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Animal Model |
From the Neuroinflammation Research Laboratories of the Department
of Psychiatry,
and the Brookdale Center for
Developmental and Molecular Biology,*
Mount Sinai School of
Medicine, New York, New York; the Department of
Inflammation,
Roche-Bioscience, Palo Alto,
California; and the Department of Neurology,§
Georgetown University School of Medicine, Washington, D.C.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recent studies of the effects of NSAIDs have revealed several possible health benefits of these COX inhibitors. Regular use of NSAIDs appears to reduce the risk of colorectal cancer6-9 and, more important for the current study, may slow the course of dementia in Alzheimer's disease (AD).10 Although it is unclear whether inflammation plays a role in the progression of AD, several lines of evidence indicate that regulation of COX-2 but not COX-1 expression may be involved in some of the neuropathology associated with AD.11
In rodents, the response to kainic acid (KA)-mediated excitotoxicity, which may be a model for aspects of the hippocampal and cortical neurodegeneration observed in AD, includes a marked up-regulation of COX-2, although COX-1 remains unaffected.12 The induction of COX-2 expression parallels the appearance of neuronal apoptotic features in cell types affected by KA, and excitotoxic neuronal death in vitro is accompanied by a selective elevation in COX-2 mRNA, indicating that COX-2 may be involved in pathways leading to neuronal death.12 This information may have important implications for AD in view of the evidence that excitotoxicity may contribute to the widespread pattern of neurodegeneration in the AD brain.13 Recently we14 and others15 found elevation of expression of COX-2, but not COX-1, in neurons of the AD brain.
For the present study, we prepared transgenic mice with neuronal overexpression of the human COX-2 gene, hCOX-2, under the regulation of the rat neuron-specific enolase (NSE) promoter. We used this model to explore the role of neuronal COX-2 expression in KA-mediated excitotoxicity. Moreover, because KA excitotoxicity has profound effects on gene regulation associated with long-lasting cellular responses in rodent brain,16 we examined the expression of representative immediate early genes (IEG) as an index of cellular response to synaptic activation. We report that overexpression of neuronal COX-2 potentiates the intensity and lethality of KA-mediated seizures in heterozygous hCOX-2 transgenic mice coincident with the potentiation of hippocampal c-fos and zif-268 mRNA expression. Finally, in vitro studies revealed that glutamate excitotoxicity was also potentiated in primary cortico-hippocampal neuron cultures derived from hCOX-2 transgenic mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A 1950-bp HindIII cDNA fragment containing the entire
coding region for hCOX-217
was inserted into a unique
HindIII site located within the second exon of the rat
neuron-specific enolase (rNSE) gene. This rNSE/hCOX-2 (NHC) transgene
contains approximately 2600 bp of rNSE promoter and 5' flanking region,
all of exon 1 (nontranslated sequence), intron 1, and 6 bp of exon 2
(without an ATG starting codon) from the rat NSE
gene.18
In addition, the NHC transgene utilizes a
polyadenylation signal from the SV40 late region (see Figure 1A
).
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A 6.95-kb SalI fragment containing the NHC transgene was purified from plasmid sequences by electrophoresis through low-melting-point agarose. The transgene was further purified by centrifugation through CsCl, followed by dialysis against 10 mmol/L Tris, pH 7.4, and 0.2 mmol/L EDTA. The concentration was adjusted to 5 Fg/ml for microinjection. The purified transgene fragment was used for microinjection of one-cell mouse eggs as previously described.19 C57Bl/6 x C3H (B6C3) F1 hybrid mice (Taconic Farms, Germantown, NY) were used as the source of fertilized eggs at the pronuclear stage.
Genotyping
Genomic DNA was isolated from tail skin as previously
described.19
Transgenic mice produced from injection of
the NHC SalI fragment were identified by dot blot
hybridization of tail skin DNA samples with a random-primed, 930-bp
EcoRI fragment, which contains the entire SV40 sequence
present in the NHC transgene (see Figure 1A
). The transgenic founder
mice identified in this fashion were mated with nontransgenic, B6C3
F1 hybrid mice to produce heterozygous transgenic offspring for
establishment of the individual lines, as well as analyses of
expression.
