J. Exp. Med.,
Volume 188, Number 2, July 20, 1998 327-340
Cerebral Ischemia Enhances Polyamine Oxidation:
Identification of Enzymatically Formed 3-Aminopropanal as
an Endogenous Mediator of Neuronal and Glial Cell Death
By
Svetlana
Ivanova,***
Galina I.
Botchkina,*
Yousef
Al-Abed,
Malcolm
Meistrell III,*
Franak
Batliwalla,
Janet M.
Dubinsky,
Constantino
Iadecola,§
Haichao
Wang,*§§
Peter K.
Gregersen,
John W.
Eaton,
and
Kevin J.
Tracey*¶
From the * Laboratory of Biomedical Science, The Picower Institute for Medical Research, Manhasset,
New York 11030; the Department of Physiology and the § Department of Neurology, University of
Minnesota, Minneapolis, Minnesota 55455; the Department of Pediatrics, Baylor College of Medicine,
Houston, Texas 77030; the ¶ Department of Surgery, North Shore University Hospital-New York
University Medical School, Manhasset, New York 11030; the ** Department of Experimental
Pathology and Laboratory Medicine, Albany Medical College, Albany, New York 12208; and the Division of Biology and Human Genetics, Department of Medicine, and the §§ Department of
Emergency Medicine, North Shore University Hospital-New York University Medical School,
Manhasset, New York 11030; and the Laboratory of Organic Chemistry, The Picower Institute for
Medical Research, Manhasset, New York 11030
 |
Abstract |
To elucidate endogenous mechanisms underlying cerebral damage during ischemia, brain
polyamine oxidase activity was measured in rats subjected to permanent occlusion of the middle cerebral artery. Brain polyamine oxidase activity was increased significantly within 2 h after
the onset of ischemia in brain homogenates (15.8 ± 0.9 nmol/h/mg protein) as compared with
homogenates prepared from the normally perfused contralateral side (7.4 ± 0.5 nmol/h/mg protein) (P <0.05). The major catabolic products of polyamine oxidase are putrescine and
3-aminopropanal. Although 3-aminopropanal is a potent cytotoxin, essential information was
previously lacking on whether 3-aminopropanal is produced during cerebral ischemia. We
now report that 3-aminopropanal accumulates in the ischemic brain within 2 h after permanent
forebrain ischemia in rats. Cytotoxic levels of 3-aminopropanal are achieved before the onset
of significant cerebral cell damage, and increase in a time-dependent manner with spreading
neuronal and glial cell death. Glial cell cultures exposed to 3-aminopropanal undergo apoptosis
(LD50 = 160 µM), whereas neurons are killed by necrotic mechanisms (LD50 = 90 µM). The
tetrapeptide caspase 1 inhibitor (Ac-YVAD-CMK) prevents 3-aminopropanal-mediated apoptosis in glial cells. Finally, treatment of rats with two structurally distinct inhibitors of
polyamine oxidase (aminoguanidine and chloroquine) attenuates brain polyamine oxidase activity, prevents the production of 3-aminopropanal, and significantly protects against the development of ischemic brain damage in vivo. Considered together, these results indicate that
polyamine oxidase-derived 3-aminopropanal is a mediator of the brain damaging sequelae of cerebral ischemia, which can be therapeutically modulated.
Key words:
stroke;
infarction;
spermine;
apoptosis;
caspase
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Introduction |
Cerebral ischemia, a leading cause of disability and mortality world-wide, is mediated by a cascade of molecular
cytotoxins that kill potentially viable cells in the brain. The
polyamines spermine, spermidine, and putrescine, which are
among the most abundant molecules in mammalian brain,
have been implicated in the pathogenesis of ischemic brain
damage (1). Polyamine biosynthesis is increased after the
onset of cerebral ischemia, due to an ischemia-mediated induction of ornithine decarboxylase, a key synthetic enzyme
in the polyamine biosynthetic pathway (9). Spermine
was recently linked to the development of glutamate-mediated cytotoxicity, because it can bind to the NR1 subunit
of the NMDA receptor and potentiate glutamate-mediated
cell damage (14). Administration of experimental therapeutics that inhibit ornithine decarboxylase prevents the
development of ischemic brain damage, suggesting that the
accumulation of polyamines in the ischemic brain occupies an important role in the pathogenesis of stroke (9).
Somewhat paradoxically, however, brain spermine and
spermidine levels decrease during cerebral ischemia (5, 18,
19). This decline of tissue spermine and spermidine levels is
accompanied by an increase in brain putrescine levels (13,
19). Furthermore, intracerebral putrescine levels correlate significantly with the volume of dead brain, suggesting
that putrescine may be an endogenous molecular marker
for the extent of ischemia-induced damage. Notably, putrescine does not interact with the N-methyl-D-aspartate
(NMDA) receptor, and does not potentiate its function.
Therefore, we reasoned that a possible explanation for
these results could be found in the catabolism of polyamines
via the "interconversion pathway", which is dependent
upon the activity of tissue polyamine oxidase (20, 22).
This ubiquitous enzyme, which is present in high levels in
brain and other mammalian tissues, cleaves spermine and
spermidine via oxidative deamination to generate the end
products putrescine and 3-aminopropanal (22, 26).
3-Aminopropanal is widely known for its cytotoxicity to
primary endothelial cells, fibroblasts, and a variety of transformed mammalian cell lines (29). 3-Aminopropanal has
also been implicated as a mediator of programmed cell death
in murine embryonic limb buds, and may contribute to the
development of necrosis in some tumors (34, 35). Inhibition
of polyamine oxidase with aminoguanidine, a well-characterized inhibitor, blocks the generation of 3-aminopropanal in cell cultures after the addition of spermine, and prevents subsequent cytotoxicity (30, 36, 37). The LD50 concentration of 3-aminopropanal to cells is similar to that of
glutamate excitotoxicity to neurons (38). In contrast, putrescine is not cytotoxic to cells (even in the millimolar
range) but its rate of production through polyamine oxidation correlates with the rate of formation of the cytotoxin
(3-aminopropanal). Reasoning that increased production of
3-aminopropanal might therefore mediate cytotoxic brain
damage during cerebral ischemia, we investigated the activity
of polyamine oxidase, and measured the levels of 3-aminopropanal in an animal model of cerebral infarction.
