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Published in final edited form as: Brain Res Bull. 2007 Mar 20;73(1-3):108–113. doi: 10.1016/j.brainresbull.2007.02.015

Gene expression of cyclooxygenase-1 and Ca2+-independent phospholipase A2 is altered in rat hippocampus during normal aging

Saba Aïd 1, Francesca Bosetti 1,*
PMCID: PMC1945113  NIHMSID: NIHMS24082  PMID: 17499644

Abstract

Brain aging is associated with inflammatory changes. However, data on how the brain arachidonic acid (AA) metabolism is altered as a function of age are limited and discrepant. AA is released from membrane phospholipids by phospholipase A2 (PLA2) and then further metabolized to bioactive prostaglandins and thromboxanes by cyclooxygenases (COX) -1 and -2. We examined the phospholipase A2 (PLA2)/cyclooxygenase (COX)-mediated AA metabolic pathway in the hippocampus and cerebral cortex of 4, 12, 24 and 30 month-old rats. A 2-fold increase in brain thromboxane B2 level in 24 and 30 months was accompanied by increased hippocampal COX-1 mRNA levels at 12, 24, and 30 months. COX-2 mRNA expression was significantly decreased only at 30 months. Hippocampal Ca2+-independent iPLA2 mRNA levels were decreased at 24 and 30 months without any change in Ca2+-dependent PLA2 expression. In the cerebral cortex, mRNA levels of COX and PLA2 were not significantly changed. The specific changes in the AA cascade observed in the hippocampus may alter phospholipids homeostasis and possibly increase the susceptibility of the aging brain to neuroinflammation.

Keywords: aging, cyclooxygenases, calcium-independent phospholipase A2, hippocampus, thromboxane, prostaglandin

1. Introduction

Aging is associated with increased inflammatory responses and vulnerability of neurons to degeneration [17, 18, 22, 39]. Some authors have raised the possibility that inflammation may occur during normal aging and increase the vulnerability to neurodegenerative disorders such as Alzheimer’s disease (AD) [7, 20, 26]. Since the arachidonic acid (AA) cascade plays a key role in neuroinflammation [37], we thought it of interest to identify the changes that occur in this pathway during physiological aging.

AA (20:4n-6) is released from membrane phospholipids by phospholipase A2 (PLA2) enzymes. The PLA2 family can be generally divided into two groups: the Ca2+-independent PLA2 (iPLA2) and the Ca2+-dependent PLA2, which includes secretory (sPLA2) and cytosolic PLA2 (cPLA2) [24]. iPLA2 is thought to mediate homeostatic phospholipid remodeling through fatty acid deacylation/reacylation reactions [49] and may also be involved in cellular signaling [5]. In contrast to cPLA2, which releases predominantly AA, iPLA2 has been suggested to selectively release docosahexaenoic acid (DHA; 22:6n-3), a n-3 polyunsaturated fatty acid highly concentrated in brain membranes [42, 43]. Once released, AA is then converted to bioactive prostaglandins and thromboxanes by cyclooxygenase (COX) enzymes. Two isoforms of COX have been described: COX-1, constitutively expressed in most tissues [30], but also induced by certain inflammatory stimuli in peripheral tissues [46], and COX-2, generally induced by inflammatory stimuli including cytokines, hormones, and mitogens [44], but also constitutively expressed in the central nervous system, especially in pyramidal neurons of hippocampal and cortical circuits [50].

Learning and memory deficits have been well documented in aged F344 rats [14, 21, 41, 45]. Activated microglia and astrocytes [16] and elevated levels of pro-inflammatory cytokines [10, 18, 51] have been described in the rodent brain during normal aging. However, the involvement of COX-mediated AA metabolism in this “pro-inflammatory-status” remains unclear [4, 23, 38]. Aging-related changes in COX-mediated AA metabolism are suggested by age-dependent spatial memory deficits and increased neuronal apoptosis and astrocytic activation in transgenic mice overexpressing COX-2 [2]. We have previously shown in the Rhesus monkey an age-dependant decrease in the levels of cPLA2 protein in the cerebellum and of COX-2 protein in the frontal pole [47]. Taken together, these data suggest that AA metabolism may be disturbed in normal aging. We chose two morphologically and functionally distinct regions, the hippocampus and the cerebral cortex, that have been shown to be affected by the aging process [14, 35]. We assessed age-related changes in the metabolism of AA, with a particular focus on the PLA2/COX pathway across a range of 4 ages (4, 12, 24, and 27–30 months).

