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Experimental Biology and Medicine 228:517-524 (2003)
© 2003 Society for Experimental Biology and Medicine

HEME OXYGENASE

Regional Rat Brain Distribution of Heme Oxygenase-1 and Manganese Superoxide Dismutase mRNA: Relevance of Redox Homeostasis in the Aging Processes

Claudia Colombrita*,{ddagger}, Vittorio Calabrese*,{ddagger}, Anna Maria Giuffrida Stella{ddagger}, Francesca Mattei*, Daniel L. Alkon* and Giovanni Scapagnini*,{dagger},1

* Blanchette Rockefeller Neurosciences Institute, West Virginia University, Rockville, Maryland 20850;
{dagger} Institute of Neurological Sciences, CNR, 95123 Catania, Italy; and
{ddagger} Section of Biochemistry and Molecular Biology, Department of Chemistry, Faculty of Medicine, University of Catania, 95125 Catania, Italy

Abstract

Increasing evidence supports the notion that reduction of cellular expression and activity of antioxidant proteins and the resulting increase of oxidative stress are fundamental causes in the aging processes and neurodegenerative diseases. In the present study, we evaluated, in the brains of young and aged rats, the gene expression profiles of two inducible proteins critically involved in the cellular defense against endogenous or exogenous oxidants: heme oxygenase-1 (HO-1) and manganese superoxide dismutase-2 (SOD-2). SOD-2 is an essential antioxidant and HO-1 has been reported to be very active in regulating cellular redox homeostasis. Deregulation of these enzymes has been extensively reported to play a crucial role in the pathogenesis of neurodegenerative disorders. To measure the regional distribution of HO-1 and SOD-2 transcript levels in the rat brain, we have developed a real time quantitative reverse transcription-polymerase chain reaction protocol. Although these two genes presented a highly dissimilar range of expression, with SOD-2 >HO-1, both transcripts were highly expressed in the cerebellum and the hippocampus, showing in a different scale a strikingly parallel distribution gradient. To further investigate the regional brain expression of these mRNAs, we performed in situ hybridization using specific riboprobes. In situ hybridization results showed that both transcripts were highly concentrated in the hippocampus, the cerebellum and some specific regions of the brain cortex. We have also quantified, by reverse transcription-polymerase chain reaction, the brain expression of HO-1 and SOD-2 mRNAs in middle aged (12 months) and aged (28 months) rats. We found that the hippocampus of aged rats presents a significant down regulation of SOD2 mRNA expression and a parallel upregulation of HO-1 mRNA compared with young (6 months) and middle-aged rats. Furthermore, in the cerebellum of the aged rats, we detected a parallel significant upregulation of both HO-1 and SOD-2 transcripts. These regional age-dependent differences may help to explain the increased susceptibility to oxidative damage in these two brain areas during aging.

Key Words: aging • antioxidant • neuron

Increased production of free radical species has been related to the development of numerous pathologies, including neurodegenerative diseases, vascular dysfunction, carcinogenesis, and aging (1). Irrespective of the source and mechanisms that lead to the oxidative challenge, mammalian cells have developed highly refined inducible systems against a variety of stressful stimuli. Each one of these systems, when appropriately activated, has the possibility to restore cellular homeostasis and to rebalance redox equilibrium (2). The brain has a limited ability to withstand oxidative stress because of its high content of easily oxidizable substrates, such as polyunsatured fatty acids and catecolamins, and because it has relatively low levels of antioxidants, such as glutathione and vitamin E (3). Heme oxygenase-1 (HO-1) and manganese superoxide dismutase (SOD-2) are two inducible proteins capable of protecting the brain from oxidative damage, and their expression has been demonstrated to be critical in modulating the response of neurons to various kinds of stress (4, 5).

