Free Radical Biology & Medicine 47 (2009) 1049–1056
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Free Radical Biology & Medicine
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Original Contribution
Neuronal NOS and cyclooxygenase-2 contribute to DNA damage in a mouse model ofParkinson disease
Tuan Hoang a, Dong-Kug Choi b,1, Makiko Nagai b, Du-Chu Wu b, Tetsuya Nagata b, Delphine Prou b,Glenn L. Wilson c, Miquel Vila b,2, Vernice Jackson-Lewis b, Valina L. Dawson d, Ted M. Dawson d,Marie-Françoise Chesselet a, Serge Przedborski b,c,e,⁎a Department of Neurology and Department of Neurobiology, University of California at Los Angeles, Los Angeles, CA 90095, USAb Department of Neurology, Columbia University, New York, NY 10032, USAc Department of Cell Biology and Department of Neuroscience, University of South Alabama, Mobile, AL 36688, USAd Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Departments of Neurology and Neuroscience Johns Hopkins University School of Medicine, Baltimore, MD 21205, USAe Department of Pathology and Cell Biology and Center for Motor Neuron Biology and Disease, Columbia University, New York, NY 10032, USA
⁎ Corresponding author. Department of Neurology, CNY 10032, USA. Fax: +1 212 342 3663.
E-mail address: [email protected] (S. Przedborski1 Present address: Department of Biotechnology, Konk2 Present address: Vall d’Hebron Research Institute-ICRE
DNA damage is a proposed pathogenic factor in neurodegenerative disorders such as Parkinson disease. Toprobe the underpinning mechanism of such neuronal perturbation, we sought to produce an experimentalmodel of DNA damage. We thus first assessed DNA damage by in situ nick translation and emulsionautoradiography in the mouse brain after administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP; 4×20 mg/kg, ip, every 2 h), a neurotoxin known to produce a model of Parkinson disease. Here weshow that DNA strand breaks occur in vivo in this mouse model of Parkinson disease with kinetics and atopography that parallel the degeneration of substantia nigra neurons, as assessed by FluoroJade labeling.Previously, nitric oxide synthase and cyclooxygenase-2 (Cox-2) were found to modulate MPTP-induceddopaminergic neuronal death. We thus assessed the contribution of these enzymes to DNA damage in micelacking neuronal nitric oxide synthase (nNOS), inducible nitric oxide synthase (iNOS), or Cox-2.We found thatthe lack of Cox-2 and nNOS activities but not of iNOS activity attenuated MPTP-related DNA damage. We alsofound that not only nuclear, but alsomitochondrial, DNA is a target for theMPTP insult. These results suggest thatthe loss of genomic integrity can be triggered by the concerted actions of nNOS and Cox-2 and provide furthersupport to the view that DNA damage may contribute to the neurodegenerative process in Parkinson disease.
Compromised genome integrity has emerged as a critical patho-genic event in neurodegenerative diseases [1]. Genetic mutations inmolecular pathways responsible for genome maintenance have beenlinked to accelerated aging phenotypes accompanied by widespreadneurodegenerative changes [1]. DNA damage-linked neuronal dys-function (also called senescence) and death have been invoked in theneurodegenerative processes underlying common sporadic braindisorders such as Parkinson disease (PD) [1]. Providing a majorimpetus to this hypothesis are the demonstrations that enzymes suchas cyclin-dependent kinases [2], tumor suppressor protein p53 [3],and poly(ADP-ribose) polymerase-1 (PARP-1) [4,5], whose activationis known to be initiated by DNA damage, were all found to be
olumbia University, New York,
).uk University, South Korea.A-CIBERNED, Barcelona, Spain.
ll rights reserved.
instrumental in the death of nigrostriatal dopaminergic neurons, thesubpopulation of neurons most affected in PD.
Here, we show that DNA strand breaks occur in the substantia nigrapars compacta (SNpc) in the mouse model of PD produced by theneurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) [6].The occurrence of DNA damage in this model of PD is associated withthe activation of PARP and follows the kinetics and topography ofdegenerating of SNpc neurons. Previously, we have shown that enzymessuch as cyclooxygenase-2 (Cox-2), neuronal nitric oxide synthase (nNOS),and inducible NOS (iNOS) contribute to SNpc dopaminergic neurodegen-eration in theMPTPmodel of PD [7–9]. The use of knockoutmice deficientin Cox-2, nNOS, or iNOS allowsus to showhere that both Cox-2 andnNOS,but not iNOS, contribute to the observed DNA damage. Finally, wedemonstrate that not only nuclear DNA, but also mitochondrial DNA, isdamaged by the insult, which is interesting in light of the presumedpathogenic role of mitochondrial defects in PD [6]. Although our resultsare correlative, we hypothesize that the loss of genome integritydocumented in this study may contribute to the degenerative process inthis model of PD and perhaps in PD itself.