2. Materials and methods

2.1 Animals

The study was approved by the National Institutes of Health (NIH) Animal Care and Use Committee in accordance with NIH guidelines on the care and use of laboratory animals. Male Fischer-344 rats, 4, 12, 24 and 27–30 month-old (NIA-sponsored colony at Harlan Sprague-Dawley, Indianapolis, IN) were housed at least one week in our animal facility, maintained at 25°C with a 12 hr light/dark cycle, with free access to food and water. All rats were killed with an overdose of sodium pentobarbital (100 mg/kg, i.p.). Then, rats used to measure prostaglandin levels were subjected to high-energy head-focused microwave irradiation (4.8 kW, 3.4 sec, Cober Electronics, Stanford, CT, USA) to stop metabolism, as reported [8, 9, 33]. Rats used for mRNA analysis were decapitated, hippocampus and cerebral cortex were rapidly dissected out on ice, immediately frozen in −50°C 2-methylbutane, and stored at −80°C until used.

2.2 Determination of brain prostaglandin E2 (PGE2) and thromboxane B2 (TXB2) concentration

After microwave fixation, the amount of the brain extract required to perform analysis did not allow measuring prostaglandin levels in individual brain regions without pooling samples from different animals. Therefore, we measured PGE2 and TXB2 concentration in the whole brain. Microwaved brains were weighed and extracted with 18 volumes of hexane: 2-propanol (3:2, by volume) using a glass Tenbroeck homogenizer. The prostanoids were purified from the lipid extract as described by Radin [34] and the concentrations of PGE2 and TXB2 were determined using specific enzyme-linked immunosorbent assay kits (Oxford Biomedical, Oxford, MI, USA), as previously reported [8]. According to the manufacturer, the sensitivity of the PGE2 and TXB2 assays are 0.2 ng/ml and 0.009 ng/ml at 80% B/Bo, respectively.

2.3 Real-Time Quantitative PCR

Total RNA was extracted from hippocampus or cerebral cortex using RNeasy Lipid Tissue Midi Kit (Qiagen, Valencia, CA, USA) as directed by the manufacturer. Five μg of total RNA was reverse transcribed using a High Capacity cDNA Archive kit (Applied Biosystems, Foster City, CA, USA). Five μg of each RNA sample was incubated similarly in the absence of reverse transcriptase to ensure that PCR products resulted from amplification from the specific mRNA rather than from genomic DNA contamination.

The levels of gene expression of COX-1, COX-2, cPLA2, iPLA2, glial fibrillary acidic protein (GFAP) were measured by real-time quantitative RT-PCR, using the ABI PRISM 7000 Sequence Detection System (Applied Biosystems). Specific primers and probes were purchased from the available Assays-on-Demands (Applied Biosystems). We used glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an endogenous control, as its expression has been shown to remain unchanged during aging [40]. Data were analyzed using sequence detection systems software (Applied Biosystems). Results were expressed as the amount of target gene normalized to the endogenous control (GAPDH) and relative to the 4 month-old rats using the ΔΔCT method [25].

2.4 Statistical analysis

Results are expressed as mean ± SEM. Statistical analysis was performed using One-Way ANOVA followed by post-hoc Bonferroni test. P < 0.05 was considered statistically significant.

3. Results

3.1 TXB2 levels are specifically increased in the aged brain

Levels of TXB2, a stable product of the very short half-lived TXA2, were increased by 2 fold [F(3,19) = 4.89, p = 0.010] in 24 and 30 month-old group compared to the 4 month-old group (Fig. 1A). PGE2 levels (Fig. 1B) showed a trend towards an increase at 24 months, which did not reach statistical significance among the different groups [F(3,19) = 2.71, p = 0.072].

Fig. 1.

Fig. 1

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Thromboxane B2 (A) and prostaglandin E2 (B) levels in the microwaved brain of 4, 12, 24 and 30 month-old F344 rats. Data (mean ± SEM, n= 5–6) are expressed as the percentage of the 4 month-old rats. p values are expressed as the comparison of aged rats with 4-month-old rats using One-Way ANOVA (*p<0.05, **p<0.01; post-hoc Bonferroni test).

3.2 COX-1 mRNA expression is upregulated whereas COX-2 mRNA expression is downregulated in the hippocampus during aging

Hippocampal COX-1 mRNA expression (Fig. 2A) was increased by 20% at 12 months and by 27% at 24 and 30 months compared to the 4 months group [F(3,19) = 28.03, p < 0.0001]. In contrast, hippocampal COX-2 mRNA expression (Fig. 2B) was statistically reduced by 15% in the 30 months group compared to the 4 months group [F(3,19) = 4.47, p = 0.015]. COX mRNA expressions (Table 1) were not changed in the cerebral cortex of all age groups examined [COX-1: F(3,19) = 0.37, p = 0.773; COX-2: F(3,19) = 2.97, p = 0.058].