Manganese superoxide dismutase is a nuclear-encoded 23-kDa protein that belongs to a group of three genetically distinct enzymes highly conserved in mammals. Although the other two isoforms of superoxide dismutase (SOD-1 and SOD-3), having both copper and zinc as metal cofactors, are mainly constitutive and localized, respectively, in cytoplasm or in the extracellular fluid, SOD-2 is a highly inducible mitochondrial protein. SOD-2 is synthesized within the cytosol and imported to the mitochondrial matrix, where it converts superoxide anion to hydrogen peroxide. The latter is subsequently metabolized to water by catalase or glutathione peroxidase. In this way, SOD-2 acts as a fundamental defense against reactive oxygen species within mitochondria. Increased levels of SOD-2 protein, mRNA, and enzymatic activity have been amply documented in the rodent brain as a consequence of normal aging, ischemia, hyperoxia, and pro-oxidant drug exposure (6, 7). SOD-2 expression is also reportedly augmented in the substantia nigra of subjects with idiopathic Parkinson’s disease, Alzheimer’s-diseased hippocampal astrocytes, and in the spinal cord of patients with amyotrophic lateral sclerosis. (8–10). Oxidative stress has been implicated in each of these conditions and is thought to be, directly or indirectly, the cause for provoking a compensatory SOD-2 response in brain (11). Nevertheless, the extracellular factors and intracellular signaling pathways mediating the upregulation of SOD-2 in aging and neurodegenerative disorders remain to be fully understood. In addition to the direct effects of pro-oxidant species, proinflammatory cytokines, lipopolysaccharide, hormones, heavy metals, and nitric oxide donors have been demonstrated to induce the SOD-2 gene in various cell types (12). Intracellular messengers implicated in SOD-2 gene regulation include the nuclear transcription factors, nuclear factor-kB and activator protein-1, protein kinase C, and possibly endogenous tumor necrosis factor-{alpha} (13).

Heme oxygenase-1 is a 32-kDa member of the stress protein superfamily that catalyzes the rate-limiting step in heme degradation in brain and other tissues. To date, three isoforms of heme oxygenase have been identified: the inducible HO-1 (14), the constitutive HO-2 (15), and the less active HO-3 (16), which has been cloned only in the rat and probably represents the result of a species-specific gene retrotransposition (17). Although the biological role of HO proteins remains to be completely elucidated, their relevance in cellular stress responses has been widely demonstrated in a variety of tissues, including the brain (18, 19). The HO-1 gene contains a heat shock element in its promoter region and is rapidly induced upon exposure to heme, metal ions, sulfhydryl compounds, UV light, and various pro-oxidants (20–22). The efficacy of HO-1 in promoting cytoprotection resides primarily in the intrinsic ability of its metabolic products (e.g., carbon monoxide [CO] and bilirubin) to exert potent antioxidant and anti-inflammatory activities (23, 24). In the brain, astrocytes strongly express HO-1 in response to injury, and the HO pathway has been shown to act as a fundamental defensive mechanism for neurons exposed to an oxidant challenge (25–28). Deregulation of the HO system has been associated with pathogenesis in several neurodegenerative disorders, including Alzheimer’s disease and multiple sclerosis (29–31).

Previous studies have correlated the expression and activity of HO-1 to that of SOD-2, suggesting a reciprocal influence between these two genes (32). Recently, we demonstrated that HO-1 mRNA expression is physiologically detectable and shows a characteristic regional distribution (17) despite the fact that HO-1 protein expression is almost null in the naïve rat brain (33). Here, we have used a real-time reverse transcription polymerase chain reaction (RT-PCR) approach to quantitatively assess the mRNA expression profiles of HO-1 and SOD-2 in various rat brain areas. To further investigate the regional expression of these two genes, we have also performed in situ hybridization using specific riboprobes.

A large body of evidence suggests that the aging process may be related to loss of antioxidant protection and to an augmented susceptibility to oxidative stress (1, 2). In the present study, we assessed, by quantitative RT-PCR, the regional brain levels of HO-1 and SOD-2 mRNA in rats at different ages (6, 12, and 28 months) to determine whether the gene expression profiles of these antioxidant enzymes vary in an age-related manner.

Materials and Methods

Animals.
Young (6 months), middle-aged (12 months), and aged (28 months) male Wistar rats (Harlan-Sprague-Dawley, Indianapolis, IN), were used. Animals were housed, three per cage, in standard conditions (lights on from 0700 to 1900 hr) with food and water freely available. After decapitation, brains were quickly removed and the following areas were dissected: cerebral cortex, hippocampus, striatum, and cerebellum. Dissection was performed according to a standardized procedure (34), in a cold anatomical chamber, and following a protocol that allows a maximum of a 50-sec time variability for each sample across the animals. Animals were used only when strictly necessary and adequate measures were taken to minimize unnecessary pain and discomfort to the animal in accordance with NIH guidelines for the care and use of laboratory animals.

