate. We observed

that Bcl-2 completely blocked the superoxide accumulation

induced by 10 lM glutamate, a concentration at which Bcl-2also decreased the neurotoxicity (Fig. 2; superoxide produc-

tion was maximal at this time point). In addition, Bcl-2

caused a trend towards decreased superoxide accumulation at

20 lM glutamate, a concentration at which Bcl-2 was not

protective. Surprisingly, despite this effect, bcl-2 overexpres-

sion had no effect on the extent of lipid peroxidation induced

by 10 lM glutamate (Fig. 3).

We then examined the effects of bcl-2 overexpression on

the actions of the ROS generator, adriamycin. Bcl-2 signi-

ficantly decreased adriamycin-induced neurotoxicity, causing

an approximate 35% increase in the LD50 (Fig. 4). As would

be expected, adriamycin markedly increased superoxide

accumulation (Fig. 5; note that the scale on the x-axis differs

from Fig. 2), hydrogen peroxide accumulation (Fig. 6; as

Fig. 1 Glutamate-induced neurotoxicity in neuron-enriched hippo-

campal cultures treated with either control vector (m) or Bcl-2 (d).

Bcl-2 caused a significant decrease in neurotoxicity (p < 0.02,

F ¼ 6.04, d.f. ¼ 1/113, two-way ANOVA). n ¼ averaged 10/data point,

derived from three different weekly culture preparations.

Fig. 2 Effects of glutamate and of differing viral vectors on superoxide

accumulation in neuron-enriched hippocampal cultures. Cultures were

exposed to indicated concentrations of glutamate and either the con-

trol (bgal) or bcl-2-expressing vector. Increasing glutamate concen-

trations produced increasing superoxide accumulation in control

cultures (p < 0.02 by one-way ANOVA), but not in Bcl-2-treated cultures

(n.s). When exposed to 10 lM glutamate, Bcl-2-treated cultures had

significantly less accumulation than did control cultures (*p < 0.05,

Tukey test following two-way ANOVA). n ¼ 22–23/group, derived from

five different weekly culture preparations.

Fig. 3 Effects of glutamate and of differing viral vectors on lipid per-

oxidation in neuron-enriched hippocampal cultures. Cultures were

exposed to 10 lM glutamate and were infected with either control or

bcl-2-expressing vector. Glutamate caused a highly significant

increase in lipid peroxidation, regardless of vector treatment

(*p < 0.001, comparing bgal/0 glutamate with bgal/10 glutamate, or

Bcl-2/0 glutamate with Bcl-2/10 glutamate; Tukey’s post-hoc test fol-

lowing two-way ANOVA). Viral vector treatment, however, had no effect

on the extent of lipid peroxidation (n.s., by two-way ANOVA). n ¼ 23–28/

data point, taken from five different weekly culture preparations.



noted in the Methods section, similar measures were not

carried out with glutamate, because the DCF assay is

disrupted by the pH changes caused by the excitotoxin),

and lipid peroxidation (Fig. 7).

Despite the protective effects of Bcl-2 against adriamycin

neurotoxicity, we observed no effects of overexpression on

these ROS-related endpoints. Bcl-2 did not decrease adria-

mycin-induced superoxide accumulation (Fig. 5); as a pos-

itive control, under those same conditions, such

accumulation was significantly blunted by the calcium-

chelating effects of EDTA. Of note, the positive effects of

Bcl-2 on glutamate-induced superoxide accumulation were

demonstrable at a single time point (Fig. 2). The failure of

Bcl-2 to decrease adriamycin-induced superoxide accumula-

tion was not due to having chosen the wrong single time

point, as this was demonstrable over a range of times

(Fig. 5b). Similarly, Bcl-2 had no effect on adriamycin-

induced hydrogen peroxide accumulation, under conditions

where EDTA was effective (Fig. 6). Moreover, overexpres-

sion had no effect on the extent of lipid peroxidation under

conditions where EDTA was protective (Fig. 7).

We then examined whether Bcl-2 could protect against a

necrotic insult whose damaging effects were unlikely to

involve the generation of ROS. Specifically, we tested a

model of permanent anoxia. Five to six hours of anoxia was

significantly damaging to neurons in cultures transfected

with control vector (Fig. 8). In contrast, Bcl-2 overexpres-

sion provided complete protection.