We now report that cerebral ischemia mediates the induction of brain polyamine oxidase activity, and that the cytotoxic
end product 3-aminopropanal accumulates in the brain at levels that are lethal to neurons and glial cells. In glial cells, 3-aminopropanal mediates apoptosis by activation of a caspase
1-dependent signaling pathway, whereas in neurons it causes
necrotic cell death. Inhibition of polyamine oxidase activity
with structurally distinct compounds prevents the formation
of 3-aminopropanal and provides significant protection
against the development of cerebral damage after permanent cerebral artery occlusion in rats. This is the first direct
evidence that polyamine oxidase-derived 3-aminopropanal
is a potential therapeutic target in cerebral ischemia.
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Materials and Methods |
Animal Model of Permanent Middle Cerebral Artery Occlusion.
All procedures involving animals were conducted in conformity
with institutional guidelines and under the approval of the Animal Care and Use Committee of North Shore University Hospital-New York University Medical School. Middle cerebral infarction was performed as previously described in detail (39, 40).
In brief, the ipsilateral common carotid artery was ligated and divided, the middle cerebral artery was coagulated and divided distal to the lenticulostriate branch, and the contralateral common
carotid was occluded for 1 h. The onset of ischemia in these experiments was defined as the time the middle cerebral artery was
cut. For measurement of infarct volume, the animals were killed
at the times indicated and fresh brain sections were prepared (1 mm) and immersed in 2,3,5-triphenyltetrazolium chloride (TTC)1 in
154 mM NaCl for 30 min at 37°C, and total cerebral infarct volume was measured by computerized quantitative planimetry as previously described (39). Similar measurements of stroke
volume were obtained in separate experiments using planimetric
analysis of brain sections stained with hematoxylin and eosin (data
not shown).
Polyamine Oxidase Assay.
Polyamine oxidase in brain homogenate was assayed as previously described (25, 26, 42). In brief, 2 h
after occlusion of the middle cerebral artery, a 4-mm-thick coronal section of ipsilateral hemisphere encompassing the zone of ischemia (beginning 3 mm caudal from the frontal pole) was manually homogenized on ice in 1.5 ml of Hanks media containing
1 mM PMSF, and was centrifuged at 43,000 g for 30 min. Brain
polyamine oxidase activity in the homogenates was determined
by addition of spermine to the homogenate at time zero (50 µl of
a 1 mM stock solution added per 1 ml of supernatant). Where indicated in some experiments the enzyme inhibitors aminoguanidine or chloroquine (50 µM-5 mM) were added 5 min before spermine. Homogenates were maintained at 37°C, and duplicate
200-µl samples were removed at time points up to 60 min after
the addition of spermine; enzyme activity in the samples was
stopped by addition of 10 µl of 60% perchloric acid (PCA). Samples for HPLC analysis to detect spermine were prepared as described below. Enzyme activity was corrected for the protein
content of the supernatants using a commercially available protein
assay (Bio-Rad Protein Assay; Bio-Rad, Hercules, CA) with BSA
(GIBCO BRL, Gaithersburg, MD) as a standard.
3-Aminopropanal.
3-Aminopropanal was prepared by hydrolysis of 3-aminopropanal diethyl acetal (145 mM; TCI America, Portland, OR) in 1.5 M of HCl for 5 h at room temperature.
The reaction mixture was applied to a column (3 × 6 cm) of
Dowex-50 (H+ form; Sigma Chemical Co., St. Louis, MO) ion
exchange resin and eluted with a step gradient of HCl (0-3 M;
160 ml; flow rate 0.7 ml/min). Fractions containing aldehyde, as
determined by the method of Bachrach and Reches (43), were
concentrated in a centrifugal evaporator at room temperature. The concentration of 3-aminopropanal was determined spectrophotometrically at 
= 531 nm, based on a reaction of aldehydes
with 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole (Purpald;
Aldrich Chemical Co., Milwaukee, WI; reference 44) with reference to a standard curve using propionaldehyde (Sigma Chemical
Co.). Acidic solutions of the aldehyde were neutralized with
NaOH to physiological pH immediately before use. Vehicle control solutions consisted of the same stoichiometric amounts of
HCl and NaOH.
Derivatization of 3-Aminopropanal with 2,4-Dinitrophenylhydrazine.
2,4-dinitrophenylhydrazine (0.5 g) in concentrated HCl/
ethanol (1:10, vol/vol; 11 ml) was refluxed for 10 s with the aqueous 3-aminopropanal. The resulting 3-aminopropionaldehyde-
2,4-dinitrophenylhydrazone derivative was precipitated at room
temperature and collected by filtration. 1H-nuclear magnetic resonance (NMR) spectroscopy (DMSO-d6 and CDCl3, 270 MHz) of
purified 2,4-dinitrophenylhydrazone derivative was employed to
confirm its structure. The NMR spectrum revealed the presence
of syn and anti isomers (1:1) with resonance at 
8.83 and 11.35. A
standard curve was generated by an HPLC assay of the dansylated
derivative of the compound (see below). An additional standard
curve was constructed to quantify recovery of the compound from
brain homogenates. In brief, a 4-mm-thick brain slice obtained from the region of the middle cerebral artery perfusion zone was homogenized manually, followed by addition of 3-aminopropanal (final concentration = 10, 100, 150, 200, 300, or 1,000 nmol/ml) in 1.5 ml of 2,4-dinitrophenylhydrazine reagent. The samples were refluxed in the 2,4-dinitrophenylhydrazine reagent for 10 s, then 20 µl of 60% PCA added to stop the reaction, followed by addition of water (200 µl). The samples were vigorously vortexed and
centrifuged at 14,000 rpm for 30 min, and the supernatant was
concentrated to near dryness in a centrifugal evaporator. Samples
were redissolved in 100 µl of water, centrifuged for 10 min at
14,000 rpm to clear precipitates, and then subjected to HPLC.