Real-Time Quantitative RT-PCR.
Total RNA from whole rat brain or dissected areas was extracted using Trizol (Sigma, St. Louis, MO) and treated with RNase-free DNase to remove any residual genomic DNA. Single-stranded cDNAs were synthesized incubating total RNA (1 µg) with SuperScript II RNase H-reverse transcriptase (200 U), oligo-(dT)12–18 primer (100 nM), dNTPs (1 mM), and RNase-inhibitor (40 U) at 42°C for 1 hr in a final volume of 20 µl. Reaction was terminated by incubating at 70°C for 10 min.

Forward (FP) and reverse (RP) primers used to amplify HO-1 and SOD-2 are listed in Table IGo. The expected amplification products for HO-1 and SOD-2 are 123 and 149-bp, respectively. To control the integrity of RNA and for differences attributable to errors in experimental manipulation from tube to tube, primers for rat phosphoglycerate kinase 1, a housekeeping gene that is consistently expressed in brain tissues, were used in separate PCR reactions and generated a 183-bp PCR product. Aliquots of cDNA (0.1 and 0.2 µg) and known amounts of external standard (purified PCR product, 102 to 108 copies) were amplified in parallel reactions using the FP and RP indicated in Table IGo. Each PCR reaction (final volume 20 µl) contained 0.5 µM of primers, 2.5 mM Mg2+ and 1 x Light cycler DNA master SYBR Green (Roche Diagnostics, Indianapolis, IN). PCR amplifications were performed with a Light-Cycler (Roche Molecular Biochemicals) using the following four cycle programs: (i) denaturation of cDNA (1 cycle: 95°C for 10 min); (ii) amplification (40 cycles: 95°C for 0 sec, 58°C for 5 sec 72°C for 10 sec); (iii) melting curve analysis (1 cycle: 95°C for 0 sec, 70°C for 10 sec, 95°C for 0 sec); and (iv) cooling (1 cycle: 40°C for 3 min). Temperature transition rate was 20°C/sec except for the third segment of the melting curve analysis where it was 0.2°C/sec. Fluorimeter gain value was 6. Real-time detection of fluorimetric intensity of SYBR green I, indicating the amount of PCR product formed, was measured at the end of each elongation phase. Quantification was performed by comparing the fluorescence of PCR products of unknown concentration with the fluorescence of the external standards. For this analysis, fluorescence values as measured in the log-linear phase of amplification were considered, using the second derivative maximum method of the Light Cycler Data Analysis software (Roche Molecular Biochemicals). Specificity of PCR products obtained was characterized by melting curve analysis followed by gel electrophoresis, visualized by ethidium bromide staining, and DNA sequencing.


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  		Table I. Sequences of Oligonucleotide Primers for HO Isoforms and PGK1 mRNAs
 