Discussion

As discussed, the role of Bcl-2 in preventing neuron death is

more complicated than the protein solely preventing apop-

tosis by antagonizing the actions of BAX. The interactions of

Bcl-2 with the mitochondrial membrane are likely to be

pivotal to its larger role. While this appears to be central to

Fig. 4 Adriamycin-induced neurotoxicity in hippocampal cultures

treated with either control vector (m) or Bcl-2 (d). Bcl-2 caused a

significant decrease in neurotoxicity (p < 0.001, F ¼ 22.68,

d.f. ¼ 1/76, two-way ANOVA). n ¼ averaged 6/data point, taken from

two different weekly culture preparations.

Fig. 5 (a) Left: Effects of adriamycin and of differing viral vectors on

superoxide accumulation in hippocampal cultures. Cultures were

exposed to 40 lM adriamycin and either the control or bcl-2-expressing

vector. Adriamycin caused a significant increase in superoxide accu-

mulation, regardless of vector treatment (***p < 0.001, comparing bgal/

0 adriamycin with bgal/40 adriamycin, or Bcl-2/0 adriamycin with Bcl-2/

40 adriamycin; Tukey’s post-hoc test following two-way ANOVA). Viral

vector treatment, however, had no effect on superoxide accumulation

(n.s., by two-way ANOVA). Right: Effects of adriamycin and of EDTA on

superoxide accumulation in hippocampal cultures. Cultures were

exposed to 40 lM adriamycin with or without 1.5 mM EDTA. Adriamycin

caused a significant accumulation of superoxide generation in the

absence of EDTA (p < 0.01, when compared with control; Tukey’s

post-hoc test), while treatment with EDTA prevented such adriamycin-

induced accumulation (*p < 0.05, Tuvkey’s post-hoc test). For

unknown reasons, adriamycin was not as effective at increasing

superoxide generation in the EDTA experiment as in the vector

experiment; (p < 0.05, when compared with 40 lM adriamycin, bgal).

n ¼ 12–13/group, from two weekly culture preparations. (b) The effects

of adriamycin and indicated vectors on superoxide accumulation at 10,

20, 30 and 40 min Adriamycin significantly increased accumulation

regardless of vector, but vector treatment did not alter accumulation

(n.s). n ¼ 12–15/data point. d, bgal; s, bgal + 40 lM adriamycin; .,

bcl-2; ,, bcl-2 + 40 lM adriamycin.



Bcl-2 preventing BAX-induced release of cytochrome c from

the mitochondria, the maintenance of mitochondrial function

is also likely to help explain the protein’s capacity to protect

against necrotic cell death (Kane et al. 1993; Papadopoulos

et al. 1998; Yang et al. 1998). This involvement of Bcl-2 in

mitochondrial function has also prompted explorations of its

Fig. 7 Left: Effects of adriamycin and of differing viral vectors on

lipid peroxidation in hippocampal cultures. Cultures were exposed to

40 lM adriamycin and either the control or bcl-2-expressing vector.

Adriamycin caused a significant increase in lipid peroxidation,

regardless of vector treatment (***p < 0.001, comparing bgal/0

adriamycin with bgal/40 adriamycin, or Bcl-2/0 adriamycin with Bcl-2/

40 adriamycin; Tukey’s post-hoc test following two-way ANOVA). Viral

vector treatment, however, had no effect on superoxide accumula-

tion (n.s., by two-way ANOVA). Right: Effects of adriamycin and of

EDTA on lipid peroxidation in hippocampal cultures. Cultures were

exposed to 40 lM adriamycin with or without 1.5 mM EDTA. Adria-

mycin caused significant lipid peroxidation in the absence of EDTA

(**p < 0.01, when compared with control; Tukey’s post-hoc test),

while treatment with EDTA caused a significant diminution of this

adriamycin effect (*p < 0.05). n ¼ 18–22, from five different weekly

culture preparations.