Northern Blot Analysis of hCOX-2 Transgene Expression
For the studies of hCOX-2 expression, half of the brain was frozen for extraction of total RNA20 and the other half was processed for in situ hybridization (ISH) assay as described below. For gel blot Northern hybridization assay, total RNA was resolved electrophoretically through 1.1% agarose, 2.2 mol/L formaldehyde gel as previously described.21 After transfer to nitrocellulose, expression of the hCOX-2 transgene was determined by hybridization with a random-primed, 1950-bp HindIII fragment containing the entire hCOX-2 cDNA fragment.
ISH Assay
Brain frozen coronal tissue sections (10 µm) were mounted on
polylysine-coated slides and stored at -70°C. For ISH, tissue
sections were postfixed in PBS containing 4% paraformaldehyde (30
minutes, room temperature), rinsed in phosphate-buffered saline (PBS),
and incubated in acetic anhydride (0.25% v/v) in 0.1 mol/L
triethanolamine, pH 8.0, for 10 minutes. After acetic anhydride
treatment, tissue sections were hybridized with
[35S]-cRNA probes (0.3 µg/ml, 2 x
109
dpm µg-1);
hCOX-2 (1.9 kb hCOX-2 derived from the NHC construct (Figure 1A)
,
c-fos, zif-268,22,23
and mouse (m) COX-2 and
mCOX-112
were transcribed from linearized cDNA probes.
After hybridization (3 hours, 50°C), stringency washes (0.1 x
SSC, 60°C), and dehydration, slides were exposed to X-ray film for 5
days for quantification. Slides were then exposed to NTB-2 emulsion
(Kodak, Rochester, NY) for approximately 12 days for microscopic
analysis of mRNA distribution; after emulsion development, tissue
sections were Nissl-counterstained. Hematoxylin and eosin
histochemistry was used to assess morphological features of apoptosis
on semiadjacent tissue sections.24
Film autoradiograms
were analyzed by computer-assisted video densitometry (Biometrics,
Nashville, TN).
Combined ISH and Immunocytochemistry (ICC)
The neuronal expression of hCOX-2 transgene was verified by combining ISH for hCOX-2 mRNA with ICC for NSE on the same brain tissue section as previously described.25 Following ISH, brain tissue sections were incubated for 30 minutes in PBS containing 10% methanol and 3% H2O2, rinsed, and permeabilized for 30 minutes at 4°C in PBS containing 0.2% Triton X-100, 0.2% normal goat serum, and 20 mg/ml L-lysine (Sigma, St. Louis, MO). Tissue sections were immunoreacted with a rabbit anti-human NSE antibody (1:1000, Dako, Carpinteria, CA). Immunoreactivities were visualized using the ABC method (Vector). Tissue samples were then dehydrated and coated with Kodak NTB2 emulsion as discussed above.
Western Blot Analysis
Brain samples were homogenized in an ice-cold buffer of Tris, pH 7.2, containing 1% NP-40, 0.5% sodium deoxycholate, 0.1% lauryl sulfate, 100 µg/ml phenylmethylsulfonyl fluorid, 3 µg/ml aprotinin, 3 µg/ml pepstatin A, 15 µg/ml propionyl-Leupeptin, and 600 µg/ml Perfabloc (Boehringer Mannheim, Indianapolis, IN), boiled, centrifuged, and electrophoresed on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as previously described.26 Proteins were transferred to nylon Transblot membrane (Biorad, Hercules, CA) and immunoreacted with a polyclonal goat anti-hCOX-2 antibody (1:500 dilution, 3 hours, room temperature) in Superblock blocking solution (Pierce, Rockford, IL). The anti-hCOX-2 antibody was raised against a synthetic peptide derived from the carboxyl region of hCOX-2 sequence (Cayman, Ann Arbor, MI). In control studies we found that anti hCOX-2 antibodies do not cross-react with COX-1 (not shown). Immunoreactivity was visualized autoradiographically using a chemiluminescence detection kit (SuperSignal, Pierce). ß-actin immunoreactivity (anti-ß-actin, Sigma 1:5000) controlled for selectivity of changes.