HPLC Detection of the Derivatization Products of 3-Aminopropanal
and 2,4-Dinitrophenylhydrazine.
A liquid chromatograph (model
1090; Hewlett-Packard, Wilmington, DE), equipped with an autosampler, photo diode-array, fluorescence detectors, and Chemstation operating software, was used for all analyses. We used
detection by fluorescence, based on the reaction of 5-dimethyl-aminonapthalene sulfonyl-chloride (dansyl chloride; Molecular
Probes, Eugene, OR; relative fluorescence intensity 280-340 out
of 430 nm) with primary and secondary amines. Dansylation was
performed by reacting 50 µl of the sample with 200 µl of 10 mg/
ml dansyl chloride solution in acetone, 200 µl of saturated Na2CO3 solution, 3 µl of 60% PCA, and 3 µl of 1-mM 1,7-diaminoheptane (Sigma Chemical Co.), followed by incubation at
65°C for 10 min. 20 µl of the resulting supernatant was injected
onto a C-4 250 × 4.6 mm column (Vydac, Hesperia, CA) with
5-µm particle size. Using a flow rate of 1.0 ml/min, runs were
initiated at 100% A (dH2O) and a linear gradient to 100% B
(methanol) performed over 45 min, followed by 5 min of 100% B
and a return to 100% A over 5 min.
For detection of the presence of 3-aminopropanal in ischemic
brain, animals were subjected to permanent middle cerebral artery occlusion and killed at the times indicated. Brain sections
corresponding to the area of focal infarction caused by middle cerebral artery occlusion (4-mm-thick located 3 mm caudal to the
frontal lobes) were quickly excised. Control brain slices were
taken from sham-operated animals. Manual homogenization was
performed in 1.5 ml of 2,4-dinitrophenylhydrazine reagent followed by concentration and HPLC analysis as described above.
The limit of detection of 3-aminopropanal with this assay is 180-
200 nmol/ml. Results are normalized for protein content using a
commercially available assay (Bio-Rad Protein Assay; Bio-Rad) and corrected for HPLC injection volume using an internal standard of 1,7-diaminoheptane.
Stereotactically Guided Microinjections of Polyamines into the Cerebral Cortex.
Male Lewis rats (270-300 g) were anesthetized and
placed in a stereotactic head frame (Stoelting Co., Wood Dale,
IL). The incisor bar was adjusted until the plane defined by the
lambda and bregma was parallel to the base plate. A microsurgical
craniotomy was performed 1.7 mm anterior to bregma, and 5 mm right of the midline, and the tip of a 29-gauge needle was advanced 2 mm deep to the dural opening. Polyamine- or 3-aminopropanal-containing solutions (25 µg) prepared in 2 µl of sterile saline (NaCl; 154 mM) were injected over 3 min, and the
needle was left undisturbed for 5 min and then removed. Animals
were killed 48 h later, and the brains were excised and sectioned in 1-mm-thick slices in the coronal plane, and then immersed for 30 min at 37°C in a solution containing 2,3,5-triphenyl-2H-tetrazolium chloride (2% in NaCl; 154 mM). Brain
infarction was visualized as areas of unstained (white) tissue which
were easily contrasted with viable tissue (stained red). Slices were
placed in buffered 10% formalin and infarct size was quantitatively assessed by planimetric analysis. In separate studies, histopathological analysis of brain sections verified the location of the
injectate and the correlation of necrosis revealed by TTC staining. Groups of three or four animals were used for each of the experimental conditions as noted.
Tissue Culture.
The glial (HTB14; reference 45) and neuronal
(HTB11; reference 46) cell lines were obtained from the American
Type Culture Collection (ATCC, Rockville, MD) and cultured
in DMEM (GIBCO BRL) containing fetal bovine serum (10%;
Hyclone, Logan, UT), sodium pyruvate (1 mM; Sigma Chemical
Co.), penicillin and streptomycin (0.5%; Sigma Chemical Co.) in a
humidified atmosphere (5% CO2; 37°C). For all experiments involving exposure to 3-aminopropanal, cells were grown in 96-well microtiter plates to 90-95% confluence and medium was replaced with fresh serum-free medium (Opti-MEM I; GIBCO
BRL) to prevent nonspecific interaction of 3-aminopropanal with serum proteins. For all experiments using a short duration of
3-aminopropanal exposure (5 min to 2 h in 96-well plates), the
cells were washed at the times indicated, and then incubated in
Opti-MEM I for up to 20 h. Where indicated, cells were pretreated with the caspase 1 inhibitor II Ac-YVAD-CMK
(BACHEM, Torrance, CA) or the caspase-3 inhibitor Ac-DEVD-CHO (Peptides International, Louisville, KY) in DMSO for 3 h
followed by addition of 3-aminopropanal for an additional 5 h.
DMSO vehicle controls were performed concurrently. Cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma Chemical Co.) as previously described (47). Data are expressed as mean ± SE; n = 3-6 wells per
condition; experiments were performed in triplicate.
TUNEL Staining by FACS®.