Fig. 6 Left: Effects of adriamycin and of differing viral vectors on

hydrogen peroxide generation accumulation (as measured with DCF

fluorescence) in hippocampal cultures. Cultures were exposed to 40 lM

adriamycin and either the control or Bcl-2-expressing vector. Adria-

mycin caused a significant increase in DCF fluorescence, regardless of

vector treatment **p < 0.01, ***p < 0.001, comparing bgal/0 adriamycin

with bgal/40 adriamycin, or Bcl-2/0 adriamycin with Bcl-2/40 adriamy-

cin, respectively; Tukey’s post-hoc test following two-way ANOVA). Viral

vector treatment, however, had no effect on superoxide accumulation

(n.s., by two-way ANOVA). Right: Effects of adriamycin and of EDTA on

hydrogen peroxide generation in hippocampal cultures. Cultures were

exposed to 40 lM adriamycin with or without 1.5 mM EDTA. Adriamycin

caused significant generation in the absence of EDTA (**p < 0.01,

when compared to control; Tukey’s post-hoc test), while treatment with

EDTA prevented such adriamycin-induced accumulation. n ¼ 6–19/

group, from three different weekly cultures.



potential role as an antioxidant. In an early version of this

view, the proximity of Bcl-2 to the mitochondria suggested

that it would be preferentially poised to quench ROS released

from mitochondria (Hockenberry et al. 1993; this view pre-

dated the discovery of Bcl–2 interactions with BAX). More

recently, there has emerged the view that Bcl-2, while not

necessarily functioning as a classical ROS quencher, may

decrease ROS production by mitochondria, secondary to its

capacity to preserve both mitochondrial potential and

function of the electron transport chain. Furthermore, Bcl-2

can decrease peroxidative damage to mitochondrial mem-

brane (Bruce-Keller et al. 1998).

As reviewed, the evidence of Bcl-2 attenuating oxidative

damage, and for such actions being critical to its overall

protective effects, is equivocal, especially in the nervous

system. Our data suggest that Bcl-2’s protective effects in

hippocampal neurons need not be heavily dependent on its

capacity to protect against the generation and/or proximal

consequences of ROS, in contrast to our prior findings in

astrocytes (Papadopoulos et al. 1998).

Glutamate neurotoxicity

We initially observed Bcl-2 to protect against glutamatergic

excitotoxicity in hippocampal cultures. This protection

involved an approximate doubling of the LD50, such that at

its most efficacious, Bcl-2 decreased neuron death more than

50% (at 10 lM glutamate). Given that herpes viral vectors

have a strong preference for infecting neurons over glia in

primary cultures (Ho et al. 1995) and that these studies utilized

neuron-enriched cultures, the protective effects of Bcl-2 were

overwhelmingly likely to be due to direct actions within

infected neurons, rather than secondary to glial effects. This

should be contrasted with our previous observation that

selective bcl-2 overexpression in astrocytes cocultured with

wild-type neurons does afford protection from combined

oxygen glucose deprivation, an injury largely dependent on

the activation of glutamate receptors (Xu et al. 1999).

These protective Bcl-2 effects agree with prior reports

showing neuroprotection by Bcl-2 against excitotoxins,

hypoglycemia and adriamycin in primary cultures derived

from a number of brain regions (Jia et al. 1996; Lawrence

et al. 1996;McLaughlin et al. 2000; Tamatani et al. 2000) and

against in vivo models of excitotoxicity, hypoxia–ischemia,

ROS generators, or mechanical trauma (Linnik et al. 1995;

Lawrence et al. 1996, 1997; Antonawich et al. 1999; Yamada

et al. 1999; Phillips et al. 2000; Shimazaki et al. 2000).

Moreover, we observed that such protection was accom-

panied by a complete block of glutamate-induced superoxide

accumulation at the time point where such accumulation is

maximal post-glutamate. Mitochondria appear to be a major

source of such accumulation during excitotoxic insults

(Dugan et al. 1995), probably secondary to the disruption

of mitochondrial potential; supporting this, we observed that

rotenone, which blocks mitochondrial superoxide produc-

tion, blocked the effects of glutamate on this endpoint. Thus,

the Bcl-2 effect is commensurate with the broadly protective

array of effects of Bcl-2 in mitochondria [nonetheless, in this

particular culture system, we observe that Bcl-2 does not

alter glutamate-induced declines in mitochondrial potential

(manuscript in preparation)]. As an alternative or additional

mechanism, Bcl-2 can increase the activity or levels of

antioxidants, or optimize their subcellular distribution (Kane

et al. 1993; Steinman 1995; Ellerby et al. 1996; Papadopo-

ulos et al. 1998; Voehringer et al. 1998; Lee et al. 2001);

insofar as this involves an increase in SOD activity, this

should lead to a reduction in superoxide accumulation.