KA-Mediated Seizures
KA-induced seizures were induced by intraventricular (ICV) infusion of KA (Sigma) in 4-month-old heterozygous hCOX-2 - NHC32 transgenic mice and control wild-type (WT) littermates, as previously described.27 KA, 1.15 nmoles/2 µl, or saline was injected in the lateral ventricle of anesthetized mice (Metofan) using a 5-µl Hamilton syringe at a rate of 1µl/minute. Behavioral changes were assessed using a method described by Kondo et al,28 modified for seizure assessment in mice. Seizures were classified into two groups: mild seizures, in which mice showed staring posture, occasional wet-dog shakes, or automatisms without any myoclonic twitches; and severe seizures, in which mice showed myoclonic twitches of the forelimbs with standing posture or generalized tonic-clonic seizures with standing postures or generalized tonic-clonic seizures with falling or wild seizure jumps.
Because death occurred within 10 minutes after ICV KA infusion in the hCOX-2 transgenic mice (see Results), for comparative analysis of changes in gene expression or neuropathology, the control WT mice were sacrificed at the same time postlesioning. After decapitation, brains were rapidly removed, immediately frozen in methylbutane cooled to -25°C, and then stored at -70°C.
Primary Neuronal Cultures
Cortico-hippocampal primary neuron cultures derived from heterozygous hCOX-2 transgenic and WT mouse embryos (embryonic day 14–16) were prepared as previously described.26 Briefly, after brain dissection, mechanical trituration, and centrifugation, neurons were seeded onto poly-D-lysine-coated 96-well plates at a density of 2 x 105 cells per well. For ISH and/or ICC studies, primary neuron cultures were plated on glass chamber slides precoated with poly-D-lysine. For glutamate neurotoxicity, primary neuron cultures were aged 10 days and then treated with 50 µmol/L glutamate (Sigma) for 24 hours. Neuronal cultures were derived from each independent embryo; treatment and analysis were made in blind. Genotyping was used for identification of hCOX-2 transgenic or WT littermate cultures. Neuronal cultures were treated with glutamate for the indicated time, and parallel cultures derived from the same embryos were simultaneously treated with drug vehicle (control). Glutamate neurotoxicity was assessed by 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay;14 changes in MTT activity for each independent embryo culture was expressed as a percentage of its own control (identical drug vehicle-treated cultures).
Statistics
Analysis used statistical analysis software (CSS, Statsoft). P values <0.05 were considered to indicate statistically significant differences (t-test). KA-induced seizures studies used n = 6–8 mice per group and the studies were replicated in 3 independent experiments. Fisher's exact test was used for statistical analysis of mortality rate associated with KA-mediated seizures. ISH studies used n = 4–5 mice per group; changes in mRNA expression were computed from two tissue sections (bilaterally) encompassing the hippocampal formation. Data from in vitro studies were averaged from 3 to 4 independent studies.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Forty-four offspring were born from eggs microinjected with the NSE-hCOX-2 transgene and DNA samples prepared from tail skin biopsies were examined to identify founder transgenic mice. A dot blot containing tail DNA samples from all of the mice was hybridized with an SV40 probe (not shown), resulting in the identification of 5 founder transgenic mice (NHC1, NHC5, NHC30, NHC32, and NHC34) that carry the randomly integrated transgene.
Establishment of Transgenic Lines
All 5 founder transgenics were mated with WT mice to obtain transgenic offspring for establishment of the transgenic lines and for analysis of hCOX-2 expression. Offspring from these matings were genotyped by tail DNA dot blot analysis using an SV40 probe to detect the NHC transgene. Only the NHC5 and NHC32 founders transmitted the transgene to multiple offspring. In contrast, transmission from the NHC1 and NHC30 founders was much lower than expected for mendelian transmission of the transgene sequences (not shown).
Tissue-Specific Expression of hCOX-2 Transgene
Total RNA was prepared from various organs isolated from NHC5 and
NHC32 transgenic offspring, as well as a WT nontransgenic littermate.
Northern blot hybridization of total RNA indicated that, as expected,
NHC5 and NHC32 transgenic mice express the hCOX-2 transgene mRNA only
in the brain (Figure 1B)
. In addition, the hCOX-2 hybridization signal
indicates that the size of the major transcript in both lines is as
predicted for the NHC gene (approximately 2500 nucleotides). A second,
higher molecular weight transcript is also observed in both lines
(Figure 1B)
. Because this analysis was performed on total cellular RNA,
the most likely interpretation is that the larger RNA represents hnRNA
in which the 1200-bp NSE intron has not been spliced out, whereas the
smaller major transcript represents the mature mRNA.
