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Myelin Basic Protein Gene Dosage Effects in the PNS
Molecular and Cellular Neuroscience 15, 343–354 (2000) doi:10.1006/mcne.1999.0829, available online at http://www.idealibrary.com on
MCN
Myelin Basic Protein Gene Dosage Effects in the PNS Candra Smith-Slatas* and Elisa Barbarese* ,1 *Combined Degree Program and Neuroscience Graduate Program and Department of Neurology, University of Connecticut Health Center, Farmington, Connecticut 06030
Myelin basic protein (MBP) plays an essential adhesive role in the formation of compact myelin in the central nervous system (CNS), but not in the peripheral nervous system (PNS). Morphologic data suggest that MBP controls the number of cytoplasmic channels or Schmidt– Lanterman incisures (SLI) present in PNS myelin. The levels of connexin-32 (Cx32) and myelin-associated glycoprotein (MAG), two components of the incisures, are inversely proportional to the levels of MBP in sciatic nerves of mice affected by the shiverer (shi) mutation, while protein zero (P0) and peripheral membrane protein 22 (PMP22), two structural components of compact myelin, remain constant. The levels of P0, PMP22, Cx32, and MAG mRNA do not vary in relationship to the levels of MBP. This indicates that MBP exerts its effect on Cx32 and MAG at a posttranscriptional level and suggests a new function for MBP in regulating gene expression in the PNS.
INTRODUCTION Myelin from the peripheral nervous system (PNS) of the homozygous mutant mouse shiverer (shi/shi) is compact despite the total absence of myelin basic protein (MBP) (Kirschner and Ganser, 1980; Mikoshiba et al., 1980; Rosenbluth, 1980). It has been postulated that the adhesive role of MBP in central nervous system myelin (CNS) is performed by protein zero (P0) in PNS myelin (D’Urso et al., 1990; Filbin et al., 1990; Martini et al., 1995). If this were the case, the level of P0 may be elevated in PNS myelin from shi/shi mice to compensate for the absence of MBP. Further morphologic studies indicated that the number of cytoplasmic channels called Schmidt–Lanterman incisures (SLI) was elevated 1 To whom correspondence and reprint requests should be addressed. Fax: (860) 679-4446. E-mail: [email protected].
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in shi/shi PNS myelin, suggesting that several components may be up-regulated in the absence of MBP (Gould et al., 1995). We sought to determine if the levels of connexin 32 (Cx32) and myelin-associated glycoprotein (MAG), two components of the incisures, and P0 were increased in shi/shi PNS myelin in comparison to wild type (wt) animals, and if the levels of these proteins correlated with the levels of MBP. In order to distinguish whether gene expression was regulated through transcription or translation, the levels of several myelin proteins cognate mRNA were measured. A comparison of morphologic and biochemical studies suggests that there is an inverse correlation between the levels of MBP and the number of SLI. In the rat sciatic nerve, the 18.5-kDa isoform of MBP is 2% of the total myelin proteins (Greenfield et al., 1973), and the number of incisures is 25 per internode (Hiscoe, 1947). In the guinea pig sciatic nerve, MBP is 16% of the total myelin proteins (Greenfield et al., 1973), and the number of incisures is 12 per internode for a fibre of equivalent diameter and internodal distance (Webster, 1964). In the CNS of anyone species, there is a positive correlation between the levels of MBP and myelin thickness (Shine et al., 1992), which may not be significant in the PNS; in the latter, there is a positive correlation between the fibre diameter and the number of SLI (Hiscoe, 1947). In the absence of MBP, in homozygous shi mice, the number of incisures is double that found in wt sciatic nerves (Gould et al., 1995), while myelin thickness is reportedly very midly reduced (Peterson and Bray, 1984) or normal (Rosenbluth, 1980). The present study uses homozygous (shi/shi) and heterozygous shi (⫹/shi) mice to examine the correlation between the level of MBP and the number of SLI in the sciatic nerves at a biochemical and molecular level because these animals differ genetically only by the number of functional MBP gene copies.
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344 The shi mutation results from a large deletion in the MBP gene with no MBP protein or messenger RNA being produced (Roach et al., 1983). It is functionally a null allele for MBP and introduction of the wt MBP gene in the genome of homozygous shi animals rescues the mutant phenotype (Readhead et al., 1987). MBP is a myelin-specific component localized to the compact regions of CNS and PNS myelin (Omlin et al., 1982). In both the CNS and the PNS, the amount of MBP is directly proportional to the number of gene copies present, and ⫹/shi myelin contains half the amount of MBP found in wt (Barbarese et al., 1983; Popko et al., 1987). SLI are cytoplasmic channels within the compact myelin sheath. They contain Schwann cell cytoplasm, a few organelles, and gap junctions composed of Cx32 proteins that link successive wrappings of myelin (Peters et al., 1991). The following proteins have been assigned to SLI: 2⬘,3⬘-cyclic nucleotide phosphohydrolase, MAG, S100, Cx32, E-cadherin, DM-20, and actin (Puckett et al., 1987; Trapp et al., 1989; Mata et al., 1990; Bergoffen et al., 1993; Fannon et al., 1995; Griffiths et al., 1995; Anderson et al., 1997; Garbern et al., 1997). P0, MBP, P2, and peripheral myelin protein-22 (PMP22) are present in the compact regions of the myelin sheath (Trapp et al., 1981; Omlin et al., 1982; Welcher et al., 1991; Snipes et al., 1992; Anderson et al., 1997; Garbern et al., 1997). There is concordance between the levels of myelin protein mRNAs and their corresponding proteins during the active period of myelination as indicated by the analysis of the steady state levels of MAG, MBP, Cx32, PMP22, and P0 mRNA in the rat sciatic nerve (Stahl et al., 1990; Gupta et al., 1990; Scherer et al., 1995; Griffiths et al., 1989; Gow et al., 1994; Kuhn et al., 1993; Snipes et al., 1992). In this paper we have used a ribonuclease protection assay (RPA) to examine the steady state mRNA levels of PMP22, Cx32, and MAG in 90-day-old shi/shi and wt animals to determine if gene expression was similarly regulated.
RESULTS Localization of Cx32 and MAG to SLI in shi/shi Sciatic Nerves In a previous study the increased density of SLI in shi/shi sciatic nerves was estimated from morphologic data (Gould et al., 1995). In order to further characterize these structures, immunofluorescence was performed to assess the localization of two of their components, Cx32 and MAG. Whole sciatic nerves were stained with
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anti-Cx32, anti-MAG, and with fluorescein-conjugated phalloidin to delineate SLI and nodes of Ranvier. The results showed that Cx32 and MAG were found in filamentous actin-positive incisures (arrowheads) in both wt and shi/shi sciatic nerves. Other compartments of the nerve such as the outer mesaxons, Schwann cell bodies (arrows), and the nodes of Ranvier, which have been reported to contain MAG and Cx32 (Trapp et al., 1989; Scherer et al., 1995), were also positively stained. All incisures-like structures identified by phalloidin labeling contained Cx32 and MAG, indicating that the supernumerary incisures in shi/shi sciatic nerves are bona fide SLI and suggesting that the levels of the two proteins should be commensurably increased in the sciatic nerves of shi/shi mice. Relationship between MBP Levels and Incisures in ⴙ/shi Mice Sciatic Nerves In order to further establish the relationship between the levels of MBP and the density of SLI, the latter was assessed in 90-day-old ⫹/shi mice sciatic nerves (n ⫽ 2) that contain half the amount of MBP present in wt sciatic nerves (see below). Fluorescein-conjugated phalloidin was used to identify the incisures. Examples of stained nerves are shown in Fig. 1. The analysis showed that ⫹/shi nerves contained 6.0 incisures per 100 m, which is intermediate between wt and shi/shi nerves of the same age (3.9 and 7.5 SLI/100 m for wt and shi/shi sciatic nerves, respectively) (Gould et al., 1995), indicating that there is an inverse correlation between the amount of MBP present in the nerve and the number of incisures. Correlation between MBP Levels and Other Myelin Proteins The relationship between the number of functional MBP genes and the level of SLI components was quantified by immunodot blot analysis of wt, ⫹/shi, and shi/shi sciatic nerve components. In a first step, the protein profile of PNS myelin from adult wt and shi/shi mice was obtained to insure the identity of the proteins recognized by the battery of antisera used. Membrane and soluble fractions of 90-day-old wt and shi/shi sciatic nerves were analyzed for the presence of myelin proteins by Western blot. Compact myelin membrane proteins P0, PMP22, and MBP, and SLI proteins MAG and Cx32 were recovered exclusively in the membrane fraction of both genotypes. Cx32 was also present in the membrane fraction of liver obtained from the same animals.