Cells were treated with 3-aminopropanal as indicated and then harvested by centrifugation
(1,500 rpm for 5 min). The pellets were fixed with 1× ORTHO
Permeafix (Orthodiagnostics, Raritan, NJ) at room temperature
for 40 min. After washing with Dulbecco's PBS containing 1%
BSA (PBS-BSA), cells were stained by the TUNEL (Tdt-mediated dUTP-biotin nick-end labeling) method using the ApopTag
Direct Fluorescein kit (Oncor, Gaithersburg, MD). Negative controls were performed using a reaction mixture devoid of TdT. A FACScan® (Becton Dickinson, Sunnyvale, CA) was used for all
analyses; 5,000-10,000 events (ungated) were collected using single color histogram for FITC.
Annexin V/Propidium Iodide Staining.
Annexin V/propidium
iodide (PI) staining was performed using a commercially available
kit (The Apoptosis Detection Kit; R&D Systems, Minneapolis,
MN). Cells were analyzed by flow cytometry within 1 h of completion of staining.
DNA Electrophoresis.
HTB11 or HTB14 cells were harvested
(2-3 × 107 cells) by centrifugation (1,000 rpm for 5 min), resuspended in a reaction buffer containing proteinase K, and incubated overnight at 55°C. RNAase was added to a final concentration of 50 µg/ml, and samples were incubated at 37°C for 1 h.
DNA was extracted three times with phenol/chloroform and two
times with chloroform and precipitated in 2 vol of 100% cold
ethanol and 0.3 M of sodium acetate (pH 5.2). DNA was resuspended in 50 µl of dH2O, fractionated by 1.5% agarose gel electrophoresis, and stained with SYBR Green I nucleic acid stain
(Molecular Probes).
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Results |
Cerebral Ischemia Enhances Brain Polyamine Oxidase Activity.
To determine the role of brain polyamine oxidase activity
in cerebral ischemia, Lewis rats were subjected to focal cerebral infarction by microsurgical occlusion of the middle
cerebral artery in a standardized model as described previously (39, 40, 48). Brain homogenates were prepared from
the anatomic region perfused by the middle cerebral artery,
and total polyamine oxidase (PAO) activity was determined
using a method described previously (25, 26, 42). Polyamine
oxidase activity was significantly higher in homogenates
prepared from ischemic hemispheres as compared with
normally perfused contralateral hemispheres (PAO activity after ischemia = 15.8 ± 0.9 nmol/h/mg protein; versus
PAO activity in sham-operated controls = 7.4 ± 0.5 nmol/h/mg protein; P <0.05; Fig. 1). The increase of
brain polyamine oxidase activity was detected within 2 h
after the onset of cerebral ischemia. Two structurally distinct inhibitors of polyamine oxidase activity (aminoguanidine and chloroquine) were used to assess specificity (26,
49). Addition of either agent to the ischemic brain homogenates dose-dependently inhibited polyamine oxidase
activity; chloroquine, IC50 = 40 µM; aminoguanidine,
IC50 = 400 µM. This indicates that within 2 h after the
onset of cerebral ischemia there is a specific induction of
brain polyamine oxidase activity, and that this activity can
be pharmacologically inhibited.
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Fig. 1.
Polyamine oxidase activity increases during cerebral ischemia, and is inhibited by aminoguanidine and chloroquine. Polyamine
oxidase activity was measured in brain homogenates prepared as described
in Methods. Data shown are mean ± SE; n = 3. Normal, sham-operated
control brain homogenate; Ischemia Vehicle, homogenate prepared 2 h after the onset of middle cerebral artery occlusion; Ischemia AG, addition of
aminoguanidine (1 mM) at time =  5 min before spermine; Ischemia
CHLQ, addition of chloroquine (1 mM) at time = 5 min before spermine. *, P <0.05 versus normal; #, P <0.05 versus ischemia vehicle.
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Cerebral Ischemia Enhances 3-Aminopropanal Production.
To obtain direct evidence that the cytotoxin 3-aminopropanal is produced during cerebral ischemia, we developed a
method to detect brain 3-aminopropanal using HPLC and
mass spectroscopy. 3-Aminopropanal was prepared by hydrolysis of the diethyl acetal and then derivatized using 2,4-dinitrophenylhydrazine (Fig. 2 a). HPLC analysis of the derivatized products revealed two peaks (at 24 and 27 min,
respectively) in equal ratio (Fig. 2 b, inset). H-NMR spectroscopy revealed the presence of anti and syn isomers, as
predicted by the structures of the principle condensation
products (Fig. 2 a). Electrospray ionization mass spectroscopy (EIMS) of the HPLC-purified products detected the
expected mass ion m/z 251 (Fig. 2 b). We then subjected
rats to permanent focal cerebral ischemia, and derivatized
brain homogenates with 2,4-dinitrophenylhydrazine. HPLC
analysis of the derivatized brain homogenate revealed the appearance of the two expected peaks, and EIMS confirmed
identity as the isomeric 3-aminopropanal-2,4-dinitrophenylhydrazone reaction products (Fig. 2 c). The 3-aminopropanal derivatization products could not be detected in
brain homogenates prepared from sham-operated, normally
perfused control animals (Fig. 3). 3-Aminopropanal became significantly elevated within 2 h after the onset of
ischemia, and increased in a time-dependent manner for at
least 25 h after the onset of ischemia (Fig. 3). The HPLC
assay we used may well have underestimated the amount of
3-aminopropanal produced in the ischemic brain, because
3-aminopropanal is a reactive molecule that can bind to the
amino and sulfhydryl groups of proteins (33, 52), thereby
decreasing its availability for derivatization and detection.
Nonetheless, after correcting the measured levels for total
brain protein (213 g/liter), brain 3-aminopropanal concentrations after ischemia reach a highly cytotoxic range (0.5-
2.7 mM). When considered with our previous observation
that cerebral ischemia mediates an early induction in the
activity of brain polyamine oxidase, these findings indicate
that this enzyme pathway continually generates 3-aminopropanal during the first 25 h after the onset of cerebral
ischemia.
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Fig. 2.