Expression of hCOX-2 protein in the NHC32-hCOX-2 mouse brain was
assessed by Western blot immunoassay (Figure 1C)
. The anti-hCOX-2
antibody used in this assay recognized an approximately 70-kd protein
species as assessed by Western blot assay of whole NHC32-hCOX-2 brain
homogenate (Figure 1C
, top panel). ß-actin immunoreactivity
controlled for equal loading (Figure 1C
, bottom panel). This result
indicates that the NHC transgene is specifically expressed in the brain
and that the mRNA transcribed from the transgene is of sufficient
length to encode the entire hCOX-2 protein.
Cell Type Expression of hCOX-2
The regional distribution of hCOX-2 mRNA expression in the mouse
brain of the NHC32-hCOX-2 transgenic mice was assessed by ISH using a
[35S]-hCOX-2 antisense cRNA probe containing
the entire hCOX-2 cDNA fragment and visualized by X-ray film
autoradiography (Figure 2A)
. ISH for
hCOX-2 mRNA combined with NSE ICC on the same tissue section confirmed
the neuronal expression of hCOX-2 mRNA in the brain of hCOX-2
transgenic mice (parietal cortex, NHC32 line; Figure 2C
); no detectable
hCOX-2 signal was found in NSE-immunopositive neurons of WT mouse brain
(Figure 2D)
.
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revealed greater than eightfold hCOX-2 mRNA
expression in parietal cortex (CTX), the pyramidal CA3-CA1 (PYR) and
dentate gyrus (DG) granule neuron layers of the hippocampal formation,
thalamus (THL), cerebellar Purkinje cell layer (PRK) of NHC32-hCOX2
transgenic mice (Figure 2A)
, relative to hCOX-2 hybridization signal in
the brain of WT control littermates (Figure 2B)
. No detectable
expression of hCOX-2 was found in corpus callosum white matter (Figure 2A
; n = 6–8 per group, 4-month-old mice;
P < 0.001). The intensity of hCOX-2 mRNA hybridization
signal in the brain of WT control littermates (Figure 2B)
approximated the intensity of the [35S]-hCOX-2
sense-strand hybridization signal on brain tissue sections of
NHC32-hCOX-2 transgenics and WT controls (not shown). hCOX-2 mRNA Expression Relative to Endogenous COX-2 mRNA and Expression of Endogenous COX-1 mRNA
Because of the constitutive expression of COX-2 mRNA in rodent
brain, we examined the relative levels of hCOX-2 mRNA expression over
endogenous murine (m) COX-2 mRNA; mCOX-2 mRNA expression was assessed
by ISH using a murine [35S]-COX-2 antisense
cRNA probe. Quantitative ISH revealed greater than three- to fourfold
hCOX-2 mRNA expression in the DG granule and pyramidal (CA3-CA1) neuron
layers of the hippocampal formation, parietal cortex, and thalamus over
the mCOX-2 mRNA hybridization signal (Figure 3A)
. In control studies, we found that
mCOX-2 mRNA expression did not differ in control WT littermates and
NHC32-hCOX-2 transgenics (not shown).
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. Neuronal Overexpression of hCOX-2 Potentiates KA-Induced Seizures in NHC32-hCOX-2 Transgenic Mice
Neuronal hCOX-2 overexpression potentiated seizures and increased
mortality during response to ICV (lateral ventricle) KA infusion. There
was marked increase in the intensity of KA-mediated seizures in NHC32
hCOX-2 transgenic mice compared to WT control littermates (Table 1)
. Vigorous status epilepticus, leading
to accelerated death within 10 minutes after ICV infusion of 1.25
nmoles of KA, occurred in each of the hCOX-2 transgenic mice. Mild
seizures but no death occurred in WT control littermates within 10
minutes after ICV infusion of an equal dose of KA
(n = 6–8 per group, P < 0.004,
Fisher's exact test; Table 1
). For further comparative analysis of
changes in gene expression or neuropathology, the control WT mice were
sacrificed within 10 minutes after lesioning. No detectable seizures
were found in either hCOX-2 transgenics or WT controls following ICV
infusion of an equal volume of vehicle (2 µl, saline).