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FIG. 1. Localization of filamentous actin (fluorescein-conjugated phalloidin), Cx32, and MAG proteins in sciatic nerves. Wt, shi/shi, and ⫹/shi sciatic nerves were incubated with fluorescein-conjugated phalloidin, Cx32 antibodies (1:75) (wt and shi/shi), and MAG antibodies (1:50) (wt and shi/shi), followed by a Texas red-conjugated secondary antibody (1:50). Schwann cell body (arrow); Schmidt-Lanterman incisures (arrowhead). The scale bar in each panel represents 25 m.
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FIG. 2. Western blot of sciatic nerve and liver proteins from 90-day-old wt and homozygous shi mice. Wt (8 g) and shi/shi (8 g) sciatic nerve, wt (2 g), and shi/shi (2 g) livers were probed with antibodies to P0, PMP22, MBP, MAG, and Cx32 as described under Materials and Methods. Molecular weight standards are indicated to the left.
Figure 2 shows the protein profile of the membrane fractions. As previously reported, the four main isoforms of MBP (21.5, 18.5, 17, and 14 kDa) were present in wt and ⫹/shi, and absent in shi/shi sciatic nerves (Carson et al., 1983; Mikoshiba et al., 1983). P0 and PMP22 migrated as single bands at their expected apparent molecular weights of 33 and 22 kDa, respectively, in both wt and shi/shi sciatic nerves. They were present in ⫹/shi samples as well (data not shown). MAG migrated as a single band with an apparent molecular weight of 100 kDa in both wt and shi/shi sciatic nerves. Cx32 appeared as a polymeric series in the liver and sciatic nerves consistent with published reports (Hertzberg and Skibbens, 1984). When the Cx32 antibody was presorbed with the Cx32 inhibitor peptide, no bands were detected indicating that these bands represent monomer and multimers containing Cx32 (data not shown). Levels of each protein identified in Fig. 2 were determined by immunodot blot of wt (n ⫽ 5), ⫹/shi (n ⫽ 3), and shi/shi (n ⫽ 5) sciatic nerves. As can be seen in Fig. 3, the levels of P0 and PMP22 were comparable in all genotypes. MBP was absent in shi/shi and present at half the wt concentration in ⫹/shi animals, a finding similar to that for 15-day-old animals (Barbarese et al., 1983). This difference between wt and ⫹/shi animals was
found to be significant (P ⬍ 0.05). MAG and Cx32 levels were significantly increased more than fourfold in shi/ shi sciatic nerves as compared to wt sciatic nerves (P ⬍ 0.05). A significant twofold increase of these proteins was also seen in ⫹/shi sciatic nerves as compared to wt (P ⬍ 0.05). In contrast to sciatic nerves, the level of Cx32 in liver, a tissue that does not contain MBP, was comparable in all genotypes (data not shown). These data indicate that the levels of molecular components of SLI, MAG, and Cx32 are correlated with the presence of supernumerary incisures in mice bearing one or two copies of the shi gene and inversely correlated to the amount of MBP in the nerves. Furthermore, the expression of Cx32 was not affected by the shi mutation in a tissue (liver) that does not contain MBP. These data reveal that there is a MBP gene dosage effect on the expression of MAG and Cx32 in the PNS. Steady State Levels of Myelin mRNAs in wt and shi/shi Sciatic Nerves The increase in MAG and Cx32 may result from upregulation of gene expression at the transcriptional or posttranscriptional levels. In order to distinguish between these two possibilities, the steady state levels of PMP22, MAG, and Cx32 mRNAs were measured in
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FIG. 3. Levels of myelin proteins in wt, heterozygous, and homozygous shi sciatic nerve. The relative levels of proteins were determined by immunodot blot as described under Materials and Methods in sciatic nerves of 90-day-old wt (n ⫽ 5), heterozygous (n ⫽ 3), and homozygous shi (n ⫽ 5) mice. Paired T test analyses were performed: (*) P ⬍ 0.05 when comparing wt and shi/shi, wt and ⫹/shi, and ⫹/shi and shi/shi levels of MBP, Cx32, and MAG. **P ⬎ 0.05 when comparing wt and shi/shi, wt and ⫹/shi, and ⫹/shi and shi/shi levels of P0 and PMP22.
adult wt (n ⫽ 3) and shi/shi (n ⫽ 3) mice; cyclophilin mRNA was used as an internal control to compare the levels of the myelin mRNAs. The cyclophilin, PMP22, Cx32, and MAG probes protected a 103, 315, 264, and 303–340 nucleotide fragments, respectively, in both wt and shi/shi samples (Fig. 4A). The ratios of PMP22 mRNA to cyclophilin mRNA were found to be 0.83 and 0.84 in wt and shi/shi nerves, respectively (Fig. 4B) and were not significantly different between the two genotypes. The ratios of MAG mRNA to cyclophilin mRNA were found to be 0.19 and 0.20 in wt and shi/shi nerves, respectively. The ratios of Cx32 mRNA to cyclophilin mRNA were 0.32 for wt and 0.36 for shi/shi nerves. The ratios for both mRNAs were not statistically different between wt and shi/shi sciatic nerves. These data indicate that the steady state levels of PMP22, MAG, and Cx32 mRNAs are similar in wt and shi/shi animals. This suggests that a posttranscriptional mechanism may be responsible for the increase in MAG and Cx32 proteins observed in shi/shi sciatic nerves.
Localization of MBP in Sciatic Nerves In order to understand how MBP may exert its action on the steady state levels of MAG and Cx32 proteins, we sought to determine if it was localized in Schwann cell subcellular compartments other than myelin. Figure 5A shows that MBP (green) was homogeneously distributed in the myelin sheath surrounding an axon (red) identified by the presence of filamentous actin. MBP (green) was absent from the perikaryon and the nucleus of Schwann cells or present at concentrations below the level of detection (Figs. 5B and 5C). MBP was never detected in the myelin sheath, Schwann cell perikaryon, or nucleus of sciatic nerves from shi/shi mice (Fig. 5).
