Role of polyamine metabolism in kainic acid excitotoxicity in organotypic hippocampal slice cultures

Journal of Neurochemistry, 2001, 79, 976±984

Role of polyamine metabolism in kainic acid excitotoxicity in

organotypic hippocampal slice cultures

Wei Liu,* Ruolan Liu,* Steve S. Schreiber² and Michel Baudry*

*Neuroscience Program and ²Department of Neurology, School of Medicine, University of Southern California, Los Angeles,

California, USA

Abstract

Polyamines are ubiquitous cations that are essential for cell

growth, regeneration and differentiation. Increases in poly-

amine metabolism have been implicated in several neuro-

pathological conditions, including excitotoxicity. However, the

precise role of polyamines in neuronal degeneration is still

unclear. To investigate mechanisms by which polyamines

could contribute to excitotoxic neuronal death, the present

study examined the role of the polyamine interconversion

pathway in kainic acid (KA) neurotoxicity using organotypic

hippocampal slice cultures. Treatment of cultures with

N1,N(2)-bis(2,3-butadienyl)-1,4-butanediamine (MDL 72527),

an irreversible inhibitor of polyamine oxidase, resulted in

a partial but signi®cant neuronal protection, especially in

CA1 region. In addition, this pre-treatment also attenuated

KA-induced increase in levels of lipid peroxidation, cytosolic

cytochrome C release and glial cell activation. Furthermore,

pre-treatment with a combination of cyclosporin A (an inhibitor

of the mitochondrial permeability transition pore) and MDL

72527 resulted in an additive and almost total neuronal

protection against KA toxicity, while the combination of

MDL 72527 and EUK-134 (a synthetic catalase/superoxide

dismutase mimetic) did not provide additive protection.

These data strongly suggest that the polyamine inter-

conversion pathway partially contributes to KA-induced

neurodegeneration via the production of reactive oxygen

species.

Keywords: cytochrome C, kainic acid, mitochondrial

permeability transition pore, neurodegeneration, polyamines,

reactive oxygen species.

J. Neurochem. (2001) 79, 976±984.

The polyamines, spermidine, spermine and putrescine, are

intracellular cations that are essential for cell proliferation,

regeneration and differentiation (Pegg and McCann 1982;

Seiler 1990). However, the speci®c functions of polyamines

in different cell types are not well understood. Polyamines

originate from the amino acid l-ornithine, and two major

pathways for polyamine catabolism have been identi®ed: the

interconversion pathway and the terminal polyamine cata-

bolism. In mammals, the interconversion pathway is a major

step for controlling intracellular polyamine levels. In this

pathway, spermine and spermidine are ®rst acetylated by

spermidine/spermine-N1-acetyltransferase (SSAT), and these

acetylated derivatives are then oxidized by polyamine

oxidase (PAO), regenerating spermidine and putrescine,

respectively (Seiler 1995). Several studies have found

increases in ornithine decarboxylase (ODC) activity as

well as in putrescine levels in brain following a variety of

experimental insults, such as ischemia, excitotoxicity and

traumatic brain injury (Najm et al. 1992; Paschen 1992;

Baskaya et al. 1996). Although these results suggest that

polyamines play an important role in neurodegeneration, the

mechanisms by which they could contribute to neuronal

death are still unclear. It has been proposed that activation of

the polyamine interconversion pathway plays an important

role in mediating cell apoptosis due to the generation of

hydrogen peroxide. In particular, inhibition of PAO activity

with N1,N(2)-bis(2,3-butadienyl)-1,4-butanediamine (MDL

72527) prevented apoptosis in non-neuronal cell cultures as

well as in brain (Hayashi and Baudry 1995; Ha et al. 1997;

Hu and Pegg 1997). On the other hand, MDL 72527 caused

apoptosis in hematopoietic cells (Dai et al. 1999).

Several lines of evidence have suggested that apoptosis

976 q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 79, 976±984

Received August 1, 2001; revised manuscript received September 13,

2001; accepted September 13, 2001.

Address correspondence and reprint requests to M. Baudry, Hnb124,

University of Southern California, Los Angeles, CA 90089±2520, USA.

