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(The FASEB Journal. 1998;12:439-449.)
© 1998 FASEB

RESEARCH COMMUNICATION

Aging-associated up-regulation of neuronal 5-lipoxygenase expression: putative role in neuronal vulnerability

Tolga Uza, Christine Pesolda, Patrizia Longonea, and Hari Maneva,1

a The Psychiatric Institute, University of Illinois at Chicago, Chicago, Illinois 60612, USA


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Aging is associated with neurodegenerative processes. 5-Lipoxygenase (5-LO), which is also expressed in neurons, is the key enzyme in the synthesis of leukotrienes, inflammatory eicosanoids that are capable of promoting neurodegeneration. We hypothesized that neuronal 5-LO expression can be up-regulated in aging and that this may increase the brain's vulnerability to neurodegeneration. We observed differences in the distribution of 5-LO-like immunoreactivity in various brain areas of adult young (2-month-old) vs. old (24-month-old) male rats. Greater 5-LO-like immunoreactivity was found in old vs. young rats, in particular in the dendrites of pyramidal neurons in limbic structures, including the hippocampus, and in layer V pyramidal cells of the frontoparietal cortex and their apical dendrites. The aging-increased expression of neuronal 5-LO protein appears to be due to increased 5-LO gene expression. Using a quantitative reverse transcription/polymerase chain reaction assay and 5-LO-specific oligonucleotide primers and their mutated internal standards, we observed about a 2.5-fold greater hippocampal 5-LO mRNA content in old rats. 5-LO-like immunoreactivity was also observed in small, nonpyramidal cells, which were positive for glutamic acid decarboxylase or glial fibrillary acid protein. This type of 5-LO immunostaining did not increase in the old rats. Hippocampal excitotoxic injury induced by systemic injection of kainate was greater in old rats. Neuroprotection was observed with the 5-LO inhibitor, caffeic acid. Together, these results suggest that aging increases both neuronal 5-LO expression and neuronal vulnerability to 5-LO inhibitor-sensitive excitotoxicity, and indicate that the 5-LO system might play a significant role in the pathobiology of aging-associated neurodegenerative diseases.—Uz, T., Pesold, C., Longone, P., Manev, H. Aging-associated up-regulation of neuronal 5-lipoxygenase expression: putative role in neuronal vulnerability. FASEB J. 12, 439–449 (1998)


Key Words: 5-LO • aging • kainate • excitotoxicity • DNA damage • hippocampus • inflammation • caffeic acid • leukotriene


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
5-LIPOXYGENASE (5-LO; ARACHIDONATE: OXYGEN 5-OXIDOREDUCTASE, EC 1.13.11.34)2 is the key enzyme in a cascade that leads to the formation of leukotrienes from arachidonic acid. Leukotrienes, along with the products of cyclooxygenase activity (prostaglandins and thromboxanes), belong to a family of biologically active molecules called eicosanoids. Leukotrienes are inflammatory lipid mediators that can induce a variety of responses, including chemotaxis of leukocytes and increased vascular permeability (1), as well as numerous effects in neurons that are not well understood (2, 3). The action of leukotrienes in the brain has been shown to include brain injury (4, 5). In the central nervous system, 5-LO is expressed in various regions, including the cerebellum and the hippocampus. In the latter region, the expression of 5-LO mRNA is most prominent in pyramidal neurons (6).

5-LO requires multiple factors to become fully active, including rises in intracellular calcium that cause the translocation of this enzyme from cytosol to membrane. At the membrane level, 5-LO interacts with an integral membrane protein, 5-LO-activating protein (FLAP) (7, 8). Activation of 5-LO appears to be involved in neurodegenerative processes in the brain. During the reperfusion of an ischemic brain, it was observed that neuronal 5-LO translocated from cytosol to membrane and that this was accompanied by increased production of leukotrienes (9). Moreover, ischemic neuronal injury was attenuated by 5-LO inhibition (10). An excitatory neurotransmitter, glutamate, plays a crucial role in the neurodegenerative processes operative both in acute injuries such as brain ischemia and in chronic, aging-associated illnesses such as Alzheimer's disease (for a review, see ref 11). A direct stimulation of glutamate receptors by kainate induces neuronal damage predominantly in the limbic cortex. This effect of kainate was associated with increased leukotriene production (12).

