Increased Expression of the Pro-Inflammatory Enzyme Cyclooxygenase-2 in
Amyotrophic Lateral SclerosisGabrielle Almer, MD,1 Christelle Guegan, PhD,1 Peter Teismann, PhD,1 Ali Naini, PhD,1
Gorazd Rosoklija, MD, PhD,2 Arthur P. Hays, MD,2 Caiping Chen, MD,1 and Serge Przedborski, MD, PhD1,2
Mutations in the copper/zinc superoxide dismutase (mSOD1) gene are associated with a familial form of amyotrophiclateral sclerosis (ALS), and their expression in transgenic mice produces an ALS-like syndrome. Recent observationssuggest a role for inflammatory-related events in the progression and propagation of the neurodegenerative process inALS. Consistent with this view, the present study demonstrates that, during the course of the disease, the expression ofcyclooxygenase type 2 (Cox-2), a key enzyme in the synthesis of prostanoids, which are potent mediators of inflamma-tion, is dramatically increased. In both early symptomatic and end-stage transgenic mSOD1 mice, neurons and, to alesser extent, glial cells in the anterior horn of the spinal cord exhibit robust Cox-2 immunoreactivity. Cox-2 mRNA andprotein levels and catalytic activity are also significantly increased in the spinal cord of the transgenic mSOD1 mice. Thetime course of the spinal cord Cox-2 upregulation parallels that of motor neuronal loss in transgenic mSOD1 mice. Wealso show that Cox-2 activity is dramatically increased in postmortem spinal cord samples from sporadic ALS patients.We speculate that Cox-2 upregulation, through its pivotal role in inflammation, is instrumental in the ALS neurode-generative process and that Cox-2 inhibition may be a valuable therapeutic avenue for the treatment of ALS.
Ann Neurol 2001;49:176–185
Amyotrophic lateral sclerosis (ALS) is the most frequentneuromuscular disorder in adults and is characterizedmainly by progressive muscle wasting and weakness.1
Mutations in the gene for the free-radical–scavengingenzyme copper/zinc superoxide dismutase (SOD1) areassociated with a familial form of ALS (FALS) 2 that isclinically and pathologically indistinguishable from themost common sporadic form of this fatal neurodegen-erative disorder. Moreover, transgenic mice that expressmutant SOD1 (mSOD1) develop an adult-onset para-lytic condition that reproduces the clinical and patholog-ical hallmarks of ALS.3–5 We6 and others7,8 have foundthat the level of mSOD1 expression markedly modulatesthe age of onset of symptoms but has no or minimaleffect on the type of symptoms or the rate of progressionof the disease. This suggests that, while mSOD1 is apivotal factor in the initiation of motor neuron disease,additional factors contribute to the propagation of theneurodegenerative process. The elucidation of such fac-tors is of major importance because it may open newtherapeutic avenues aimed at stopping or slowing theprogression of ALS.
There is mounting evidence that inflammatory-related events could be among the factors that promote
progression and propagation of motor neuron degener-ation in ALS. For instance, in addition to the dramaticloss of motor neurons, which predominates in the an-terior horn, the gray matter of the spinal cord is alsothe site of a robust glial reaction in both humans andtransgenic mice.9–13 Although gliosis may in some in-stances be associated with beneficial effects, many moresituations come to mind in which gliosis may be dele-terious.14,15 Consistent with this view, we have dem-onstrated that inducible nitric oxide synthase (iNOS) isupregulated in activated microglia in the spinal cord oftransgenic mSOD1 mice,13 whereby surrounding cells,especially neurons, can be flooded with high amountsof iNOS-derived NO and other reactive oxygen species(ROS).16 Factors such as the pro-inflammatory cyto-kine interleukin-6 (IL-6) and interleukin-1b (IL-1b)may also mediate some of the glial-related deleteriouseffects. IL-6 and IL-1b levels are elevated in the cere-brospinal fluid17 and spinal cord, respectively, of ALSpatients.18 IL-1b and tumor necrosis factor-a levels arealso increased in the spinal cord of experimental mod-els of ALS,18,19 and inhibition of IL-1b activation isassociated with clinical benefits in transgenic mSOD1mice.20
From the Departments of 1Neurology and 2Pathology, ColumbiaUniversity, New York, NY.
Received Apr 19, 2000, and in revised form Jul 18. Accepted forpublication Sep 6, 2000.