In Situ Hybridization.
Brains from decapitated 6-month-old Wistar rats (n = 3) were rapidly removed and frozen at –70°C. Coronal sections of 12-µm thickness were cut on a cryostat at –20°C, thaw mounted onto silanated glass slides (Digene, Gaithersburg, MD), and stored at -70°C until hybridization. The sections were fixed in 4% paraformaldehyde in 1xPBS buffer (pH 7.3), acetylated, dehydrated in serial alcohol solutions, dilapidated in chloroform, partially redehydrated, and air-dried. In situ hybridization was performed as described previously (11). A 315-bp fragment of cDNA corresponding to bases 561–867 of the previously characterized rat HO-1 mRNA (GenBank accession n.: NM_012580) was synthesized by PCR from rat brain RNA using specific primers (FP: 5'-CGCATGAACACTCTGGAGAT–3'; RP: 5'-CATGGCATAAATTCCCACTG–3'). A 319-bp fragment of cDNA corresponding to bases 287–605 of the previously characterized rat SOD2 mRNA (GenBank accession n.: NM_017051) was synthesized by PCR from rat brain RNA using specific primers (FP: 5'-GGCCAAGGGAGATGTTACAA–3'; RP: 5'-CCACAGGCCTTATTCCACTG–3'). These fragments, designed to specifically detect transcripts encoding, respectively, HO-1 and SOD-2, were subcloned into the PCR 4Blunt-TOPO vector (Invitrogen, Carlsbad, CA). The orientation of the inserts was determined by DNA sequencing. After linearization with the restriction enzymes PmeI and NotI and SpeI and NotI, respectively, the HO-1 and SOD-2 cDNA fragment-inserted vectors were used as templates for riboprobe synthesis. The sense and antisense riboprobes were synthesized with T3 or T7 RNA polymerase (Ambion, Austin, TX), respectively, in the presence of 1 µg cDNA (HO-1/SOD-2) template, 0.5 mM ATP, 0.5 mM CTP, 0.5 mM GTP, and 2 µM [{alpha}-35 S] UTP (>1000 Ci/mmol, NEN). The transcribed products were purified on Sephadex G-25 spin columns (Roche, Indianapolis, IN), and probe labeling was assessed by scintillation counting. Fifty microliters of the HO-1 and SOD-2 riboprobes (106 cpm/µl) were applied, respectively, to each slide holding three sections and hybridized in a mixture containing 20 mM Tris-HCl, pH 7.4, 1 mM EDTA, 300 mM NaCl, 50% formamide, 10% dextran sulfate, 1x Denhardt’s solution, 100 µg/ml salmon sperm DNA, 250 µg/ml yeast total RNA, 250 µg/ml yeast tRNA, 100 mM DTT, 0.1% sodium dodecyl sulfate (SDS), 0.1% NTS. Hybridization was carried out at 55°C for 24 hr. Sections incubated with the sense probe during hybridization were used as negative controls. After high-stringency post hybridization washes and RNase treatment, brain sections were dehydrated in graded ethanol. Slides were then exposed to film (hyperfilm Bmax; Amersham, Piscataway, NJ) for 24 hr for the generation of autoradiograms. Hybridization with the sense riboprobe led to no detectable signal (data not shown).

Results

Quantification of HO-1 and SOD-2 mRNA in Different Rat Brain Areas by Real-Time RT-PCR.
We selected specific primer pairs (Table IGo) to amplify HO-1 and SOD-2 mRNAs from rat brain cDNA (Fig. 1AGo). Known amounts of purified PCR products were used as external standards (102 to 108 copies/reaction) to produce two standard curves for HO-1 and SOD-2 (Fig. 1BGo). All samples were measured in triplicate and variations were typically within 10%. Expression of the genes showed a wide regional variability in the different brain areas examined. As shown in Table IIGo, SOD2 mRNA was expressed in levels that were several times higher than HO-1. SOD-2 was particularly expressed in the cerebellum and in the hippocampus, followed by the cortex and the striatum. Although HO-1 mRNA was expressed in lower levels than SOD-2, it was detectable in the cerebellum, the hippocampus, the striatum and the cortex Comparing the two different expression profiles, nevertheless was evident that these genes follow, in a different scale, a strikingly parallel distribution pattern (Fig. 2Go).



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  		Figure 1. Real-time quantification of HO-1 and SOD-2 mRNA levels by RT-PCR. Specific primers for HO-1 and SOD-2 were used to amplify rat brain RNA (A). Total RNA from different brain regions and known amounts of external standards (purified PCR product, 102 to 108 copies) were amplified in parallel reaction. Fluorimetric intensity of SYBR green I, indicating the amount of PCR product formed was measured at the end of each elongation phase. Quantification was performed by comparing the fluorescence of PCR products of unknown concentration with the fluorescence of the external standards (B). Fluorescence values measured in the log-linear phase of amplification were measured by the second derivative maximum method and used to produce standard curves that in turn were used to estimate the concentration of unknown samples. The specificity of the products amplified was evaluated by melting curve analysis.