Significant changes in 5-LO activity/expression have been correlated with aging. For example, it was found that expression of the 5-LO protein and the activity of the 5-LO enzyme are greater in alveolar macrophages obtained from old as opposed to young cattle (13) and that the generation of 5-LO products increases with aging in both mice and humans (14). On the other hand, aging is also associated with neurodegenerative processes, such as Alzheimer's disease, which include an inflammatory component in their pathobiological mechanism (15–17). Not only is the brain pathology of the elderly characterized by chronic neurodegeneration, but the aging brain appears to be more vulnerable when exposed to acute insults such as stroke (18). Although anti-inflammatory treatments are being considered for both acute and aging-associated neurodegenerations, the cyclooxygenase rather than the lipoxygenase pathway is being considered as the main target (19, 20).

In this work, we hypothesized that neuronal 5-LO expression can be up-regulated in aging and that this may increase the brain's vulnerability to neurodegeneration. We found confirmatory evidence for this hypothesis in experiments performed in adult young (2-month-old) and old (24-month-old) male rats.


   MATERIAL AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Studies were performed with 2- and 24-month-old male Fisher 344 rats. The animals were housed under conditions of controlled temperature (23±2°C) and illumination (14 h light/10 h darkness; darkness commenced at 18:00). Experiments were performed between 10:00 and 16:00. The experimental protocol was approved by the Institutional Animal Care and Use Committee.

Drug treatment
Kainate (KA; Sigma, St. Louis, Mo., 10 mg/kg) was dissolved in phosphate-buffered saline and the pH was adjusted to 7.4 by the addition of NaOH. This solution was injected into young or old rats intraperitoneally. The corresponding controls received saline. Rats that expressed and survived generalized seizures triggered by KA (for details, see ref 21) were killed 24 or 72 h after KA injection and their brains were analyzed.

The 5-LO inhibitor caffeic acid (Sigma) (22) was dissolved in saline and 50 mg/kg was injected intraperitoneally four times: 20 min prior to KA, immediately after KA, and 1 h and 2 h after KA.

Immunocytochemical procedures
Rats were anesthetized with equithesin and perfused through the heart with 0.9% saline in 50 mM phosphate buffer, followed by 4% buffered paraformaldehyde. Brains were removed, kept overnight in the same fixative, and then in the 30% sucrose at 4°C until processing (maximum of 2 wk). They were sectioned on a sliding microtome to a thickness of 20 µm (400-µm apart, starting at level -1.8, according to ref 23). Sections were stored at -20°C in a cryoprotectant solution consisting of ethylene glycol, glycerol, and phosphate buffer.

5-LO immunoreactivity
Free-floating sections were washed with Tris-buffered saline (TBS, 0.1 M, pH 7.4) and pretreated with 3% goat serum and 0.1% Triton X-100 in TBS for 20 min, followed by 1% bovine serum albumin (BSA) blocking rinses. Sections were incubated overnight at 4°C with a polyclonal rabbit 5-LO antiserum (1:200; LO-32 antiserum, kindly provided by Dr. J. Evans, Merck Frosst, Canada). To check the specificity of the 5-LO antiserum, control sections were treated with nonimmunized rabbit serum or incubated without the primary antibody. Primary and secondary antibodies were diluted in TBS containing Triton-X 100. Samples were washed repeatedly in TBS containing 1% goat serum, incubated with biotinylated goat anti-rabbit IgG (4 µl/ml, Vector, Burlingame, Calif.), washed repeatedly with TBS, and incubated with avidin and biotinylated horseradish peroxidase complex (ABC Elite kit, Vector) in TBS. Secondary and tertiary reactions were carried out at room temperature for 1 h. Peroxidase immunoreactivity was developed by 3,3'-diaminobenzidine tetrahydrochloride (DAB; Sigma) in Tris buffer (0.1M, pH 7.4) containing D-glucose (7 mM), ammonium chloride (7.5 mM), and glucose oxidase (0.7 U/ml).

GFAP immunoreactivity
Astrocytes were identified by their immunoreactivity for glial fibrillary acidic protein (GFAP) (24). Sections were incubated with a monoclonal mouse anti-GFAP antibody (1:500; Boehringer-Mannheim, Mannheim, Germany) overnight at 4°C, followed by incubation with the biotinylated goat anti-mouse secondary antibody (1:200; Vector).

GAD immunoreactivity
The brain samples were treated with an antibody (GAD67, 1:2000, Chemicon, El Segundo, Calif.) that recognizes the active form of glutamic acid decarboxylase (GAD), the enzyme responsible for synthesis of {gamma}-aminobutyric acid (GABA). The primary antibody was incubated overnight at 4°C, followed by incubation with the secondary biotinylated goat anti-rabbit antibody (1:250; Vector).

