The FASEB Journal express article 10.1096/fj.02-0231fje. Published online September 19, 2002.
Decreased GFAP-mRNA expression in spinal cord of cobalamin-deficient rats Valerio Magnaghi†,‡, Daniela Veber*,‡, Alberto Morabito§, Francesca R. Buccellato*,‡, Roberto C. Melcangi†,‡, and Giuseppe Scalabrino*,‡ *Institute of General Pathology and †Department of Endocrinology, ‡Center for Excellence on Neurodegenerative Diseases, and §Institute of Biometrics and Medical Statistics, University of Milan, Milan, Italy Corresponding author: G. Scalabrino, MD Institute of General Pathology University of Milan, Via Mangiagalli, 31, 20133 Milan, Italy. E-mail:
[email protected] ABSTRACT We have demonstrated previously that chronic vitamin B12 [cobalamin (Cbl)] deficiency preferentially affects glial cells in the rat central nervous system (CNS) and severely affects peripheral glial cells independently of and concomitantly with the central neuropathy. In this study, we determined the mRNA levels for myelin basic protein (MBP) and glial fibrillary acidic protein (GFAP) in different CNS areas of rats made Cbl-deficient by total gastrectomy, as well as the mRNA levels for glycoprotein Po and peripheral myelin protein (PMP)22 in the sciatic nerve. GFAP-mRNA levels were significantly decreased in the spinal cord (SC) and hypothalamus, but not in the cortex, hippocampus, or striatum of totally gastrectomized (TGX) rats. No differences in GFAP protein levels were found in the SC and hypothalamus of the TGX rats treated or not with Cbl. MBP-mRNA levels were significantly decreased only in the hypothalamus, and the levels of mRNA for both glial markers returned to normal with Cbl replacement therapy. The levels of mRNA for the various myelin proteins in the sciatic nerve were not modified by Cbl deficiency. These results demonstrate that: a) the neurotrophic action of Cbl in rat CNS occurs in a zonal manner; and b) Cbl deficiency does not affect myelin synthesis (with the sole exception of the hypothalamus). Key words: CNS • hypothalamus • subacute combined degeneration • vitamin B12
I
n the adult human central nervous system (CNS), prolonged deficiency of vitamin B12 [hereafter referred to as cobalamin (Cbl)] leads to typical histologic lesions, which have been described extensively and are classically referred to as subacute combined degeneration (SCD), particularly affecting the spinal cord (SC) (1–3). The ultrastructural hallmarks of SCD are intramyelinic edema and interstitial edema in CNS white matter, again particularly SC white matter, and gliosis (1–3). Both intramyelinic edema and interstitial edema account for the widespread but uneven spongy vacuolation previously seen by using optical microscopy in the SC of Cbl-deficient (Cbl-D) rats (4, 5). SCD has therefore been classified as a pure myelinolytic disease with no apparent loss of myelin, and consequently it should no longer be considered a demyelinating disease stricto sensu (3, 6). Understanding the pathogenesis of SCD is of considerable importance because Cbl deficiency is fairly common in adult humans. Furthermore,
some adult Cbl-D patients may have serious neurological dysfunction, even without recognizable hematological abnormalities (7). We have been working on the pathogenesis of SCD from an experimental point of view for a number of years. We first succeeded in reproducing SCD-like lesions in the CNS of rats made Cbl-D as a result of total gastrectomy (TG) (4, 5). The totally gastrectomized (TGX) rats are deprived of intrinsic factor immediately after TG and subsequently develop permanent Cbl deficiency, thus reproducing the pathogenetic events of human Cbl deficiency as occur in pernicious anemia (4, 5). Secondly, we provided experimental evidence of peripheral neuropathy in rats made Cbl-D by means of TG or a prolonged Cbl-D diet, concomitant with and independent of the central neuropathy (8). We have also recently clarified the pathogenesis of the SCD induced in the CNS (and especially the SC) of TGX rats by demonstrating that the myelin lesions are caused by the locally increased production of the neurotoxic agent, tumor necrosis factor (TNF)-α (9), combined with the locally decreased production of the neurotrophic agent epidermal growth factor (EGF) (10, 11). We have also shown that the shift in the physiological equilibrium between neurotoxic and neurotrophic agents in the CNS of TGX rats (in favor of the former) is etiologically linked to their Cbl-D status, as it was substantially corrected by postoperative Cbl treatment (9, 10). There are still, however, some critical issues in the pathogenesis of SCD that remain to be elucidated, including the role of glial cells (2). In this context, it may be important that TGX rats show a marked increase in SC gray matter astrocytes (5), positive for their most classical marker glial fibrillary acidic