E-mail: [email protected]

Abbreviations used: KA, kainic acid; Cyto C, cytochrome C; CSA,

cyclosporin A; PAO, polyamine oxidase; mPTP, mitochondrial perme-

ability transition pore; ROS, reactive oxygen species; MDL 72527,

N1,N(2)-bis(2,3-butadienyl)-1,4-butanediamine; OHC, organotypic

hippocampal slice cultures.

plays a primary role in excitotoxic cell death induced by

kainic acid (KA) (Filipkowski et al. 1994; Morrison et al.

1996). Recently, the release of cytochrome C (Cyto C) from

mitochondria into the cytosol has been shown to be a critical

event in mediating neuronal apoptosis induced by a variety

of stimuli (Reed et al. 1998; Kroemer 1999; Desagher and

Martinou 2000). The exact mechanism by which Cyto C is

released is poorly understood, but several pathways have

been proposed, including the pro-apoptotic protein Bax,

the mitochondrial membrane permeability transition pore

(mPTP), and reactive oxygen species (ROS) (Zoratti and

Szabo 1995; Atlante et al. 2000; Brenner and Kroemer

2000). Inhibition of mPTP by cyclosporin A (CSA)

prevented Cyto C release in hepatoma cells (Petronilli

et al. 2001). Interestingly, spermine caused the leakage of

Cyto C into the cytosol from mitochondrial matrix in

leukemia cells (Stefanelli et al. 1998). To understand the

role of the polyamine interconversion pathway in KA

excitotoxicity, we examined the effects of the PAO inhibitor

MDL 72527 on KA toxicity, Cyto C release and lipid

peroxidation in organotypic hippocampal slice cultures

(OHC). As changes in polyamines following lesions have

also been proposed to regulate gliosis (Zini et al. 1990; Zoli

et al. 1993) and since glial activation during excitotoxic

insults may contribute to neuronal vulnerability (Porter and

McCarthy 1995; Simantov et al. 1999), we also investigated

the effects of MDL 72527 on glial activation. The results

indicate that the polyamine interconversion pathway parti-

cipates in KA-induced neuronal death via production of

ROS and Cyto C release.

Materials and methods

Materials

MDL 72527 was a generous gift from Hoechst Marion Roussel

Inc. (Bridgewater, NJ, USA). Modi®ed essential medium (MEM)

medium was obtained from Gibco Inc. (Rockville, MD, USA).

Monoclonal antibodies against glial ®brillary acidic protein

(GFAP) were purchased from Boehringer Mannheim Inc. (Indian-

apolis, IN, USA). Anti-cytochrome C monoclonal antibodies were

purchased from PharMingen Inc. (San Diego, CA, USA). Avidin±

biotin complex (ABC) kit and biotinylated goat anti-mouse IgG

antibodies were purchased from Vector Laboratories (Burlingame,

CA, USA). Cyclosporin A (CSA) was obtained from RBI Inc.

(Natick, MA, USA), and EUK-134 was obtained from Eukarion,

Inc. (Bedford, MA, USA). Nitro-blue tetrazolium (NBT) and

5-bromo-4-chloro-3-indolyl-phosphate toluidine (BCIP) were pur-

chased from Bio-Rad (Hercules, CA, USA). Other reagents were

purchased from Sigma Chemical Co. (St Louis, MO, USA).

Preparation of organotypic hippocampal slice cultures

Hippocampal slice cultures were prepared as described (Stoppini

et al. 1991). Brie¯y, transverse slices (400 mm thick) were

prepared from the hippocampi of 6±8-day-old rats, using a

McIlwain tissue slicer, and were placed on a membrane insert

(Millicell-CM, Millipore Co., Bedford, MA, USA). The culture

medium was Gibco MEM containing: HEPES 30 mm, d-glucose

30 mm, glutamine 3 mm, NaHCO3 5 mm, MgCl2 2.5 mm,

l-ascorbate 0.5 mm, CaCl2 2 mm, 1 mg/mL insulin and 20%

horse serum. Cultures were maintained at 358C in a humidi®ed

incubator with 5% CO2, and the medium was changed every 2 or

3 days. The effects of KA and other agents were tested in mature

cultures, 20±25 days in vitro (DIV).

