Neurochemical Research, Vol. 28, No. 2, February 2003 (© 2003), pp. 341–345
S100B Protein and 4-Hydroxynonenal in the Spinal Cord of Wobbler Mice* Valentina Corvino,1 Rita Businaro,2 Maria Concetta Geloso,1 Paolo Bigini,3 Valentina Cavallo,1 Elena Pompili,2 Tiziana Mennini,3 Lorenzo Fumagalli,2 and Fabrizio Michetti1,4 (Accepted August 23, 2002)
S100B is a calcium-binding protein that, in the nervous system, is mainly concentrated in glial cells. Although its biological role is still unclear, the protein is hypothesized, at high concentrations, to act in the pathogenesis of neurodegenerative processes, possibly through oxidative stress mechanisms. To investigate this hypothesis we studied the spinal cord of wobbler mice, an animal model of motor neuron degeneration. Using immunocytochemistry, we detected an overexpression of S100B in astrocytes of the cervical spinal cord of these animals. We also confirmed this finding by reverse transcriptase polymerase chain reaction. In the same spinal cord regions, scattered neurons appeared to be immunostained for 4-hydroxynonenal–modified proteins, an indicator of lipid peroxidation. This finding constitutes a sign of oxidative stress– induced neurodegeneration.
KEY WORDS: S100B protein; 4-hydroxinonenal; wobbler mice; neurodegeneration; oxidative stress.
INTRODUCTION
the participation of oxidative stress in wobbler mice spinal cord neuropathology rests mainly on indirect evidence (7), it is believed to play an important role in motor neuron degeneration in ALS and its animal model, consisting of transgenic mice overexpressing human mutated SODI (8–12). An increased number of reactive astrocytes has also been observed in the neighborhood of degenerating neurons and in the whole spinal cord both in gray and white matter (13– 15); an active role for astrocytes in the course of wobbler disease has also been hypothesized (13,14,16). S100B is a calcium-binding protein that, in the nervous system, is most abundant in glial cells. Although the biological role of this protein appears to be multifaceted and not fully clarified, it is believed to act as a cytokine, displaying a neurotrophic effect at physiological concentrations, but being neurotoxic at high concentrations, and in this way participating in the pathophysiology of degenerative disorders, including Alzheimer’s disease (17,18). In this respect, evidence
The wobbler mouse (1) is an autosomal recessive mutant considered a useful model for human inherited motor neuron diseases, including amyotrophic lateral sclerosis (ALS) and infantile spinal muscular atrophy (ISMA) (2,3). The mutant mice develop progressive muscular atrophy associated with motor neuron degeneration, markedly in the cervical ventral horn of the spinal cord (2,4), accompanied by upper limb weakness, shakiness, and unsteady gait (5,6). While * Special issue dedicated to Dr. Anders Hamberger. 1 Institute of Anatomy, Catholic University, Rome, Italy. 2 Department of Cardiovascular Sciences, University “La Sapienza”, Rome, Italy. 3 Istituto di Ricerche Farmacologiche Mario Negri, Milano, Italy. 4 Address reprint requests to: Professor Fabrizio Michetti, Institute of Anatomy, Catholic University, L.go F. Vito 1, 00168 Rome, Italy. Tel: ⫹39 0630155848; Fax ⫹39 0630154813; E-mail:
[email protected]
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342 has been shown in vitro that S100B plays a role in the induction of oxidative stress (19,20). The present study investigates in the spinal cord of wobbler mice the behavior of S100B and of 4hydroxynonenal (HNE), a membrane lipid peroxidation product which is considered a causative factor in oxidative stress–induced cell damage (21).