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Materials and methods
Animals and MPTP treatment
For this study, we used 10-week-old male C57BL mice (CharlesRiver Laboratories, Wilmington, MA, USA) as well as nNOS and iNOSknockout mice and their wild-type littermates (from the Dawsonlaboratory at Johns Hopkins University) and Cox-2 knockout mice andtheir wild-type littermates (from Taconic Farms, Hudson, NY, USA). Allmice (n=4–8 per group) received four intraperitoneal injections ofMPTP–HCl (18–20 mg/kg of free base; Sigma–Aldrich, St. Louis, MO,USA) in saline at 2-h intervals in one day and were sacrificed atselected time points (ranging from 0 to 7 days) after the last injectionof MPTP. Control mice received saline only. MPTP handling and safetymeasures were in accordance with Columbia University and ourpublished guidelines [10,11]. This protocol was in accordance with theNIH guidelines for use of live animals and was approved by theInstitutional Animal Care and Use Committees of Columbia UniversityMedical Center and Johns Hopkins University School of Medicine.
Tyrosine hydroxylase immunostaining and counts
At selected time points, three or four mice per time point weredecapitated and whole brains harvested and processed as before [12].Coronal sections (10 μm) encompassing the entire midbrain (i.e.,bregma −3.4 mm to bregma −3.8 mm [13]) were cut on a cryostatand tyrosine hydroxylase (TH) immunostaining was performed usinga polyclonal anti-TH antibody (1:1000; Calbiochem, San Diego, CA,USA) following the procedure reported before [12] with minormodifications. At the end of the TH immunostaining, sections werecounterstained with thionin. The numbers of TH-positive cells werecounted manually using light microscopy from at least four repre-sentative planes of the SNpc for each time point. The total number ofTH-positive cells was calculated for each side, averaged for eachanimal, and expressed per section.
DNA strand break by in situ nick translation (ISNT) and emulsionautoradiography
This assay was performed as previously reported [14] using [35S]dATP and endonuclease-free DNA polymerase I. At selected time points,mice were decapitated and whole brains harvested and immediatelyfrozen indry-ice-cooled isopentane. As above,midbrain coronal sections(10 μm)were cut on a cryostat, but here they were thaw-mounted ontogelatin-coated slides and stored at−80 °C until processed. Briefly, afterexposure to formamide and acetylation, tissue sections were incubatedwith [35S]labeled dATP, unlabeled nucleotides, and endonuclease-freeDNA polymerase I. Adjacent sections incubated in the absence of DNApolymerase I were used as negative controls. All sections were coatedwith Kodak NTB3 emulsion (VWR Scientific, Bridgeport, NJ, USA) andcounterstained with hematoxylin and eosin. The criteria for ISNTlabeling included the presence of silver grains over a cell with a visiblenucleus and in a number at least five times greater than nonspecificlabeling. The latter was defined as the number of silver grains in anadjacent, equally sized background area. The numbers of ISNT-labeledcells were counted following the same procedure as described above forTH. Then, for each time point, the numbers of ISNT-labeled cells werenormalized to the number of TH-positive cells.
PARP activity and in situ histochemistry
Ventral midbrain, striatum, and cerebellum from mice 0 to 7 dayspost-MPTP were homogenized, and 20 μg of protein extract from eachsample was used to determine PARP activity using a commercial kit(Trevigen, Gaithersburg, MD, USA). The latter is based on themeasurement of [32P]NAD incorporation into nuclear acceptor
proteins following the manufacturer's instructions. Tissue extractsfrom PARP-1 knockout mice, as well as from wild-type mice treatedwith the PARP-1 inhibitor 3-aminobenzamide, were used for negativecontrols. At the end of the incubation period (10 min, 25 °C), thereaction was stopped by the addition of ice-cold 20% trichloroaceticacid (TCA). The TCA-insoluble precipitates were washed three timeswith ice-cold 10% TCA and resuspended in liquid scintillation cocktail,and the incorporation of [32P]ADP-ribose into TCA-precipitableproteins was quantified by scintillation spectroscopy.