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. In pilot titration studies, potentiation of KA-mediated seizures in NHC32-hCOX-2 transgenic mice was found after ICV infusion of KA at doses between 0.75 and 3 nmoles. Mild or no detectable seizures were found in both hCOX-2 transgenics and WT control littermates after ICV infusion of KA at doses below 0.75 nmoles (not shown). Similar potentiation of KA-induced seizures occurred in NHC5-hCOX-2 transgenic mice (data not shown).
Potentiation of KA-Mediated Induction of c-fos and zif-268 mRNA Expression in NHC32-hCOX-2 Transgenic Mice
In NHC32-hCOX-2 transgenic mice, generalized status epilepticus,
characterized by intense tonic-clonic seizures and death by 10 minutes
after KA (1.25 nmoles), coincided with potentiation of c-fos
mRNA expression in neuronal layers the hippocampal formation,
and parietal cortex relative to KA-injected control WT littermates.
Quantitative ISH autoradiography revealed greater than three- to
fourfold (n = 4, P < 0.05)
potentiation of c-fos mRNA expression in the granule neuron
layer of the DG ipsilateral to the lesion side compared to WT controls
(Figure 4, A and B
; Figure 5A
, left panel). Similarly, there was
>1.5-fold potentiation (P < 0.05) of
c-fos mRNA expression in the CA3-CA1 pyramidal layer of the
hippocampal formation (Figure 4, A and B
; Figure 5B
, left panel) and in
the parietal cortex (Figure 4, A and B
Figure 5C
, left panel),
ipsilateral to the lesion side, compared to WT controls.
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; Figure 5A
, right
panel). On the contrary, no detectable potentiation of
zif-268 mRNA expression was found in the CA3-CA1 pyramidal
neuron of the hippocampal formation, compared to WT control littermates
(Figure 4, C and D
; Figure 5B
, right panel), as assessed by ISH assay
on tissue sections adjacent to those used for c-fos mRNA. No
detectable potentiation of zif-268 mRNA hybridization signal
was found in parietal cortex of hCOX-2-NHC32 transgenic mice compared
to control WT littermates (Figure 4, C and D
; Figure 5C
, right panel). In response to ICV infusion of drug vehicle (saline) in NHC-32-hCOX-2 transgenic mice, we found no detectable potentiation of hippocampal and cortical c-fos and zif-268 mRNA hybridization signal relative to control WT littermates (not shown). Moreover, hematoxylin and eosin histochemistry was used to identify neurons with pyknotic condensed nuclei in neuronal layer of the hippocampal formation and cerebral cortex. No overt sign of neurodegeneration was found in either hCOX-2 transgenics or WT control littermates within 10 minutes after ICV KA (not shown).
Neuronal hCOX-2 Overexpression Potentiates Glutamate-Mediated Excitotoxicity in Vitro
Ten-day-old primary cortico-hippocampal neuron cultures generated
from control WT embryos resulted in 31 ± 2% impairment of redox
activity after exposure to glutamate (50 µmol/L, 24 hours) relative
to vehicle-treated control cultures, as assessed by MTT assay. However,
glutamate treatment in parallel cortico-hippocampal neuronal cultures
generated from hCOX-2 (NHC32) transgenic embryos resulted in 46 ±
3% impairment of redox impairment relative to vehicle-treated control
cultures. Thus, overexpression of COX-2 in cortico-hippocampal neurons
potentiated glutamate-mediated redox impairment by approximately 50%
relative to WT controls (n = 5–6 independent
cultures per group, P < 0.004; Figure 6A
)
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To generate our transgenic mice, a modified rat NSE promoter was used to regulate expression of hCOX-2 in neurons. The rat NSE promoter has been successfully used by multiple groups to selectively direct the overexpression of heterologous proteins in neurons of transgenic mice.18,29-37 Previous studies reported the use of a rat NSE promoter consisting only of a 1.8-kb sequence located 5' end to the first exon; this 1.8-kb promoter fragment has been found to regulate selective neuron-specific expression, but also with some minor expression in other tissues.29-35
In this study, in addition to a longer segment of the promoter and 5'
flanking region (2.6 kb), the rat NSE regulatory fragment also included
exon 1, intron 1, and 6 bp of exon 2 (Figure 1A)
. Intron 1 contains a
putative neuronal regulatory element,38
and this larger
rat NSE promoter construct was previously used to direct specific
expression of heterologous proteins to neurons in transgenic
mice.18,36,37
The results presented in this study confirm
the cell type specificity determined by the larger NSE promoter, which
includes intron 1; transgene expression in the NHC32-hCOX-2 line
examined was restricted to the brain, whereas ISH revealed that the
detectable brain expression is essentially only neuronal.