Fig. 2.

Fig. 2

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COX-1 and COX-2 mRNA expression in the hippocampi (A-B) of 4, 12, 24 and 30 month-old F344 rats. Data (mean ± SEM, n=5–6) are normalized to the level of the internal control, GAPDH and are expressed as the percentage of the 4 month-old rats. p values are expressed as the comparison of aged rats with 4-month-old rats using One-Way ANOVA (*p<0.05, ***p<0.001; post-hoc Bonferroni test).

Table 1.

mRNA expression of genes involved in the PLA2/COX pathway in the rat cerebral cortex during aging using quantitative real–time PCR.

Genes1 4 months (n=6) 12 months (n=6) 24 months (n=6) 30 months (n=5)
COX-1 100 ± 2 100 ± 5 105 ± 9 108 ±7
COX-2 100 ± 5 126 ±12 100 ± 3 97 ± 8
cPLA2 101 ± 5 113 ± 6 118 ± 6 105 ± 7
iPLA2 100 ± 3 96 ± 4 90 ± 6 88 ± 6
GFAP 101 ± 6 122 ± 4 206 ± 12 *** 241 ± 11 ***
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1

Data (mean ± SEM, n=5–6) are normalized to the level of the internal control, GAPDH, and are expressed as the percentage of values in the 4 month-old rats. p values are expressed as the comparison of aged rats with 4-month-old rats using One-Way ANOVA

***

p<0.01; post-hoc Bonferroni test.

3.3 Hippocampal iPLA2 (VI) mRNA expression is downregulated in aged rats

Hippocampal iPLA2 mRNA expression (Fig. 3A) was decreased by 15% at 24 and 30 months compared to the 4 months group [F(3,19) = 4.67, p = 0.013]. Hippocampal cPLA2 mRNA expression level (Fig. 3B) was not changed in all age groups examined [F(3,19) = 0.32, p = 0.81], neither did cortical cPLA2 [F(3,19) = 1.89, p = 0.165] and iPLA2 mRNA expression levels [F(3,19) = 1.37, p = 0.283] (Table 1).

Fig. 3.

Fig. 3

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iPLA2 (VI) and cPLA2 mRNA expression in the hippocampi (A-B) of 4, 12, 24 and 30 month-old F344 rats. Data (mean ± SEM, n=5–6) are normalized to the level of the internal control, GAPDH and are expressed as the percentage of the 4 month-old rats. p values are expressed as the comparison of aged rats with 4-month-old rats using One-Way ANOVA ( *p<0.05; post-hoc Bonferroni test).

3.4 GFAP mRNA expression is increased in hippocampus and cerebral cortex during aging

To confirm aging-related astrocytic activation, we measured the expression of GFAP, a specific marker of astrocytes. Hippocampal GFAP mRNA expression (Fig. 4) was increased by 46% at 24 months and by 72 % at 30 months compared to the 4 months group [F(3,19) = 31.21, p < 0.0001]. Cortical GFAP mRNA expression (Table 1) was increased by 2 fold at 24 months and by 2.4 fold at 30 months compared to the 4 months group [F(3,19) = 60.74, p < 0.0001].

Fig. 4.

Fig. 4

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GFAP mRNA expression (B) in the hippocampi of 4, 12, 24 and 30 month-old F344 rats. Data (mean ± SEM, n=5–6) are normalized to the level of the internal control, GAPDH, and are expressed as the percentage of the 4 month-old rats. p values are

4. Discussion

In this study, we found that brain TXB2 level was increased by 2 fold in 24 and 30 month-old rats compared to 4 month-old rats and we also showed that parts of the AA cascade are specifically altered in the hippocampus during normal aging: namely 1) COX-1 expression is increased, 2) COX-2 expression is decreased, and 3) iPLA2, but not cPLA2, expression is decreased. In contrast, we did not find any significant change in the expression of COX or iPLA2 in the cerebral cortex from aged rats.