 

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  		Table II. Transcript Levels for HO Isoforms in Different Rat Brain Areas Assessed by Real-Time Quantitative RT-PCR
 


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  		Figure 2. HO-1 and SOD2 mRNA expression profiles in different brain areas. Brain expression of HO-1 (A) and SOD-2 (B) transcripts relative to the expression of phosphoglycerate kinase 1 (mean ± SEM) for the different areas considered. Abbreviations: CX, cortex; HI, hippocampus; CB, cerebellum; ST, striatum.

 
Distribution of HO-1 and SOD-2 mRNA in the Rat Brain.
To further investigate the regional expression of HO-1 and SOD-2 mRNA we performed in situ hybridization with specific riboprobes. In situ hybridization results showed that the HO-1 and the SOD-2 mRNA signals were highly concentrated in the hippocampus, the cerebellum and some specific regions of the brain cortex (Fig. 3Go). In the hippocampus, the CA3, the CA2, the CA1, and the dentate gyrus showed the highest HO-1 and SOD-2 expression. The habenular nucleus also showed a high level of HO-1. By contrast, elevated concentrations of SOD-2 mRNA were present in the red nucleus. In the brain cortex, SOD-2 transcript levels were significantly higher in the retrospenial granular and agranular cortex and in the visual cortex compared to the HO-1 mRNA level. Relatively high levels of HO-1 mRNA were present in the auditory and the entorhinal cortex. Elevated concentrations of HO-1 and SOD-2 mRNA signals were also seen in the granule cell layer of the cerebellar cortex. In particular, high concentrations of SOD2 mRNA were detected in the inferior olive and in the pyramidal tract.



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  		Figure 3. Localization of HO-1 and SOD-2 mRNA in the adult rat brain by in situ hybridization. A 315-bp riboprobe for HO-1 and a 319-bp riboprobe for SOD-2 labeled with [{alpha}-35 S] UTP were synthesized and hybridized with coronal sections of the rat brain. After hybridization, the labeled HO-1 (A–C) and SOD-2 (D–F) mRNA signals were revealed with autoradiography. Abbreviations: AC, auditory cortex; CA1, CA2, CA3, pyramidal layers of the hippocampus; CCX, cerebellar cortex, CX, cortex; DG, dentate gyrus; EC, entorhinal cortex; HB, habenular nucleus; IO, inferior olive; RC, retrospenial cortex; RD, red nucleus; VC, visual cortex; PY, pyramidal tract.

 
Age-Related Differential Expression of HO-1 and SOD-2 in the Rat Brain.
We then investigated the differential mRNA expression of HO-1 and SOD-2 in brain regions of rats at different ages by using a quantitative RT-PCR approach. HO-1 and SOD-2 mRNA expression levels of young (6 months), middle-aged (12 months), and aged (28 months) rats were estimated in the following different brain regions: cortex, hippocampus, cerebellum, and striatum. As shown in Figure 4Go, no significant differences in HO-1 and SOD-2 mRNA levels were detectable between young and middle-aged rats for all the areas examined. In aged rats, however, significantly increased HO-1 mRNA levels were observed in the cerebellum followed by the hippocampus, but not in the cortex and in the striatum, compared with HO-1 mRNA levels in 6 and 12 months old rats. SOD-2 mRNA levels were also significantly increased in the cerebella of aged rats, but significantly decreased in the hippocampus, compared with SOD-2 mRNA expression in 6- and 12-months-old rats.



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  		Figure 4. HO-1 (A) and SOD-2 (B) mRNA expression profiles in different brain areas from young (6 months) middle-aged (12 months), and aged rats (28 months). Each histogram represents the mean of three animals. *P < 0.05 in 28 vs 8 and 12 months. Abbreviations: CX, cortex; HI, hippocampus; CB, cerebellum; ST, striatum.

 
Discussion

The purpose of this study was to describe the localization and steady-state levels of HO-1 and SOD-2 mRNA in the rat brain, using the recently introduced real-time PCR technique, which provides a sensitive and rapid method for gene expression quantification (35). Previous descriptive studies on SOD-2 and HO-1 gene distributions in the rat brain were performed using methods characterized by a lower sensitivity and a narrow range of linearity (6, 36). Results from these studies showed a clear expression signal for SOD-2, whereas HO-1 was shown to be minimally detectable in the brain and only in some cell types (36).