DISCUSSION There is no apparent increase in the amount of P0 in PNS myelin of shi/shi mice to compensate for the ab-
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FIG. 4. Ribonuclease protected fragments in wt and homozygous shi sciatic nerve. (A) PMP22, MAG, and Cx32 (top), and cyclophilin (bottom) mRNA protected fragments in wt and homozygous shi sciatic nerves. (B) Ratios of PMP22, MAG, and Cx32 mRNA levels to cyclophilin mRNA levels in wt and shi/shi sciatic nerves. The amount of total RNA used was 0.75, 2.5, and 2.5 g for PMP22, MAG, and Cx32, respectively. **P ⬎ 0.05, when comparing wt and shi/shi, paired 2-tail T test. Wt, n ⫽ 3; shi/shi, n ⫽ 3.
sence of MBP. There is, however, an increase in the amount of MAG and Cx32 proteins, which is inversely proportional to the amount of MBP present in myelin. This up-regulation of gene expression is a posttranscriptional event for both MAG and Cx32. These results are consistent with morphologic data that show that PNS myelin from shi/shi mice is normal in compaction, lamellar structure, and periodicity (Kirschner and Ganser, 1980; Mikoshiba et al., 1980; Rosenbluth, 1980), yet has an increased number of SLI (Gould et al., 1995). The increase in two of the proteins normally found in SLI, Cx32 and MAG, is proportional to the increase in the density of incisures in ⫹/shi and shi/shi sciatic nerves.
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The level of MBP appears to be the primary factor in regulating Cx32 expression in Schwann cell. This is suggested by the findings that the expression of Cx32 in liver, a tissue that does not express MBP (Paul, 1995; Spray and Dermietzel, 1995; Bruzzone et al., 1996) is unaffected by the shi mutation, and by the observed gene dosage effect of MBP on MAG and Cx32 in heterozygous shi animals. E-cadherin, which is expressed in several tissues, but is only present in myelin in PNS-myelinated fibers where it is confined to the paranodes, SLI, and inner and outer loops (Fannon et al., 1995), is also elevated in shi/shi sciatic nerves (data not shown). Other nonmyelin components of the SLI such as actin may be elevated as well. Increase in actin gene expression could be detected in shi/shi sciatic nerves but was small and not highly significant (data not shown), most likely due to the large pool of actin present in axons. MBP is part of a larger gene, Golli, that generates several products expressed in neural and nonneural tissues (Campagnoni et al., 1993; Landry et al., 1993; Pribyl et al., 1993). Some Golli proteins, more prominently the BG21 isoform, continue to be expressed in the PNS of shi/shi mice in the absence of MBP (Landry et al., 1997). However, the presence of BG21 is not apparent in Schwann cells (Campagnoni, personal communication), and it could not be documented that the level of BG21 was elevated in dorsal root ganglionic cells (Landry et al., 1997), making it unlikely that another product from the Golli-mbp gene family is responsible for the effects described here. Myelin gene expression is regulated predominantly at the transcriptional level during development and regeneration as shown by the parallel increases in steady state levels of mRNA and protein in the CNS and the PNS (Carson et al., 1983; Milner et al., 1985; Gupta et al., 1988; Trapp et al., 1988; Griffiths et al., 1989; LeBlanc et al., 1990; Wiktorowicz and Roach, 1991; Gow et al., 1992; Snipes et al., 1992; Kuhn et al., 1993; Scherer et al., 1995). In contrast, our results indicate that a posttranscriptional, translational, or posttranslational mechanism regulates the level of MAG and Cx32 proteins in adult PNS. It is possible that MBP acts as a negative translational regulator for the expression of certain myelin proteins. This type of regulation has been reported in other systems (Chu et al., 1993; Chu and Allegra, 1995). We have postulated that in order for MBP to act at the level of translation would require that it be localized in the perikaryon rather than in the myelin sheath where it functions as an adhesive protein (Rosenbluth, 1980; Omlin et al., 1982). The presence of MBP in compartments other than myelin has been described in
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FIG. 5. Localization of MBP in sciatic nerves. Wt (A, B, C), and shi/shi sciatic nerves were incubated with antibodies to MBP (1:100) followed by fluorescein-conjugated secondary antibody (1:75) and Texas red-conjugated phalloidin. All panels except for C are simultaneous representations of the green and red channels. C represents the green (MBP) channel only of B. Schwann cell body (asterisk); Schmidt–Lanterman incisures (arrowhead). The scale bar in each panel represents 5 m.
oligodendrocytes in the CNS (Hardy et al., 1996; Pedraza, 1997; Staugaitis et al., 1990), but not in Schwann cells. In this study, we report that Schwann cells have no detectable levels of MBP in their nucleus or cytoplasm. This is not unexpected considering that MBP is present at much lower levels in the PNS than in the CNS, and may be below the level of detection, and that the geometry of the Schwann cells, i.e., flattened along the length of the axon does not permit to distinguish subcellular compartments as easily as in the oligoden-
drocyte. It is also possible that the histochemical conditions required to visualize MBP in the myelin sheath are deleterious to the detection of MBP in the cell body. These caveats not withstanding, our results suggest that MBP may exert its effect while in the myelin compartment. This is a possibility given the fact that MBP gene dosage affects several gene products and may do so in an indirect manner. PNS-myelinated fibers that regenerate after injury have an increased number of SLI due to shorter inter-
350 nodes (Ghabriel and Allt, 1980; Hiscoe, 1947). The levels of MBP transcripts and protein that accumulate in the regenerated fibers, however, is comparable to that of uninjured animals suggesting that they are not influenced by that morphologic peculiarity (Gupta et al., 1988; LeBlanc and Poduslo, 1990). The increase in MAG and Cx32 levels and in the number of incisures where they are found may represent an adaptive mechanism to low levels or absence of MBP. A recent study has demonstrated that gap junctions in the incisures provide a radial pathway for the rapid diffusion of small molecules from the outer to the inner myelin layers (Balice-Gordon et al., 1998). Thus, the role of SLI has been postulated to be in the delivery of nutrients to the sheath and in removal of toxic metabolites. It is possible that the stability of the myelin sheath is compromised in the absence of MBP, and that removal of components needs to be accelerated, hence the increase in the number of incisures. In that context, the increase in incisures, and therefore in Cx32 and MAG, in shi/shi sciatic nerves would represent a part in a general response aimed at maintaining the integrity of the MBP-deficient myelin sheath by facilitating more active transport of nutrients and removal of toxic metabolites (Paul, 1995; Scherer et al., 1995). However, our earlier study indicates that the increase in SLI in shi/shi sciatic nerves is apparent as soon as myelin starts being made, suggesting that MBP must affect the initial levels of incisures components, rather than their levels after myelin has become compact (Gould et al., 1995). Furthermore, there is no evidence that shi/shi myelin is degenerating, and the number of Schwann cells which increases in regenerating fibers and leads to shorther myelin internodes (Ghabriel and Allt, 1980) appears to be the same in shi/shi and wt mice (Gould et al., 1995). Thus, a more likely explanation is that MBP directly or indirectly regulates the expression of Cx32 and MAG and possibly other myelin and nonmyelin components as well during normal development. This would represent a new role for this protein traditionally considered to be solely a structural element of the myelin membrane. MBP could act to affect the stability or the turnover rate of proteins of the SLI, or could act as an inhibitor of protein translation. There is no evidence that MBP is an RNA binding protein that could directly regulate the translation of Cx32 mRNA or MAG mRNA. Nor is there any data on possible regulatory sites in Cx32 or MAG mRNA. There are several mechanisms for the control of translation initiation in animals (Gray and Wickens, 1998) and one or more can be at play for either Cx32 or MAG mRNA. Analysis of Cx32 mRNA with an RNA folding program reveals the presence of putative stem–
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loop structures with a single-stranded RNA bulge similar to those which have been implicated in negative translational regulation of mRNAs such as ferritin and transferrin mRNAs (Chu et al., 1993; Burd and Dreyfuss, 1994; Chu and Allegra, 1995; McCarthy and Kollmus, 1995; Henderson and Kuhn, 1997; Muckenthaler and Hentze, 1997). It seems unlikely, however, given the complexity of the SLI, that MBP regulates all their components directly or by the same mechanism.