Address correspondence to Dr Przedborski, Departments of Neurol-ogy and Pathology, BB-307, Columbia University, 650 West 168thStreet, New York, NY 10032. E-mail: [email protected]
Expression of cyclooxygenase type 2 (Cox-2) hasemerged as an important determinant of cytotoxicityassociated with inflammation.21,22 As reviewed byO’Banion,22 Cyclooxygenase is a rate-limiting enzymein prostanoid synthesis, and, to date, two distinct Coxisoenzymes have been purified and molecularly cloned:cyclooxygenase type 1 (Cox-1) and Cox-2. Cox-1 isconstitutively expressed in many cells of the brain,where it produces prostanoids that are involved inphysiological functions. In the normal brain, Cox-2 issignificantly expressed only in specific subsets of fore-brain neurons that are primarily glutamatergic in na-ture,23 which suggests a role for Cox-2 in the postsyn-aptic signaling of excitatory neurons. However, underpathological conditions, especially associated with aninflammatory reaction, Cox-2 expression in the centralnervous system (CNS) can increase significantly, asdoes the level of its products, which are responsible formany of the cytotoxic effects of inflammation. Thus,our success in unraveling the role of inflammation inALS as well as in developing neuroprotective therapiesthat target inflammation is contingent on our elucida-tion of which pathways contribute to the inflammatoryreaction seen in the spinal cord of ALS. In the presentstudy, we demonstrate that the expression of Cox-2,but not of Cox-1, and the level of prostaglandin E2
(PGE2), one of the main products of Cox-2 in medi-ating inflammatory effects,22 are markedly increased inthe spinal cord of transgenic mSOD1 mice. We alsoshow that the level of PGE2 is significantly elevated inhuman postmortem spinal cord samples from sporadicALS. Thus, our results indicate that Cox-2 is upregu-lated in both the transgenic mSOD1 mouse model ofALS and in human ALS, and, given its pivotal role inprostanoid-mediated inflammatory effects, it may bespeculated that Cox-2 appears to be a valuable thera-peutic target for ALS.
Materials and MethodsAnimalsTwo lines of hemizygote transgenic mice were used: (1) lineG1H, which carries the point mutation Gly 3 Ala at codon93 of the human SOD1 gene and expresses about 18 copiesof human mSOD1 gene,3 and (2) line N1029, which carriesmore than 10 copies of human wild-type SOD1 gene.3 Onpostnatal day 14, genotyping was performed as previouslyreported.6 After being genotyped, animals were divided intotransgenic mSOD1 mice, transgenic wild-type SOD1 mice,and nontransgenic mice (i.e., nontransgenic littermates fromtransgenic mSOD1 mice). All studies were performed onasymptomatic ('6-week-old), early symptomatic (117 6 6days [mean 6 SD]), and end-stage (165 6 2 days of age)mice as defined previously.24 The nontransgenic littermateswere sacrificed at 110 and 168 days of age while transgenicwild-type SOD1 mice at 168 days of age only.
Total RNA Preparation and Reverse TranscriptasePolymerase Chain Reaction AnalysisTotal RNA from both spinal cord and cerebellum of the dif-ferent groups of mice was prepared using the RNeasy kit(QIAGEN, Inc, Valencia, CA) according to the manufactur-er’s instructions. First, strand cDNA was synthesized usingSuperScript II RNase H reverse transcriptase (GIBCO BRL,Gaithersburg, MD) according to the manufacturer’s instruc-tions. Then, 1 ml of cDNA template was amplified by poly-merase chain reaction in a 20 ml total reaction volume con-taining 18 ml of Supermix (GIBCO BRL) and 4 to 10 pmolof each specific primer. The Cox-2 primer sequences were59-GAAGGGACACCCTTTCACAT-39 (forward) and 59-ACACTCTATCACTGGCATCC-39 (reverse). The Cox-1primer sequences were 59-GTGCAGCACTTGAGTGGC-TATT-39 (forward) and 59-GAGCCCCCATCTCTATC-ATACTT-39 (reverse). As internal control, b-actin cDNA wasco-amplified using primer sequences 59-CTTTGATGTCAC-GCACGATTTC-39 (forward) and 59-GGGCCGCTCTAG-GCACCAA-39 (reverse). All primers were mouse specific andintron spanning and were designed based on reported se-quences available from the GENEBANK database. The PCRproducts were all of the expected size, and their proper iden-tities were confirmed by automatic sequencing using an ABIPrism-310 Genetic Analyzer (Perkin Elmer, Norwalk, CT).For Cox-2, the PCR thermal program was 94°C for 3.5 min-utes, 32 cycles of 94°C (1 minute), 58°C for 1 minute, 72°Cfor 1 minute, and 72°C for 8 minutes. For Cox-1, the thermalprogram was 95°C for 7 minutes, 28 cycles of 95°C (1minute), 52°C for 1 minute, 72°C for 2 minutes, and 72°Cfor 7 minutes. For b-actin, the program was 94°C for 5 min-utes, 28 cycles of 94°C (45 seconds), 60°C for 45 seconds,and 72°C for 1 minute, and 72°C for 7 minutes. Under thesePCR conditions, each PCR amplification was within the ex-ponential amplification range for quantification of eachmRNA. To control for the specificity of the PCR reaction, wealso performed the reaction using normal mouse genomicDNA and mRNA subjected to cDNA synthesis without re-verse transcriptase. Neither reaction produced Cox-2, Cox-1,or b-actin PCR products. After amplification, the productswere separated on a 1.5% agarose gel containing 0.03%ethidium bromide. Bands were then visualized under ultravi-olet illumination, and gels were photographed with Polaroid-665 positive-negative films. Negatives were scanned on anHP-4C Scanjet, and bands were quantified by using NIH-Image 1.62 software.