For PARP histochemistry, fresh-frozen cryostat-cut sections(14 μm) collected at the same levels as for ISNT were fixed in ethanoland permeabilized with Triton X-100. This assay is based on the in situdemonstration of biotinyl-ADP ribose incorporation into nuclearacceptor proteins using 6-biotin-17-NAD+ (bio-NAD; Perkin–Elmer,Boston, MA, USA). The histochemical method was performed asdescribed previously [15]. Incorporated biotinwas detected by avidin:peroxidase-conjugated biotin complex, and color was developed with3,3′-diaminobenzidine. The sections were counterstained with thio-nin or subjected to immunostaining using a rat anti-dopaminetransporter (DAT) antibody (Chemicon, Temecula, CA, USA), andcolor was developed with an SG peroxidase substrate kit (VectorLaboratories, Burlingame, CA, USA). The percentages of SNpc PARP+/DAT+ and PARP+/DAT− cells were determined by counting manually~50 PARP+ cells from at least four representative planes of ventralmidbrain per mouse killed at 6 h after the last MPTP injection.
FluoroJade histochemistry
To assess the time course of neurodegeneration in the SNpc, micewere anesthetized and then perfused with saline and 4% paraformal-dehyde at selected time points ranging from 0 to 7 days after the lastMPTP injection. Brains were removed, postfixed overnight in the samefixative, and frozen in dry-ice-cooled isopentane. nNOS knockoutmiceand their wild-type littermates were also used at 10 and 24 h after thelast MPTP injection as above. Cryostat-cut brain sections (30 μm)weremounted onto gelatin-coated slides before staining with 0.001%FluoroJade in 0.1% acetic acid as previously described [16]. As above,the total number of FluoroJade-labeled cells was calculated for eachside, averaged for each animal, and expressed per section.
The ISNT-labeled sections were three times thinner than theFluoroJade-labeled sections. In our pilot studies, we defined therelationship between FluoroJade counts and tissue thickness. Fivemice were injected with MPTP as detailed above and killed at 2 dayspost-MPTP (i.e., the peak of FluoroJade-positive SNpc cells). Then, brainswere harvested, hemisected, and processed as above for FluoroJade,except that one-half of the midbrain was sectioned at 30 μm and theother half was sectioned at 10 μm. The number of FluoroJade-labeledcells per section at 30 μm was 96.9±6.5 and at 10 μm was 34.5±1.8(mean±SEM). Based on these results, we divided the FluoroJade countsby 2.81 to facilitate the comparisonbetween the ISNTand the FluoroJadeSNpc cell counts. Then, for each time point, the numbers of FluoroJade-positive cell were normalized by the number of TH-positive cells.
Mitochondrial DNA and Southern blot analysis
Ventral midbrain, striatum, and cerebellum were taken from miceinjected with either saline or MPTP at selected time points rangingfrom 0 to 7 days after the last injection. Tissues were homogenizedand treated with proteinase K overnight at 37 °C. After 1/4 vol of 5 MNaCl was added, high-molecular-weight DNA was extracted withequal volumes of Sevag's solution (24:1 chloroform:isoamyl alcohol),followed by precipitation with ammonium acetate and ethanol.Resuspended DNA was digested with XhoI and RNase. Digestedsamples were precipitated, resuspended in TE buffer, and quantified.Samples containing 3 μg of total DNAwere heated (20 min, 65 °C) andthen cooled at room temperature. NaOH was then added to a final
Fig. 1. MPTP causes DNA damage and neuronal degeneration. (A) By 10 h after the lastinjections of MPTP, numerous clusters of silver-grain deposits, as evidenced by DNAstrand breaks, are seen within the SNpc area and to a lesser extent within the VTA,confined over cells with large nuclei (inset), consistent with them being neurons. (B)Similar findings are found for the cellular FluoroJade labeling in both ventral midbrain
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concentration of 0.1 M, and samples were incubated (15 min, 37 °C).Samples were then mixed with alkaline loading dye, loaded onto ahorizontal 0.6% alkaline agarose gel, and electrophoresed. DNA wasthen transferred to Zeta-Probe GT nylon membranes (Bio-RadLaboratories, Hercules, CA, USA). The membranes were cross-linkedand hybridized with a 32P-labeled mouse mitochondrial DNA-specificPCR-generated probe using primers corresponding to 1924–1953(forward) and 2505–2473 (reverse) of mouse mtDNA and usingTaKaRa LA Taq (Takara Bio, Inc., Shiga, Japan). The amplified fragmentwas gel purified and labeled with [32P]dATP using the Random PrimedDNA Labeling Kit (Roche Diagnostics, Indianapolis, IN, USA). Bands ofinterest were analyzed and quantified using FluorChem 8800 (AlphaInnotech, San Leandro, CA, USA).