Finally, the effects of Bcl-2 on availability of substrates such

as GSH or GSSG are unknown, although the glutamatergic

excitotoxin kainic acid does not alter levels of either in this

culture system (Patel et al. 2002).

Glutamate-induced superoxide production appears to be an

important contributor to the subsequent neuron death (Dugan

et al. 1995) and administration or overexpression of a variety

of antioxidants (SOD, vitamin E, 21-amino steroids, oxy-

purinol, glutathione peroxidase) can decrease glutamatergic

injury in the brain (Acosta et al. 1987; Monyer et al. 1990;

Chan et al. 1991; Lin and Phillis 1991; Lafon-Cazal et al.

1993). Therefore, the decreased accumulation could help

explain the neuroprotective actions of Bcl-2. Thus, while there

is little evidence that Bcl-2 is acting in this case as a classical

quencher of ROS, some of its protective functionsmay revolve

around it indirectly decreasing ROS accumulation.

Our data also indicate that the glutamate-induced super-

oxide accumulation does not necessarily lead to peroxidative

damage to lipid membranes, as Bcl-2 reduced the former

without altering the latter. Excitotoxin-induced peroxidative

damage to cell membranes probably reflects ROS generation

in the cytosol and membrane itself. This would typically be

secondary to calcium-induced activation of xanthine oxidase,

phospholipase A2, and NOS, and the generation of hydrogen

peroxide, hydroxyl radicals, and peroxynitrites. In addition,

Fig. 8 Percentage neuron loss in cultures made permanently anoxic

and treated with control vector expressing bgal alone, or experimental

vector expressing Bcl-2. ***p < 0.001, by t-test. n ¼ 20–22/group, from

four different weekly culture preparations.



superoxide can be generated in the cytosol by oxygenases,

and thus could readily contribute to oxidative damage to the

cell membrane. However, our rotenone data suggest that the

superoxide generated by glutamate in our insult model is

predominately mitochondrial in origin. Thus, a change in

superoxide generation from mitochondria is not likely to

impact the endpoint of lipid peroxidation in the cell

membrane. It has been speculated that Bcl-2 can protect

mitochondrial membrane from lipid peroxidation (Bruce-

Keller et al. 1998). Such peroxidation could be generated by

the superoxide and be diminished by Bcl-2 in the present

case; however, any such peroxidation would be in amounts

below the level of detection in our assay.

Our data also demonstrate that in this model, glutamate-

induced lipid peroxidation is not likely to be playing an

obligatory role in the neurotoxicity, insofar as bcl-2

overexpression reduced neurotoxicity without altering the

extent of peroxidation. This is in contrast to the situation in

ischemic brain injury, in which lipid peroxidation is thought

to play a more central role in the neuron death (Traystman

et al. 1991; Chan 1996; Liu et al. 1998). However, it is

quite plausible that the peroxidative damage could impair

functional recovery in surviving neurons. In support of this,

under a number of circumstances, bcl-2 overexpression can

spare neurons from insult-induced death, but not from

insult-induced dysfunction (McLaughlin et al. 2000; Dumas

et al. 2000).

Adriamycin neurotoxicity

Bcl-2 can decrease the toxicity of a number of insults that are

heavily or entirely oxidative in nature, such as adriamycin,

paraquat, hydrogen peroxide, or 6-OHDA (Oh et al. 1995;

Lawrence et al. 1996; Lezoualc’h et al. l996; Marin et al.

1996; Bruce-Keller et al. 1998; ; Hochman et al. 1998; Yang

et al. 1998Yamada et al. 1999; Luc Cadet et al. 2000). We

examined whether Bcl-2 could protect against an insult that

is overwhelmingly oxidative in nature, and if any such

protection arose as a result of reducing ROS accumulation or

oxidative damage. We utilized adriamycin (doxorubicin), a

potent pro-oxidant commonly used in the treatment of

malignant tumors, which is toxic to cultured neurons in the

lM range. As expected, the neurotoxin generated a consid-

erable oxidative challenge; at its approximate LD50 (40 lM),adriamycin caused a 200% increase in superoxide accumu-

lation (in contrast, glutamate, at its LD50 of 10 lM, caused a15% increase).