Kainic acid treatment and assessment of neuronal injury

KA (50 mm) was applied for 3 h after mature cultures were

incubated in serum-free culture medium overnight. After treatment,

cultures were allowed to recover for 24 h in fresh, serum-free

medium containing 4.6 mg/mL of the ¯uorescent dye propidium

iodide (PI). MDL 72527 (100 mm) was applied in cultures

overnight before KA application and re-applied for 24 h immedi-

ately after treatment. In some groups, either CSA (100 mm) or

EUK-134 (5 mm) was applied alone, or together with MDL 72527.

Previous studies addressed the principle of PI staining and its

usefulness for evaluating neuronal damage in hippocampal cultures

(Vornov et al. 1991; Bruce et al. 1995; Vornov et al. 1998). In the

current study, toxicity was observed by microscopic evaluation

of PI uptake 24 h after KA treatment. Results were scored

semiquantitatively with a scale of 1±5, with 1 � no toxicity, 5 �maximum toxicity. Sections were also photographed. Another

measurement of cellular injury consisted in determining LDH

release in the culture medium (Koh and Choi 1987). Data are

generally reported as means ^ SEM from the indicated number of

independent experiments, and each treatment consisted of three to

four replicate plates of cultures. In preliminary studies, applications

of those inhibitors did not interfere with measurement of PI uptake

or LDH activity.

Western blots

After assessment of PI uptake and LDH release 24 h after KA

treatment, cytosolic fractions were prepared from the slices.

Fourteen to sixteen cultured hippocampal slices were pooled and

homogenized in a buffer containing: HEPES 20 mm, pH 7.5,

sucrose 250 mm, KCl 10 mm, MgCl2 1.5 mm, EDTA 1 mm, EGTA

1 mm, dithiothreitol (DTT) 0.5 mm, phenylmethyl sulfonyl ¯uoride

(PMSF) 0.5 mm, 2 mg/mL leupeptin and 2 mg/mL antipain.

Homogenates were centrifuged at 1000 g for 10 min at 48C and

the supernatants were then centrifuged at 8000 g for 30 min at 48C.

The supernatants were further centrifuged at 100 000 g for 60 min

at 48C, and the resulting supernatant was used as the cytosolic

fraction. Aliquots from each sample (15 mg prot./lane) were

subjected to sodium dodecyl sulfate polyacrylamide gel electro-

phoresis (SDS-PAGE) and transferred onto nitrocellulose mem-

branes as described previously (Liu et al. 1996). The membranes

were probed with primary antibodies (1 : 250) at RT overnight,

then were incubated with appropriate IgG, and the antigen/antibody

complexes were visualized with NBT/BCIP. Immunoblots were

scanned and the digitized images were quantitatively analyzed by

densitometry with ImageQuant program providing peak areas. Data

were expressed as percentage of controls.

Lipid peroxidation

Lipid peroxidation was assessed by using the thiobarbituric

acid-reactive substances (TBARS) assay as described previously

(Ohkawa et al. 1979; Pike et al. 1997). Cultured slices (six slices

Role of polyamines in kainate excitotoxicity 977

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 79, 976±984

per group) were sonicated in a buffer containing 2.5% SDS,

6.25 mm deferoxamine and 12.5 mm probucol. After addition of

15 : 1 n-butanol/pyridine into the sample, TBARS were isolated

from the organic layer that was formed from the reaction mixture

by centrifugation (4000 g, 10 min, RT). Levels of TBARS

were quanti®ed by spectro¯uorometry (excitation/emission �515/553 nm). Each assay included a standard curve of 1,1,3,3-

tetramethoxypropane for comparison; absorbance data of 1,1,3,3-

tetramethoxypropane were linear within the tested range and

spanned the observed values of cell lysates. Data were corrected

according to protein concentration, and expressed as a percentage

of untreated control values.

Evaluation of glial activation

After assessment of PI uptake and LDH release, hippocampal slice

cultures were ®xed in 2% (w/v) paraformaldehyde (PFA), sectioned

on a cryostat (12 mm) and the sections mounted onto microscope

slides. The sections were incubated with anti-GFAP monoclonal

antibodies (1 : 50) at 48C overnight, and the immunoreactivity

was detected by the avidin±biotin±peroxidase method as per

the manufacturer's protocol with DAB as the chromogen. No

immunoreactivity was detected in sections incubated in the absence

of primary antibody. Additional slice cultures (eight slices per

group) were collected and homogenized in buffer as described

above. Homogenates were centrifuged at 1000 g for 10 min at 48C,

and equal amount of supernatant proteins were processed for

Western blots as described above using the same anti-GFAP

antibody.