Statistical analysis
All counts were performed by an individual blinded to thetreatment conditions and time points. All values are expressed asmeans±SEM. Differences among means were analyzed using one- ortwo-way ANOVA with time, treatment, and/or genotype as theindependent factors. When ANOVA showed significant differences,comparisons between means were examined using the Newman–Keuls post hoc test, except for the instances in which all groups werecompared against the control group (i.e., saline or time 0); in thesecases, we used the Dunnett post hoc test. When only two groups werecompared, Student's t test was used. In all analyses, the nullhypothesis was rejected at the 0.05 level.
Results
Time course and topography of MPTP-mediated DNA damage
To demonstrate whether the demise of nigrostriatal dopaminergicneurons is associated with a loss of genome integrity, we first assessedthe occurrence of double and single DNA strand breaks in selectedbrain regions after the administration of the neurotoxin MPTP to mice[6]. DNA strand breaks were assessed at the single-cell level by ISNTwith radiolabeled nucleotides and emulsion autoradiography [14,17].As previously reported [12], we found that the regimen of MPTP usedhere causes ~60% neuronal death in the SNpc by 7 days after the lastinjection (Table 1); the SNpc is the brain area that houses the cellbodies of the nigrostriatal dopaminergic pathway and is the brainstructure most affected in PD [6]. The selected time points encom-passed the entire phase of neurodegeneration that follows thisregimen of MPTP injection [18,19]. SNpc TH-positive neuronal countscan be found in Table 1.
Using ISNT, evidence of DNA damagewas found in the SNpc and, toa lesser extent, in other brain regions such as the ventral tegmentalarea (VTA) of MPTP-injectedmice (Fig.1A). Based on hematoxylin andeosin counterstaining, which provided a light labeling of cellular
Table 1SNpc TH-positive neuron counts post-MPTP
Mean±SEM(n=4 mice/time point)
Saline control 151.8±10.90 h 152.0±11.63 h 148.7±12.46 h 132.5±10.68 h 110.0±9.810 h 91.6±11.612 h 78.3±11.21 day 62.5±10.42 days 39.8±5.44 days 45.2±10.27 days 44.6±9.4
SNpc neuronal counting was performed as outlined under Materials and methods.
regions. The inset in (B) shows the neuronal morphology of the FluoroJade-labeledcells. (C) Except for the 12-h time point, the time course of the SNpc ISNT labelingafter MPTP injection is very similar to (D), that of the FluoroJade labeling. Data aremeans±SEM of four or five mice per time point after the last injections of MPTP andnormalized to the number of TH-positive SNpc neurons (see Table 1 for actual values).⁎Different from t=0 (pb0.05, Dunnett's post hoc test). Scale bar, 200 μm (A and B)and 20 μm in both insets.
cytoplasm and nucleic acid materials, we were able to determine thatmost of the ISNT-positive cells were large and exhibited a neuronalmorphology (Fig. 1A, inset). Through a time-course study (Fig. 1C), wefound that the number of ISNT-positive cells in the SNpc variedsignificantly (F9,38=4.61, pb0.014) over time after MPTP administra-tion. A few ISNT-positive neurons in the brains of these mice weredetected as early as 6 h post-MPTP (Fig. 1C). Thereafter, their numbersquickly rose, reaching a first peak at 10 h and an even higher, secondpeak at ~48 h post-MPTP (Fig. 1C). By day 7 post-MPTP, there was
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typically ≤10 ISNT-labeled cell per section detected in any of thestudied brain regions (Fig. 1C). During the time course, the greatestnumber of ISNT-labeled cells was consistently found in the SNpc(Fig. 1A). Roughly half as many ISNT-labeled neurons were detectedin other dopaminergic brain regions such as the VTA. Consistentwith MPTP relative specificity for ventral midbrain dopaminergicneurons, no more than 3 ISNT-labeled cells per section weredetected in nondopaminergic brain regions (e.g., hippocampus:2.7±0.1 cell/section at 24 h post-MPTP, n=5).