Western Blot AnalysisTotal proteins from spinal cord and cerebellum were isolatedin 10 volumes [weight per volume (w/v)] of 50 mM Tris-HCl, pH 7.0; 150 mM NaCl; 5 mM ethylenediaminetet-raacetic acid (EDTA); 1% sodium dodecyl sulfate (SDS); 1%NP-40; and protease inhibitors (Mini Cocktail, Roche Diag-nostics, Indianapolis, IN). Protein concentration was deter-mined using a BCA kit (Pierce, Rockford, IL). After boilingin 13 Laemmli’s buffer, 25 to 70 mg of protein was loadedonto 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a nitrocellulose membrane, andblocked with 5% nonfat dry milk in 13 Tris-buffered salinesolution (TBS) containing 0.05% Tween-20 for 1 hour. In-
Almer et al: Cyclooxygenase-2 Induction in ALS 177
cubation with one of the following primary antibodies wasperformed at room temperature for 2 hours: 1:9,000 goatanti–Cox-1 (Santa Cruz Biotechnology, Santa Cruz, CA),1:2,000 rabbit anti–Cox-2 (Santa Cruz Biotechnology), or1:15,000 mouse anti–b-actin (Sigma, St Louis, MO). Incu-bation with either a secondary anti-goat–, anti-rabbit–, oranti-mouse–conjugated horseradish peroxidase antibody wasperformed at room temperature for 1 hour. After washing in13TBS and 0.05% Tween-20, blots were exposed to SuperSignal Ultra chemiluminescence (Pierce) and Kodak Be-taMax film. Then, films (Kodak BioMax MS) were scannedon a HP-4C Scanjet, and bands were quantified by using theNIH-Image 1.62 software.
Immunohistochemical StudiesAll mice were anesthetized (pentobarbital, 35 mg/kg intra-peritoneally) and perfused intracardially with 25 ml of nor-mal saline solution followed by 75 ml of 4% (w/v) parafor-maldehyde (PF) in 0.1 M phosphate buffer (PB) (4% PF/PB, pH 7.1). Spinal cord and cerebellum were then dissectedout on ice, postfixed by immersion in 4% PF/PB (for 4hours at 4°C), cryoprotected in 30% (w/v) sucrose in PB,and frozen by immersion in isopentane cooled on dry ice.Frozen spinal cord and cerebellum samples were cut (30 mm)in a cryostat. Twenty to 40 serial sections from cervical (C7)and lumbar (L3) levels were collected from the spinal cord,and a few transversal sections were collected from the cere-bellum. Spinal segments were identified by location of spinalroots and by the characteristic morphologic features of thespinal cord. For all immunostainings, sections were collectedfree floating in ice-cold PB and then successively rinsed (3times for 5 minutes each) in 0.1 M Tris-HCl (pH 7.4)buffer containing 9 gm/L NaCl (TBS), incubated in 5%normal serum (NS) in TBS containing 0.1% Triton-X100(TBS-Tx; for 60 minutes, 25°C), and incubated with one ofthe primary antibodies: Cox-2 (1:500, polyclonal; CaymanChemical, Ann Arbor, MI), glial fibrillary acidic protein(GFAP, 1:500, monoclonal; Boehringer Mannheim, India-napolis, IN), specific nuclear protein (NeuN, 1:5,000,monoclonal; Chemicon, Temecula, CA), or macrophage anti-gen complex-1 (MAC-1, 1:1,000, monoclonal; Serotec, Ra-leigh, NC). After three 5-minute rinses in TBS, sections weresuccessively incubated for 1 hour at 25°C in biotinylated-conjugated polyclonal anti-rabbit or anti-mouse antibody(1:200), rinsed three times for 5 minutes each in TBS, incu-bated in horseradish peroxidase–conjugated avidin-biotin com-plex (Vector, Burlingame, CA), rinsed three times for 5 min-utes each in TBS, and incubated in diaminobenzidine-H2O2.Then, all sections were mounted on glass slides and counter-stained with thionin prior to being dehydrated in alcohols,cleared in xylene, and coverslipped. On selected sections, dou-ble immunohistochemistry was performed as described previ-ously,25 using diaminobenzidine with and without nickel in-tensification and VECTORt VIP substrate (Vector).
Prostaglandin E2 Tissue ContentThe content of PGE2 in tissue extracts from mouse and hu-man samples was measured by an immunoassay kit (CaymanChemical) according to the manufacturer’s instructions. Fro-zen tissue samples from spinal cord and cerebellum were ho-
mogenized in 10 volumes (w/v) of ice-cold 0.1 M phosphatebuffer (pH 7.4) containing 1 mM EDTA and 100 mg/mlindomethacin (Sigma, St Louis, MO). After homogenizationand prior to centrifugation (at 16,500 3 g for 25 minutes at4°C), homogenates were spiked with 10,000 dpm of [3H]-8-iso-prostaglandin F2a (PGF2a, specific activity 5 Ci/mmol;Cayman Chemical) as an internal control. The resulting su-pernatants were collected, acidified with formic acid to pH4, and recentrifuged (at 16,500 3 g for 15 minutes at 4°C)to eliminate any precipitate formed after acidification. Sam-ples were then purified by applying aliquots of 500 ml ofeach clear supernatant to preconditioned C18 column (Sep-Pak Light; Waters, Milford, MA). After rinses with 5 ml wa-ter and 5 ml hexane, PGE2 was eluted with 5 ml ethyl ace-tate containing 1% methanol. Then, 500 ml of each eluatewas taken for counting radioactivity emitted by the residualradiolabeled PGF2a. For each sample, the difference in dis-integrations per minute (dpm) before and after purificationwas used to calculate the efficiency of purification. Then, thesolvent was evaporated from the remaining eluate under ni-trogen, and PGE2 was reconstituted in 450 ml of reactionbuffer (Cayman Chemical). After a 1:10 dilution in reactionbuffer, PGE2 was measured according to the 4°C methoddescribed in the manufacturer’s instructions. The tissue con-tents of PGE2 were determined using a standard curve madeof different known amounts of PGE2 standard (CaymanChemical) and were expressed in picograms per milligram oftissue after correction for the efficiency of the purificationaccording to the manufacturer’s instructions.
