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Arachidonic acid (AA) signaling is upregulated in the caudate-putamen and frontal cortex of unilaterally 6-hydroxydopamine (6-OHDA) lesioned rats, a model for asymmetrical Parkinson disease. AA signaling can be coupled to D2-like receptor initiated AA hydrolysis from phospholipids by cytosolic phospholipase A2 (cPLA2) and subsequent metabolism by cyclooxygenase (COX)-2. In unilaterally 6-OHDA- and sham-lesioned rats, we measured brain expression of cPLA2, other PLA2 enzymes, and COX-2. Activity and protein levels of cPLA2 were significantly higher as was COX-2-protein in caudate-putamen, frontal cortex and remaining brain on the lesioned compared to intact side of the 6-OHDA lesioned rats, and compared to sham brain. Secretory sPLA2 and Ca2+-independent iPLA2 expression did not differ between sides or groups. Thus, the tonically increased ipsilateral AA signal in the lesioned rat corresponds to upregulated cPLA2 and COX-2 expression within the AA metabolic cascade, which may contribute to symptoms and pathology in Parkinson disease.
Parkinson disease is a progressive neurodegenerative disorder that results from cell death in the substantia nigra and loss of dopaminergic nerve terminals in the basal ganglia (caudate-putamen) [1, 2]. L-DOPA or dopamine receptor agonists relieve some of the symptoms but do not stop disease progression .
Animal models of Parkinson disease have been developed to elucidate disease pathophysiology and to design pharmacotherapy. One such model is the rat in which a selective monoaminergic toxin, 6-hydroxydopamine (6-OHDA), has been injected into the substantia nigra or medial forebrain bundle of one hemisphere [4–7]. Injection causes rapid local neuronal death  and motor deficits within 1 week, including rotation in response to the dopaminergic D1/D2 receptor agonist, apomorphine .
The unilateral 6-OHDA lesioned rat demonstrates reduced levels of presynaptic tyrosine hydroxylase, decreased dopamine synthesis, and reduced expression of the presynaptic dopamine reuptake transporter (DAT) in the ipsilateral basal ganglia, but increased numbers and mRNA levels of postsynaptic dopamine D2-like receptors [10–15]. D2 receptors can be coupled by a G-protein to Ca2+-dependent cytosolic phospholipase A2 (cPLA2), which selectively hydrolyzes arachidonic acid (AA, 20:4n-6) from synaptic membrane phospholipids [16–19]. cPLA2 is localized at post-synaptic sites, and is in turn functionally coupled to cyclooxygenase (COX)-2 at these sites [20, 21].
Using quantitative autoradiography, we reported increased resting-state AA signaling in unanesthetized rats with a chronic unilateral 6-OHDA lesion of the substantia nigra, in ipsilateral basal ganglia and anterior cerebral cortical regions [22–24]. These regions also demonstrated elevated AA signaling responses when the rats were given quinpirole, a D2-like receptor agonist [23, 24]. The quinpirole-induced changes could be blocked by pre-administration of a D2-receptor antagonist, consistent with cPLA2 activation mediated through D2-like receptors.
These observations suggest that the AA metabolic cascade in brain regions ipsilateral to a chronic substantia nigra lesion is tonically upregulated in association with increased D2-like receptor expression. Such upregulation could lead to multiple biological effects and produce eicosanoid products that influence Parkinson symptoms [25–28]}. We decided to test this hypothesis in unilaterally 6-OHDA-lesioned rats by measuring bilateral regional brain expression of AA-selective cPLA2-(Group IVA), secretory sPLA2 (Group IIA), docosahexaenoic acid (DHA 22-6n-3)-selective Ca2+-independent iPLA2 (Group VIA), and COX-2 [20, 21, 29–32].
This study was approved by the Animal Care and Use Committee of the National Institute of Child Health and Human Development (Protocol 03-012). Experiments were conducted following the Guide for the Care and Use of Laboratory Animals (NIH Publication 86-23). Male unilaterally 6-OHDA lesioned Fischer-CDF rats were purchased from Charles River Laboratories (Wilmington, MA). At 8.6 ± 0.3 weeks of age, the rat was anesthetized with 43 mg/kg ketamine and 8.77 mg/kg xylazine, and the left substantia nigra was lesioned by a 3-min infusion of 12 µg/4 µl 6-OHDA in 0.9% saline (w/v) at the following coordinates from the bregma: AP = −1.5, ML = +1.8, and DV = −7.5 mm . Another group of rats, which had been injected with saline (0.9% NaCl) using the same stereotaxic procedures, was used as “sham” controls.