One possible mechanism by which neuronal COX-2 overexpression may influence the neuronal response to KA-induced seizures and glutamate excitotoxicity in vitro may involve the control of transcription factor DNA-binding activities. We found that neuronal hCOX-2 overexpression in NHC32-hCOX-2 transgenic mice potentiate the expression of the IEG transcription factors c-fos and zif-268 in subsets of hippocampal neurons in response to KA, compared to WT littermates. This regulation of gene expression might occur through paracrine and autocrine signaling mediated by prostaglandins (PGs) through PG receptors, which are members of the G-protein-coupled family of receptors. In non-neuronal cells, the signals depend on the specific agonist and the receptor, but include changes in cyclic AMP, turnover of phosphatidylinositol, and activation of both protein kinase C (PKC) and mitogen-activated protein (MAP) kinase.39 Thus, it is possible that overexpression of hCOX-2 mRNA in neurons may increase sensitivity or lower the threshold level of excitation to KA through changes in gene expression. We also note that there is evidence suggesting a direct link between COX-2 and c-fos and zif-268 expression in excitotoxicity. All three are rapidly transcribed during the response to KA-induced seizures40 and inhibition of intracellular platelet activating factor (PAF),41 whereas limiting KA-mediated induction of zif-268 expression also attenuates COX-2 induction.40
The lack of potentiation of zif-268 mRNA expression in the hippocampal CA3-CA1 pyramidal layers and parietal cortex of hCOX-2 transgenic mice versus WT littermates in response to KA-induced seizures is puzzling; it may indicate a regional variation in the time course of changes in gene expression in this model system. In support of this hypothesis we note that a common feature of these responses to excitotoxic insult is the concerted induction of IEG with a distinct time course of activation.42,43
COX-1 and COX-2 are key enzymes in the generation of prostanoids including PGs. Both enzymes catalyze two functional reactions: a cyclooxygenase reaction to form the endohydroperoxide PGG2, and an iron heme-mediated peroxidase activity to reduce PGG2 to PGH2.11 PGH2 is the precursor to all other PG. The primary source of arachidonate for COX is thought to be membrane phospholipid; arachidonic acid (AA) is released from esterified phospholipid by the action of phospholipase A2. Substances that increase intracellular Ca2+, such as glutamate,44 also activate phospholipase A245 to make available the substrate AA for COX reactions. Thus, agents increasing neuronal intracellular free Ca2+ in presence of high levels of COX-2 may lead to higher levels of prostanoid synthesis. Because several of the prostanoid products of AA metabolism generated by COX may also control the activity of excitatory and inhibitory amino acid pathways,46 it is plausible that overexpression of neuronal COX-2 might also modulate glutamate mediated responses through excitotoxic mechanisms. In support of this hypothesis there is evidence that (N-methyl-D-aspartate (NMDA)-mediated neuronal death is diminished in a dose-dependent manner by COX-2 inhibitors in primary neuronal cultures.47 However, we note that there is also evidence that PGs may protect from glutamate toxicity in cortical neurons under certain conditions.48
Free radicals are intermediate products in COX-mediated PG synthesis, and lipid peroxides influence the activity of COX.49 Moreover, the role of free radicals in NMDA receptor-mediated glutamate excitotoxicity is well established in various experimental conditions.50,51 Thus, given that oxidative stress seems to be involved in mechanisms of excitotoxicity,52,53 it is not unexpected that neuronal COX-2 activity may contribute to neurodegeneration via oxidative mechanisms11 ; this COX-2 influence may not necessarily be closely linked to PG production.54 Oxidative stress resulting from COX-2 activity may have important implications for neurodegenerative diseases such as AD, in which an elevated glutamatergic tone, also defined as disinhibition syndrome, may be responsible for a widespread pattern of neurodegeneration.13 The evidence of potentiation of glutamate-mediated redox impairment in primary neuron cultures from the hCOX-2 transgenic mice suggests that COX-2 overexpression indeed may be involved in mechanisms of oxidative stress in vitro. Future studies will clarify the role of neuronal COX-2 expression in neurodegenerative pathways such as caspase activation and apoptosis in vitro and in vivo.