Statistical analysis

All experimental data were statistically analyzed by using analysis

of variance (anova) followed by post hoc analysis, and the level of

statistical signi®cance was de®ned as p , 0.05.

Results

Neuronal protection by the PAO inhibitor MDL 72527

As previously reported (Bruce et al. 1995) application of

KA (50 mm) for 3 h in OHC produced a prominent increase

in PI uptake assessed 24 h later in hippocampal CA1,

CA3 and dentate gyrus, while untreated cultures (Control)

showed no noticeable ¯uorescence (Figs 1 and 2). In addi-

tion, LDH activity in culture medium increased by about six

times in KA-treated groups as compared to control slices

(Fig. 3). Pre-treatment with the PAO inhibitor, MDL 72527,

signi®cantly reduced KA-induced increase in PI uptake and

LDH release (by 40 and 43%, respectively) (Figs 1±3 and

Table 1).

Effects of co-treatment with MDL 72527 and cyclosporin

A, and MDL 72527 and EUK-134 on KA-mediated

excitotoxicity in OHC

Further examination of PI uptake in hippocampal sub®elds

showed that pre-treatment with MDL 72527 produced

more protection in CA1 than in CA3 (Figs 1 and 2). We

Fig. 1 Effects of MDL 72527 on KA-induced PI uptake in OHC.

Organotypic hippocampal slice cultures were loaded with PI

(4.6 mg/mL) and examined under ¯uorescent microscopy (2.5�) as

described in Methods. Cont: untreated cultures; KA: 50 mM for 3 h;

KA 1 MDL: MDL 72527 (100 mM) 1 KA (50 mM). Representative

images (magni®cation � 2.5�).

Fig. 2 Semi-quantitative analysis of the

effects of MDL 72527, EUK-134, and CSA

on KA-induced PI uptake in OHC. PI uptake

images of OHC similar to those shown in

Fig. 1 were semiquantitatively analyzed as

described in Methods. Data represent

means ^ SEM of six experiments. CONT:

untreated cultures; KA: 50 mM for 3 h;

KA/MDL: MDL 72527 (100 mM) 1 KA

(50 mM); KA/CSA: Cyclosporin A (100 mM) 1

KA (50 mM); KA/MDL 1 CSA: (100 MDL

72527 (100 mM) 1 Cyclosporin A (100 mM) 1

KA (50 mM); KA/MDL 1 EUK: MDL 72527

(100 mM) 1 EUK-134 (5 mM) 1 KA (50 mM).

* statistically signi®cant from KA-treated

alone group ( p , 0.05). ²Statistically signi®-

cant from single-agent pre-treated groups

( p , 0.05). VStatistically signi®cant in CA1

sub®eld from CA3 sub®eld within KA/MDL

group ( p , 0.05). Data for the KA/CSA

alone group were from Liu et al. (2001).

978 W. Liu et al.


previously observed that the blocker of the mitochondrial

permeability transition pores, CSA, also provided partial

neuronal protection against KA, especially in CA3 (Liu et al.

2001). We proposed the hypothesis that there may be

different neuronal death pathways mediating KA neuro-

toxicity in different neuronal populations (Liu et al. 2001).

Thus, we examined the effects of pre-treatment with a

combination of MDL 72527 and CSA on KA neurotoxicity.

Co-treatment with both agents almost completely prevented

KA-induced PI uptake as well as LDH release (Figs 2 and 3

and Table 1).

We previously reported that pre-treatment with EUK-134,

a superoxide dismutase/catalase mimetic, signi®cantly pro-

tected neurons from KA-induced oxidative stress (Rong et al.