(a) 1H-NMR spectroscopy (DMSO-d6 and CDCl3, 270 MHz) of the products of reacting 3-aminopropanal with 2,4-dinitrophenylhydrazine. NMR revealed the presence of anti and syn isomers with resonance at 8.83 and 11.35. (Inset) Structure of the principle condensation products. (b)
Electrospray ionization mass spectrum of synthetic dansylated 3-aminopropanal-2,4-dinitrophenylhydrazone. 3-aminopropanal was derivatized with 2,4-dinitrophenylhydrazine and dansyl chloride, and the reaction products were subjected to HPLC as outlined in Materials and Methods. The inset shows
the HPLC profile of the separable geometric isomers. Note that the EIMS of the HPLC-purified fractions revealed the expected molecular ion at m/z
251. (c) EIMS of derivatized ischemic brain homogenate. Animals were subjected to permanent focal cerebral ischemia and after 25 h, brain tissue was obtained for homogenization and derivatization as described in Materials and Methods. The inset shows the HPLC profile, and the mass spectrum confirms
the expected molecular ion of the HPLC-purified fractions at m/z 251.
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Fig. 3.
Brain 3-aminopropanal levels increase during cerebral
ischemia. Brain 3-aminopropanal levels were measured by HPLC in rats
subjected to permanent focal cerebral ischemia. 3-Aminopropanal was
not detected in sham-operated controls. Note that 3-aminopropanal tissue
levels increased markedly within 2 h after middle cerebral artery occlusion, and continued to increase for at least 25 h. Data shown are mean ± SE, n = 3 animals/group. *, P <0.05 versus t = 0 h by analysis of variance.
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Production of 3-Aminopropanal Precedes the Development of
Significant Brain Cell Death.
We next examined whether
the enzymatic formation of 3-aminopropanal preceded the
onset of ischemic cell death. Accordingly, the volume of
dead brain was measured by staining brain sections with the
vital dye TTC. For the first 3 h of ischemia, cells in the region of the occluded middle cerebral artery were observed to be largely viable (total volume of cell death = 2 ± 2 mm3). Histological examination of hematoxylin and eosin-
stained brain sections (data not shown) confirmed that cells
were morphologically intact and had not yet developed degenerative changes at a time when 3-aminopropanal levels
were already significantly increased (Fig. 3). Over the next
25 h, spreading cell death developed in association with increasing 3-aminopropanal levels (infarct volume at 25 h = 71 ± 24 mm3; versus infarct volume at 3 h = 2 ± 2 mm3;
P <0.05). These findings give evidence that 3-aminopropanal accumulates during the early response to cerebral ischemia, and precedes the development of progressive, spreading brain cell death.
Brain Damage Is Mediated by Intracortical Microinjection of
Spermine, Spermidine, or 3-Aminopropanal, but Not by Putrescine.
Since polyamine oxidase activity is present in
normal mammalian brain (Fig. 1 and references 22, 25), we
wished to investigate whether increased extracellular levels
of substrate (e.g., spermine or spermidine) would induce
local cell death. Accordingly, spermine and spermidine
were administered into rat cerebral cortex by direct stereotactic microinjection, and the volume of cell death was measured by TTC staining brain sections. We observed
significant cortical cell death after spermine or spermidine
administration, but not after that of putrescine, a polyamine
that cannot be degraded by polyamine oxidase (Fig. 4 a).
Microinjection of 3-aminopropanal mediated significant
cell death in the cerebral cortex (Fig. 4 a). The quantity of
3-aminopropanal administered (25 µg/injection) is similar
to the amounts endogenously produced during ischemia (~350 µM assuming a volume of distribution of a typical
middle cerebral artery infarction in this model). Systemic
administration of the polyamine oxidase inhibitors (chloroquine or aminoguanidine) conferred significant protection
against the development of spermine-mediated intracortical
damage (Fig. 4 b), suggesting that polyamine oxidase activity is necessary to mediate the cytotoxicity of extracellular spermine. Intracortical administration of aminoguanidine
also conferred significant protection against intracortical
spermine-mediated cell death (Fig. 4 b), indicating that the
cerebroprotective effects of enzyme inhibition occur locally
in brain, and not via some unanticipated peripheral drug
action. Of note, aminoguanidine failed to significantly attenuate the direct cytotoxicity of intracortical 3-aminopropanal (Fig. 4 b), suggesting that the protective mechanism of aminoguanidine against spermine cytotoxicity is through
inhibition of polyamine oxidase activity, and not through
direct inhibition of 3-aminopropanal. Thus, increased extracellular levels of spermine, spermidine, or 3-aminopropanal (but not putrescine) are cytotoxic to cerebral cortical
cells in vivo.
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Fig. 4.
Brain damaging effects of intracortically administered
polyamines, and protection with polyamine oxidase inhibitors. (a) Brain
damage mediated by intracortical microinjection of spermine, spermidine,
and 3-aminopropanal, but not putrescine. Data shown are volume of
brain damage (mm3) as measured by integrating the area of negative TTC
staining over the entire brain hemisphere in animals injected with the
polyamines shown; mean ± SE, n = 6-8/group. *, P <0.05 versus vehicle. (b) Aminoguanidine and chloroquine protection against intracortical
spermine toxicity. All animals received intracortical spermine (25 µg in 2 µl)
by stereotactically guided microinjection. Experimental animals were
treated with aminoguanidine or chloroquine simultaneously with the intracortical spermine injectate in the following doses: systemic aminoguanidine was 320 mg/kg intraperitoneal 30-min pretreatment followed by subsequent doses of 110 mg/kg intraperitoneally each 8 h after
intracortical spermine; intracortical aminoguanidine was administered by
a single dose (320 mg/kg) given simultaneously with intracortical spermine; chloroquine was administered by a single intraperitoneal dose (25 mg/kg) 30 min before the spermine injection. Data shown are infarct volume (mm3) assessed quantitatively 48 h after the intracortical spermine injection (mean ± SE, n = 6-8/group). *, P <0.05 versus spermine/vehicle.