Human Postmortem Samples and Cox-2 ActivityThe content of PGE2 was also assessed in human samplesfrom sporadic ALS patients. The tissues were processed asdescribed above for mice, and the assay was performed iden-tically because no adaptation was necessary.
The spinal cord and cerebellar cortex samples used origi-nated from 5 patients with ALS and 8 neurological patientswith no spinal cord or cerebellar pathologic conditions ob-tained from the Brain Bank of the Department of Pathologyat Columbia University. In the ALS group, the cause ofdeath was pneumonia (n 5 1) and respiratory failure (n 54). In the control group, the cause of death was respiratoryfailure (n 5 5), stroke (n 5 1), subarachnoid hemorrhage(n 5 1), and diabetic ketoacidosis (n 5 1). At autopsy,brains and spinal cords were removed and processed forbanking as previously described.26 Half of the brain andblocks from various levels of the spinal cord were placed in10% buffered formalin and were subjected to neuropatho-logical examination. The other half of the brain and the restof the spinal cord blocks were immediately frozen on dry iceand stored at 280°C until used. Prior to assay, small frozensamples ('100 mg) of the anterior horn of the cervical spi-nal cord and of cerebellar cortex were cut from the frozensamples. There was no significant difference between thetime of death to autopsy or between the time of autopsy toassay for the ALS and control groups (Table). The clinicaldiagnosis of ALS was confirmed pathologically in all 5 ALSpatients. In these spinal cords, dramatic neuronal loss wasobserved in the anterior horn, with mild to moderate gliosis,whereas no remarkable pathological changes were noted in
178 Annals of Neurology Vol 49 No 2 February 2001
the cerebellar cortex. In all control brains, no remarkablepathological changes were noted either in spinal cord or incerebellum. For the ALS patients, the mean age (6 standarddeviation [SD]) of onset was 63.4 6 14.0 years, with themean duration of disease being 3.0 6 1.0 years and rangingfrom 2 to 4 years. None of the ALS patients had a familyhistory of the illness. None of the ALS patients or controlsubjects was treated by chronic administration of anti-inflammatory medication or Cox inhibitors.
StatisticsFor each experiment, at least 5 mice per group were studied.All values are expressed as means 6 the standard error of themean (SEM) unless stated otherwise. Differences betweenmeans were analyzed using a two-tail Student t test. Differ-ences among means were analyzed using one-way ANOVAwith disease stage as the independent factor. When ANOVAshowed significant differences, pair-wise comparisons be-tween means were assessed by Newman-Keuls post hoc test-ing. Prior to performing Student’s t test or ANOVA, datasets were tested for their equality of variance and their nor-mal distribution. If either or both of these conditions wereviolated, the appropriate nonparametric test was used. In allanalyses, the null hypothesis was rejected at the 0.05 level.All statistical analyses were performed using SigmaStat forWindows (version 2.0, Jandel Corporation, San Rafael, CA).
ResultsProgression of the DiseaseConsistent with our previous studies,6 the first behav-ioral abnormalities in the animals, which occurred byapproximately 14 weeks of age, were a fine tremor inand posturing of at least one limb when the animal washeld in the air by the tail. Then, weakness and atrophyprogressed, with the end stage occurring around 8weeks from the time of symptom onset. At that point,the animals were approximately 22 weeks old and wereso severely paralyzed that they could not eat, drink, ormove freely, and thus were killed.
Upregulation of Cox-2 mRNA in TransgenicmSOD1 MiceTo determine whether Cox-1 and Cox-2 were inducedduring the neurodegenerative process in ALS, we com-pared the abundance of mRNA for both isoforms inspinal cord samples of different groups of mice. Cox-2mRNA levels significantly differed among the groups
of mice studied (F[4,24] 5 23.8, p , 0.001). In spinalcord of early symptomatic and, to a lesser extent,asymptomatic and end-stage transgenic mSOD1 mice,Cox-2 mRNA was more abundant than in spinal cordof nontransgenic mice (Fig 1). Transgenic wild-typeSOD1 mice aged-matched with end-stage transgenicmSOD1 mice exhibited no significant alteration in spi-nal cord Cox-2 mRNA levels (see Fig 1). Likewise, nosignificant change in the expression of Cox-2 mRNAwas seen in unaffected brain regions of transgenicmSOD1 mice, such as the cerebellum. The Cox-2/b-actin mRNA ratio in early symptomatic transgenicmSOD1 mice was 101.2 6 13.5 (mean 6 SEM; n 55) and that in nontransgenic mice was 107.9 6 6.3(n 5 5, Student’s t test, p 5 0.7).