Temporal relationship between MPTP-induced DNA damage andneuronal death
To establish the temporal relationship between DNA damage andneuronal death, a separate set of tissue sections from MPTP-injectedmice killed at the same time points as above were stained withFluoroJade, a fluorochrome allowing for the sensitive and reliablehistochemical localization of degenerating neurons [16]. Like thenumber of ISNT-positive neurons, the number of FluoroJade-stainedneurons (Fig. 1B and inset) varied in a time- (F9,38=41.61, pb0.001)and region-dependent manner (Fig. 1D). Consistent with the ISNTresults, the first FluoroJade-positive cells were also detected by 6 h
Fig. 2. MPTP administration activates PARP in dopaminergic neurons. (A) PARP activity riseinjections of MPTP. At the level of the ventral midbrain, activation of PARP is seen after (C, E, Gsaline injections. ⁎Different from t=0 (pb0.05, Dunnett's post hoc test). Scale bar, 300 μm
post-MPTP, and the largest number of dying neurons was again foundin the SNpc (Fig. 1B). Furthermore, the overall time course ofFluoroJade-positive cell numbers was comparable to that of theISNT-positive cell numbers (Fig. 1C and D). Several FluoroJade-stainedneurons were also seen in the VTA (Fig. 1B), whereas nonewas seen inany other region of the brain, including the hippocampus, at any of thestudied time points. Thus, evidence of cell degeneration, evidenced byFluoroJade, occurs in the brains of MPTP-intoxicated mice with ananatomical distribution and a timeline consistent with the known cellspecificity and time course of degeneration caused by this regimen ofMPTP [12].
MPTP-mediated DNA damage is associated with PARP activation
DNA damage activates the DNA-repair and protein-modifyingenzyme PARP-1 in many cells, including neurons [20]. Again, asmentioned above for the other assays, PARP activity measures inwhole tissues from ventral midbrain varied over time (F9,21=9.72,pb0.001) (Fig. 2A). In MPTP-injected mice, ventral midbrain PARPactivity began to increase by 3 h, peaked between 6 and 9 h (300%increase), and then slowly subsided back to control activity by 2 daysafter MPTP injections (Fig. 2A). PARP catalytic activity in the
s soon after the last injections of MPTP and returns to baseline by 2 days after the last, I) MPTP injections in (E, G) neurons, which expressed DAT (I), but not after (B, D, F, H)(B, C), 75 μm (D–I).
Fig. 3. Contribution of NOS isoforms and Cox-2 in MPTP-induced DNA damage. (A) At24 h after the last injections of MPTP, nNOS−/− and Cox-2−/−, but not iNOS−/−,mice exhibit significantly fewer ISNT-labeled SNpc neurons compared to theirrespective wild-type littermates. (B) Likewise, at 6 h after the last injections of MPTP,nNOS−/− mice show lower ventral midbrain PARP activity and, (C) at 24 h after thelast injections of MPTP, fewer SNpc FluoroJade-labeled neurons compared to theirnNOS+/+ littermates. Values are means±SEM (n=3–5). ⁎Significantly different(Newman–Keuls post hoc test, Pb0.01) from wild-type littermates.
Fig. 4. MPTP damages mitochondrial DNA. (A) Southern blot analysis of striatal tissuereveals a tail of higher intensity caused by DNA common deletions and a lower intensityof the intact full-length 16.3-kb band evidencing intact mitochondrial DNA, at 8 h afterthe last injections of MPTP. The presented pattern was confirmed in three independentexperiments. (B) The reduction of the intact full-length 16.3-kb band at 8 h after the lastinjections of MPTP is significant only in nNOS+/+ mice and not in the nNOS−/−
littermates. Values are means±SEM (n=3–5). ⁎Significantly lower (Newman–Keulspost hoc test, Pb0.05) than all other groups.
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cerebellum was low and not significantly altered by MPTP injection(not shown). To determine whether PARP was activated in SNpcdopaminergic neurons, we performed in situ PARP histochemistrycombined with immunostaining for DAT at 6 h after the last injectionof saline or MPTP. In saline-injected controls, no PARP histochemicallabeling was detected (Fig. 2B, D, F, and H). In contrast, this double-staining procedure revealed PARP activation in several ventralmidbrain cells (Fig. 2C and E), all exhibiting a definite neuronalmorphology (Fig. 2G) and nearly all expressing DAT (Fig. 2I): of ~50PARP+ SNpc cells per animal, 96±4% were PARP+/DAT+ and 4±2%were PARP+/DAT− (n=3). No PARP activity or histochemical labelingwas detected in ventral midbrains frommice deficient in PARP-1 (datanot shown). Thus, after the administration of MPTP, there is a rapid
and transient activation of PARP-1 within dopaminergic neurons,specifically in the ventral midbrain.