Two weeks after lesioning, the rats were shipped to our animal facility, which has a constant temperature, humidity, and light cycle (6:00 AM – 6:00 PM), and were given free access to food pellets (NIH-31 18-4 diet, Zeigler Bros, Gardners, PA, USA) and water. To assess the efficacy of the 6-OHDA lesion, each lesioned rat was tested 24 ± 4 days later for its response to S-(+)-apomorphine HCl, 0.5 mg/kg i.p. [24, 33]. Only rats that completed at least 100 contralateral rotations in 20 min were studied. Such rats are reported to have increased expression of post-synaptic D2 receptors , and loss of tyrosine hydroxylase immunoreactivity in the ipsilateral basal ganglia and substantia nigra [6, 11, 24, 35].
14.1 ± 0.7 week-old sham (n = 10) and 6-OHDA (n = 11) lesioned rats (5.1 ± 0.4 week after 6-OHDA lesion) were anesthetized with Nembutal® (50 mg/kg, i.p.) and decapitated. The brain was removed and dissected bilaterally into caudate-putamen, cortical-regions, and remaining brain including the hippocampus, thalamic and subthalamic nuclei, brain stem and cerebellum. Dissected tissue was frozen quickly in 2-methyl butane at −40°C and stored at −80°C. The frozen tissue later was homogenized using a Teflon-glass homogenizer in three volumes of ice-cold buffer containing 10 mmol/L HEPES, pH 7.5, 1 mmol/L EDTA, and 0.34 mol/L sucrose with a protease inhibitor cocktail (Roche, Indianapolis, IN). Homogenates were centrifuged at 100,000 × g for 1 h at 4°C (Beckman L8-80M Ultracentrifuge, Fullerton, CA, USA). Supernatants corresponding to the cytosolic fraction were assayed for PLA2 activities and subjected to Western blotting. The protein concentration of the cytosolic fraction was determined using Bio-Rad Protein Reagent (Bio-Rad, Hercules, CA).
Activities of individual PLA2 enzymes were measured in the cytosolic fraction as described elsewhere [36, 37], with slight modifications of extraction procedures. For cPLA2, a portion of the cytosolic fraction was incubated in 100 µM 1-palmitoyl-2-arachidonoyl-sn-glycerol-3-phosphorylcholine (Avanti, Alabaster, AL) and phosphatidylinositol 4,5-bisphosphate (97:3), containing approximately 100,000 cpm of 1-palmitoyl-2-[1-14C] arachidonoyl-sn-glycerol-3-phosphorylcholine (Perkin- Elmer, Boston, MA) and 4,5 biphosphatidylinositol (Avanti) in 400 µM triton X-100 mixed micelles containing 100 mM Hepes, pH 7.5, 80 µM calcium, 2 mM dithiothreitol and 0.1 mg/ml fatty acid free bovine serum albumin. For iPLA2, a portion of the cytosolic fraction was incubated in 100 µM 1-palmitoyl-2-palmitoyl-sn-glycerol-3-phosphorylcholine (Avanti) containing approximately 100,000 cpm of 1-palmitoyl-2-[1-14C] palmitoyl-sn-glycerol-3-phosphorylcholine (Amersham, Buckinghamshire, UK) in 400 µM Triton X-mixed micelles in 100 mM HEPES, pH 7.5, 5 mM EDTA, 2 mM DTT, and 1 mM ATP. For sPLA2 Group IIA, a portion of the cytosolic fraction was incubated in 100 µM 1-palmitoyl-2-linoleoyl-sn-glycerol-3-phosphorylethanolamine (Avanti) and 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphorylserine (Avanti) (1/1) in the form of small unilamellar vesicles containing approximately 100000 cpm of 1-palmitoyl-2-[1-14C] linoleoyl-sn-glycerol-3-phosphorylethanolamine (Amersham) in 100 mM HEPES, pH 7.5, with 1 mM calcium and 1 mg/ml bovine serum albumin. Reactions were started by adding reagent to cytoplasm extracts for 30 min at 40°C in a shaking bath. Reactions were terminated by adding Dole’s reagent (2-propanol: heptane:0.5 M sulfuric acid, 400:100:20, by volume) followed by vortexing. Released [1-14C] fatty acids were extracted with heptane and water. One ml of heptane was loaded on a bond elute reservoir with a frit preloaded with silicic acid. The unesterified fatty acids were eluted from the silicic acid by adding diethyl ether, with the help of a vacuum. Radioactivity of the eluant was determined by liquid scintillation counting and enzyme activity was calculated after correcting for the background of blank samples. All samples were run in triplicate and values are expressed in pmol/min/mg of protein.