In contrast to Cox-2, no significant (F[4,24] 5 0.8,p 5 0.6) alteration was found in Cox-1 mRNA expres-sion among the various groups of mice studied (seeFig 1).
Increase of Cox-2 Protein and Enzymatic Activity inTransgenic mSOD1 MiceWe next determined whether the observed change inthe spinal cord expression of Cox-2 mRNA in trans-genic mSOD1 mice was associated with detectable al-teration of Cox-2 protein level and enzymatic activity,as assessed by PGE2 content. We found that the spinalcord content of both Cox-2 protein and PGE2 in-creased significantly over the course of the degenerativeprocess (F[4,24] , 6, p , 0.004; Figs 2, 3). Con-versely, no significant change was observed in the spi-nal cord content of either Cox-2 protein or PGE2 intransgenic wild-type SOD1 mice (see Figs 2, 3) or ofCox-1 protein in the spinal cord of transgenic mSOD1mice at any of the disease stages studied (see Fig 2).Likewise, no significant change in Cox-2 protein orPGE2 content was seen in the cerebellum of transgenicmSOD1 mice. The Cox-2/b-actin protein ratio was9.7 6 0.6 in end-stage transgenic mSOD1 mice (n 55) and 10.2 6 0.8 in nontransgenic mice (n 5 5, Stu-dent’s t test, p 5 0.8). Cerebellar PGE2 content was90 6 21 pg/mg tissue in end-stage transgenic mSOD1mice (n 5 5) and 109 6 18 pg/mg tissue in nontrans-genic mice (n 5 5, Student’s t test, p 5 0.5).
Cox-2–Positive Cells in Spinal Cord of TransgenicmSOD1 MiceTo determine the cellular origin of Cox-2 upregulationin the spinal cord of transgenic mSOD1 mice, we per-formed immunohistochemical studies for Cox-2. Innontransgenic littermates, light but definite Cox-2 im-munoreactivity was seen in both the gray and the whitematter of the spinal cord (Fig 4A). In the gray matter,the most intense Cox-2 immunoreactivity was observedin the dorsal horn and especially in layers 1 to 3 of thespinal cord (see Fig 4A). Immunostaining occurred pri-
Table. Clinical Data
Control Subjects ALS Subjects
No. of patients 8 5Age at time of death (yr) 61.4 6 15.7 66.4 6 14.0Sex 7 women, 1 man 3 women, 2 menPostmortem delay (hr) 10.9 6 4.9 8.4 6 0.9
Values represent mean 6 standard deviation. Differences between ALSand control subjects were tested by Student’s t test; none of the testresults showed any significant difference (p . 0.05).
Almer et al: Cyclooxygenase-2 Induction in ALS 179
marily in the neuropil and to a lesser extent in a fewfaintly stained cells with neuronal morphologic features(Figs 4C, 4G). As previously described,27,28 Cox-2–pos-itive neurons displayed immunoreactivity over the cyto-plasmic and nuclear areas, which is consistent with theknown subcellular localization of Cox-2,29 as well as in
Fig 1. Spinal cord Cox-2 and Cox-1 mRNA expression. Pho-tograph of representative gel (top panel) and its correspondingquantification (bar graphs) reveal increased spinal cord Cox-2mRNA expression in asymptomatic (Asympt.), early symptom-atic (Onset) and end-stage (End-St.) transgenic mSOD1 micecompared to age-matched nontransgenic (Non-Tg) controlmice. Cox-2 mRNA expression in transgenic wild-type SOD1(WT-Tg) mice is similar to that in age-matched nontrans-genic mice. Conversely, there is comparable spinal cord Cox-1mRNA expression among the different groups of mice studied.Experiments were performed as described in Methods. Data(means 6 SEM) are from 5 mice per group and are represen-tative of at least two independent experiments. Ratios are Cox-2/b-actin; and Cox-1/b-actin arbitrary optical density multi-plied by 100. **p , 0.01 and *p , 0.05 (Newman-Keulspost hoc test) compared to nontransgenic mice.
Fig 2. Spinal cord Cox-2 and Cox-1 protein expression. Pho-tographs of representative gel and its corresponding quantifica-tion (bar graphs) reveal increased spinal cord Cox-2 proteinexpression, not in asymptomatic (Asympt.), but in early symp-tomatic (Onset), and end-stage (End-St.) transgenic mSOD1mice compared to age-matched nontransgenic (Non-Tg) con-trol mice. Cox-2 protein expression in transgenic wild-typeSOD1 (WT-Tg) mice is similar to that in age-matched non-transgenic mice. Conversely, there is comparable spinal cordCox-1 protein expression among the different groups of micestudied. Experiments were performed as described in Methods.Data (means 6 SEM) are from 5 mice per group and arerepresentative of at least two independent experiments. Ratiosare Cox-2/b and Cox-1/b-actin arbitrary optical density time100. **p , 0.01 and *p , 0.05 (Newman-Keuls post hoctest) compared to nontransgenic mice.