EXPERIMENTAL PROCEDURE Animals. Wobbler mice (NFR/wr strain, NIH, Animal resources, Bethesda, MD USA) were bred at the Consorzio Mario Negri Sud, S. Maria Imbaro, Italy. The mice were maintained at a temperature of 21 ⫾ 1°C with relative humidity of 55% ⫾ 10% and a 12-hr light /dark cycle. Food (standard pellets) and water were supplied ad libitum. Phenotypical screening was performed to separate the wobbler mice(wr/wr) from their normal littermates, which were used as controls. The animals were killed at 12 weeks of age (plateau stage of this disease). Procedures involving animals and their care were conducted in conformity with the institutional guidelines that are in compliance with national and international laws and policies (EEC Council directive 86/609, 0J L 358, 1 Dec. 12, 1987; NIH Guide for the Care and use of Laboratory Animals, U.S. National Research Council, 1996). For immunocytochemistry, 6 animals (3 wobbler, 3 control) were anesthetized with Equithesin (1% phenobarbital /4% (vol/ vol) chloral hydrate, 30 l/10 g IP.) and transcardially perfused with 20 ml saline followed by 50 ml of sodium phosphate–buffered 4% paraformaldehyde solution. The spinal cord was removed and postfixed in 4% PBS paraformaldehyde for 48 hr and then embedded in paraffin. For RNA extraction and reverse transcriptase polymerase chain reaction (RT-PCR) procedure, 6 mice (3 wobbler, 3 control) were sacrificed by decapitation. Spinal cords were forced out intact using physiological buffer from a syringe. The cervical region was dissected and stored at ⫺70°C. Immunocytochemistry. Seven-micrometer sections were hydrated and subjected to antigen retrieval in microwaves (6 min, 500 W) in 10 mM, pH 6, citrate buffer. After several rinses in PBS, the sections were incubated with 3% H2O2 in methanol for 15 min at room temperature to block endogenous peroxidase. After a series of rinses in PBS, the sections were incubated in normal goat serum 3% for 1 hr at room temperature. Sections were then incubated overnight in anti-S100B polyclonal antibody (DAKO A/S, Denmark), anti-GFAP polyclonal antibody (DAKO A/S, Glostrup, Denmark), or anti-HNE polyclonal antibody (Calbiochem, San Diego, CA), all diluted 1:1000 in PBS 0.01 M, pH 7.4. The sections were washed in PBS and treated with the anti-rabbit biotinylated secondary antibody (1:200) for 1 hr at room temperature, followed by incubation with an avidin-biotinylated horseradish peroxidase complex (ABC Elite, Vector Laboratories, Burlingame, CA) for 1 hr at room temperature. Peroxidase labeling was visualized by incubation of the sections in 0.01% 3,3⬘-diaminobenzidine (DAB, Sigma, St. Louis, MO). Controls were performed by omitting the primary antibody. RT-PCR. Samples of 10–20 mg were homogenized in 1 ml total RNA Fast at 0–4°C using a motor-driven grinder (Sigma, St. Louis, MO) homogenizer. Total RNA was obtained from homogenates of cervical tracts of the spinal cords of wobbler and control mice. A single-step method (RNA Fast isolation system, Molecular Systems,
Corvino et al. San Diego, CA) was used for RNA extraction. Total RNA was reverse transcribed into cDNA; the reaction was carried out in a final volume of 20 l containing 2 pmole of the reverse primer, 1 g of total RNA, 12 l of DEPC-treated distilled water (this mixture was heated to 70°C for 10 min), 4 l of 5⫻ First Strand Buffer (250 mM Tris-HCl at room temperature, 375 mM KCl, 15 mM MgCl2), 2 l of 0.1M DTT, and 1 l 10 mM dNTP mix. Samples were incubated at 42°C for 2 min and then 1 l (200 units) of Superscript II, RNase H– Reverse Transcriptase (Invitrogen, Life Technologies, Carlsbad, CA USA) was added to each tube. The synthesis of the first strand of cDNA was carried out at 42°C for 50 min, and the reaction was then inactivated by heating to 70°C for 15 min. The cDNA was amplified in a 50-l reaction mixture containing 5 l of 10⫻ PCR buffer (200 mM Tris-HCl, pH 8.4, 500 mM KCl), 1.1 l 10 mM dNTP mix, 1 l amplification primer 1(10 pM), 1 l amplification primer 2 (10 pM), 0.5 l Platinum Taq DNA Polymerase (2.5 units, final concentration) (invitrogen, Life Technologies, USA), 1 l cDNA, 39.5 l DEPC-treated distilled water. Samples were heated to 94°C for 2 min, and 30 cycles were carried out in three temperature steps: 94°C for 30 s, 57°C for 30 s, and 72°C for 1 min. Ten microliter of PCR product (398 bp) was electrophoresed on 1.5% agarose gel and visualized after ethidium bromide staining. The densitometry of fluorescent bands was used to determine the level of mRNA encoding S100B. Quantitation was performed using a PC-assisted CCD camera (Gel Doc 2000 system/Quantity One software, Bio-Rad Laboratories, Hercules, CA), and the values obtained for S100B were normalized to the mRNA content of glycerol-phosphate dehydrogenase (GAPDH) a typical reference constitutive transcript. Primers for the RT-PCR of mouse S100B mRNA were designated on the basis of the structure of S100B mRNA, 5⬘ primer:5⬘-GCTGAAGAAGTCAGAACTGAAG-3⬘; 3⬘ primer: 5⬘-CGAAGTGCTAACTTAAAGCAGC-3⬘.