Proteins from cytoplasmic (50 µg) extracts were separated on 4–20% SDS-polyacrylamide gels (PAGE) (Bio-Rad), and then were electrophoretically transferred to a nitrocellulose membrane. Cytoplasmic protein blots were incubated overnight in TBS buffer containing 5% nonfat dried milk and 0.1% Tween-20, with specific primary antibodies (1:200 dilution) for group IVA cPLA2, group IIA sPLA2 and group VIA iPLA2 (Santa Cruz Biotech, Santa Cruz, CA) and for cyclooxygenase-2 (COX-2) (1:500) (Cell Signaling, Beverly, MA). The cytoplasmic protein blots were incubated with appropriate HRP-conjugated secondary antibodies (Bio-Rad), visualized using a chemiluminescence reaction (Amersham, Piscataway, NJ) and exposed within 5 min on X-Ray film (XAR-5, Kodak Rochester, NY). Optical densities of immunoblot bands were measured using Alpha Innotech Software (Alpha Innotech, San Leandro, CA) and were normalized to β-actin (Sigma) to correct for unequal loading and for measuring the protein expression level. Membrane was reprobed using stripping buffer (Sigma) for repeated use. All experiments were carried out twice with 6 or 8 independent samples. Values are expressed as percent of control.
Data are expressed as means ± SEM. Statistical analysis was performed by a one-way ANOVA with Tukey’s multiple comparison tests. Statistical significance of differences was set at P < 0.05.
In the “sham” animals, protein and activity levels of cPLA2, sPLA2, iPLA2 and COX-2 protein, as well as the ratio of phosphorylated to total cPLA2 protein, did not differ significantly between the lesioned and intact hemisphere. Data from both hemispheres therefore were combined and will be referred to as “sham” data. In the lesioned animals, data are presented for both intact and lesioned sides, since 6-OHDA was injected unilaterally.
Compared to sham brain, cPLA2 activity was increased significantly on the lesioned side in the caudate-putamen (66%), cortex (55%) and remaining brain (28%) (Table 1). Compared to the intact side, cPLA2 activity was increased in the caudate-putamen (62%), and cortex (33%) on the lesioned side.
cPLA2 protein was increased significantly ipsilateral to the lesion in the caudate-putamen (40%), cortex (49%) and remaining brain (35%) compared to sham, and in the cortex (27%) compared to intact side (Table 2). The ratio of phosphorylated to total cPLA2 protein was not changed significantly in the caudate-putamen (ratio = 1.04), cortex (ratio = 0.96) or remaining brain (ratio = 0.90), compared to the ratio in the sham brain (Table 2).
Compared to the sham brain or the intact side, activities of iPLA2 and of sPLA2 were not significantly different on the lesioned side in the caudate-putamen, cortex or remaining brain (Table 1). Protein levels of the two enzymes also did not differ significantly between the groups (Table 2).
Compared to sham, COX-2 protein was increased significantly on the lesioned side in the caudate-putamen (54%), cortex (43%) and remaining brain (37%)(Table 3). Compared to intact side, COX-2 protein was increased significantly on the lesioned side in the caudate-putamen (45%), and remaining brain (34%).
The substantia nigra of adult male rats was lesioned unilaterally by an injection of 6-OHDA. Five weeks after lesioning, statistically significant increases were demonstrated in cPLA2 Group IVA activity and protein levels and in COX-2 protein in the caudate-putamen, cortex and remaining brain on the lesioned compared to the intact side or sham-operated brain. No statistically significant difference was found in sPLA2 Group IIA or iPLA2 Group VIA activity or protein level between the two sides of lesioned or sham brain. Efficacy of unilateral lesioning was confirmed by showing contralateral rotation in response to i.p. apomorphine.