1999). Therefore, we examined the effects of a combination

of MDL 72527 and EUK-134 on KA toxicity. We ®rst tested

different concentrations of EUK-134, and found that 5 mm

EUK-134 was optimal to produce a signi®cant degree of

protection against KA-induced toxicity. At this concen-

tration, EUK-134 provided the same amount of neuro-

protection as MDL 72527 with both PI uptake and LDH

Table 1 Protection by various treatments against KA-induced pathological changes in organotypic hippocampal cultures

Treatment

Pathological marker KA/MDL KA/CSA KA/EUK KA/MDL 1 CSA KA/MDL 1 EUK

PI uptake

CA1 67% ^ 3% 34% ^ 5% 36% ^ 6% 97% ^ 8% 70% ^ 2%

CA3 41% ^ 11% 64% ^ 3% 44% ^ 7% 86% ^ 6% 46% ^ 11%

DG 76% ^ 9% 47% ^ 6% 68% ^ 4% 99% ^ 4% 77% ^ 1%

LDH activity 44% ^ 7% 37% ^ 9% 24% ^ 6% 94% ^ 7% 41% ^ 4%

Cyto C release 31% ^ 11% 56% ^ 7% 41% ^ 9% 90% ^ 12% 43% ^ 12%

Organotypic hippocampal slice cultures were pretreated overnight with MDL 72527 (100 mM), CSA (100 mM), EUK 134 (5 mM), MDL 72527 1 CSA,

or MDL 1 EUK 134, before a 3-h treatment with KA (50 mM). 24 h later, PI uptake in CA1, CA3 or DG, LDH release in the medium or Cyto C

release in the cytoplasm was measured. Data are expressed as percent reduction of the effects of KA alone and are means ^ SEM of 4±6

experiments.

Fig. 4 Effects of various pre-treatments on KA-induced Cyto C

release. Cytosolic fractions were prepared from OHC after KA treat-

ment as described in Methods. (a) Representative western blots

from untreated (CONT), KA-treated (KA), MDL 72527-pretreated

(KA/MDL), EUK-134-pretreated (KA/EUK), MDL 72527 and CSA

co-pre-treated (KA/MDL 1 CSA), as well as MDL 72527 and EUK

134 co-pre-treated cultures. (b) Quantitative analysis of blots similar

to representatives in (a). Blots were scanned and the intensities of

bands were quanti®ed and represented as means ^ SEM. *Signi®-

cantly different from KA-treated group ( p , 0.05), and ²different

from a single agent pre-treated cultures ( p , 0.05).

Fig. 3 Effects of MDL 72527, EUK-134 and CSA on KA-induced

LDH release in OHC. LDH activity released in the culture medium

was measured as described in Methods. Data are expressed as

means ^ SEM of six experiments. The labels are the same as in

legend for Fig. 2. *Statistically different from KA-treated alone

cultures ( p , 0.05). ²Statistically different from a single-agent pre-

treated culture. Data for the KA/CSA alone group were from Liu et al.

(2001).



release (Table 1). However, and in contrast to the effects

of MDL 72527 and CSA co-treatment, MDL 72527 and

EUK-134 cotreatment did not produce additive protection

against KA neurotoxicity (Figs 2 and 3 and Table 1).

Attenuation of KA-induced Cyto C release by

pre-treatment with MDL 72527

Cyto C, a constitutive enzyme residing within the mitochon-

drial matrix, can be released into the cytosol under various

conditions to trigger a cascade of caspase activation and cell

apoptosis (Liu et al. 1996; Brenner et al. 2000). Since

polyamines have been shown to interact with several

mitochondrial functions (Jensen et al. 1987; Toninello

et al. 1990; Rustenbeck et al. 1998), we assessed the effects

of MDL 72527 on KA-induced cytosolic Cyto C release.

Cyto C levels were increased in cytosolic fractions prepared

from KA-treated OHC as compared to control by ®vefold

(Fig. 4). Pre-treatment with MDL 72527 partially reduced

Cyto C release by 31% (Fig. 4 and Table 1). Similarly, pre-

treatment with CSA also partially blocked KA-induced

Cyto C release by 56%. Consistent with the observations on

neuronal protection, a combination pre-treatment of CSA

and MDL 72527 almost completely blocked Cyto C

release (by 90%) (Fig. 4). In contrast, EUK134/MDL

72527 pre-treatment reduced Cyto C release by 44%

(Fig. 4 and Table 1).

Attenuation of KA-induced lipid peroxidation


KA-induced neuronal death is accompanied by the forma-

tion of ROS, although the sources of free radicals are

unclear. To test the hypothesis that the polyamine inter-

conversion pathway may contribute to ROS formation, we

examined the effects of MDL 72527 on lipid peroxidation

products measured with the TBARS assay. KA treatment

produced an increase in lipid peroxidation, which was

partially but signi®cantly reduced by MDL 72527 pre-

treatment at both 6 and 24 h after KA treatment (by 55 and

45%, respectively) (Fig. 5). Interestingly, the increase in

lipid peroxidation was larger at 6 h than at 24 h after KA

treatment, suggesting that this event occurs relatively early

after KA addition.