EXPERIMENTAL METHODS Animals. Wt and ⫹/shi mice with the same genetic background (C3HeB/FeJ) were obtained from the Jackson Laboratories (Bar Harbour, ME). The latter were mated to produce shi/shi animals. Mice were euthanized according to a protocol approved by the committee for Laboratory Animal Care at the University of Connecticut Health Center. Sciatic nerves were removed and fixed in 4% buffered paraformaldehyde solution, or snap frozen in liquid nitrogen, and stored at ⫺75°C. Livers were removed and processed in the latter manner. Immunofluorescence. Sciatic nerves were desheathed and teased into fiber bundles. For staining with antiMBP, the nerves were permeabilized with a solution of 5% acetic acid in methanol for 10 min. All nerves were further incubated in Buffer A (0.1% Triton X-100, 5% normal goat serum, 0.1 M NaCl, 0.05 M Tris–HCl, pH 7.6) for 30 min at room temperature, with gentle rocking. Incubations in primary and secondary antibodies diluted in Buffer A were done overnight at 4°C with rocking. Each incubation with antibodies was followed by four 10 min washes in Buffer B (0.05% Triton X-100, 0.1 M NaCl, 0.05 M Tris–HCl, pH 7.6) at room temperature with rocking. Nerves were otherwise processed according to standard protocols and examined by confocal fluorescence microscopy. Mouse anti-Cx32 (R5.21C) was used at 1:75 dilution. Mouse anti-MAG was used at 1:50 dilution. Mouse anti-MBP (Sternberger Monoclonals Inc., Baltimore, MD) was used at a 1:100 dilution. Texas-red-conjugated goat anti-mouse secondary antibody was used at 1:50 dilution. Fluoresceinconjugated goat anti-mouse secondary antibody was used at 1:75 dilution. Phalloidin staining and SLI counts. Two fixed sciatic nerves from two ⫹/shi mice were incubated in fluorescein-conjugated phalloidin (Sigma Chemical Co., St. Louis, MO) (1:70 dilution in PBS) overnight at 4°C and examined by confocal fluorescence microscopy. A total of 209 myelinated axons were sampled, and the
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number of SLI was determined as described in Gould et al. (1995). Sciatic nerves from wt and shi/shi mice were incubated with Texas red-conjugated phalloidin (Sigma Chemical Co., St. Louis, MO) (1:70 dilution in PBS) overnight at 4°C. Tissue preparation. The detergent-independent isolation of sciatic nerves and liver membranous fraction was performed according to the method of Hertzberg (Hertzberg, 1984). Protein determination. Protein content was determined by the method of Lowry (Lowry et al., 1951) using the BioRad DC Protein Assay kit (Bio-Rad Laboratories, Hercules, CA). Western blot. Protein samples were run on 8 –16% polyacrylamide gradient gels (Novex, San Diego, CA) according to the method of Laemmli (1970) and transferred to supported nitrocellulose according to the method of Towbin (Towbin et al., 1979). Immunodetection was performed with antibodies to myelin components and horseradish peroxidase-conjugated secondary antibodies. Antibodies were used at the following dilutions: mouse anti-MAG (1/1000), rabbit anti-P0 (1/ 3000), rabbit anti-PMP22 (1/2000), mouse anti-MBP (1/ 1000), mouse anti-Cx32 (1/1000), horseradish-conjugated donkey anti-rabbit (1/1000), and horseradishconjugated sheep anti-mouse (1/1000). Enhanced chemiluminescence reagents (Supersubstrate kit, Pierce Chemical Co., Rockford, IL) were used to detect immune complexes. Immuno-dot blot and quantitative analysis. Solubilized membrane fractions from 90-day-old wt, ⫹/shi, and shi/shi sciatic nerves having the same protein concentration were serially diluted and blotted in duplicate directly onto supported nitrocellulose. Immunodetection was performed as for Western blot. The immune complexes visualized on X-ray films were photographed with a digital camera, and densitometry and quantification were done using the Molecular Dynamics ImageQuaNT analysis system (Molecular Dynamics, Sunnyvale, CA). The intensity levels of P0, PMP22, MBP, MAG, and Cx32 proteins were measured in wt, ⫹/shi, and shi/shi samples, and those in wt samples were set arbitrarily to 1. The amounts of the proteins from ⫹/shi and shi/shi were expressed relative to the wt values. Protein levels were determined in at least five independent experiments for wt and shi/shi samples and in at least three independent experiments for ⫹/shi samples. Each sample consisted of two sciatic nerves from the same animal. The mean and standard deviation were calculated for each protein and a paired T test was performed using Microsoft Excel (Microsoft Corp.).
351 RNA isolation. Total RNA was isolated from sciatic nerves with Tri Reagent (Sigma BioSciences, St. Louis, MO) according to the manufacturer’s instructions. Each preparation consisted of two sciatic nerves crushed in liquid nitrogen and suspended in 1 ml of Tri Reagent. The yield of RNA averaged around 1 g per sciatic nerve. RNA samples were stored at ⫺75°C until use. Ribonuclease protection assay (RPA). RPA was performed using the Hybspeed RPA kit from Ambion (Ambion, Austin, TX) according to the manufacturer’s instructions. The protected fragments were separated by electrophoresis on 6% TBE-Urea gels (Novex, San Diego, CA) and detected by autoradiography. Films were photographed with a digital camera, and quantification by densitometry was done using Molecular Dynamics ImageQuaNT, Version 4.2 (Molecular Dynamics, Sunnyvale, CA). At least three independent RPA determinations were done for each mRNA in wt and shi/shi sciatic nerve samples. The mean and standard deviation of the ratios of myelin mRNA to control mRNA intensity values were calculated and a paired T test was performed using Microsoft Excel (Microsoft Corp.). cDNA templates. The following cloned DNAs were used to prepare the antisense RNA probes: a 515-bp rat PMP22 cDNA inserted in the HindIII site of pRc/CMV (D’Urso, personal communication); a 2,049-bp rat LMAG cDNA inserted in the ApaI site of Bluescript KS⫹ (Salzer et al., 1987); and a 1458-bp rat Cx32 cDNA inserted in the KpnI site of pGEM3Zf⫹ (Werner et al., 1991). Antisense RNA probes. Template for cyclophilin was obtained from Ambion (Ambion Inc., Austin, TX). The cyclophilin antisense probe was 165 nucleotides long and protected a 103-nucleotide fragment. The PMP22 containing plasmid was cut with XmnI and SP6 polymerase was used to make a 424 nucleotide probe, which protected a 315-nucleotide fragment. The Cx32 containing plasmid cut with KpnI and T7 polymerase was used to make a 281-nucleotide probe, which protected a 264-nucleotide fragment. The L-MAG containing plasmid cut with EcoRI and T3 polymerase was used to make a 361-nucleotide probe, which protected a 341-nucleotide fragment of L-MAG and a 303-nucleotide fragment of S-MAG. These two fragments could not be resolved in the gel system used. In vitro transcription of [␣- 32P]UTP (800 Ci/mmol) (DuPont NEN, Boston, MA) labeled antisense RNA probes and RNA markers was performed with the Maxiscript In Vitro Transcription kit from Ambion (Ambion Inc., Austin, TX), according to the manufacturer’s instructions. Labeled probes were gel purified
352 using 6% TBE-Urea gels (Novex, San Diego, CA). The RNA Century Marker Plus Template set was from Ambion. Antibodies, plasmids, and reagents. Mouse antiMAG antibody was a generous gift from Dr. R. Quarles (NIH, Bethesda, MD). Rabbit anti-P0 was a generous gift from Dr. M. Filbin (Hunter College, NY, NY). Rabbit anti-PMP22 was a generous gift from Dr. H. W. Muller (Laboratory for Molecular Neurobiology, Department of Neurology, University of Dusseldorf, Germany). Mouse anti-MBP was purchased from Sternberger Monoclonals Inc. (Baltimore, MD). Mouse antiCx32 (R5.21C) was obtained from The Developmental Studies Hybridoma Bank (University of Iowa, IA). Mouse and rabbit anti-Cx32 were purchased from Zymed Laboratories (San Francisco, CA). An inhibitor peptide for the Cx32 antibodies was also purchased from Zymed Laboratories. Texas red-conjugated goat anti-mouse IgG was purchased from Accurate Chemical & Scientific Corp. (Westbury, NY). Fluorescein-conjugated goat anti-mouse IgG was purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Horseradish peroxidase-conjugated donkey antirabbit, sheep anti-rat, and sheep anti-mouse antibodies were purchased from Amersham Life Sciences (Buckinghamshire, England). Rat PMP22 cDNA was a generous gift from Drs. H. W. Muller and D. D’Urso (Heinrich-Heine-University, Dusseldorf, Germany). Rat L-MAG cDNA was a generous gift from Dr. J. Salzer (New York University School of Medicine, New York, NY). Rat Cx32 cDNA was a generous gift from Dr. R. Werner (University of Miami School of Medicine, Miami, FL). Except where otherwise noted all reagents were from Sigma Chemical Co. (St. Louis, MO).