180 Annals of Neurology Vol 49 No 2 February 2001
processes (see Fig 4C). In the white matter, the immu-nostaining was evenly distributed over the ventral, lat-eral, and dorsal funiculus of the spinal cord (see Fig4A) and was due mainly to Cox-2–positive fibers andsmall scattered cells resembling oligodendrocytes (Fig4E). In asymptomatic transgenic mSOD1 mice, Cox-2immunostaining in the spinal cord was similar to thatseen in nontransgenic control mice. However, in bothearly symptomatic and end-stage transgenic mSOD1mice, Cox-2 immunostaining was strikingly different.For instance, Cox-2 immunoreactivity in both the grayand the white matter of the spinal cord was muchmore intense than in nontransgenic control micethroughout, except in layers 1 to 3, where immuno-staining appeared unchanged (Fig 4B). In the graymatter, increased immunostaining was most noticeablein the anterior horn and corresponded both to neuropiland to cells with different morphologic features (Fig4D). Although the number of motor neurons in theanterior horn was decreased and that of glial cells wasincreased, we found, using double immunostainingprocedures, that the majority of the Cox-2–positivecells were neurons (Fig 5A), whereas only some werereactive astrocytes (Fig 5C), and even fewer were acti-vated microglia (Fig 5D). In the posterior horn, mostCox-2–positive neurons exhibited the same faint im-munoreactivity as seen in the nontransgenic controlmice (Fig 4H), whereas most of those in the anteriorhorn exhibited a much more robust immunoreactivity,as seen in the nontransgenic control mice (Fig 4D). Inthe white matter, Cox-2 immunostaining was also in-creased and corresponded to intensely immunoreactive
Fig 4. Representative color photomicrographs illustrating the ex-pression of Cox-2 in the spinal cord. In nontransgenic controlmice, several cells with neuronal morphologic features (see highermagnification in C) are immunoreactive for Cox-2 (A). In end-stage transgenic mSOD1 mice, although there is a loss of Cox-2–positive neurons, the remaining neurons exhibit increased immu-noreactivity (B and D). In these mice, a few Cox-2–positivereactive astrocytes and rare Cox-2–positive activated microglialcells (D) are seen. In the white matter of nontransgenic mice,scattered small Cox-2–positive cells resembling oligodendrocytes areseen (E), whereas in end-stage transgenic mice, Cox-2–positivecells are mainly reactive astrocytes (F). Cox-2 immunoreactivity inthe posterior horn of nontransgenic (G) and transgenic mice (H)is comparable. Sample preparation and immunohistochemicalanalysis were performed as described in Methods using 1:500anti–Cox-2 antibody (Cayman Chemical). Scale bars 5 200 mmin A,B; 50 mm in C–H.
Fig 3. Bar graph representing the spinal cord content of PGE2
in asymptomatic (Asympt.), early symptomatic (Onset), andend-stage (End-St.) transgenic mSOD1 mice and age-matchednontransgenic (Non-Tg) and transgenic wild-type (WT-Tg)control mice. Experiments were performed as described in theMethods. Data (means 6 SEM) are from 5 to 11 mice pergroup. **p , 0.01, and *p , 0.05 (Newman-Keuls post hoctest) compared to nontransgenic mice.
Almer et al: Cyclooxygenase-2 Induction in ALS 181
fibers and numerous cells (Fig 4F), including severalreactive astrocytes (Fig 5B).
No difference in Cox-2 immunoreactivity was ob-served in the cerebellum between end-stage transgenicmSOD1 and age-matched nontransgenic mice or inthe spinal cord between 4-month-old transgenic wild-type SOD1 mice and age-matched nontransgenic mice.In the absence of anti–Cox-2 antibody, none of the spi-nal cord immunostaining described above could be seen.
Increase of Cox-2 Activity in Human PostmortemSamplesTo assess whether the change in Cox-2 activity ob-served in the transgenic mouse model of ALS was alsopresent in the human condition, we measured PGE2
contents in postmortem spinal cord samples from spo-radic ALS patients. Consistent with our observations intransgenic mice, we found that ALS samples had sig-nificantly higher PGE2 content than did control sam-ples (Fig 6). As in transgenic mice, no significant
change in the content of PGE2 was seen in the cere-bellum of ALS patients (ALS samples 5 39.1 6 1.6pg/mg tissue; control samples 5 38.7 6 2.0 pg/mgtissue; n 5 5 per group; Student’ t test; p 5 0.9).