The upregulation of COX-1 mRNA expression appeared quite early in adulthood (12 months) and therefore can be viewed as part of the normal aging process. In contrast, COX-2 mRNA level was downregulated only at 30 months. Previous studies have examined hippocampal and cortical COX-2 mRNA expression in different strains of rats aged from 3 to 27 months and reported no change [4, 23]. Therefore, COX-2 gene expression appears to become affected only in the advanced phase of the aging process. COX-1 upregulation as a function of age may have different implications. First, it may be responsible for the increased brain levels of TXB2, an AA-derived prostaglandin, via the subsequent metabolism of COX and thromboxane synthase. Indeed, evidence indicates that thromboxane synthase preferentially couples with COX-1 for TXB2 production [11]. Although brain microvessels also contribute to COX-1 derived TXB2 synthesis, since microvessels represent only 0.1 % of the whole brain [48] the increase in TXB2 that we observed in this study is unlikely significantly contributed by the cerebral circulation. However, since in this study TXB2 and PGE2 levels were determined in the whole brain, it would be interesting to further examine the regional distribution of the changes in prostaglandin during normal aging. An increase in COX activity has been reported in the cerebrum of F344 rats at 24 months versus 6 month-old [4], corresponding approximately to the age at which we found increased levels of TXB2 in this study. Since COX-1 is mainly localized in glia, as opposed to COX-2 which is mainly in neurons [13, 50, 52], the upregulation of COX-1 could be associated with the aging-related glial activation. In particular, the age-related increase of GFAP mRNA that we show has been well documented in human and rodent brain [19, 29, 32]. Glial activation may contribute to neuronal dysfunction as both microglia and astrocytes become activated early during physiological aging [12, 16]. It remains unclear why only the hippocampus is responsive to the age-related astrocytic activation which occurred in several brain regions. It is unlikely that COX-1 upregulation in the hippocampus alone could account for the increase of TXB2 level in the whole brain. It is possible that other cerebral regions that we did not examine may have an upregulated COX-1 expression. Another possibility is that increased hippocampal COX-1 mRNA expression is accompanied by an upregulation in thromboxane synthase activity in the hippocampus and/or in other regions. Even though age-related changes occur in the whole brain, the aging process can exhibit regional specificity, the hippocampus being especially sensitive [6, 14].

Only few reports have addressed changes in brain prostanoid production with aging. Using brain microwaving to stop lipid metabolism in vivo [3, 9], we demonstrated an increased level of TXB2 in the brains of 24 and 30 month-old animals compared to 4 month-old rats. The same tendency was observed for PGE2 levels, although it did not reach statistical significance. Prostaglandins have been shown to have numerous effect on astrocytes in vitro including upregulating GFAP expression [31]. Thus, it should be further clarified if the astrocytic activation observed during aging is either the consequence or the cause of altered AA metabolism. COX-1 upregulation with aging might play a role in altering the neuroinflammatory response and increasing vulnerability to neurodegenerative diseases with a marked inflammatory component [28].

We also found a decrease in the Ca2+-independent iPLA2 mRNA levels in the hippocampus of 24 and 30 month-old rat. In contrast to cPLA2, which preferentially cleaves AA from membrane phospholipids, iPLA2 is thought to show selectivity towards docosahexaenoic acid (DHA) [42]. DHA, the major n-3 polyunsaturated fatty acid found in cerebral membranes, plays a crucial role in physiological functions such as neurotransmission, membrane fluidity, ion channel and regulation of enzyme activity and gene expression [1], and it may protect against cognitive decline [27]. The decrease in iPLA2 expression observed in this study is consistent with evidence that the DHA content in cerebral phospholipids is decreased during normal aging [15] and that cortical iPLA2 is downregulated in DHA-deprived rats [36]. Considering the role of iPLA2 in regulating homeostatic phospholipid levels [49], the downregulation of iPLA2 expression during aging might alter phospholipid homeostasis by decreasing the release of DHA in the hippocampus. Therefore, the age-related decrease of AA and DHA in membrane phospholipids [15], coupled with the downregulation of iPLA2, could contribute to increase the ratio of free AA/DHA and consequently alter the brain prostanoid profile. These changes may exert profound effects in the hippocampus, a region highly vulnerable to the aging process [6, 14].

In summary, our results indicate that the mRNA expression of three important enzymes in the AA metabolic pathway, COX-1, COX-2, and iPLA2, is altered in the hippocampus during normal aging. These changes may alter brain phospholipids homeostasis and possibly increase the susceptibility of the aging brain to neuroinflammation. Future studies should address the functional consequences of the changes in gene expression described here by examining protein expression and activity of the AA cascade enzymes, as well as regional distribution of COX-1 within all brain regions during aging.

Acknowledgments

This work was supported by the Intramural Research Program of the National Institute on Aging, National Institutes of Health.

Footnotes

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