MPTP-related oxidative stress implication in DNA damage andneuronal death
Wenext sought to determine the underlyingmechanism leading toDNAdamage in this PDmodel. Among the previously identified factorsinvolved in MPTP neurotoxicity, we elected to study DNA damage byperforming an ISNT assay in MPTP- and saline-injected mice deficientin Cox-2 or in nNOS, themain isoform of NOS in the nervous system. Inboth Cox-2−/− and nNOS−/− mice, the numbers of neurons positivefor ISNT were dramatically smaller than in their wild-type littermates,at 24 h post-MPTP (Fig. 3A); a similar attenuationwas seen at the 10-htime point (data not shown). In contrast to Cox-2 and nNOS ablation,iNOS deficiency did not significantly decrease MPTP-induced SNpcDNA damage as assessed by ISNT, neither at 10 h (data not shown) norat 24 h post-MPTP (Student's t test: t8=−0.30, P=0.78) (Fig. 3A).
Given that the most prominent effect on the number of ISNT-positive cells in the MPTP model was observed for nNOS ablation,we also compared PARP activity and FluoroJade labeling betweennNOS+/+ and nNOS−/− mice at selected time points. Six hours afterMPTP administration, nNOS+/+ mice exhibited significantly higherventral midbrain PARP activity than their nNOS−/− counterparts(Fig. 3B). Furthermore, 24 h after MPTP administration, nNOS+/+
mice exhibited a significantly higher number of FluoroJade-positivecells than their nNOS−/− counterparts (Fig. 3C). Thus, these resultsindicate that DNA damage in the SNpc results from an in vivooxidative insult after MPTP administration and that neuronal-
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derived NO and apparently not glial-derived NO contributes to theloss of genome integrity in this model of PD.
Mitochondrial DNA is also damaged by MPTP administration
Mitochondrial DNA damage associated with respiratory chaindeficiency has been documented within SNpc dopaminergicneurons from PD patients [21]. To test whether mitochondrialDNA is damaged after MPTP administration, the integrity of theentire mitochondrial genome was assessed by Southern blotanalysis [22] at various time points post-MPTP injection (Fig. 4A).This experiment revealed evidence of mitochondrial DNA damage(i.e., presence of a tail) primarily in the striatum between 8 and 10h post-MPTP (Fig. 4A). As expected, together with the tail seen onthe gel, there was also at 8 h after the last MPTP injection a 63%reduction in intensity of the band corresponding to the intact, full-length 16.3-kb mitochondrial DNA compared to saline-injectedcontrols (Fig. 4B). Note that at this time point, striatal dopaminelevels and TH activity are already reduced in MPTP-injected mice[23]. At none of the studied time points was damaged mitochon-drial DNA detectable in ventral midbrain or cerebellum (data notshown). To determine whether the damage to the mitochondrialDNA in the striatum was also provoked by a NO-mediated insult,the same Southern blot analysis was performed in mice deficient innNOS (Fig. 4B). Compared to the wild-type littermates, nNOS−/−
mice showed significantly less (two-way ANOVA, pb0.05) mito-chondrial DNA damage at 8 h post-MPTP (Fig. 4B).