In unanesthetized unilaterally-lesioned rats, at baseline (in response to saline) and following administration of the D2-like receptor agonist quinpirole, quantitative autoradiography showed increased AA signaling (measured as increased AA incorporation coefficients k*) in ipsilateral caudate-putamen and frontal cortex, [22–24]. These increases are consistent with evidence of increased density of postsynaptic D2-like receptors, which can be coupled to AA-selective cPLA2 [18, 19], in brain regions ipsilateral to a chronic substantia nigra lesion [16, 18, 19, 22, 24, 27, 38–41], and suggest tonic activation of these receptors. In contrast, protein and activity levels of sPLA2 and iPLA2 were unchanged on the lesioned side compared with the intact side. sPLA2 mediates membrane fusion during presynaptic release of neurotransmitters, whereas iPLA2 selectively hydrolyzes DHA compared to AA from membrane phospholipids [16, 29, 32, 42].
The 6-OHDA lesioned rat is considered a model for late-stage asymmetrical Parkinson disease, which is associated with an elevated D2 receptor density, reduced presynaptic dopamine uptake and a reduced DAT density in the affected caudate-putamen [43, 44]. Additionally, Ca2+-dependent PLA2 activity was reported increased in the putamen but not in other regions of the postmortem Parkinson disease brain . Based on our current and prior observations on the unilaterally lesioned rat [22–24], we predict that baseline and D2 receptor agonist-induced AA signaling would be increased in the caudate-putamen and frontal cortex of Parkinson patients. This could be tested directly using [1-11C]AA and positron emission tomography [46–49].
In normal brain, COX-2 is functionally coupled and coevolved with cPLA2 at post-synaptic neuronal membranes [20, 21, 31, 50–52]. The new finding of increased ipsilateral expression of both cPLA2 and COX-2, and earlier evidence of increased ipsilateral baseline and D2-agonist initiated AA signaling [22–24], argue that the AA cascade  initiated at D2-like receptors is upregulated in the unilaterally 6-OHDA lesioned rat model of Parkinson disease.
Microglial activation was reported in both the rat substantia nigra and striatum (but not the cortex) 4 weeks after lesioning the substantia nigra with 6-OHDA, suggesting neuroinflammation , and neuroinflammation can be accompanied by upregulated AA metabolic enzymes . On the other hand, in a chronic low-dose lipopolysaccharide-infusion rat model of neuroinflammation, associated with microglial activation, expression of both cPLA2 and sPLA2 was increased [54–56], possibly through a common effect of microglial-derived cytokines on the NF-κB transcription factor of both enzymes . Thus, while our results most likely reflect the effects of excess tonic D2-like receptor-mediated activation of cPLA2 and COX-2, associated with increased AA signaling, they may represent neuroinflammation to some extent. Additionally, D2-like receptor mediated activation of the two enzymes in the rat model might eventually increase risk for neuroinflammation and disease progression.
With regard to Parkinson disease, it generally is reported that activated microglia are localized in the substantia nigra on postmortem, but there is disagreement about whether activated astrocytes are found there or elsewhere in the postmortem brain [58–60]. CSF measurements show elevated levels of microglia-derived cytokines, interleukin (IL)-1 and IL-6 . In one report, the activity of Ca2+-stimulated PLA2 was normal except for a significant 26% increase in the putamen, which was ascribed to either decreased dopaminergic striatal input or to a dopamine nerve terminal degenerative process .
Epidemiological studies provide contradictory results regarding neuroprotection by nonsteroidal anti-inflammatory drugs in Parkinson disease patients [62–64], but the general impression is that they are not effective. In one recent study involving 6512 participants at risk, NSAIDs did not decrease risk of Parkinson disease , and a recent meta-analysis of published data supported this conclusion .
An upregulated AA cascade associated with D2-like receptor supersensitivity, if it also exists in Parkinson disease, may contribute to disease symptoms. Unesterified AA and its eicosanoid metabolites have been shown to affect multiple brain processes, including membrane excitability  long term potentiation , gene transcription , membrane fluidity , neurite outgrowth , cerebral blood flow , and as precursors of reactive oxygen species [26, 72]. Thus, cPLA2 and COX-2 enzymes might be considered as targets in the treatment of Parkinson disease.
In summary, protein and activity levels of cPLA2 and the protein level of COX-2 were increased in caudate-putamen and frontal cortex ipsilateral to a 5-week 6-OHDA lesion of the substantia nigra in rats, a model of late stage asymmetrical Parkinson disease. These increases correspond to reported upregulated AA signaling in the regions, at baseline and following pharmacological D2 receptor stimulation, elevated expression of post-synaptic D2 receptors, and reduced presynaptic DAT and tyrosine hydroxylase immunoreactivity. An upregulated AA cascade thus may exist in Parkinson disease and contribute to its symptoms.
This research was supported entirely by the Intramural Program of the National Institute on Aging.