DiscussionIn the present study, we found that the expression ofCox-2 mRNA and protein was markedly increased inthe spinal cord of symptomatic transgenic mSOD1mice at about 14 weeks of age and older (see Figs 1, 2).As in ischemic brain damage,30 the upregulation wasrestricted to Cox-2 and did not involve the closely re-lated prostaglandin-synthesizing enzyme Cox-1 (seeFigs 1, 2). Moreover, Cox-2 alteration was specific foraffected brain regions, because cerebellum, which is de-void of neuropathological changes in this model,8 didnot show any evidence of Cox-2 upregulation. Thistemporal and regional specificity of Cox-2 upregulationis likely not due to an insertional consequence of thetransgene, because mSOD1 in these mice is expressed
Fig 5. Representative color photomicrographs illustrating the expression of Cox-2 in specific cell types of the spinal cord of end-stagetransgenic mice. (A) Cox-2 immunoreactivity (black staining indicated by the small arrow) is expressed in neurons, as evidenced byits co-expression with the neuronal marker NeuN (light-brown staining indicated by the long arrow). (B, C) Cox-2 immunoreac-tivity (light-brown staining indicated by the small arrow) in both the white (B) and gray matter (C) is also expressed in astrocytes,as evidenced by its co-expression with the astrocytic marker GFAP (black staining indicated by the long arrow). (D) Infrequently,Cox-2 immunoreactivity (dark-blue staining indicated by the small arrow) is expressed in microglial cells, as evidenced by its co-expression with the microglial marker MAC-1 (light-brown staining indicated by the long arrow). Samples and double-immunohistochemical analysis were performed as described in Methods using 1:500 anti–Cox-2 antibody (Cayman Chemical),1:5000 anti-NeuN antibody (Chemicon), 1:500 anti-GFAP antibody (Boehringer Mannheim), and 1:1000 anti–MAC-1 antibody(Serotec). Scale bars 5 20 mm.
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throughout the brain and in all cells from birth.3,31
The transgenic mSOD1 mice used here have about afourfold higher SOD1 activity than do nontransgenicmice.31 Consequently, it is important to emphasizethat alterations of Cox-2 expression were related to thecytotoxic effects of the mutant protein and not to in-creased SOD1 activity, because age-matched transgenicwild-type SOD1 mice, also with about a fourfold in-crease in SOD1 activity,31 did not exhibit changes inspinal cord Cox-2 expression.
Immunohistochemical studies demonstrated thatCox-2 protein was also upregulated in the spinal cord oftransgenic mSOD1 mice, occurring mainly in motorneurons of the anterior horn see (Figs 4, 5). Many of theCox-2–positive neurons were devoid of pathologicalchanges, but some were clearly distorted and smaller,probably corresponding to more advanced injury of neu-rons. To a lesser extent, Cox-2–positive cells in the graymatter were nonneuronal and corresponded primarily toreactive astrocytes and occasionally to activated micro-glial cells (see Figs 4, 5). The neuronal and nonneuronalinduction of Cox-2 observed in transgenic mSOD1mice is consistent with that documented in several otherbrain injury settings.22,27,28 However, it is interestingto note that, despite the strong microglial reaction inthe spinal cord of symptomatic transgenic mSOD1mice,11,13 reactive astrocytes but not activated microglialcells were the frequent site of Cox-2 induction. Relevantto this discrepancy are the in vitro demonstrations thatIL-1b, whose level is markedly increased in the spinalcord of transgenic mSOD1 mice,18 causes a rapid androbust induction of Cox-2 in astrocytes32 but fails to doso in microglial cells.33
We also demonstrate that the Cox-2 mRNA and pro-tein upregulation was associated with increased tissuecontent of PGE2 (see Fig 3), one of the main Cox-2reaction products. This finding is compelling evidencethat the increased Cox-2 mRNA and protein levels andimmunoreactivity seen in the spinal cord of the trans-genic mSOD1 mice correspond to the expression of acatalytically active form of the protein. The transgenicmSOD1 mouse is a faithful model of the mSOD1-linked familial form of ALS,34 but how good this exper-imental model is for sporadic ALS is unclear. It is thusreassuring to see that the tissue content of PGE2 is, as inthe transgenic mice, markedly increased in human post-mortem spinal cord samples from sporadic ALS patients(see Fig 6). This result provides further credibility forthe transgenic mSOD1 mouse model of ALS and dem-onstrates that Cox-2 upregulation is probably a consis-tent neuropathological feature of ALS, regardless ofwhether the disease is linked to mSOD1. The findingthat PGE2 levels in ALS patients were increased in thespinal cord but not in the cerebellum supports our con-tention that the difference in PGE2 between ALS pa-tients and control subjects (see Fig 6) is due to the dis-
ease, as opposed to agonal events. it is also worthmentioning that five of the eight controls died from res-piratory failure (see Methods) Like most of the ALS pa-tients, 5 of the 8 control subjects died of respiratory fail-ure (see Methods), and yet they exhibited low spinalcord PGE2 levels relative to those of ALS patients.