protein (GFAP) (12–14), and also show ultrastructural evidence of activation of glial cells in both the peripheral nervous system (PNS) (8) and CNS (15), whereas neurons do not show any morphological alteration (15). For the present study, our hypothesis was that chronic Cbl deficiency might affect biochemical activities characteristic of the glial cells of the CNS and PNS of TGX rats. To this end, we determined the mRNA levels for one of the major myelin proteins, myelin basic protein (MBP) (16, 17), and the mRNA levels for GFAP in different CNS areas. Moreover, the mRNA levels for the two major myelin proteins of the PNS, glycoprotein Po and peripheral myelin protein (PMP)22 (16, 17), were evaluated in sciatic nerve. Furthermore, when abnormalities in the mRNA levels for these myelin proteins were observed, we determined whether or not they were corrected by correction of the Cbl deficiency. Last, the GFAP protein levels in the CNS areas, in which we found a decrease in GFAP-mRNA levels (see results), were analyzed by means of Western blots. MATERIALS AND METHODS Animals and in vivo treatments Adult non-inbred male albino Sprague-Dawley rats (Charles River Italia, Calco, Italy), weighing 250 g at the beginning of the experiment, were housed as previously described (4, 5). Some rats underwent TG in order to induce experimental SCD, as previously reported (4, 5). Body temperature was maintained at 37°C over the period of the surgical procedures and recovery from anesthesia (i.e., until normal locomotor activity was observed). Laparotomized (LPT)
animals served as controls. Short-term post-operative treatment with antibiotics was given as previously described (4, 5). The perioperative mortality rate and the mean change in body weight of the TGX rats during the investigation period were as previously published for our laboratory (4, 5). When administered, Cbl was injected subcutaneously once a week at 150 µg/100 g body weight, for the first two post-operative months (mo). The rats were killed by decapitation at the times shown in the figures, with different CNS areas (cerebellum, cortex, hypothalamus, hippocampus, striatum, and SC) and the sciatic nerve rapidly removed and stored at –80° C for subsequent Northern or Western blot analyses. At the time of death, all of the rats were the same age. Procedures involving animals and their care were conducted in conformity with the institutional guidelines and in compliance with national and international laws and policies (EEC Council Directive 86/609, OJ L 358, 1 Dec., 12, 1987; NIH Guide for the Care and Use of Laboratory Animals, NIH Publication No. 86-23, 1985). RNA preparation Tissues were homogenized in 4 M guanidium isothiocyanate (containing 25 mM sodium citrate, pH 7.5, 0.5% sarcosyl and 0.1% 2-mercaptoethanol), and total RNA was isolated by phenolchloroform extraction according to the method of Chomczynski and Sacchi (18). Quantification was performed by absorption at 260 nm, and the RNA was re-precipitated in ethanol. RNA probes The probes were made with [32P]CTP as the labeled nucleotide. In particular, levels of GFAP were determined with a pBluescript-SK plasmid containing a 2.7kb insert of GFAP, which, after linearization with HindIII, was used as a template to generate an antisense cRNA probe by using T3 RNA polymerase. The levels of MBP were determined with a pBluescript-SK plasmid containing a 660 bp insert of MBP; the plasmid was linearized with EcoRI and used for in vitro transcription with T7 RNA polymerase to generate an antisense cRNA probe. The levels of Po were analyzed with a pBluescript-SK plasmid containing a 297bp insert of Po; after linearization with ClaI, the plasmid was used for in vitro transcription with T3 RNA polymerase to generate an antisense cRNA probe. Finally, the levels of PMP22 were analyzed with pRc/CMV plasmid containing a 524 bp insert of PMP22; after linearization with XmnI, the plasmid was used as a template to generate an antisense cRNA probe using SP6 RNA polymerase. The pTRI-28S-rat plasmid (Ambion, Austin, TX) was used to generate an antisense cRNA probe to 28S rRNA (internal standard) with T3 RNA polymerase. Northern blot analysis Northern blot analyses of GFAP-, MBP-, Po-, and PMP22-mRNA levels were performed as previously described (19, 20). In brief, total RNA was separated on 1% agarose-formaldehyde gel and then transferred to a nylon membrane (Z-probe, BioRad, Milan, Italy) with a 10× solution containing 300 nM sodium chloride, 30 nM sodium citrate (SSC). The blots were prehybridized at 65°C for 4 h, the buffer changed and the membranes hybridized overnight in buffer containing 50% formamide, 4× solution containing 0.6 M sodium chloride, 40 nM sodium dihydrophosphate, 4 nM EDTA, 2× Denhardt’s solution, 0.5% sodium dodecyl sulfate (SDS),