Gold immunolabeling
To demonstrate the colocalization of 5-LO and GFAP- or GAD-positive cells, double labeling was performed using a combination of gold immunolabeling (25) and DAB immunostaining. After incubation with the 5-LO primary antibody, sections were treated with gold-labeled secondary antibody (goat anti-rabbit) diluted 1:200 in 1% BSA; after several rinses with 1% BSA and water, the sections were incubated for 12 min with a silver enhancing solution. Thereafter, the same sections were double labeled with GFAP or GAD antibodies as described earlier.

Quantitative reverse transcription/polymerase chain reaction (RT/PCR) assay of mRNA content
RNA isolation from brain tissue
Hippocampal samples were dissected and frozen on dry ice. Frozen tissue was homogenized in 4 M guanidine isothiocyanate, 50 mM Tris/HCl (pH 7.4), and 25 mM EDTA-Na2 and total RNA was isolated by CsCl2 ultracentrifugation, as described previously (26). The yield of total RNA was determined by measuring the absorbance of an aliquot of the precipitated stock at 260/280 nm. After each extraction, one sample was run by RT-PCR without adding reverse transcriptase enzyme in order to check for possible DNA contamination.

Oligonucleotides
Amplification primers were synthesized on the Model 381A DNA synthesizer (Applied Biosystems, Foster City, Calif.) by using phosphoramidite chemistry, leaving the terminal dimethoxytrityl group intact. All primers were purified by reverse-phase chromatography, using oligonucleotide purification columns (Applied Biosystems), following the manufacturer's manual. The primer pairs were designed to allow amplification of 488 to 1002 bp for 5-LO (27) and 177 to 474 bp for cyclophilin (cyc) (28). Each primer contained a comparable G/C content to minimize variability in hybridization efficiency at the annealing temperature ( Table 1). The specificity of the cyclophilin products was checked by sequencing the amplified area, using the Sequenase Version 2.0 DNA Sequencing Kit (USB/Amersham, Arlington Heights, Ill.). The identity conformation of the 5-LO product was checked by restriction site analysis using AvaI and HinfI, which produced fragments of the expected size (107 and 409 bp, and 278 and 238 bp, respectively; data not shown).


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  		Table 1. 5-Lipoxygenase (5-LO) and cyclophilin (cyc) PCR amplification primers and internal standardsa

Synthesis and cloning of internal standards
Internal standard templates were generated by site-directed mutagenesis using PCR overlap extension, as previously described (29). Each standard was designed to introduce a BglII restriction site midway between the amplification primers so that the digestion of the amplicon would generate two fragments of approximately equal molecular size. As illustrated in Table 1, the single-strand internal primers were designed and synthesized so that the restriction site was introduced with only a minimal number of base substitutions. The internal primers were designed such that there was a 24-bp overlap of the primary PCR products ( Table 1). Each internal standard was synthesized in two PCR steps, starting with a cDNA template reverse-transcribed from the total adult rat hippocampus RNA. The first PCR step: different concentrations of heat-denatured linear starting template (from 1 to 100 ng) were amplified with 1 µmol of either the 5' external and 3' internal or the 3' external and 5' internal primers. PCR was performed with 1.5 U of Hot Tub DNA polymerase in a 100 µl reaction volume containing 200 µM dNTPs (deoxynucleotide triphosphates), 1.5 mM MgCl2, 50 mM Tris-HCl (pH 9.0), 20 mM ammonium sulfate, and 15 mM KCl. Amplicons from the first PCR step were extracted and purified from low melting point agarose. The second PCR step: increasing and equivalent amounts of the two amplicons from the first PCR were pooled; a second PCR reaction was performed with the two external primers containing the cloning sites EcoRI and HindIII. The final product was purified from low melting point agarose and digested with BglII to verify the presence of the restriction site. After digestion with EcoRI-HindIII, the material was extracted from low melting point agarose and cloned into the corresponding sites of pGem-3Z by using standard cloning methodology (30). In vitro cRNA synthesis: The internal standard templates were linearized either with SspI, which cuts 601 bp downstream of the EcoRI 3' cloning site (for 5-LO), or with PvuII, which cuts 269 bp downstream of the HindIII 3' cloning site (for cyc). The cRNA corresponding to the sense strand was synthesized using 4–8 µg of the linearized template and either Sp6 (for 5-LO) or T7 (for cyc) RNA polymerase, using an in vitro transcription kit. Typically, we obtained 30–40 µg of cRNA when using the manufacturer's recommended conditions for large-scale cRNA synthesis. Aliquots of the stock cRNA were used to obtain reproducible concentration measurements based on the optical density at the wavelength 260/280 nm.