Discussion
This study demonstrates that DNA damage in the form of strandbreaks arises in cell groups located within the ventral midbrain afterMPTP administration. Although no double labeling was performed,the positive cells illustrated in Fig. 1 were all located within theanatomical boundaries of the SNpc and the VTA and were ISNT andFluoroJade positive after the injections of MPTP, a toxin that fails todamage nondopaminergic elements in the ventral midbrain. Thesecharacteristics make it almost certain that the labeled cells aredopaminergic neurons. Although our findings are correlative, thedemonstration of genomic and mitochondrial DNA damage bydifferent techniques in an experimental model of PD raises thepossibility that these alterations may participate in the neurodegen-erative process. We thus speculate that the occurrence of DNA strandbreaks in the MPTP model of PD provides further support to thehypothesis that the increased levels of DNA damage markers such as8-hydroxyguanine and DNA deletions/rearrangements found inpostmortem PD samples may be pathogenically significant [21,24–26]. However, at this point, we cannot exclude with certainty that theobserved ISNT labeling is a consequence rather than a cause of theSNpc neurodegeneration. Arguing against the idea that the occurrenceof DNA damage evidenced here by ISNT is a mere, nonspecificconsequence of dying neurons are the following two observations.First, as indicated under Results, after MPTP administration, smallnumbers of cells with a low, but unambiguous ISNT+ signal wereconsistently counted in the hippocampus, whereas, after MPTPadministration, no FluoroJade+ cells (or any suppressed-silver-stained cells; data not shown) were detected in this region of thebrain. Thus, it is unlikely that the occurrence of the ISNT+ signal is amere reflection of the dying process. Incidentally, the fact that someextranigrostriatal neurons exhibited DNA damage without dying tothe same extent as SNpc neurons (as assessed by FluoroJade labeling)is consistent with the idea that a threshold of DNA damage may haveto be reached to provoke cellular senescence and death. Second, PARPactivation used here as a faithful marker of DNA damage clearlypreceded the main wave of neurodegeneration (this finding isdiscussed below in more detail).
Remarkably, the number of ISNT-positive neurons in the SNpcvaried over the studied period in a biphasic fashion. This suggests thatMPTP causes genotoxicity in SNpc dopaminergic neurons by morethan one mechanism, these mechanisms exerting their respectivedeleterious effects on DNA in a sequential manner. Consistent withthis view, and despite the fact that nNOS and Cox-2 mediatedopaminergic neurodegeneration in MPTP mice [7,9] probably bydistinct mechanisms, are our observations that the deletion of bothenzymes did attenuate DNA damage. Alternatively, it is also possiblethat SNpc neurons respond unevenly to the genotoxic effects of MPTP,as different neurons may have different arsenals of protection as wellas mechanisms of DNA damage surveillance and repair [27]. This mayalso explainwhy, at some time points, an unusually large variability inSNpc ISNT+ cell number was consistently observed, which cannot beexplained by technical reasons given our inclusion of quality controlsamples in each experiment.
In damaged cells, PARP binds to DNA strand breaks and catalyzesthe covalent attachment of ADP-ribosyl polymers, derived from thehydrolysis of NAD+, onto a variety of nuclear proteins including PARPitself [28]. Here, PARP activity was used as a means of confirming theoccurrence of DNA damage in the MPTP mouse model of PD, as theactivity of this enzyme correlates linearly with the number of strandbreaks [29,30]. Although PARP activity did increase markedly afterMPTP administration, which confirmed the occurrence of DNAdamage in this model of PD, the kinetics of PARP activity after MPTPinjection differed from that of ISNT in two striking aspects. First, PARPactivity rose before any detectable ISNT labeling, which is consistentwith the sensitivity of PARP to minute amounts of DNA damage [28].Second, PARP activity normalized well before evidence of DNAdamage disappeared. The latter discrepancy may find its explanationin the following facts. During PARP-mediated poly(ADP-ribosyl)ationof nuclear proteins, PARP itself becomes poly(ADP-ribosyl)ated, whichdecreases its affinity for damaged DNA as well as its catalytic activity[31]. Moreover, in vivo, the cellular level of the poly(ADP-ribosyl)polymer relies on the opposing actions of PARP and poly(ADP-ribose)glycohydrolase (PARG), the former synthesizing the polymer and thelatter degrading it [28]. The apparent premature normalization ofPARP activity may thus be due to the combined effects of theautomodification of PARP, which inhibits the enzyme, and theactivation of PARG, which catabolizes poly(ADP-ribosyl) polymers.Alternatively, it has also been reported that, in some instances, PARPactivation may be caused by MAPKs such as ERKs, irrespective of DNAdamage [32]. Although the possibility of DNA damage independent ofPARP activation remains to be established in in vivo models of humandiseases, this idea is certainly worth considering here. Thus, furtherstudies will be required to test these different possibilities.