100 µg/ml salmon sperm DNA, and [32P]cRNA antisense probes to GFAP, MBP, Po, or PMP22. The following day, the membranes were washed once at room temperature for 20 min in 2× SSC-0.1% SDS, and 2–3 times at 65°C in 0.1× SSC-0.1% SDS. The blots hybridized for MBP, Po, or PMP22 were then exposed to the autoradiographic films, with intensifying screens at –80°C for 2–10 h; the blots hybridized for GFAP were exposed under the same conditions for 10–24 h. Membranes were subsequently hybridized with the [32P]cRNA antisense probe to the 28S rRNA. The hybridization and washing conditions were as described above, with an exposure time of 2– 6 h. Calculation of mRNA levels The levels of mRNA for GFAP, MBP, Po, and PMP22 were calculated from the peak densitometric area of the autoradiograph as measured by an LKB Laser Densitometer. Different exposure times were used to ensure that the autoradiographic bands were in the linear range of intensity. Western blot analysis The cytoskeletal-enriched fraction was prepared as described by Dahl et al. (21) with slight modifications. Briefly, the hypothalamus and SC from the LPT, TGX, and TGX- and Cbl-treated rats were rapidly removed 2 and 6 months after surgery, frozen on dry ice, and stored at –80°C. The tissues were homogenized for 1 min at 4°C in a polytron homogenizer by using a cytoskeletal extraction buffer (1:4 w/v) whose composition was 1% Triton X-100, 0.6 M KCl, 10 mM MgCl2, 2 mM EDTA, 1 mM ethylene glycol-bis(β-aminoethyl)-N,N,N’,N’-tetraacetate (EGTA), 1 mM phenylmethyl sulfonyl fluoride, 0.5 mg/ml p-tosyl-L-arginine methyl ester hydrochloride in Ca2+ and Mg2+-free phosphate buffered saline, pH 7.1, at 4°C. This extraction buffer contained a protease inhibitor cocktail of aprotinin (1 µg /ml), pepstatin A (1 µg /ml), and leupeptin (250 µg /ml). Homogenization was followed by the addition of 0.1 mg/ml DNase I (type II, Sigma, St. Louis, MO) and prolonged for an additional 1 min. The cytoskeletal residues were pelleted at 12,000 g for 2 min. The supernatants were removed, incubated for 30 min at 4°C, and centrifuged at 48,000 g for 30 min. The protein content was quantified as previously described (4). The samples were immediately frozen in dry ice and stored at –80°C before Western blot analysis. The cytoskeletal extracts containing 20 µg of protein were dissolved in SDS-gel sample buffer (10 mM sodium phosphate, pH 7.2 , 1% SDS, 20 mM dithiotreitol, 10% glycerol and bromophenol blue) and heated for 10 min in a boiling water bath. They were then electrophoresed on 12% sodium phosphate SDS-PAGE. Polyclonal antibodies to GFAP (Dako Corporation, Glostrup, Denmark) were used at a final dilution of 1:1000. Immunoreactive bands were visualized by using a chemoluminescent substrate (SuperSignal West, Pierce, Rockford, IL) and were quantified by densitometry (LKB Laser Densitometer). Studies of blood components Red cell count, hematocrit, hemoglobin concentration, and the levels of Cbl and folate in sera were determined from blood collected from anesthetized animals by cardiac puncture just before killing, as previously described (5, 8). Anemia and very-low-serum Cbl levels were observed in TGX rats, as routinely observed in our laboratory (5, 8). In agreement with our previous
observations, post-operative administration of Cbl to TGX rats following the scheme above reported significantly increased serum Cbl levels above those of non-replaced TGX animals (5, 8). Statistical analyses The homoscedasticity of the variances was tested by Bartlett’s test (22), which never reached the level of statistical significance. To normalize the variable ratio of the arbitrary densitometric units for each protein to the arbitrary densitometric units for the corresponding 28S, ratios were logarithmically transformed. The transformed ratios were analyzed by one-way analysis of variance (ANOVA), taking into account the duration of the experiment (0, 2, and 4 months), the treatment (Cbl deprivation). The post-operative Cbl therapy was also taken into account as appropriate. Bonferroni’s correction (23) was used to avoid false positive statistical levels. For each statistical analysis, an α level of 0.05 or less was considered statistically significant. RESULTS The results of the densitometric analyses of the Northern blots of GFAP- and MBP-related mRNAs in the SC of TGX rats (after normalization against 28S rRNA) are shown in Figure 1A. These data demonstrate that the mRNA levels of GFAP are decreased significantly 2 and 4 months after TG, whereas those of MBP are unchanged 2 months after TG. Furthermore, Figure 1A also shows that the decrease in SC GFAP-mRNA is prevented by Cbl replacement therapy over the first two post-operative months. Figure 1B shows a representative Northern blot in which the decrease in GFAP-mRNA but not in MBP-mRNA is shown, in line with the densitometric analyses shown in Figure 1A. GFAP-mRNA levels remained