Quantitative analyses of 5-LO and cyc mRNAs: competitive RT-PCR
Decreasing concentrations of either 5-LO or cyc internal standard cRNA were added to 1 µg of the total RNA isolated from hippocampal samples. The RNA/cRNA mixtures were denaturated at 80°C for 6 min and then reverse transcribed with cloned Moloney murine leukemia virus reverse transcriptase (Gibco, BRL, Chagrin Falls, Ohio; 200 U) in RT buffer containing 50 mM Tris/HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 1 mM dNTPs (Gibco, BRL), using random hexamers (Pharmacia Biotech, Piscataway, N.J.; 5 mM) and ribonuclease inhibitor (HPRI) (Amersham; 28 U) in a volume of 20 µl. The RT mixture was incubated at 37°C for 60 min to promote cDNA synthesis. The reaction was terminated by heating the samples at 98°C for 5 min and the mixture was quick-chilled on ice. As a control, in all assays one RT reaction was performed in the absence of RNA. Competitive PCR amplification: After termination of the RT reaction, cDNA aliquots containing reverse transcribed material were amplified with Hot Tub DNA polymerase (Amersham) in the Thermal Cycler (Perkin Elmer, Oak Brook, Ill., 9600). The amplification mixture contained cDNA, 0.5 µM specific primer pairs, 200 µM dNTPs, 1.5 mM MgCl2, 50 mM Tris-HCl (pH 9.0), 20 mM ammonium sulfate, 15 mM KCl, and 1.5 U of Hot Tub polymerase in a 100 µl volume. Trace amounts of [32P]dCTP (Amersham; 0.5–1 µCi/sample) were included during the PCR step for subsequent quantification. The PCR mixture was amplified for 30 cycles with denaturation (94°C, 15 s), annealing (60°C, 30 s), and elongation (72°C, 30 s) amplification steps. The reaction was terminated with a 5-min final elongation step. After amplification, aliquots were digested with BglII in triplicate and run by agarose gel electrophoresis (for example, see Fig. 5A).



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  		Figure 5. Quantitative RT-PCR assay of 5-LO mRNA in the hippocampus of young and old rats. Quantitative RT-PCR assay was performed using specific internal standards (see Material and Methods for details). The photographs (A) show examples of typical gels obtained after competition with 5-LO or cyc probes and internal standards. 5-LO gel: upper band, 515 bp 5-LO RNA + internal standards; lower bands, 242 + 273 bp digested internal standard cRNA pieces. Cyc gel: upper band, 298 bp cyc RNA + internal standards; lower bands, 144 + 154 bp digested internal standard cRNA pieces. In each gel, triplicate samples were analyzed: triplets of five different (80, 60, 40, 20, 10 pg), decreasing concentrations of internal standards. The graph (B) shows the result of quantitative analysis: absolute amounts of 5-LO and cyc mRNAs were assayed in the hippocampi of four rats per group (see Results) and are expressed as 5-LO/cyc ration. Bars are the mean ± SEM; P < 0.05 (Student's t test).

To quantitate the amount of the product corresponding to the RT and amplified mRNA, the ethidium bromide-stained bands were excised and the radioactivity was determined by Cerenkov counting (31). The results are expressed as 5-LO or cyc mRNA attomol/µg total RNA and as the ratio 5-LO/cyc mRNA. Statistical analysis was performed using the Student's t test (significance was accepted at P<0.05).