Given the genotoxic potential of oxidative stress [33], it is tantalizingto consider whether the results obtained here with mice deficient inCox-2 and nNOS may be linked to damage caused by reactive oxygen(ROS) and nitrative species. Aside from producing extracellularproinflammatory prostanoids, Cox-2 can generate ROS during theperoxidase catalysis of prostaglandin G2 conversion to prostaglandinH2 [34]. Upon donation of electrons to Cox, co-substrates such asdopamine become oxidized to dopamine quinone [35], which is highlyreactivewithglutathione, aminoacids suchascysteinyl, andDNA [36]. Inour hands, there was no evidence for a proinflammatory role of Cox-2after MPTP administration to mice [7]. However, there was a markedincrease in Cox-2-dependent protein cysteinyl-dopamine content [7], afingerprint for protein cysteinyl attack by dopamine quinone [35].Thus, these data suggest that Cox-2 does promote oxidative events inMPTP-injected mice. Also relevant for a potential role for oxidation hereis our previous demonstration that MPTP, in mice, markedly increaseslevels of 3-nitrotyrosine in affected brain regions [37]. This post-translational modification probably reflects the alteration of tyrosineresidues by peroxynitrite, a reactive species resulting from the combina-tion of superoxide and NO [33]. It is intriguing to note, however, that
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although ablation of iNOS attenuates neurodegeneration and thenitrative modification of proteins with pathogenic significance for PDsuch as parkin [8,38], mice deficient in iNOS exhibit the same extent ofDNA damage as their wild-type counterparts. NOS activity in tissuessuch as ventral midbrain derives mainly from nNOS and, to a muchlesser extent, from iNOS [8]. It is therefore possible that only a profoundreduction in NO bioavailability for peroxynitrite formation, as caused bynullifying nNOS, is required before any meaningful abatement of thebiochemical reaction thatdamagesDNA is observed. In lightof the abovediscussion,we believe that it is indeed possible for both Cox-2 and nNOSto act in concert to oxidatively damage DNA in the MPTPmouse model.
All neurons possess both a nuclear and a mitochondrial genome;damage to either one may carry dramatic consequences for cellfunction and survival as discussed below. Because neuronal mitochon-dria are few in number in the soma [39] and MPTP intoxicatesessentially dopaminergic neurons, it is likely that the observed ISNTlabeling in ventral midbrain reflects damage primarily to nuclear DNAin dopaminergic neurons. Supporting this view is our lack of detectionof mitochondrial DNA damage in ventral midbrain at all of the studiedtime points by Southern blot analysis using a specific mitochondrialprobe. Conversely, neuronal mitochondria are mainly found in nerveterminals [39], making it not surprising that mitochondrial DNAdamage was detected in the striatum, where SNpc dopaminergicneuron nerve terminals are found. Thus, our data suggest that, afterMPTP injury and in PD, the neurodegenerative processes may beassociated with both nuclear and mitochondrial DNA damage. Thestriatal mtDNA damage detected by alkaline Southern blot seems to betransient, starting as early as 6 h and disappearing by 10 h after MPTP.Because equal amounts of total DNA from tissue homogenates wereloaded onto the gel, this would suggest that mtDNA is rapidly repairedafter strand breaks are produced. This finding was rather unexpectedbecause nuclear DNA damage had not yet peaked at 10 h, which raisesthe possibility that the apparent distinct kinetics of loss of nuclear andmitochondrial DNA integritymay be due to different repair capabilitiesor to the cellular localization of the insult (i.e. cell body versusterminals). Relevant to the latter aspect, it has been demonstrated thatdopaminergic neurodegeneration of the terminals precedes that of thecell bodies after MPTP administration [40].
In conclusion, this work demonstrates that the parkinsonian toxinMPTP causes DNA damage as previously reported in PD autopsymaterials. The cascade of deleterious events discussed here, togetherwith previously identified noxious events such as activation of theapoptotic machinery [41], supports the potential significance of DNAdamage in the MPTP model and in PD as well.
Acknowledgments
The authors thank Mr. Matthew Lucas and Michael Shelley forassistance in preparing themanuscript, as well as Dr. Csaba Szabó for hisuseful discussionabout thePARPdata. The authors are supportedbyNIH/NINDSGrants RO1NS38586andNS42269, R21NS062180,NS064191, P01ES016732, P50NS38370, P50NS38377, P01NS11766-27A2, P50NS38367,and U54 ES12078; U.S. Department of Defense grants (W81XWH-08-1-0522,W81XWH-08-1-0465, and DAMD17-03-1); the ParkinsonDiseaseFoundation (New York, NY, USA); the Thomas Hartman Foundation ForParkinson's Research, theMDA/Wings-over-Wall Street, the Marie CurieExcellence Grant and International Reintegration Grant (EuropeanCommission), “La Caixa” Foundation (Spain), Fondo de InvestigaciónSanitaria (Instituto de Salud Carlos III, Spain).
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