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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 19, 2002 as doi:10.1096/fj.02-0231fje. |
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,
,2
* Institute of General Pathology; and
Department of Endocrinology;
Center for Excellence on Neurodegenerative Diseases; and
Institute of Biometrics and Medical Statistics, University of Milan, 20133 Milan, Italy
2Correspondence: Institute of General Pathology, University of Milan, Via Mangiagalli, 31, 20133 Milano (MI), Italy. E-mail: giuseppe.scalabrino{at}unimi.it
SPECIFIC AIM
In the adult human central nervous system (CNS), prolonged deficiency of vitamin B12 [hereafter referred to as cobalamin (Cbl)] leads to typical histological lesions, which have been extensively described and are classically referred to as subacute combined degeneration (SCD), particularly affecting the spinal cord (SC). There are still, however, some critical issues in the pathogenesis of SCD that remain to be elucidated, including the role of glial cells. It may be important that our experimental model of human SCD, i.e., the totally gastrectomized (TGX) rat (which lacks Cbl), shows a marked increase in SC gray matter astrocytes, positive for their most classical marker glial fibrillary acidic protein (GFAP), and also shows ultrastructural evidence of activation of glial cells in both the peripheral nervous system (PNS) and CNS, whereas neurons show no morphological alteration. We previously provided experimental evidence of peripheral neuropathy in TGX rats concomitant with and independent of the central neuropathy.
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 mRNA levels for one of the major myelin proteins, myelin basic protein (MBP), and mRNA levels for GFAP in different CNS areas. mRNA levels for the two major myelin proteins of the PNS, glycoprotein Po and peripheral myelin protein (PMP)22, were evaluated in sciatic nerve. 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, GFAP protein levels in the CNS areas, in which we found a decrease in GFAP-mRNA levels, were analyzed by means of Western blots.
PRINCIPAL FINDINGS
1. Cbl positively regulates the GFAP-mRNA expression in the rat SC
Results of the densitometric analyses of the Northern blots of GFAP- and MBP-related mRNAs in the SC of TGX rats demonstrate that mRNA levels of GFAP are significantly decreased 2 and 4 months after total gastrectomy (TG), whereas those of MBP are unchanged 2 months after TG (Fig. 1
A). Figure 1A
also shows that the decrease in SC GFAP-mRNA is prevented by Cbl replacement therapy over the first 2 postoperative 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 Fig. 1A
. GFAP-mRNA levels remained unchanged in the cortex, hippocampus, and striatum of 2 month TGX rats (not shown). Western blot analysis showed that GFAP protein levels remained unchanged in the SC of TGX rats (data not shown).
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2. Cbl positively regulates the expression of the GFAP-and MBP-mRNA in the rat hypothalamus
The results presented in Fig. 2
demonstrate that GFAP- and MBP-mRNA in the hypothalamus both decreased significantly in TGX rats 2 months after TG and that this decrease can be prevented by Cbl replacement therapy over the first 2 months after TG. MBP-mRNA levels in cortex, hippocampus, and striatum are not changed by Cbl-deficient (Cbl-D) status, i.e., 2 months after TG (not shown).
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3. Chronic Cbl deficiency does not affect myelin synthesis in the sciatic nerve of the rat
Densitometric analysis of the Northern blots of the main peripheral myelin protein (P0, PMP22, and MBP) shows that chronic Cbl deficiency did not affect their mRNA levels in the sciatic nerve of 2 month TGX rats (not shown).
CONCLUSIONS
The major finding of the present study is that GFAP-mRNA levels are positively regulated by Cbl in some parts (the SC and hypothalamus) of rat CNS. 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-
protein in different parts of the CNS of TGX rats: both the decrease in EGF synthesis and the increase in TNF- protein (not followed by any histological signs of apoptosis) in the SC were corrected by post-operative Cbl administration. The fact that a Cbl-mediated decrease in GFAP-mRNA levels was only seen in the SC and hypothalamus clearly means that the neurotrophic action of Cbl in rat CNS occurs in a zonal manner. Given that the SC is the CNS area most affected by chronic Cbl deficiency in humans and rats, 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, and it has therefore been inferred that GFAP plays an essential role in maintaining the structure of CNS white matter, especially in the SC. In keeping with this, we previously demonstrated that administration of Cbl to TGX rats for the first 2 postoperative months greatly decreased the severity of spongy vacuolation and the increase in GFAP-positive astrocytes and increased the number of GFAP-positive astrocytes in the SC white matter. However, other authors have reported that mice lacking GFAP do not reveal 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 and that specific astroglial subpopulations exist in rat CNS. Nevertheless, the findings of this and our previous study of glial cells in the SC of TGX rats lead us to conclude that chronic Cbl deficiency mainly affects SC astrocytes.
It is still difficult to say whether the decrease in GFAP-mRNA levels is caused directly by Cbl deficiency or mediated by a decrease in CNS EGF synthesis and EGF levels in the cerebrospinal fluid, as we have observed both in Cbl-D rats. It has been shown that some hormones (e.g., insulin, corticosteroids, and sex steroids), growth factors (e.g., EGF, fibroblast growth factor, and transforming growth factor ß1) and cytokines interleukin-1 (IL–1) and IL-6 regulate GFAP synthesis in rodent brain and cultured rat astrocytes.
In terms of the response of the main myelin genes to the Cbl deficiency induced by TG, 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 levels of mRNA for P0 and PMP22 in the PNS. Our results agree with what has been reported by other authors 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 and MBP gene products have functions other than those of myelin proteins (e.g., they can act as cell adhesion molecules, like P0). Therefore, the degenerative Cbl-D neuropathy in the CNS and PNS of TGX rats cannot reasonably be attributed to abnormal myelin synthesis. According to these results, we propose a schematic diagram of the hypothetical sequence of events whereby a permanent withdrawal of Cbl is associated with the myelinolysis in SC white matter (Fig. 3
).
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0231fje; to cite this article, use FASEB J. (September 19, 2002) 10.1096/fj.02-0231fje 
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