ACKNOWLEDGMENTS This work was supported by NIH Grant NS19943 (E.B.), and by a graduate fellowship from the U. of CT. Health Ctr. (C.S.-S). We thank Drs. D’Urso, M. Filbin, H. W. Muller, R. Quarles, and J. Salzer for their generous gifts of reagents, and Dr. D. Oliver (U. Conn. Health Ctr. Farmington, CT) for his comments on the manuscript. We also thank Frank Morgan and Maureen Pons for technical assistance. The Cx32 antibody developed by Dr. Daniel Goodenough was obtained form the Developmental Studies Hybridoma Bank maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242, under contract NO1-HD-7-3263 from the NICHD.
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354 Scherer, S. S., Deschenes, S. M., Xu, Y., Grinspan, J. B., Fischbeck, K. H., and Paul, D. L. (1995). Connexin32 is a myelin-related protein in the PNS and CNS. J. Neurosci. 15: 8281– 8294. Shine, H. D., Readhead, C., Popko, B., Hood, L., and Sidman, R. L. (1992). Morphometric analysis of normal, mutant, and transgenic CNS: Correlation of myelin basic protein expression to myelinogenesis. J. Neurochem. 58: 342–349. Snipes, G. J., Suter, U., Welcher, A. A., and Shooter, E. M. (1992). Characterization of a novel peripheral nervous system myelin protein (PMP-22/SR13). J. Cell Biol. 117: 225–238. Spray, D. C., and Dermietzel, R. (1995). X-linked dominant CharcotMarie-Tooth disease and other potential gap-junction diseases of the nervous system. Trends Neurosci. 18: 256 –262. Stahl, N., Harry, J., and Popko, B. (1990). Quantitative analysis of myelin protein gene expression during development in the rat sciatic nerve. Mol. Brain Res. 8: 209 –212. Staugaitis, S. M., Smith, P. R., and Colman, D. R. (1990). Expression of myelin basic protein isoforms in nonglial cells. J. Cell. Biol. 110: 1719 –1727. Towbin, H., Staehelin, T., and Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedures and some applications. Proc. Natl. Acad. Sci. USA 76: 4350 – 4354.
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Trapp, B. D., Andrews, S. B., Wong, A., O’Connell, M., and Griffin, J. W. (1989). Co-localization of the myelin-associated glycoprotein and the microfilament components, F-actin and spectrin, in Schwann cells of myelinated nerve fibres. J. Neurocytol. 18: 47– 60. Trapp, B. D., Hauer, P., and Lemke, G. (1988). Axonal regulation of myelin protein mRNA levels in actively myelinating Schwann cells. J. Neurosci. 8: 3515–3521. Trapp, B. D., Itoyama, Y., Sternberger, N. H., Quarles, R. H., and Webster, H. D. (1981). Immunocytochemical localization of P0 protein in Golgi complex membranes and myelin of developing rat Schwann cells. J. Cell Biol. 90: 1– 6. Webster, H. deF. (1964). The relationship between Schmidt-Lantermann incisures and myelin segmentation during Wallerian degeneration. Annals N.Y. Acad. Sci. 122: 29 –38. Welcher, A. A., Suter, U., DeLeon, M., Snipes, G. J., and Shooter, E. M. (1991). A myelin protein is encoded by the homologue of a growth arrest-specific gene. Proc. Natl. Acad. Sci. USA 88: 7195–7199. Werner, R., Levine, E., Rabadan-Diehl, C., and Dahl, G. (1991). Gating properties of connexin32 cell– cell channels and their mutants expressed in Xenopus oocytes. Proc. R. Soc. Lond. 243: 5–11. Wiktorowicz, M., and Roach, A. (1991). Regulation of myelin basic protein gene transcription in normal and shiverer mutant mice. Dev. Neurosci. 13: 143–150. Received August 9, 1999 Revised November 1, 1999 Accepted November 24, 1999
MCN
Myelin Basic Protein Gene Dosage Effects in the PNS Candra Smith-Slatas* and Elisa Barbarese* ,1 *Combined Degree Program and Neuroscience Graduate Program and Department of Neurology, University of Connecticut Health Center, Farmington, Connecticut 06030
Myelin basic protein (MBP) plays an essential adhesive role in the formation of compact myelin in the central nervous system (CNS), but not in the peripheral nervous system (PNS). Morphologic data suggest that MBP controls the number of cytoplasmic channels or Schmidt– Lanterman incisures (SLI) present in PNS myelin. The levels of connexin-32 (Cx32) and myelin-associated glycoprotein (MAG), two components of the incisures, are inversely proportional to the levels of MBP in sciatic nerves of mice affected by the shiverer (shi) mutation, while protein zero (P0) and peripheral membrane protein 22 (PMP22), two structural components of compact myelin, remain constant. The levels of P0, PMP22, Cx32, and MAG mRNA do not vary in relationship to the levels of MBP. This indicates that MBP exerts its effect on Cx32 and MAG at a posttranscriptional level and suggests a new function for MBP in regulating gene expression in the PNS.
INTRODUCTION Myelin from the peripheral nervous system (PNS) of the homozygous mutant mouse shiverer (shi/shi) is compact despite the total absence of myelin basic protein (MBP) (Kirschner and Ganser, 1980; Mikoshiba et al., 1980; Rosenbluth, 1980). It has been postulated that the adhesive role of MBP in central nervous system myelin (CNS) is performed by protein zero (P0) in PNS myelin (D’Urso et al., 1990; Filbin et al., 1990; Martini et al., 1995). If this were the case, the level of P0 may be elevated in PNS myelin from shi/shi mice to compensate for the absence of MBP. Further morphologic studies indicated that the number of cytoplasmic channels called Schmidt–Lanterman incisures (SLI) was elevated 1 To whom correspondence and reprint requests should be addressed. Fax: (860) 679-4446. E-mail: [email protected].