unchanged in the cortex, hippocampus, and striatum of 2 mo-TGX rats (not shown); whereas both GFAP- and MBP-mRNA in the hypothalamus significantly decreased in TGX rats two months after TG (Fig. 2). This decrease in hypothalamic mRNA levels can be prevented by Cbl replacement therapy over the first two months after TG (Fig. 2). The changes in MBP-mRNA levels in the hypothalamus seem to be a peculiarity of this cerebral structure. MBP-mRNA levels in cortex, hippocampus, and striatum are not changed by Cbl-D status (not shown). Densitometric analysis of Northern blots of the main peripheral myelin proteins (Po, PMP22, and MBP) shows that Cbl deficiency did not affect their mRNA levels in the sciatic nerve (not shown). Immunoblot analysis for GFAP protein Western blot analysis showed that GFAP protein is present in the SC and hypothalamus of LPT, TGX, and TGX- and Cbl-treated rats (not shown). Densitometric analysis did not reveal any differences in either CNS area in any of the experimental groups (data not shown). This means that there is no correlation between GFAP-mRNA and protein levels in the SC and hypothalamus of TGX rats treated or not with Cbl.
DISCUSSION The major finding of the present study is that GFAP-mRNA levels are regulated by Cbl in some parts of rat CNS (the SC and hypothalamus). In TGX rats (which lack Cbl), there is a sustained decrease in the levels of GFAP-mRNA, which is prevented by post-operative Cbl treatment. This type of regulation seems to be very similar to that previously found for EGF-related mRNAs and TNF-α protein in different parts of the CNS of TGX rats: both the decrease in EGF synthesis and the increase in TNF-α protein [not followed by any histological signs of apoptosis (9)] in the SC were corrected by post-operative Cbl administration (9, 10). The fact that a Cbl-mediated decrease in GFAP-mRNA levels was seen only in the SC and hypothalamus clearly suggests the neurotrophic regional action of Cbl in rat CNS. Given that the SC is the CNS area most affected by chronic Cbl deficiency in both man and rat (2, 5, 7), it is tempting to speculate that decreased levels of GFAP-mRNA play a role in the markedly damaged SC induced by Cbl deficiency. The CNS of mice lacking GFAP shows severe morphological damage, particularly of the white matter (24), and it has therefore been inferred that GFAP plays an essential role in maintaining the structure of CNS white matter, especially in the SC (25). In keeping with this, we have demonstrated previously that the administration of Cbl to TGX rats for the first two postoperative months greatly decreased the severity of spongy vacuolation and increased the number of GFAP-positive astrocytes in the SC white matter (5). However, other authors (26, 27) have reported that mice lacking GFAP do not show any detectable abnormalities in the anatomy of the brain and cerebellum; i.e., in CNS portions other than the SC. It is widely recognized that the precise function of GFAP remains unclear (13, 25), and that specific astroglial subpopulations exist in rat CNS (28–30). Nevertheless, the findings of this and our previous study (15) on glial cells in the SC of TGX rats lead us to conclude that chronic Cbl deficiency mainly affects SC astrocytes. We have previously demonstrated an increase in GFAP immunostaining (5) and ultrastructural signs of astroglial activation (15), both of which persist over the course of the disease. They all seem to be characteristic of experimental SCD, because they are corrected by post-operative Cbl replacement treatment (5, 15). Parenthetically, a second type of GFAP-positive and myelin marker-positive glia has been tentatively identified as a nonmyelinating oligodendrocyte (31), and so it is conceivable that Cbl deficiency may also affect this type of glial cell. In a previous paper (5), we reported an increase in GFAP-immunoreactive astrocytes in the gray matter of the SC of TGX rats. At first sight, this result may seem to be in contrast with the decrease in GFAP-mRNA levels reported here, but this is not necessarily the case because it is well known that enhanced GFAP immunohistochemical staining frequently does not correlate with the content of GFAP protein and/or GFAP-mRNA levels (13, 25, 32, 33). The reason for this dichotomy in astrocytes is essentially unknown, although it may reflect a change in the accessibility of epitopes recognized by anti-GFAP antibodies (25). In the present study there is another dichotomy: the lack of correlation between GFAP-mRNA and protein levels in the tested CNS areas of TGX rats treated or not with Cbl. This result supports the existence of a difference in the post-transcriptional control of GFAP gene expression between normal and Cbl-D CNS. Alternatively, the stability of GFAP-mRNAs may differ in the CNS of the rats belonging to the different experimental groups.