The mechanisms responsible for Cox-2 induction inALS remain to be elucidated. In the normal central ner-vous system, Cox-2 is restricted to certain regions,23,28
including the spinal cord, at least in rodents.27 In agree-ment with this observation, we have found detectablelevels of Cox-2 mRNA and protein (see Figs 1, 2) andfaint but definite Cox-2 immunoreactivity in the spinalcord of nontransgenic control mice (see Fig 4). In ex-perimental seizures and following the local injection ofkainic acid into the brain, Cox-2 is upregulated in neu-rons, an effect blocked by the N-methyl-D-aspartate re-ceptor antagonist MK-801.28,35 These observations sug-gest that Cox-2 expression may be induced by activationof glutamate receptors. In ALS, several abnormalities inglutamate metabolism have been described,36 which pre-sumably could increase extracellular glutamate. It is thuspossible that activation of glutamate receptors partici-pates in ALS-related Cox-2 induction. Alternatively, thetime course of Cox-2 induction closely follows the tem-poral profile of IL-1b increased production in thesemice.18 Because cytokines such as IL-1b are known toinduce Cox-2 expression,22 it is also possible that cyto-kines contribute to ALS-related Cox-2 induction. Trans-forming growth factor b1 and platelet-activating factor,whose production and release are stimulated in severalbrain injury settings,37 are two other potent Cox-2–in-ducing factors.38 In light of all these examples, it is mostlikely that the induction of Cox-2 observed in ALS re-sults, not from the action of a single factor, but, rather,from a combination of factors.
It is interesting to note that Cox-2 and iNOS are in-duced over a similar time period during the neurodegen-eration in transgenic mSOD1 mice.13 Co-induction ofCox-2 and iNOS has also been reported in models ofinflammation.22 Cox-2 promoter shares many featureswith iNOS promoter.39,40 Accordingly, it is not surpris-ing that Cox-2 and iNOS are coordinately regulated indisease states associated with inflammation. In addition,iNOS-derived NO enhances Cox-2 catalytic activity andthus its production of pro-inflammatory prostaglan-dins.30 Moreover, the combination of iNOS-derivedNO and superoxide generated by Cox-2 can lead to theformation of the highly reactive tissue-damaging per-oxynitrite, which is thought to play a major role in sev-eral neurodegenerative diseases, incuding ALS.41 There-fore, Cox-2 activation could be one mechanism bywhich NO exerts its pathogenic effect on the ALS spinalcord.42,43
Aside from its interaction with NO, other factorsmay be responsible for the cytotoxicity of Cox-2. One
Almer et al: Cyclooxygenase-2 Induction in ALS 183
likely mechanism is related to production of ROS,which are considered one of the major determinants ofALS neurodegeneration.44–46 Indeed, ROS are formedby the peroxidase step of the Cox reaction in whichprostaglandin G2 is converted to prostaglandin H2.22
Phospholipase A2, which generates the precursor sub-strates for Cox by hydrolyzing phospholipids,47 is alsoupregulated in the spinal cord of transgenic mSOD1mice.48 This finding suggests that, during the neuro-degenerative process, thanks to the upregulation ofphospholipase A2, there may be no shortage of sub-strates, making the Cox-2 pathway an important routefor free radical production in the ALS spinal cord.Cox-2 enzymatic activity can also mediate tissue dam-age by producing pro-inflammatory prostanoids suchas PGE2.21 A third mechanism by which Cox-2 couldcontribute to cell death is related to the induction ofapoptosis by PGE2 in some cell systems.49,50 Thisraises the possibility that Cox-2 contributes to the de-mise of motor neurons in ALS by promoting apoptosis,which is an active form of cell death. This prospect isof particular interest, because we have demonstratedpreviously that mSOD1 stimulates apoptosis in vitroand that several molecular pathways of apoptosis areactivated in transgenic mSOD1 mice during the neu-rodegenerative process.6,18,31 Morphological evidencethat motor neurons are, at least in part, dying by apop-tosis has also been demonstrated in the spinal cord ofsporadic human ALS cases.51
Collectively, our data provide evidence of a markedupregulation of Cox-2 in ALS and suggest a pivotalrole for Cox-2–derived prostanoids in the cascade ofdeleterious events that ultimately lead to spinal cordmotor neuronal death in transgenic mSOD1 mice andin human ALS. Therefore, in light of the present data,
we suggest that inhibition of Cox-2 may be a valuabletarget for the development of new therapies for ALSaimed at slowing the progression and propagation ofthe disease. Consistent with this view is the recentdemonstration that the chronic administration of thenonselective Cox inhibitor acetylsalicylate delays the ap-pearance of motor deficit in transgenic mSOD1 mice.52
This observation provides major impetus to the use ofCox inhibitors and warrants further studies using potentselective Cox-2 inhibitors to better define the potentialrole of this type of therapeutic strategy for ALS.
This study was supported by the Muscular Dystrophy Association, theALS Association, Project-ALS, the National Institute of NeurologicalDisorders and Stroke (grants R01 NS38586, R29 NS37345, and P50NS38370), the US Department of Defense (grant DAMD 17–99-1–9471), the Lowenstein Foundation, the Smart Foundation, and theParkinson’s Disease Foundation. GA is recipient of the Erwin Schro-dinger Scholarship of the Austrian Research Funds, and CG is a re-cipient of a scholarship from the INSERM (France).
We thank Ms Norma Romero for assisting in the genotyping andthe tissue sample preparation and to the members of Dr WilliamSmith’s laboratory (Michigan State University, East Lansing, MI)for their guidance in the performance of Cox-1 and Cox-2 Westernblot analyses.
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