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Intracortical Administration of 3-Aminopropanal Induces Apoptosis and Necrosis.
We performed histopathologic examination of brain sections 24 h after intracortical 3-aminopropanal microinjection and observed localized degenerative
changes surrounding the injection zone (Fig. 5 A). Cells
within the 3-aminopropanal injection site were necrotic, as
evidenced by eosin-positive staining. Moreover, in the same
region we also observed cells undergoing programmed cell death, as evidenced by TUNEL-positive staining (Fig. 5, B
and C). These changes were not observed in the injection
zone when either vehicle or putrescine was administered
(data not shown). Direct intracortical administration of
spermine also caused cell necrosis and apoptosis, and these
findings were significantly inhibited by administration of
aminoguanidine (data not shown). Thus, the accumulation
of extracellular 3-aminopropanal induces brain cell damage
in vivo, which occurs through both necrosis and programmed cell death.
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Fig. 5.
Histology of 3-aminopropanal-induced cell death.
Animals were anesthetized and
perfused intracardially with 100 ml saline followed by 200-300
ml of 4% formaldehyde in PBS.
Brains were removed, postfixed
for 2 hr in the same fixative, and
then incubated overnight in 30%
sucrose at 4°C. Ten micrometer
sections were prepared and airdried. TUNEL staining was performed as outlined in Methods.
(A) Low magnification microphotograph demonstrating the
site of microinjecting needle
(large arrow) and areas of 3-aminopropanal-induced prominent
cell death (small arrows). (B) High
power microphotograph taken
within the marked area showing
coexistence of apoptosis (TUNEL-positive cell on the right) and
necrosis (eosin-stained cell on the
left). (C) High magnification of
area indicated in A showing the
extent of cellular degeneration.
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3-Aminopropanal Induces Apoptosis in Glial Cells, but Necrosis in Neurons.
To investigate directly the cytotoxic signaling mechanisms of 3-aminopropanal, we exposed cultured human glial (HTB14) and neuronal (HTB11) cell
lines to 3-aminopropanal. After 20 h of incubation, the
LD50's for 3-aminopropanal were 160 ± 10 µM for the
glial cell line and 90 ± 20 µM for the neuronal cell line (HTB11). 3-Aminopropanal was somewhat more cytotoxic in primary rat astroglial cell cultures (LD50 = 80 ± 9 µM). A time-course study revealed that 3-aminopropanal
exposure for as little as 5 min was significantly cytotoxic to
neuronal cells, but a longer exposure was required to mediate significant cytotoxicity in glial cells (Table 1). This delayed onset suggested that glial cell death might be dependent upon apoptosis-mediated pathways, and in agreement
with this possibility, we observed apoptosis-specific DNA
fragmentation after exposure of glial cells to 3-aminopropanal (Fig. 6 a). We obtained additional evidence of apoptosis
by flow cytometric detection of DNA strand breaks using
the TUNEL method (53). In these experiments, 76% of
the glial cells stained TUNEL-positive after 13 h of exposure to 160 µM of 3-aminopropanal (Fig. 6 b), whereas vehicle-treated control cells were uniformly negative (Fig. 6 c).
Multiparameter flow cytometry revealed that glial cells exposed to 3-aminopropanal exhibited a decrease in cellular forward light scatter and an increase in side scatter, in
agreement with typical cell shrinkage, chromatin condensation, and nuclear fragmentation. Apoptosis of glial cells was
also confirmed by subdiploid staining with propidium iodide and annexin V/PI (data not shown).
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Fig. 6.
Apoptosis in glial cells exposed to 3-aminopropanal. (a)
DNA gel electrophoresis of glial-like and neuronal-like cells exposed to
3-aminopropanal for 13 h as described in Materials and Methods; 7.5 µg
of DNA was loaded per lane for the glial cells (lanes B and C), and 15 µg
was loaded per lane for the neuronal cells (lanes D and E). Lane A, 1 kb
DNA size marker; lane B, 3-aminopropanal-treated (160 µM); lane C,
vehicle control; lane D, vehicle-treated; lane E, 3-aminopropanal-treated
(90 µM). (b) FACS® histogram showing TUNEL staining of 3-aminopropanal-treated glial cells (160 µM, 13 h) as outlined in Materials and
Methods. As indicated by the marker, 76% of the cell population stained
TUNEL positive. (c) FACS® histogram of vehicle-treated glial cells revealed no evidence of apoptosis (0.13% TUNEL-positive cells). (d)
FACS® histogram showing TUNEL staining of 3-aminopropanal-treated
neuronal cells (90 µM, 13 h) as outlined in Materials and Methods. As indicated by the marker, only 5.02% of the cell population of stained
TUNEL positive, though a forward/side scatter of the same cell population revealed 55.7% cell death upon electronic gating (data not shown).
(e) FACS® histogram of vehicle-treated neuronal cells showed no evidence of apoptosis (0.53% TUNEL-positive cells). Electronic gating for
forward/side scatter showed 9.1% cell death under these conditions. ( f )
Annexin V FITC/propidium iodide (PI) staining of 3-aminopropanal-
treated neuronal cells, as above, revealed no evidence of apoptosis (1.8%
apoptotic cells). Necrotic cells comprised 68.97% of the cell population.
(g) Annexin V FITC/propidium iodide staining of vehicle-treated neuronal cells revealed no evidence of apoptosis (1.02% of the cell population)
or necrosis (1.58% of the cell population).
|
|
In contrast to the results in the glial cell line, 3-aminopropanal did not induce apoptosis in neuronal cell cultures
(HTB11) using similar experimental methods. DNA electrophoresis of 3-aminopropanal-treated neurons revealed
no evidence of chromosomal DNA degradation (Fig. 6 a).