In conclusion, this study demonstrated a possible causative role for neuronal COX-2 expression in excitotoxicity. This model system will allow a systematic examination of the role of COX-2 in mechanisms of neurodegeneration that involve excitatory amino acid pathways. Ongoing laboratory studies exploring the role of selective and nonselective COX-2 inhibitors on KA-mediated responses in NHC32-hCOX-2 transgenics will clarify the causative role of COX-2 in mechanisms of excitotoxicity.
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Footnotes |
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Supported by National Institute on Aging grants AG13799, AG14239, and AG14766 to G. M. P.
Accepted for publication May 22, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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H. C. Salzberg-Brenhouse, E.-Y. Chen, D. F. Emerich, S. Baldwin, K. Hogeland, S. Ranelli, D. Lafreniere, B. Perdomo, L. Novak, T. Kladis, et al. Inhibitors of Cyclooxygenase-2, but Not Cyclooxygenase-1 Provide Structural and Functional Protection against Quinolinic Acid-Induced Neurodegeneration J. Pharmacol. Exp. Ther., July 1, 2003; 306(1): 218 - 228. [Abstract] [Full Text] [PDF]
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 L. Acarin, B. Gonzalez, and B. Castellano Decrease of Proinflammatory Molecules Correlates With Neuroprotective Effect of the Fluorinated Salicylate Triflusal After Postnatal Excitotoxic Damage Stroke, October 1, 2002; 33(10): 2499 - 2505. [Abstract] [Full Text] [PDF]
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P. Ciceri, Y. Zhang, A. F. Shaffer, K. M. Leahy, M. B. Woerner, W. G. Smith, K. Seibert, and P. C. Isakson Pharmacology of Celecoxib in Rat Brain after Kainate Administration J. Pharmacol. Exp. Ther., September 1, 2002; 302(3): 846 - 852. [Abstract] [Full Text] [PDF]
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 A. Basu, J. K. Krady, M. O'Malley, S. D. Styren, S. T. DeKosky, and S. W. Levison The Type 1 Interleukin-1 Receptor Is Essential for the Efficient Activation of Microglia and the Induction of Multiple Proinflammatory Mediators in Response to Brain Injury J. Neurosci., July 15, 2002; 22(14): 6071 - 6082. [Abstract] [Full Text] [PDF]
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M. Mirjany, L. Ho, and G. M. Pasinetti Role of Cyclooxygenase-2 in Neuronal Cell Cycle Activity and Glutamate-Mediated Excitotoxicity J. Pharmacol. Exp. Ther., May 1, 2002; 301(2): 494 - 500. [Abstract] [Full Text] [PDF]
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K. I. Andreasson, A. Savonenko, S. Vidensky, J. J. Goellner, Y. Zhang, A. Shaffer, W. E. Kaufmann, P. F. Worley, P. Isakson, and A. L. Markowska Age-Dependent Cognitive Deficits and Neuronal Apoptosis in Cyclooxygenase-2 Transgenic Mice J. Neurosci., October 15, 2001; 21(20): 8198 - 8209. [Abstract] [Full Text] [PDF]
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L. Ho, D. Purohit, V. Haroutunian, J. D. Luterman, F. Willis, J. Naslund, J. D. Buxbaum, R. C. Mohs, P. S. Aisen, and G. M. Pasinetti Neuronal Cyclooxygenase 2 Expression in the Hippocampal Formation as a Function of the Clinical Progression of Alzheimer Disease Arch Neurol, March 1, 2001; 58(3): 487 - 492. [Abstract] [Full Text] [PDF]
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 C. Iadecola, K. Niwa, S. Nogawa, X. Zhao, M. Nagayama, E. Araki, S. Morham, and M. E. Ross Reduced susceptibility to ischemic brain injury and N-methyl-D-aspartate-mediated neurotoxicity in cyclooxygenase-2-deficient mice PNAS, January 30, 2001; 98(3): 1294 - 1299. [Abstract] [Full Text] [PDF]
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 C. Petersen, S. Petersen, L. Milas, F. F. Lang, and P. J. Tofilon Enhancement of Intrinsic Tumor Cell Radiosensitivity Induced by a Selective Cyclooxygenase-2 Inhibitor Clin. Cancer Res., June 1, 2000; 6(6): 2513 - 2520. [Abstract] [Full Text]
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