We then observed that bcl-2 overexpression decreased

adriamycin neurotoxicity, although to a lesser extent than

against glutamate. Despite these neuroprotective effects,

Bcl-2 had no effect on the accumulation of superoxide,

hydrogen peroxide at a range of time points, or on the extent

of lipid peroxidation. This dissociation between protecting

from ROS while not decreasing a measure of oxidative

damage is reminiscent of the finding that in a peripheral cell

line, Bcl-2 decreased hydrogen peroxide toxicity without

decreasing oxidative damage to lipids, DNA or protein (Lee

et al. 2001). As discussed, the ability of Bcl-2 to lessen

glutamate-induced superoxide accumulation is, most parsi-

moniously, a consequence of the protein’s actions at

mitochondria. Commensurate with this, we observe that

Bcl-2 also blocks cytochrome c release from mitochondria in

this insult model (manuscript in preparation). The lack of an

effect of Bcl-2 against the far greater superoxide accumula-

tion induced by adriamycin suggests either that (i) the

superoxide is derived from mitochondria but exceeds Bcl-2’s

capacity to constrain such accumulation, and/or (ii) the

superoxide is predominately derived from non-mitochondrial

sites not subject to Bcl-2’s effects.

Recent work has emphasized the potential role of nitro-

sylative rather than oxidative damage in cell death. Along

these lines, in a case where Bcl-2 protected against hydrogen

peroxide without decreasing oxidative damage, it decreased

3-nitrotyrosine levels (Lee et al. 2001). Thus, the same may

hold in the present study.

ROS accumulation is thought to be one of the signals

initiating injury-induced apoptosis (e.g. the translocation of

BAX to the mitochondria). Our data suggest that Bcl-2

reduces adriamycin neurotoxicity by one of two routes. First,

it may protect downstream of the oxidative realm, with

blocking of cytochrome c release being the most implicated,

but not sole, possible mechanism. Second, it is currently not

known whether adriamycin causes nitrosylative damage and

whether such damage can also initiate apoptosis. If so, Bcl-2

might be blocking the nitrosylation pathway. Thus, while

protecting against an ROS generator, such protection may not

be centered in the oxidative realm, a point emphasized

previously (Oh et al. 1995).

Anoxic neurotoxicity

Glutamatergic excitotoxicity represents a model in which

ROS generation is likely to contribute at least somewhat to

damage, while adriamycin toxicity is overwhelmingly oxi-

dative in nature. Anoxia, in contrast, represents an insult in

which ROS play a minimal role, if any, in the neuron death.

As noted, Bcl-2 protects against insults under anaerobic

conditions in peripheral cell types (Jacobson and Raff 1995;

Shimizu et al. 1995), and this has been strongly interpreted

as evidence against Bcl-2’s protective actions being solely

antioxidant in nature. We observed that Bcl-2 overexpression

blocked the toxicity induced by permanent anoxia in these

cultures. Broadly, this suggests the same non-oxidative facets

of Bcl-2 actions within the CNS.

In conclusion, these data suggest a mixed picture

concerning the antioxidant actions of Bcl-2. The glutamate

data suggest that, while Bcl-2 can have antioxidant actions,

they may not impact one of the major endpoints of

oxidative damage. The adriamycin data, moreover, suggest

that, while Bcl-2 can protect against a classical ROS



generator, such protection may arise from actions either

lateral to, or downstream of, specific oxidative events.

Finally, the anoxia data show that Bcl-2 can protect against

an insult likely to have little or no elements of ROS

generation. Thus, in this model system, the antioxidant

actions of Bcl-2 may not be either necessary or sufficient to

explain its protective actions.

Acknowledgements

Funding was provided by NIH PO1 NS27520 (RG and RS), a

TRDRP State of California grant (RS), the International Anesthesia

Research Society (RG) and a URO Grant (CB). Technical

assistance was provided by Martin Brown, Nick Denko, Adrian

Dunn, Pedram Ghafourifar, Mark Mattson, Stefano Sensi and John

Weiss.

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Neuroprotective effects of bcl-2 overexpression in hippocampal cultures: interactions with pathways of oxidative damage