In addition, we observed no increase in TUNEL positivity under these conditions, although a forward/side scatter
analysis revealed significant cell death after 3-aminopropanal treatment (55.7%), but not in vehicle-treated controls
(9.1%). There was also no evidence of apoptosis as measured with annexin V, a method used to detect loss of cell
membrane phospholipid asymmetry that can be associated
with apoptosis (Fig. 6, f and g). Apoptosis could be induced
in neuronal cells by exposure to camptothecin (15 µg/ml
for 20 h; reference 54) as assessed by TUNEL and Annexin V methods (data not shown), indicating that the absence of
apoptosis after 3-aminopropanal exposure was not due to
some unanticipated generalized cellular defect in this neuronal cell line. Thus, in contrast to glial cells, exposure of
neurons to 3-aminopropanal causes primarily necrotic cell
death.
Inhibition of Caspase 1 Prevents 3-Aminopropanal-induced
Apoptosis in Glial Cells.
The cysteine proteases caspase 1 and caspase 3 have been implicated in the cellular signaling
pathways mediating apoptosis during cerebral ischemia (55-
59). To investigate whether these proteases were required
for the induction of apoptosis by 3-aminopropanal in glial
cells, HTB14 cells were treated for 3 h with a tetrapeptide caspase 1 inhibitor (Ac-YVAD-CMK), or with a caspase 3 inhibitor (Ac-DEVD-CHO), followed by a 5-h treatment
with 3-aminopropanal. Treatment with the caspase 1 inhibitor, but not the caspase 3 inhibitor, conferred dose-dependent inhibition of 3-aminopropanal-induced cell death (Fig.
7, a and b). These data give evidence for a specific role of
the caspase 1 proteases in the 3-aminopropanal-induced signaling that mediates apoptosis in glial cells.
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Fig. 7.
Inhibition of caspase
1 but not of caspase 3 blocks 3-aminopropanal-induced glial apoptosis. Cells were pretreated with
(a) the caspase 1 inhibitor (Ac-YVAD-CMK) or (b) the caspase 3 inhibitor (Ac-DEVD-CHO) at
concentrations 0.4 (triangles) or 40 µM (circles) for 3 h, followed by
treatment with 3-aminopropanal
for an additional 5 h, and then
were analyzed for cell viability by
the 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide
assay. Controls consisted of
DMSO-treated cells (squares) to
assess for nonspecific solvent effects. Data are mean ± SE, n = 3 wells/experiment.
|
|
Administration of Polyamine Oxidase Inhibitors In Vivo Attenuates 3-Aminopropanal Production and Protects against Brain
Damage, even after the Onset of Cerebral Ischemia.
The mechanism of ischemic brain cell damage proposed here predicts
that administration of polyamine oxidase inhibitors during cerebral ischemia in vivo will reduce both the accumulation of 3-aminopropanal and the volume of cerebral infarction. Accordingly, we measured these end points after administering two structurally distinct polyamine oxidase
inhibitors to rats in the standardized model of permanent
middle cerebral artery occlusion. We reported previously
that aminoguanidine administered after the onset of cerebral ischemia (320 mg/kg intraperitoneally 15 min after
ischemia) significantly reduces the volume of cerebral damage (40). In this study we administered aminoguanidine by
this established treatment protocol and observed that it effectively prevented the increase of brain 3-aminopropanal
levels (Table 2). In agreement with the proposed mechanism of inhibiting polyamine oxidase, the administration of
the structurally distinct enzyme inhibitor chloroquine also
conferred effective cerebroprotection against ischemic cell damage, even when the administration was delayed 15 min
after occlusion of the middle cerebral artery (Table 2). We
previously reported that the protective effects of aminoguanidine are not attributable to altering peripheral cardiovascular parameters that influence the volume of brain
damage (40). In this study, physiological parameters determined before and during ischemia (blood pressure, heart
rate, body temperature, and arterial blood gases) did not
differ among groups treated with vehicle or chloroquine (data not shown). Thus, the cerebroprotective effects of
chloroquine cannot be attributed to altering the peripheral
cardiovascular response to cerebral ischemia.
Previously, Zhang et al. reported that iNOS is upregulated 24-48 h after cerebral ischemia, and that delayed administration of aminoguanidine can prevent secondary
NO-mediated brain damage in a delayed therapeutic window (60). Our results here indicate that polyamine oxidase
activity is upregulated much earlier after cerebral ischemia
(within 2 h), and that early administration of aminoguanidine inhibits the generation of 3-aminopropanal. Although
the most direct interpretation of our results is that two
structurally distinct inhibitors of polyamine oxidase prevent ischemic damage by preventing the formation of 3-aminopropanal, we nonetheless performed a series of additional
experiments to exclude other possibilities.
First, we wished to exclude the unlikely possibility that
chloroquine protection occurred through an unanticipated
inhibition of iNOS. Addition of even suprapharmacological amounts of chloroquine (1 mM) failed to inhibit iNOS
activity measured in murine macrophage-like RAW264.7
cell lysates (control iNOS activity = 13,254 ± 250 DPM/ µg protein; versus chloroquine iNOS activity = 11,755 ± 883 DPM/µg protein; P >0.05). We also wished to exclude the unlikely possibility that aminoguanidine or chloroquine might protect cells by directly inactivating the cytotoxicity of 3-aminopropanal. Cell cytotoxicity was
measured in the presence of inhibitors, and we observed
that the LD50 for 3-aminopropanal after overnight incubation in HTB11 cells was similar whether or not aminoguanidine or chloroquine were added (data not shown).