It is still difficult to say whether the decrease in GFAP-mRNA levels is caused directly by Cbl deficiency or is mediated by a decrease in CNS EGF synthesis and EGF levels in the cerebrospinal fluid, as we have observed both in Cbl-D rats (10). It has been shown that some hormones (e.g., insulin, corticosteroids, and sex steroids) (19, 34–38), growth factors (e.g., EGF, fibroblast growth factor, and transforming growth factor-β1) (34, 39, 40) and cytokines [interleukin (IL)-1 and IL-6] (34) regulate GFAP synthesis in rodent brain and cultured rat astrocytes. The in vivo up-regulation of GFAP-mRNA levels by Cbl is the opposite of the downregulation by corticosteroids observed in vivo in some areas of rat brain, in which GFAP transcription is inhibited (19, 34–36, 38). Furthermore, a Cbl-mediated effect on GFAP-mRNA degradation cannot be ruled out. In terms of the response of the main myelin genes to the Cbl deficiency induced by TG, the levels of mRNA for the main CNS myelin protein (MBP) are generally unaffected by Cbl deficiency in TGX rats (with the sole exception of the hypothalamus), as are the levels of mRNA for Po and PMP22 in the PNS. Our results are in agreement with a previous report by Deacon et al. (41), who found no change in the methylation of MBP in the brain of Cbl-D rats, although MBP is present in various neuron subpopulations of rat CNS (42), and MBP gene products have functions other than those of myelin proteins; e.g., they can act as cell adhesion molecules, like P0 (43–46). Therefore, the degenerative Cbl-D neuropathy in both the CNS and PNS of TGX rats cannot reasonably be attributed to abnormal myelin synthesis. All of these findings further support the view that experimental Cbl-D neuropathy is a pure myelinolytic neurodegenerative disease (6) with neither ultrastructural signs of remyelination in the CNS of Cbl-D rats (15) nor any changes in the main classes of neurolipids in the SC (3). ACKNOWLEDGMENTS G.S. is greatly indebted to J.W. Funder (Melbourne, Australia), for his critical reading of the manuscript and helpful discussions, and is also very grateful to P. Mantegazza (Rector of the Universiy of Milan), for his continued interest in this research. This study was supported by grants from the Ministero dell’Università e della Ricerca Scientifica e Tecnologica (MURST 60%, Rome, Italy) to G.S. and R.C.M., and from the University of Milan (“Progetto Speciale”) to G.S. G.S. would also like to acknowledge the kind supply of antibiotics used in this study by Aventis S.p.A. (Milan, Italy) and by Hoechst Italia (Milan, Italy). Finally, G.S. would like to thank E. Mutti for editorial assistance. REFERENCES 1.
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Fig. 1
Figure 1. Effect of vitamin B12 (cobalamin, Cbl) deficiency and Cbl replacement therapy on the levels of glial fibrillary acidic protein (GFAP)- or myelin basic protein (MBP)-mRNAs in the spinal cord of totally gastrectomized (TGX) rats killed postoperatively at the indicated times (mo = months). A) Results of the densitometric analysis of the Northern blot autoradiography of GFAP- or MBP-mRNAs. Data are expressed as arbitrary densitometric units (ADU); the values are means (columns) ± SEM (vertical bars). The number of animals is given in parentheses. **P