Quantitation of kainate-induced damage: TUNEL and Nissl assays
These assays were performed as described elsewhere (32, 33). Briefly, DNA damage was detected in situ by using the immunocytochemical terminal deoxynucleotidyl transferase (TdT) mediated dUTP-biotin nick end labeling (TUNEL) technique (ApopTag, Oncor). After sections were mounted onto silanized slides (Oncor, Gaithersburg, Md.), they were rehydrated and exposed to proteinase K (Boehringer-Mannheim, 20 mg/ml, 15 min). The endogenous peroxidase activity was quenched with 2% H2O2 and slides were incubated with working-strength TdT enzyme, followed by working-strength stop/wash buffer. After adding of antidigoxigenin-peroxidase, peroxidase activity was detected with 3,3'-diaminobenzidine tetrahydrochloride. Adjacent sections of tissue were stained with thionin, using standard methods (Nissl staining) to identify neuronal loss. Quantitative analysis of TUNEL- or Nissl-stained sections was performed using a computer-assisted morphometry; the results are expressed either as a number of TUNEL-positive cells or as a percent loss of Nissl staining (32–34). Statistical analysis was performed with the Student's t test. Significance was accepted at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
5-LO immunostaining in brain sections from young and old rats
Staining of the hippocampal brain sections with 5-LO antiserum revealed a striking difference between young and old rats. Although in both age groups, 5-LO-like staining was prominent in the cell bodies of hippocampal pyramidal neurons ( Fig. 1 shows examples of this staining in CA1 and CA3 neurons), only in old rats did we observe very intense 5-LO-like staining in the apical dendrites of those cells that project to stratum radiatum and lacunosum moleculare. Moreover, we observed similar differences between young and old rats in the distribution of 5-LO-like immunostaining in coronal sections of amygdaloid nuclei and the entorhinal and frontoparietal cortices: only in old rats did we observe a high expression of 5-LO in the principal pyramidal cells and their apical dendrites ( Fig. 2 and Fig. 3). In the frontoparietal cortex of old rats, 5-LO was highly expressed in layer V pyramidal cells and their apical dendrites ( Fig. 3).



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  		Figure 1. 5-LO immunostaining in the hippocampus reveals a different distribution of 5-LO-like immunoreactivity in young (2 months) vs. old (24 months) male Fisher 344 rats. Left panels (A, C) show results obtained in samples from young rats; right panels (B, D) are from old rats. A, B) CA1 field of the hippocampus; C, D) CA3 field. Note that in the hippocampus of young rats, 5-LO-like immunostaining is restricted to pyramidal cell bodies, whereas in the old rats 5-LO-like immunostaining is also highly expressed in the apical dendrites of pyramidal cells. No positive immunoreactivity was observed when 5-LO antiserum was omitted from the staining procedure, and the pattern of 5-LO-like immunostaining was not observed with the nonimmunized serum (not shown). Magnification: CA1, objective 20x; CA3, objective 10x. Similar results were obtained in three rats per group.



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  		Figure 2. Increased dendritic 5-LO-like staining in the limbic cortex of old rats; brain sections taken at the Bregma 4.80 mm (23) from young (A) or old (B) rats. The drawing indicates areas of the lateral entorhinal cortex (LEnt), the amygdalopiriform transition area (APir), and the posteromedial cortical amygdaloid nucleus (PMCo). In old rats (B), 5-LO-like immunopositivity is highly expressed in the apical dendrites of the principal pyramidal cells. The density of small 5-LO positive cells is slightly lower in old than in young rats. Magnification: objective 5x.



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  		Figure 3. Appearance of 5-LO antiserum-positive apical dendrites and cell bodies in layer V pyramidal cells in the frontoparietal cortex of old rats. Cortical layers (I–VI) are indicated on the left. A) 2 month old; B) 24 month old. Note the strong soma and dendritic 5-LO-like staining in panel B (an example is indicated by the arrow). In both young and old rats, 5-LO-like staining is also present in small, nonpyramidal cells scattered throughout the cortical layers. Magnification: objective 5x. Similar results were obtained in three rats per group.

In the amygdala, the entorhinal and frontoparietal cortices, and to a lesser extent in hippocampal sections, 5-LO-like immunoreactivity was also localized in small, nonpyramidal cells scattered throughout the cortical layers. This type of 5-LO staining did not increase in old rats. In fact, the number of small 5-LO positive cells was about 20–30% lower in the amygdala of old rats (see example in Fig. 2). A double staining with 5-LO and GFAP or GAD primary antibodies and with gold (for 5-LO) and peroxidase (for GFAP or GAD) secondary antibodies revealed that the nonpyramidal 5-LO-like immunolabeling is present in GFAP-positive glia-like cells and GAD-positive, GABA neuron-like cells ( Fig. 4). Although the number of these small 5-LO-positive cells in representative samples of the amygdala was fewer in old than in young rats, the number of GFAP-positive cells did not decrease in old rats; on the contrary, we observed more GFAP-positive cells in old rats (not shown).