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in shi/shi PNS myelin, suggesting that several components may be up-regulated in the absence of MBP (Gould et al., 1995). We sought to determine if the levels of connexin 32 (Cx32) and myelin-associated glycoprotein (MAG), two components of the incisures, and P0 were increased in shi/shi PNS myelin in comparison to wild type (wt) animals, and if the levels of these proteins correlated with the levels of MBP. In order to distinguish whether gene expression was regulated through transcription or translation, the levels of several myelin proteins cognate mRNA were measured. A comparison of morphologic and biochemical studies suggests that there is an inverse correlation between the levels of MBP and the number of SLI. In the rat sciatic nerve, the 18.5-kDa isoform of MBP is 2% of the total myelin proteins (Greenfield et al., 1973), and the number of incisures is 25 per internode (Hiscoe, 1947). In the guinea pig sciatic nerve, MBP is 16% of the total myelin proteins (Greenfield et al., 1973), and the number of incisures is 12 per internode for a fibre of equivalent diameter and internodal distance (Webster, 1964). In the CNS of anyone species, there is a positive correlation between the levels of MBP and myelin thickness (Shine et al., 1992), which may not be significant in the PNS; in the latter, there is a positive correlation between the fibre diameter and the number of SLI (Hiscoe, 1947). In the absence of MBP, in homozygous shi mice, the number of incisures is double that found in wt sciatic nerves (Gould et al., 1995), while myelin thickness is reportedly very midly reduced (Peterson and Bray, 1984) or normal (Rosenbluth, 1980). The present study uses homozygous (shi/shi) and heterozygous shi (⫹/shi) mice to examine the correlation between the level of MBP and the number of SLI in the sciatic nerves at a biochemical and molecular level because these animals differ genetically only by the number of functional MBP gene copies.
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344 The shi mutation results from a large deletion in the MBP gene with no MBP protein or messenger RNA being produced (Roach et al., 1983). It is functionally a null allele for MBP and introduction of the wt MBP gene in the genome of homozygous shi animals rescues the mutant phenotype (Readhead et al., 1987). MBP is a myelin-specific component localized to the compact regions of CNS and PNS myelin (Omlin et al., 1982). In both the CNS and the PNS, the amount of MBP is directly proportional to the number of gene copies present, and ⫹/shi myelin contains half the amount of MBP found in wt (Barbarese et al., 1983; Popko et al., 1987). SLI are cytoplasmic channels within the compact myelin sheath. They contain Schwann cell cytoplasm, a few organelles, and gap junctions composed of Cx32 proteins that link successive wrappings of myelin (Peters et al., 1991). The following proteins have been assigned to SLI: 2⬘,3⬘-cyclic nucleotide phosphohydrolase, MAG, S100, Cx32, E-cadherin, DM-20, and actin (Puckett et al., 1987; Trapp et al., 1989; Mata et al., 1990; Bergoffen et al., 1993; Fannon et al., 1995; Griffiths et al., 1995; Anderson et al., 1997; Garbern et al., 1997). P0, MBP, P2, and peripheral myelin protein-22 (PMP22) are present in the compact regions of the myelin sheath (Trapp et al., 1981; Omlin et al., 1982; Welcher et al., 1991; Snipes et al., 1992; Anderson et al., 1997; Garbern et al., 1997). There is concordance between the levels of myelin protein mRNAs and their corresponding proteins during the active period of myelination as indicated by the analysis of the steady state levels of MAG, MBP, Cx32, PMP22, and P0 mRNA in the rat sciatic nerve (Stahl et al., 1990; Gupta et al., 1990; Scherer et al., 1995; Griffiths et al., 1989; Gow et al., 1994; Kuhn et al., 1993; Snipes et al., 1992). In this paper we have used a ribonuclease protection assay (RPA) to examine the steady state mRNA levels of PMP22, Cx32, and MAG in 90-day-old shi/shi and wt animals to determine if gene expression was similarly regulated.
RESULTS Localization of Cx32 and MAG to SLI in shi/shi Sciatic Nerves In a previous study the increased density of SLI in shi/shi sciatic nerves was estimated from morphologic data (Gould et al., 1995). In order to further characterize these structures, immunofluorescence was performed to assess the localization of two of their components, Cx32 and MAG. Whole sciatic nerves were stained with
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anti-Cx32, anti-MAG, and with fluorescein-conjugated phalloidin to delineate SLI and nodes of Ranvier. The results showed that Cx32 and MAG were found in filamentous actin-positive incisures (arrowheads) in both wt and shi/shi sciatic nerves. Other compartments of the nerve such as the outer mesaxons, Schwann cell bodies (arrows), and the nodes of Ranvier, which have been reported to contain MAG and Cx32 (Trapp et al., 1989; Scherer et al., 1995), were also positively stained. All incisures-like structures identified by phalloidin labeling contained Cx32 and MAG, indicating that the supernumerary incisures in shi/shi sciatic nerves are bona fide SLI and suggesting that the levels of the two proteins should be commensurably increased in the sciatic nerves of shi/shi mice. Relationship between MBP Levels and Incisures in ⴙ/shi Mice Sciatic Nerves In order to further establish the relationship between the levels of MBP and the density of SLI, the latter was assessed in 90-day-old ⫹/shi mice sciatic nerves (n ⫽ 2) that contain half the amount of MBP present in wt sciatic nerves (see below). Fluorescein-conjugated phalloidin was used to identify the incisures. Examples of stained nerves are shown in Fig. 1. The analysis showed that ⫹/shi nerves contained 6.0 incisures per 100 m, which is intermediate between wt and shi/shi nerves of the same age (3.9 and 7.5 SLI/100 m for wt and shi/shi sciatic nerves, respectively) (Gould et al., 1995), indicating that there is an inverse correlation between the amount of MBP present in the nerve and the number of incisures. Correlation between MBP Levels and Other Myelin Proteins The relationship between the number of functional MBP genes and the level of SLI components was quantified by immunodot blot analysis of wt, ⫹/shi, and shi/shi sciatic nerve components. In a first step, the protein profile of PNS myelin from adult wt and shi/shi mice was obtained to insure the identity of the proteins recognized by the battery of antisera used. Membrane and soluble fractions of 90-day-old wt and shi/shi sciatic nerves were analyzed for the presence of myelin proteins by Western blot. Compact myelin membrane proteins P0, PMP22, and MBP, and SLI proteins MAG and Cx32 were recovered exclusively in the membrane fraction of both genotypes. Cx32 was also present in the membrane fraction of liver obtained from the same animals.
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FIG. 1. Localization of filamentous actin (fluorescein-conjugated phalloidin), Cx32, and MAG proteins in sciatic nerves. Wt, shi/shi, and ⫹/shi sciatic nerves were incubated with fluorescein-conjugated phalloidin, Cx32 antibodies (1:75) (wt and shi/shi), and MAG antibodies (1:50) (wt and shi/shi), followed by a Texas red-conjugated secondary antibody (1:50). Schwann cell body (arrow); Schmidt-Lanterman incisures (arrowhead). The scale bar in each panel represents 25 m.
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FIG. 2. Western blot of sciatic nerve and liver proteins from 90-day-old wt and homozygous shi mice. Wt (8 g) and shi/shi (8 g) sciatic nerve, wt (2 g), and shi/shi (2 g) livers were probed with antibodies to P0, PMP22, MBP, MAG, and Cx32 as described under Materials and Methods. Molecular weight standards are indicated to the left.