Attenuation of KA-induced glial activation by


Twenty-four hours after KA treatment, GFAP immuno-

reactivity was increased mostly in strata radiatum and

lacunosum moleculare, in cells that exhibited a glial

morphology (Fig. 6a). Under higher magni®cation, GFAP-

immunoreactive cells displayed a classic morphology of

activated astrocytes with numerous processes and stellate

cell bodies (Fig. 6b). Levels of GFAP were estimated by

western blots and were increased by 50% following KA

treatment (Fig. 6c). Pre-treatment with MDL 72527 attenu-

ated KA-induced increase in GFAP immunoreactivity and

levels by 50% (Fig. 6).

Fig. 5 Effects of KA and MDL 72527 on lipid peroxidation in OHC.

Cultures were treated with (KA/MDL) or without (KA) pre-treatment

with MDL 72527. Lipid peroxidation was measured at 6 and 24 h

after KA treatment using TBARS method as described in Materials

and methods. Raw data were ®rst corrected according to protein

concentration, and expressed as percentage of control values

(means ^ SEM). *Signi®cantly different from KA-treated group

( p , 0.05). CONT represents untreated cultures.

Fig. 6 Effects of KA and MDL 72527 on GFAP immunoreactivity

and levels in OHC-GFAP immunocytochemistry was performed as

described in Methods 24 h after treatment of cultures with KA

(50 mM) for 3 h. (a) Low magni®cation of representative hippocampal

slices (Bar � 400 mm). (b) High magni®cation of representative

hippocampal slices (Bar � 30 mm). CONT is an example of

untreated slice cultures. KA is an example from KA-treated cultures,

and MDL/KA is an example from cultures pretreated with MDL

72527. (c) Western blots analysis for GFAP under the same condi-

tions. Top: Representative western blots from untreated (CONT),

KA-treated (KA), MDL 72527-pretreated (KA/MDL). Bottom: Quanti-

tative analysis of blots similar to representatives in (a). Blots were

scanned and the intensities of bands were quanti®ed; data represent

means ^ SEM of four experiments. *Signi®cantly different from

KA-treated group ( p , 0.05).

980 W. Liu et al.


Discussion

Since the OHC model preserves several characteristics of

the intrahippocampal circuitry observed in vivo, application

of KA in OHC mimics excitotoxicity of KA in intact

animals, and thus provides a good alternative method to

study mechanisms of KA-induced neuronal apoptosis.

One advantage of this system is to be able to apply

known concentrations of antagonists against putative death-

promoting factors to examine the roles of these factors in

KA neurotoxicity. Recently, MDL 72527, a speci®c irre-

versible inhibitor of PAO, has been reported to prevent

apoptosis in non-neuronal cell lines (Hu and Pegg 1997),

while it may induce apoptosis in some cells through

lysosomotropic effects (Dai et al. 1999). Our results indicate

that MDL 72527 treatment provides signi®cant, albeit

partial, neuronal protection against KA toxicity. The pro-

tection was more pronounced in CA1 than in CA3, and was

associated with decreases in KA-induced Cyto C release in

the cytosol, lipid peroxidation and activation of glial cells.

Therefore, the polyamine interconversion pathway plays an

important role in mediating KA neurotoxicity, particularly

in CA1. Pre-treatment with a combination of MDL 72527

and CSA provided additive neuronal protection in the whole

slice, suggesting that there may be multiple mechanisms

responsible for KA excitotoxicity in different hippocampal

sub®elds.