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  		Figure 4. Double labeling with 5-LO/GFAP (A) or 5-LO/GAD (B) antibodies in the entorhinal cortex of old rats. 5-LO-like immunoreactivity was visualized using gold immunolabeling; GFAP and GAD antibodies were visualized using DAB. Examples are shown in panels A and B: note the colocalization of black granules (gold labeling of 5-LO antiserum) with the brown DAB staining (GFAP or GAD). Magnification: objective 100x.

5-LO mRNA expression in the hippocampus of young and old rats
To investigate whether the more widespread neuronal distribution of 5-LO immunoreactivity (particularly in the hippocampus) found in old vs. young rats is associated with increased 5-LO expression in old animals, we developed a quantitative RT-PCR assay of 5-LO mRNA. We also assayed the cyclophilin mRNA content as a measure of a constitutive gene expression and used its values to normalize the 5-LO mRNA content. Figure 5A shows examples of gel photographs of RT-PCR products of RNA analysis with specific 5-LO and cyclophilin amplification primers and their respective internal standards. Not only was the absolute amount of hippocampal 5-LO mRNA greater in old than in young rats (attomol/µg total RNA: young = 41±5.6; old = 95±6.1), but a comparison of corresponding 5-LO/cyclophilin ratios confirmed this difference ( Fig. 5B).

Effect of a 5-LO inhibitor, caffeic acid, on kainate-induced excitotoxicity
Area CA3 of the hippocampus is the most epileptogenic structure of the brain; along with the entorhinal and piriform cortices, the CA3 pyramidal neurons appear to be the most vulnerable cells to kainate-induced excitotoxic damage (for a review, see ref 35). Recently, it was shown that indomethacin, a cyclooxygenase inhibitor, potentiated whereas the cycloxygenase/lipoxygenase inhibitor BW755C reduced kainate-triggered damage in the brain regions noted above (36). We tested the effect of a 5-LO inhibitor, caffeic acid (22), against kainate-triggered excitotoxicity. Table 2 shows that this 5-LO inhibitor significantly reduced neuronal loss in the CA3 hippocampal field. Although caffeic acid prolonged latency in the behavioral response to kainate injection, both control and caffeic acid-treated rats expressed similar generalized tonic-clonic seizures.


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  		Table 2. Protective effect of the 5-LO inhibitor caffeic acid against kainate-induced excitotoxicitya

Excitotoxic action of kainate in young and old rats
In male Fisher 344 rats, intraperitoneal administration of 10 mg/kg kainate induced seizures with comparable latency in both young and old (young = 71±5; old = 62±12 min; mean ± SEM; n=14 per group). Mortality within 72 h was greater in the old group: young 2/14; old 5/14. The extent of kainate-induced limbic damage was significantly greater in old than in young rats. Figure 6 shows an example of the TUNEL assay (DNA damage) in the CA3 hippocampal field of a kainate-injected old rat. Quantitative analysis of the damage to limbic structures 72 h after kainate revealed significantly greater injury in old than in young rats ( Fig. 6). Similarly, the extent of kainate-induced Nissl loss in the CA3 was greater in old than in young rats (not shown).



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  		Figure 6. TUNEL assays of kainate-induced neuronal damage in the limbic areas of young and old rats. Kainate was injected intraperitoneally (10 mg/kg; animals were killed 72 h later). Left panel shows an example of TUNEL staining after kainate injection into an old rat (arrows point to TUNEL-positive cells). No TUNEL-positive cells were observed in saline-injected controls (not shown). Right panel shows the results of quantitative TUNEL assay. Quantitative analysis of TUNEL labeling was performed in the hippocampal CA3 field, entorhinal cortex (EC), and amygdala (A) of five rats per group. Results are the mean ± SEM; P < 0.05 in comparison with the corresponding young group (Student's t test).

Although typically no damage due to systemic kainate administration is observed in the rat frontoparietal cortex, when using the TUNEL assay we noted distinct cell damage in layer V pyramidal cells in the old rats treated with kainate. These are the pyramidal glutamatergic neurons where we also identified the appearance of intense neuronal 5-LO staining in the old rats ( Fig. 7).