We also wished to exclude the unlikely possibility that the
mechanism of aminoguanidine protection is not mediated
via altering the sensitivity of cells to the cytotoxicity of
glutamate. When aminoguanidine was added to primary
neuronal cultures treated with NMDA we observed no
significant attenuation of cytotoxicity (Table 3). We also
addressed whether 3-aminopropanal mediates cell death
through induction of iNOS activity. Addition of iNOS inhibitors (L-NMMA or aminoguanidine) to 3-aminopropanal-treated glial cells failed to attenuate the development of
TUNEL positivity as measured by FACS® (data not
shown).
Although we have excluded a number of plausible alternative mechanisms through which aminoguanidine might
protect against cerebral ischemia, it remains theoretically
possible that other nonspecific activities of chloroquine
might additionally contribute to the observed protection
against infarction (i.e., inhibition of free radical formation,
phospholipase activity, or protein synthesis). However, these
mechanisms are not supported by our direct observations
that (a) inhibiting polyamine oxidase activity reduces the
formation of cytotoxic concentrations of 3-aminopropanal; (b) 3-aminopropanal cytotoxicity cannot be blocked with
aminoguanidine or chloroquine; and (c) either chloroquine
or aminoguanidine prevent the brain damaging effects of
either intracortical spermine or ischemia.
| |
Discussion |
Four closely related lines of evidence therefore support
the role of 3-aminopropanal as a cytotoxic mediator of
brain damage in cerebral ischemia. First, cerebral ischemia
mediates an early induction of polyamine oxidase activity.
Second, the cytotoxic enzyme product 3-aminopropanal
accumulates during the early response to cerebral ischemia,
but is not produced in normally perfused controls. Third,
3-aminopropanal production in the ischemic brain increases before the onset of significant cellular degeneration, with tissue 3-aminopropanal levels rising further during the
period of progressive cell death. Fourth, 3-aminopropanal
is a potent cytotoxin that activates apoptosis via a caspase
1-dependent mechanism in glial cells and necrosis in neurons. Considered together, these data offer an explanation
for the correlation between brain levels of putrescine, a stable end-product of terminal polyamine oxidation, and infarct volume (5, 18, 61), since catabolism of spermine and
spermidine by polyamine oxidase produces both a stable,
nontoxic end product (putrescine) and a potent cytotoxin
(3-aminopropanal). The latter product mediates cell death,
and the former accumulates in correlation to the extent of
damage.
Previous observations suggest that polyamines can prevent
apoptosis in neuronal cultures (62, 63), or can amplify
glutamate-mediated cell cytotoxicity (14). Cell survival in
the ischemic zone is likely to be critically dependent upon
the balance between the direct effects of polyamines and the
cytotoxic effects of 3-aminopropanal. We have yet to address the contribution to brain damage of an alternative
pathway of polyamine catabolism, the acetylation pathway, but it is reasonable to speculate that this pathway could
provide an additional source of potentially toxic aldehyde
products, e.g., 3-acetamidopropanal (64, 65). There has
been some controversy as to whether both 3-aminopropanal and 3-acetamidopropanal can produce acrolein in vivo,
a known mediator of cytotoxicity and apoptosis (66, 67). It
is likely that several products of polyamine oxidation further augment the cytotoxicity of 3-aminopropanal. When
considered together, these observations add credence to the previously uninvestigated hypothesis that enhanced polyamine
oxidation during cerebral ischemia is deleterious.
Our results now suggest the following mechanism of
brain cell death during cerebral ischemia: dead and dying
cells in the densely h
ypoxic core release stores of intracellular spermine and
spermidine, which are catabolized by polyamine oxidase.
The resultant production of 3-aminopropanal causes apoptosis in surrounding glial cells, and necrosis of neurons,
which in turn release more spermine and spermidine as
substrate for polyamine oxidase. This cytotoxic mechanism
spreads to involve a larger volume of potentially viable cells
surrounding the ischemic core. It is likely that 3-aminopropanal is positioned proximally in the mediator cascade elicited by cerebral ischemia, which includes the excitatory
amino acids, activated oxygen species, nitric oxide, TNF,
IL-1, IL-6, and platelet-activating factor (48, 60, 68). It
is interesting to reconsider previous observations that expression of dominant negative mutants of IL-1
converting
enzyme (caspase 1) protects against the development of apoptosis during cerebral ischemia (58, 76). Based on our results, it is now plausible that inhibition of caspase 1 activity
prevents the damaging effects of 3-aminopropanal. Further,
we previously reported that TNF synthesis is upregulated during the first 12 h of brain ischemia, and that TNF participates in the mediation of brain damage (48). It will now
be interesting to explore further the influence of decreasing
brain spermine levels after ischemia, because spermine is a
direct inhibitor of TNF synthesis in human peripheral
blood mononuclear cells (77). We have recently found that
centrally administered 3-aminopropanal directly stimulates
intracerebral TNF synthesis (data not shown), indicating
that 3-aminopropanal may stimulate this component of the
ischemic cytotoxic cascade.
Polyamines and polyamine oxidase are ubiquitous in
mammalian tissues (78). This poses the intriguing possibility that the mechanism of polyamine oxidative tissue damage proposed here might be invoked in other ischemic
conditions, like myocardial infarction and tumor necrosis.
| |
Footnotes |
Address correspondence to Kevin J. Tracey, The Picower Institute for Medical Research, 350 Community
Dr., Manhasset, NY 11030. Phone: 516-562-9476; Fax: 516-562-2356; E-mail: ktracey{at}picower.edu
Received for publication 10 February 1998 and in revised form 6 May 1998.
Abbreviations used in this paper
EIMS, Electrospray ionization mass spectroscopy;
iNOS, inducible nitric oxide synthase;
NMDA, N-methyl-D-aspartate;
NMR, nuclear magnetic resonance;
PAO, polyamine oxidase;
PCA, perchloric acid;
TTC, 2,3,5-triphenyltetrazolium chloride;
TUNEL, Tdt-mediated dUTP-biotin nick-end labeling.
| |
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Abstract
Introduction