By performing the quantitative analysis of HO-1 and SOD-2 transcripts, we have found that both mRNAs are widely expressed in the whole brain, with SOD-2 mRNA at a higher level than HO-1. Although the two transcripts showed different concentration gradients, they followed a parallel pattern of expression in the brain areas examined, with particular elevations in the hippocampus and in the cerebellum. By performing in situ hybridization, we found that both HO-1and SOD-2 mRNA signals were highly concentrated in the hippocampus, particularly in the CA1, CA2, and CA3 areas and in the dentate gyrus, in the granule cell layer of the cerebellum, and in some specific regions of the brain cortex

A parallel expression of HO-1 and SOD-2 could be extremely relevant in the regulation of intracellular redox homeostasis, since both of these enzymes counteract oxidative challenges. The fact that HO-1 and SOD-2 are expressed more in certain areas, such as the hippocampus, might indicate that these regions are particularly exposed to endogenous or stress-related redox perturbations (2, 37).

Although previous reports have not found detectable levels of HO-1 protein in the normal brain (33, 36), here, we show measurable levels of HO-1 mRNA in all the brain regions examined. This evidence may suggest the possible existence of a cellular reserve of HO-1 transcript quickly available for protein synthesis and a post-transcriptional regulation of its expression. In this regard, there are new lines of evidence suggesting the existence in the central nervous system of a post-transcriptional control for other inducible genes (38) and this mechanism seems to be critical for brain functions. In addition to regulating the translation efficacy of HO-1 mRNA, other types of post-transcriptional modifications have been suggested for this molecule (39) and may contribute to the fine regulation of its expression. More interestingly, the expression pattern shown by HO-1 mRNAs overlaps with the brain distribution of guanylate cyclase (40), the main CO functional target. Increasing evidence implicates CO as a chemical messenger that can influence physiological and pathological processes in the central and peripheral nervous system (41, 42).

Accumulation of free radicals as a principal cause of age-related neurodegeneration, is suggested by studies that show a high level of protein damage due to oxidative challenges in pathological conditions of accelerated aging in humans and rodents (43, 44). Other studies have demonstrated the accumulation of free radical induced damage in several organs during normal aging (45) or an increased susceptibility to free radical–induced damage as age increases (46). Different regional activities of antioxidant systems and variable metabolic rates can lead to a region-specific accumulation of oxidative damage, and such differences can increase the vulnerability of specific brain regions to age-dependent oxidative stress. Several studies have been carried out to establish the age-dependent changes in activities of antioxidant enzymes (47, 48). It is hypothesized that decreased capacity in DNA repair and/or antioxidant defenses increases brain region vulnerability to oxidative damage and is responsible for age-associated deficits in cellular function, particularly in postmitotic cells such as neurons (49).

Previous studies have described SOD-2 enzymatic activity declining with increased age in certain areas of rodent brain, especially in the hippocampus (50). This decrease in activity has been attributed to inactivation of the enzyme by free radicals. Other studies have demonstrated changes in HO-1 protein expression related to age (51). Here, for the first time, we have shown an age-related decrease of SOD-2 mRNA expression in the rat hippocampus. However, hippocampal HO-1 expression significantly increased in aged rats compared to the young. This overexpression may reflect a reaction to an excess of oxidative perturbation, possibly related to the SOD-2 downregulation. Downregulation of SOD-2 expression and activity, in fact, could have catastrophic consequences for the mitochondria, the neurons and, ultimately, the entire brain. Thus, the unbalance between SOD-2 and HO-1 can be a critical factor in the brain aging process, and our data suggest that the hippocampus is one of the most susceptible areas of the brain for oxidative damages during aging. In addition, the parallel over-expression of HO-1 and SOD-2 in the cerebellum of aged rats might be one of the reasons for the relative sparing of this area in pathological changes of brain disorders associated with oxidative stress and aging.

Footnotes

1 To whom request for reprints should be addressed at Blanchette Rockefeller Neurosciences Institute, JHU, Academic and Research Building, 9601 Medical Center Drive, Rm. 351, Rockville, MD 20850-3332. E-mail: gscapag{at}brni-jhu.org Back

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