RESULTS The immunocytochemical study using anti-GFAP and anti-S100B was performed on cervical spinal cord, where neurodegenerative processes and astrogliosis are most pronounced in wobbler mice. GFAP staining detected many immunolabeled astrocytes in wobbler mice (not shown). There was evidence of diffuse gliosis, as expected. S100B staining also detected a diffuse population of immunolabeled cells exhibiting astroglial morphology. S100B-positive cells also appeared to be more abundant in wobbler than in control mice, although quantitative studies were not performed (Fig. 1). The cDNA, obtained by reverse transcription from total RNA of the cervical tract of spinal cords of wobbler and control mice, was amplified by PCR. An amplified product of 398 bp corresponding to the S100B mRNA was obtained. The mRNA for the constitutive enzyme GAPDH was examined as the reference cellular transcript (Fig. 2). The densitometric analysis of the amplified products showed that the level of S100B mRNA was significantly higher (P ⬍ 0.05 using Student’s t test; n ⫽ 6) in wobbler mice than in controls; a representative experiment is shown in Fig. 2.
S100B and 4-Hydroxynonenal in Wobbler Mice
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Fig. 1. Micrographs of transverse sections of cervical spinal cord from control (A) and wobbler mice (B) stained with anti-S100B antibody. A higher number of S100B-positive cells is detectable in the white and gray matter of wobbler mice sections than in controls. Magnification: ⫻98.
The presence of 4-hydroxynonenal (HNE)-protein adducts as lipid peroxidation products was shown in cervical spinal cord sections of wobbler mice treated with anti-HNE antibodies. Scattered HNE-stained neurons were detectable in anterior horns, whereas no reactivity could be observed in controls (Fig. 3).
DISCUSSION
Fig. 2. A representative RT-PCR analysis of S100B expression. PCR products of S100B mRNA and GAPDH were obtained from control spinal cord (respectively lanes 1 and 3) and from wobbler spinal cords (respectively, lanes 2 and 4). Band relative intensity (int /mm2), as measured by Quantity One Software, was: lane 1: 4011.63; lane 2: 9921.53; lane 3: 4324.34, lane 4: 3872.47. M, molecular marker (100 bp DNA ladder, Pharmacia).
The present data indicate S100B overexpression and immunostaining for HNE-modified proteins, an indicator of lipid peroxidation (22), in the spinal cord of wobbler mice. The overexpression of S100B in this animal model for motor neuron disease is consistent with previous data obtained in the spinal cord of ALS patients (23). On the basis of morphological features S100B also appears to be located in astrocytes, as expected. In addition, the pattern of distribution of the protein essentially reflected the distribution of the astroglial marker GFAP. We were unable to detect S100B immunoreactivity in motor neurons, as sporadically observed by Migheli et al. (23). The different retrieval procedures used in the immunocytochemical methods (24), and differences in the species used, could explain this discrepancy. Astrocytes have been hypothesized to play an active role in pathological processes of wobbler disease (13,14,16). Bearing this consideration in mind, it is tempting to speculate that overexpression of S100B is a part of this cascade of events. Findings suggesting that the protein exerts a protective effect at physiological concentrations but is neurotoxic at high local concentrations (17,18) additionally support this possibility. It may be that the overexpression of S100B in acti-
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Fig. 3. Micrograph of transverse sections of cervical spinal cord from control (A) and wobbler mice (B) stained with anti HNE-antibody. Sharply stained neurons are evident in wobbler mice sections, whereas no reaction is detectable in controls. Magnification: ⫻266.
vated glia is initially beneficial, as part of a compensatory response to neurodegeneration, whereas excessive or chronic overexpression of the protein may have deleterious consequences. The immunohistochemical detection of HNEmodified proteins offers evidence of lipid peroxidation in the spinal cord of wobbler mice. The property of HNE of binding cell proteins to form stable adducts has already been used to show the occurrence of oxidative stress–induced cell damage in pathological conditions such as Alzheimer’s disease and ALS (12,25,26). Prior to the contribution of the present data, the occurrence of oxidative stress in wobbler disease was based mainly on indirect evidence (7). Although S100B protein has been shown to induce oxidative stress in in vitro experimental systems (19,20), we are unable to identify any relationship between S100B overexpression and oxidative stress in the present animal model for motor neuron disease. Likewise, the present data do not allow us to state whether increased S100B in wobbler mice is due to the higher number of S100B-containing cells, to the upregulation of S100B in individual cells, or to both these factors. In any case, the larger quantity of S100B mRNA in wobbler mice is consistent with the higher number of cells immunostained for S100B detectable by light microscopy in these animals, and appears to be linked to an active transcription of S100B during the degenerative process. Future studies involving gene manipulation strategies and time-course of S100B mRNA transcription, and S100B protein expression and production of HNE-modified proteins during wobbler disease, will clarify whether the overexpression of S100B interacts with the cascade of
events that induces oxidative stress in motor neuron disease or is merely an epiphenomenon of reactive gliosis. ACKNOWLEDGMENTS This work was partially supported by grants from Università Cattolica del S. Cuore to F. M. We are grateful to Enrico Guadagni and Roberto Passalacqua (Università Cattolica S. Cuore) for their skilful technical assistance. P. B. is a recipient of “Fondazione M. Monzino” fellowship.
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