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  		Figure 7. Kainate induces DNA damage in layer V pyramidal cells of the frontoparietal cortex of old rats. A) 5-LO-like immunostaining in the cortex of a saline-injected old rat; B) TUNEL staining 72 h after kainate administration (note the dark nuclear staining in the pyramidal cells). Magnification: objective 20x.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
A prominent finding in this study is the distinct pattern of neuronal 5-LO-like immunoreactivity in the cortical brain structures of old vs. young Fisher 344 rats. The most evident difference between 2- and 24-month-old rats is the exceptionally widespread 5-LO-like immunoreactivity in the dendrites (mostly apical dendrites) of pyramidal neurons in the CA hippocampal fields, in dendrites of pyramidal neurons in the entorhinal cortex, and in layer V cells in the frontoparietal cortex of old but not young rats. The increase in neuronal 5-LO-like immunoreactivity appears to be due to the increased expression of the 5-LO gene, as suggested by increased 5-LO mRNA expression in the hippocampus of old rats. A stimulatory effect of aging on 5-LO expression was previously observed in alveolar macrophages (13). The mechanism of this increase has not been elucidated. In vitro, several factors are capable of stimulating 5-LO mRNA expression. For example, 5-LO mRNA expression in cell lines, can be stimulated by transforming growth factor ß1 or by 1,25-dihydroxyvitamin D3 (37, 38). Suppression of 5-LO gene expression, on the other hand, can be induced by binding of the pineal hormone melatonin to its nuclear receptor, which then binds the response element of the 5-LO gene (39, 40). Because aging is associated with a decline in melatonin secretion (41), it is possible that aging-associated melatonin deficiency lessens the tonic inhibitory action of this hormone on 5-LO gene expression and leads to increased levels of 5-LO mRNA and protein. Support for this proposal is provided by a recent observation that melatonin deficiency produced in rats by pinealectomy triggered in the hippocampus an increase in 5-LO mRNA content (42).

The localization of neuronal 5-LO we found in the hippocampus of young rats is in accordance with immunoreactivity previously described in the gerbil brain; it was observed predominantly in the cell bodies (9). Although in the study by Ohtsuki et al. (9) an acute brain ischemia caused translocation of neuronal 5-LO from cytosol to membrane, these authors did not observe the corresponding ischemia-triggered changes in localization or intensity of neuronal 5-LO immunoreactivity. Thus, it is not clear what could be guiding the 5-LO protein into the pyramidal cell dendrites of old rats, the effect observed in this study. One possibility is that the neuronal distribution of 5-LO binding protein (or proteins) is affected by aging. FLAP is required for the full activation of 5-LO. In the rat hippocampus, FLAP-like immunoreactivity was observed in pyramidal neurons in the CA fields, where it was colocalized with the 5-LO mRNA as detected by the in situ hybridization technique (6). Whether aging primarily affects the distribution of neuronal FLAP and by what mechanism have yet to be investigated. An alternative and intriguing possibility is that in neurons, as recently described in human neutrophils and HL-60 cells (43), the 5-LO protein may be present in two forms—phosphorylated and nonphosphorylated—and that these forms can be associated with cellular proteins other than FLAP. Whether this applies to neurons and whether aging may alter the rate of 5-LO phosphorylation are not known, however.

Teleologically, we can only speculate about the reason for the aging-associated increased 5-LO expression/distribution in the pyramidal neurons observed in this study. In rats, aging was shown to be associated with a decrease of the number of cells in the CA3 subfield, which correlated with working memory deficiency (44). It has been proposed that age-related dendritic remodeling may be viewed as a compensatory response to the dying of neighboring neurons (45). It is tempting to speculate that 5-LO up-regulation in aging neurons is one such compensatory response. Lammers et al. (6) proposed that neuronal 5-LO may participate in somatostatin receptor transmembrane signaling in a manner that would promote synaptic plasticity.

In the frontoparietal and the entorhinal cortices, we also observed strong 5-LO-like immunostaining in small nonpyramidal cells that were GFAP positive ( Fig. 4). This finding is consistent with previously reported studies of the capability of glia to produce leukotrienes (46). The number of the glia-like 5-LO-immunopositive cells appears to decrease with aging, although the number of GFAP-positive cells does not. Aging is known to be associated with increased GFAP expression (47). However, whereas the number of GFAP-positive astrocytes increases with aging, the GFAP volume per astrocyte decreases (48). Thus, it is possible that some astrocyte-specific changes associated with astrocyte functioning in aging are responsible for the altered 5-LO-like immunolabeling. However, not all 5-LO-positive small cells were GFAP positive; some were positive for GAD, suggesting that they may be the GABA-ergic interneurons, and others still have to be identified. It is possible that this particular population of small nonpyramidal cells is specifically affected by aging.