Figure 2 shows the protein profile of the membrane fractions. As previously reported, the four main isoforms of MBP (21.5, 18.5, 17, and 14 kDa) were present in wt and ⫹/shi, and absent in shi/shi sciatic nerves (Carson et al., 1983; Mikoshiba et al., 1983). P0 and PMP22 migrated as single bands at their expected apparent molecular weights of 33 and 22 kDa, respectively, in both wt and shi/shi sciatic nerves. They were present in ⫹/shi samples as well (data not shown). MAG migrated as a single band with an apparent molecular weight of 100 kDa in both wt and shi/shi sciatic nerves. Cx32 appeared as a polymeric series in the liver and sciatic nerves consistent with published reports (Hertzberg and Skibbens, 1984). When the Cx32 antibody was presorbed with the Cx32 inhibitor peptide, no bands were detected indicating that these bands represent monomer and multimers containing Cx32 (data not shown). Levels of each protein identified in Fig. 2 were determined by immunodot blot of wt (n ⫽ 5), ⫹/shi (n ⫽ 3), and shi/shi (n ⫽ 5) sciatic nerves. As can be seen in Fig. 3, the levels of P0 and PMP22 were comparable in all genotypes. MBP was absent in shi/shi and present at half the wt concentration in ⫹/shi animals, a finding similar to that for 15-day-old animals (Barbarese et al., 1983). This difference between wt and ⫹/shi animals was
found to be significant (P ⬍ 0.05). MAG and Cx32 levels were significantly increased more than fourfold in shi/ shi sciatic nerves as compared to wt sciatic nerves (P ⬍ 0.05). A significant twofold increase of these proteins was also seen in ⫹/shi sciatic nerves as compared to wt (P ⬍ 0.05). In contrast to sciatic nerves, the level of Cx32 in liver, a tissue that does not contain MBP, was comparable in all genotypes (data not shown). These data indicate that the levels of molecular components of SLI, MAG, and Cx32 are correlated with the presence of supernumerary incisures in mice bearing one or two copies of the shi gene and inversely correlated to the amount of MBP in the nerves. Furthermore, the expression of Cx32 was not affected by the shi mutation in a tissue (liver) that does not contain MBP. These data reveal that there is a MBP gene dosage effect on the expression of MAG and Cx32 in the PNS. Steady State Levels of Myelin mRNAs in wt and shi/shi Sciatic Nerves The increase in MAG and Cx32 may result from upregulation of gene expression at the transcriptional or posttranscriptional levels. In order to distinguish between these two possibilities, the steady state levels of PMP22, MAG, and Cx32 mRNAs were measured in
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FIG. 3. Levels of myelin proteins in wt, heterozygous, and homozygous shi sciatic nerve. The relative levels of proteins were determined by immunodot blot as described under Materials and Methods in sciatic nerves of 90-day-old wt (n ⫽ 5), heterozygous (n ⫽ 3), and homozygous shi (n ⫽ 5) mice. Paired T test analyses were performed: (*) P ⬍ 0.05 when comparing wt and shi/shi, wt and ⫹/shi, and ⫹/shi and shi/shi levels of MBP, Cx32, and MAG. **P ⬎ 0.05 when comparing wt and shi/shi, wt and ⫹/shi, and ⫹/shi and shi/shi levels of P0 and PMP22.
adult wt (n ⫽ 3) and shi/shi (n ⫽ 3) mice; cyclophilin mRNA was used as an internal control to compare the levels of the myelin mRNAs. The cyclophilin, PMP22, Cx32, and MAG probes protected a 103, 315, 264, and 303–340 nucleotide fragments, respectively, in both wt and shi/shi samples (Fig. 4A). The ratios of PMP22 mRNA to cyclophilin mRNA were found to be 0.83 and 0.84 in wt and shi/shi nerves, respectively (Fig. 4B) and were not significantly different between the two genotypes. The ratios of MAG mRNA to cyclophilin mRNA were found to be 0.19 and 0.20 in wt and shi/shi nerves, respectively. The ratios of Cx32 mRNA to cyclophilin mRNA were 0.32 for wt and 0.36 for shi/shi nerves. The ratios for both mRNAs were not statistically different between wt and shi/shi sciatic nerves. These data indicate that the steady state levels of PMP22, MAG, and Cx32 mRNAs are similar in wt and shi/shi animals. This suggests that a posttranscriptional mechanism may be responsible for the increase in MAG and Cx32 proteins observed in shi/shi sciatic nerves.
Localization of MBP in Sciatic Nerves In order to understand how MBP may exert its action on the steady state levels of MAG and Cx32 proteins, we sought to determine if it was localized in Schwann cell subcellular compartments other than myelin. Figure 5A shows that MBP (green) was homogeneously distributed in the myelin sheath surrounding an axon (red) identified by the presence of filamentous actin. MBP (green) was absent from the perikaryon and the nucleus of Schwann cells or present at concentrations below the level of detection (Figs. 5B and 5C). MBP was never detected in the myelin sheath, Schwann cell perikaryon, or nucleus of sciatic nerves from shi/shi mice (Fig. 5).
DISCUSSION There is no apparent increase in the amount of P0 in PNS myelin of shi/shi mice to compensate for the ab-
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FIG. 4. Ribonuclease protected fragments in wt and homozygous shi sciatic nerve. (A) PMP22, MAG, and Cx32 (top), and cyclophilin (bottom) mRNA protected fragments in wt and homozygous shi sciatic nerves. (B) Ratios of PMP22, MAG, and Cx32 mRNA levels to cyclophilin mRNA levels in wt and shi/shi sciatic nerves. The amount of total RNA used was 0.75, 2.5, and 2.5 g for PMP22, MAG, and Cx32, respectively. **P ⬎ 0.05, when comparing wt and shi/shi, paired 2-tail T test. Wt, n ⫽ 3; shi/shi, n ⫽ 3.
sence of MBP. There is, however, an increase in the amount of MAG and Cx32 proteins, which is inversely proportional to the amount of MBP present in myelin. This up-regulation of gene expression is a posttranscriptional event for both MAG and Cx32. These results are consistent with morphologic data that show that PNS myelin from shi/shi mice is normal in compaction, lamellar structure, and periodicity (Kirschner and Ganser, 1980; Mikoshiba et al., 1980; Rosenbluth, 1980), yet has an increased number of SLI (Gould et al., 1995). The increase in two of the proteins normally found in SLI, Cx32 and MAG, is proportional to the increase in the density of incisures in ⫹/shi and shi/shi sciatic nerves.