Increased expression and activity of ODC, the rate-

limiting enzyme in polyamine synthesis, and increased

levels of putrescine in brain were previously observed

following a variety of stimuli, including ischemia, seizure

activity and mechanical injury (Paschen et al. 1987; Najm

et al. 1992a; Baskaya et al. 1996; Schipper et al. 2000). We

also reported a positive correlation between the levels of

putrescine and the degradation of spectrin, a cytoskeleton

marker, following KA-induced seizures (Najm et al. 1992b),

results which were interpreted as indicating that the poly-

amine synthetic pathway was possibly involved in neuro-

degeneration. However, several lines of evidence are not in

agreement with this notion. The increase in ODC activity

was only transient in contrast to the prolonged increase in

putrescine levels in rat brain after systemic KA treatment

(Najm et al. 1992a). Inhibition of ODC activity did not

modify the levels of putrescine and the extent of spectrin

degradation (Najm et al. 1992a). Dissociation between ODC

activity and putrescine levels was also found in other

experimental models such as ischemia and microencephalic

rats in response to KA excitotoxic injury (Paschen et al.

1993; Contestabile et al. 1998). Moreover, overexpression

of putrescine in transgenic mice did not result in neuronal

injury (Lukkarainen et al. 1995). By contrast, the polyamine

interconversion pathway, which was ®rst described by Seiler

(1995), has been reported to be associated with apoptosis in

various types of tissues including the central nervous

system. Under normal and pathological conditions, 70% of

the levels of putrescine in rat brain originate from the

interconversion pathway, and only 30% is formed from

l-ornithine by ODC (Dogan et al. 1999). Therefore,

increased putrescine levels in brain after KA treatment are

more likely to result from activation of the interconversion

pathway than from increased ODC activity (Seiler 2000).

Consistent with this hypothesis, our results show that

inhibition of PAO activity by MDL 72527 signi®cantly

protected neurons from KA toxicity. MDL 72527 also

signi®cantly prevented neuronal death in ischemia and

mechanical injury models (Dogan et al. 1999). Although

3-aminopropanal has been shown to be cytotoxic (Ivanova

et al. 1998), there is no evidence to demonstrate formation

of acrolein, a known apoptosis inducer, by 3-aminopropanal

in vivo (Fernandez et al. 1995; Schipper et al. 2000). On the

other hand, it has been reported that apoptosis of H157

cells by generation of hydrogen peroxide from the inter-

conversion pathway can be attenuated by pre-treatment with

catalase or other antioxidants (Brunton et al. 1991; Ha et al.

1997). In the present study, we examined the effects of a

combination of MDL 72527 and EUK-134 on KA neuronal

toxicity. EUK-134 is a synthetic superoxide dismutase/

catalase mimetic that antagonizes toxicity of hydrogen

peroxide in various cell types (Doctrow et al. 1997; Melov

et al. 2000), and protected neurons from KA-induced

oxidative stress and damage in hippocampus (Rong et al.

1999). Pre-treatment with the combination of MDL 72527/

EUK-134 did not produce an additive neuronal protective

effect. These observations suggest that the polyamine inter-

conversion pathway contributes to KA-induced neuronal

death by generating hydrogen peroxide.

Activation of caspases is a crucial event in cellular apop-

tosis, and the release of soluble molecules from mito-

chondrial matrix, including Cyto C, into cytosol plays an

important role in triggering a cascade of caspase activation.

Cytosolic Cyto C was signi®cantly increased as early as 3 h

after KA treatment (R. Liu et al., unpublished results). We

previously reported that OHC pre-treatment with cyclo-

sporin A, but not FK506, provided a signi®cant degree of

neuroprotection against KA toxicity (Liu et al. 2001). As

both compounds are known inhibitors of the phosphatase

calcineurin, but cyclosporin A also blocks the mitochondrial

permeability transition pores, we concluded that KA neuro-

toxicity involved opening of mPTP (Liu et al. 2001). Pre-

treatment with either MDL 72527 or CSA reduced Cyto C

release at each time point (3, 6, 16 and 24 h) tested after KA

treatment (unpublished data). At 24 h, either agent provided

the maximum percentage of inhibition of Cyto C release.

Pre-treatment with a combination of both agents almost

completely blocked Cyto C release. These results indicate

that polyamines interfere with Cyto C release independently

of the mPTP. In addition, since the combination MDL

72527/EUK-134 incompletely prevented Cyto C release,



preventing ROS formation is necessary but not suf®cient to

completely block Cyto C release. On the other hand, ROS

formation is probably not solely due to the activation of the

polyamine interconversion pathway; however, we were

unable to ®nd any difference between MDL 72527 alone

and MDL72527/EUK-134 groups in neuronal protection as

well as in prevention of Cyto C release. This could be due to

the fact that ROS formation is only one of the factors

contributing to KA neuronal toxicity, and, that the differ-

ence between these two groups may not be large enough to

be detected by our assays. Using more sensitive methods

may help differentiate the roles of polyamines and of other

pathways in producing ROS.