Although aging-associated up-regulation of neuronal 5-LO might serve a physiological purpose, it appears it also may increase the vulnerability of the aging brain to neurodegenerative insults. Leukotrienes (5-LO products) are increased in the brain in response to ischemia (9) and glutamate receptor-mediated (e.g., kainate-triggered) excitotoxicity (12), whereas inhibitors of leukotriene formation provide protection in brain ischemia (10) and hypoxic or N-methyl-D-aspartate-induced impairment (5). Our results show that a 5-LO inhibitor, caffeic acid, reduces the excitotoxic action of kainate. Protection against kainate neurotoxicity was demonstrated previously by using a mixed cyclooxygenase/lipoxygenase inhibitor, BW755C (36). Because caffeic acid may affect other enzymes such as oxidases and peroxidases, further experiments with more specific 5-LO inhibitors are warranted.

We found that in addition to increased neuronal 5-LO expression/distribution in old rats, there was a greater brain vulnerability to kainate-induced excitotoxicity. Previously published reports indicate that aging generally increases the brain's vulnerability to acute insults, including excitotoxicity. For example, the greater vulnerability of old rats was observed after administration of kainate (49), 3-acetylpyridine (50) and ibotenate (51) or after experimental stroke (18). One study (52) found the opposite effect of kainate-induced hippocampal injury: greater damage was observed in young than in old rats. The reason for this discrepancy is not clear, but it may originate in rat strain differences.

It is likely that multiple changes occur with aging that may contribute to increased brain vulnerability (53), and that neuronal 5-LO up-regulation is only one such change. The results of this study and previous work by others strongly suggest that kainate excitotoxicity is sensitive to 5-LO inhibitors and that 5-LO products may contribute to this toxicity. We observed in old rats that a discrete cell damage occurs in layer V neurons of the frontoparietal cortex after kainate administration; these are the glutamatergic pyramidal cells that, in aged rats, express a rather distinct pattern of 5-LO up-regulation. Although it is not clear whether aging-associated 5-LO up-regulation has any physiological function, it appears that it might be responsible in part for the increased susceptibility of the brain to neurodegeneration. Thus, some similarities exist between aging and pinealectomy-produced melatonin deficiency. For example, like the old rats in this study, pinealectomized rats are more susceptible to brain injuries triggered by stroke or kainate excitotoxicity (34) and express higher levels of 5-LO mRNA in the hippocampus (42). Thus, it is possible that at least some aspects of aging-associated increased brain vulnerability are driven by the aging-associated decline in pineal functioning.

If a pattern of 5-LO expression similar to that which we observed in old rats can be documented in the aging human brain, several important implications for our understanding of neurodegenerative processes, including Alzheimer's disease, would ensue. These implications could be important for the development of novel neuroprotective treatments that would target the neuronal 5-LO pathway. Such a therapeutic approach would be complementary to other anti-inflammatory treatments currently considered for neurodegenerative diseases such as Alzheimer's.


   ACKNOWLEDGMENTS
 
This work was supported by NIH-NIA grant RO3 AG14630-01. T.U. was supported by a Fogarty International Award, F05 TW/NS5271-02. The 5-LO antiserum (LO-32 antiserum) was kindly provided by Dr. Jilly Evans, Merck Frosst, Canada. We thank Dr. Erminio Costa for invaluable discussions and suggestions and Peter Farmer for help in preparing figures.


   FOOTNOTES
 
1 Correspondence: The Psychiatric Institute, The University of Illinois at Chicago, 1601 West Taylor St., M/C 912, Chicago, Illinois 60612, USA. E-mail: HManev{at}psych.uic.edu

2 Abbreviations: FLAP, 5 lipoxygenase-activating protein; GAD, glutamic acid decarboxylase; GFAP, glial fibrillary acidic protein; KA, kainic acid; 5-LO, 5-lipoxygenase; RT-PCR, reverse transcription/polymerase chain reaction; TUNEL, terminal deoxynucleotidyl transferase (TdT) mediated dUTP-biotin nick end labeling; BSA, bovine serum albumin; TBS, Tris-buffered saline; DAB, 3,3'-diaminobenzidine tetrahydrochloride; GABA, 8-aminobutyric acid; cyc, cyclophilin; dNTP, deoxynucleotide triphosphate.

Received for publication October 8, 1997. Accepted for publication December 4, 1997.


   REFERENCES
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 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
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