The CA1-selective neuronal protection by MDL 72527

suggests that neuronal sensitivity to KA toxicity in different

hippocampal sub®elds involves different cell death mech-

anisms. It has been reported that the polyamine inter-

conversion pathway is more activated in CA1 region after

barbiturate treatment and ischemia (Paschen et al. 1990;

Dogan et al. 1999), which may explain the selective

neuronal protection of CA1 by MDL 72527. We recently

observed that blockade of KA-induced mitochondrial mem-

brane potential collapse by CSA or inhibition of caspase-3

activity by z-VAD-fmk provided more protection in CA3

than in CA1 (Liu et al. 2001). In agreement with these

results, pre-treatment with the combination MDL 72527/

CSA resulted in an almost complete protection in the whole

culture, indicating that there are different pathways medi-

ating KA excitotoxicity in different hippocampal regions.

Interestingly, pre-treatment with EUK-134 alone attenuated

KA neurotoxicity to a similar extent in CA1 and CA3,

suggesting that ROS formation, although occurring through-

out the hippocampus, is important, but not necessarily

critical, to determine the susceptibility of each hippocampal

sub®eld to KA toxicity.

Several studies have indicated an important role for p53

gene in excitotoxic neuronal apoptosis in both in vivo and

in vitro models (Hughes et al. 1997). We previously

demonstrated p53 expression in both CA1 and CA3 regions

of OHC after KA treatment (Sakhi et al. 1997). However,

results from the present study indicate that there may be

different downstream events of p53-dependent pathways

responsible for KA neurotoxicity in different populations. In

CA3, the involvement of mitochondrial dysfunction and

caspase activation in p53-dependent pathway appears to be

critical for KA-induced neuronal death (Liu et al. 2001), a

result in agreement with several reports (Gillardon et al.

1997; Camins et al. 1998; Prehn 1998). In CA1, however,

the relationship between p53 expression and polyamines

needs to be further explored. There is growing evidence that

mechanisms of apoptosis are linked to processes that control

cell division and differentiation (Liu et al. 1996). In this

regard, p53 and polyamines, are both essential for cell

growth and may therefore coordinately contribute to KA

neurotoxicity. A dual role of polyamines in cell cycle and

apoptosis has also been implicated in autoimmunity

(Brooks 1995). Furthermore, association between p53

expression, polyamines and neuronal death was also found

in microencephalic rats following systemic KA treatment

(Contestabile et al. 1998).

An increasing body of evidence indicates an association

between glial activation and KA-induced neuronal damage

(Ben-Ari 1985; Anderson et al. 1991). Since glial cells have

been extensively shown to release a variety of factors that

can enhance survival or accelerate death of neighboring

neurons, KA-induced glial reactions may be critical to

determine the fate of neurons after insults. Increased

expression of GFAP has been found to be an early event

following kindling and lesions (Steward et al. 1991;

Baldwin et al. 1998). Inhibition of the polyamine synthesis

pathway prevented the increase in GFAP immunoreactivity

caused by ischemia and mechanical injury (Zini et al. 1990;

Zoli et al. 1993). By contrast, chronic inhibition of ODC

activity resulted in more neuronal damage in the same

models, suggesting that polyamine metabolism plays a

critical role in mediating neuronal and glial reactions

following a variety of insults. In our experiments, GFAP

immunoreactivity was increased after KA treatment,

indicating hypertrophy and proliferation of astrocytes in

response to KA treatment. Inhibition of PAO activity by

MDL 72527 signi®cantly prevented astrocyte reactions,

especially in CA1 region. Therefore, we conclude that

mediation of KA-induced neuronal damage by polyamines

appears to depend upon the formation of free radicals, and

cytosolic Cyto C release within neurons and the resulting

stimulation of proliferation of astrocytes that release factors

accelerating neuronal death.

Acknowledgement

This work was supported by grant NS18527 from NINDS to

MB.

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Role of polyamine metabolism in kainic acid excitotoxicity in organotypic hippocampal slice cultures