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The level of MBP appears to be the primary factor in regulating Cx32 expression in Schwann cell. This is suggested by the findings that the expression of Cx32 in liver, a tissue that does not express MBP (Paul, 1995; Spray and Dermietzel, 1995; Bruzzone et al., 1996) is unaffected by the shi mutation, and by the observed gene dosage effect of MBP on MAG and Cx32 in heterozygous shi animals. E-cadherin, which is expressed in several tissues, but is only present in myelin in PNS-myelinated fibers where it is confined to the paranodes, SLI, and inner and outer loops (Fannon et al., 1995), is also elevated in shi/shi sciatic nerves (data not shown). Other nonmyelin components of the SLI such as actin may be elevated as well. Increase in actin gene expression could be detected in shi/shi sciatic nerves but was small and not highly significant (data not shown), most likely due to the large pool of actin present in axons. MBP is part of a larger gene, Golli, that generates several products expressed in neural and nonneural tissues (Campagnoni et al., 1993; Landry et al., 1993; Pribyl et al., 1993). Some Golli proteins, more prominently the BG21 isoform, continue to be expressed in the PNS of shi/shi mice in the absence of MBP (Landry et al., 1997). However, the presence of BG21 is not apparent in Schwann cells (Campagnoni, personal communication), and it could not be documented that the level of BG21 was elevated in dorsal root ganglionic cells (Landry et al., 1997), making it unlikely that another product from the Golli-mbp gene family is responsible for the effects described here. Myelin gene expression is regulated predominantly at the transcriptional level during development and regeneration as shown by the parallel increases in steady state levels of mRNA and protein in the CNS and the PNS (Carson et al., 1983; Milner et al., 1985; Gupta et al., 1988; Trapp et al., 1988; Griffiths et al., 1989; LeBlanc et al., 1990; Wiktorowicz and Roach, 1991; Gow et al., 1992; Snipes et al., 1992; Kuhn et al., 1993; Scherer et al., 1995). In contrast, our results indicate that a posttranscriptional, translational, or posttranslational mechanism regulates the level of MAG and Cx32 proteins in adult PNS. It is possible that MBP acts as a negative translational regulator for the expression of certain myelin proteins. This type of regulation has been reported in other systems (Chu et al., 1993; Chu and Allegra, 1995). We have postulated that in order for MBP to act at the level of translation would require that it be localized in the perikaryon rather than in the myelin sheath where it functions as an adhesive protein (Rosenbluth, 1980; Omlin et al., 1982). The presence of MBP in compartments other than myelin has been described in
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FIG. 5. Localization of MBP in sciatic nerves. Wt (A, B, C), and shi/shi sciatic nerves were incubated with antibodies to MBP (1:100) followed by fluorescein-conjugated secondary antibody (1:75) and Texas red-conjugated phalloidin. All panels except for C are simultaneous representations of the green and red channels. C represents the green (MBP) channel only of B. Schwann cell body (asterisk); Schmidt–Lanterman incisures (arrowhead). The scale bar in each panel represents 5 m.
oligodendrocytes in the CNS (Hardy et al., 1996; Pedraza, 1997; Staugaitis et al., 1990), but not in Schwann cells. In this study, we report that Schwann cells have no detectable levels of MBP in their nucleus or cytoplasm. This is not unexpected considering that MBP is present at much lower levels in the PNS than in the CNS, and may be below the level of detection, and that the geometry of the Schwann cells, i.e., flattened along the length of the axon does not permit to distinguish subcellular compartments as easily as in the oligoden-
drocyte. It is also possible that the histochemical conditions required to visualize MBP in the myelin sheath are deleterious to the detection of MBP in the cell body. These caveats not withstanding, our results suggest that MBP may exert its effect while in the myelin compartment. This is a possibility given the fact that MBP gene dosage affects several gene products and may do so in an indirect manner. PNS-myelinated fibers that regenerate after injury have an increased number of SLI due to shorter inter-
350 nodes (Ghabriel and Allt, 1980; Hiscoe, 1947). The levels of MBP transcripts and protein that accumulate in the regenerated fibers, however, is comparable to that of uninjured animals suggesting that they are not influenced by that morphologic peculiarity (Gupta et al., 1988; LeBlanc and Poduslo, 1990). The increase in MAG and Cx32 levels and in the number of incisures where they are found may represent an adaptive mechanism to low levels or absence of MBP. A recent study has demonstrated that gap junctions in the incisures provide a radial pathway for the rapid diffusion of small molecules from the outer to the inner myelin layers (Balice-Gordon et al., 1998). Thus, the role of SLI has been postulated to be in the delivery of nutrients to the sheath and in removal of toxic metabolites. It is possible that the stability of the myelin sheath is compromised in the absence of MBP, and that removal of components needs to be accelerated, hence the increase in the number of incisures. In that context, the increase in incisures, and therefore in Cx32 and MAG, in shi/shi sciatic nerves would represent a part in a general response aimed at maintaining the integrity of the MBP-deficient myelin sheath by facilitating more active transport of nutrients and removal of toxic metabolites (Paul, 1995; Scherer et al., 1995). However, our earlier study indicates that the increase in SLI in shi/shi sciatic nerves is apparent as soon as myelin starts being made, suggesting that MBP must affect the initial levels of incisures components, rather than their levels after myelin has become compact (Gould et al., 1995). Furthermore, there is no evidence that shi/shi myelin is degenerating, and the number of Schwann cells which increases in regenerating fibers and leads to shorther myelin internodes (Ghabriel and Allt, 1980) appears to be the same in shi/shi and wt mice (Gould et al., 1995). Thus, a more likely explanation is that MBP directly or indirectly regulates the expression of Cx32 and MAG and possibly other myelin and nonmyelin components as well during normal development. This would represent a new role for this protein traditionally considered to be solely a structural element of the myelin membrane. MBP could act to affect the stability or the turnover rate of proteins of the SLI, or could act as an inhibitor of protein translation. There is no evidence that MBP is an RNA binding protein that could directly regulate the translation of Cx32 mRNA or MAG mRNA. Nor is there any data on possible regulatory sites in Cx32 or MAG mRNA. There are several mechanisms for the control of translation initiation in animals (Gray and Wickens, 1998) and one or more can be at play for either Cx32 or MAG mRNA. Analysis of Cx32 mRNA with an RNA folding program reveals the presence of putative stem–
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loop structures with a single-stranded RNA bulge similar to those which have been implicated in negative translational regulation of mRNAs such as ferritin and transferrin mRNAs (Chu et al., 1993; Burd and Dreyfuss, 1994; Chu and Allegra, 1995; McCarthy and Kollmus, 1995; Henderson and Kuhn, 1997; Muckenthaler and Hentze, 1997). It seems unlikely, however, given the complexity of the SLI, that MBP regulates all their components directly or by the same mechanism.
EXPERIMENTAL METHODS Animals. Wt and ⫹/shi mice with the same genetic background (C3HeB/FeJ) were obtained from the Jackson Laboratories (Bar Harbour, ME). The latter were mated to produce shi/shi animals. Mice were euthanized according to a protocol approved by the committee for Laboratory Animal Care at the University of Connecticut Health Center. Sciatic nerves were removed and fixed in 4% buffered paraformaldehyde solution, or snap frozen in liquid nitrogen, and stored at ⫺75°C. Livers were removed and processed in the latter manner. Immunofluorescence. Sciatic nerves were desheathed and teased into fiber bundles. For staining with antiMBP, the nerves were permeabilized with a solution of 5% acetic acid in methanol for 10 min. All nerves were further incubated in Buffer A (0.1% Triton X-100, 5% normal goat serum, 0.1 M NaCl, 0.05 M Tris–HCl, pH 7.6) for 30 min at room temperature, with gentle rocking. Incubations in primary and secondary antibodies diluted in Buffer A were done overnight at 4°C with rocking. Each incubation with antibodies was followed by four 10 min washes in Buffer B (0.05% Triton X-100, 0.1 M NaCl, 0.05 M Tris–HCl, pH 7.6) at room temperature with rocking. Nerves were otherwise processed according to standard protocols and examined by confocal fluorescence microscopy. Mouse anti-Cx32 (R5.21C) was used at 1:75 dilution. Mouse anti-MAG was used at 1:50 dilution. Mouse anti-MBP (Sternberger Monoclonals Inc., Baltimore, MD) was used at a 1:100 dilution. Texas-red-conjugated goat anti-mouse secondary antibody was used at 1:50 dilution. Fluoresceinconjugated goat anti-mouse secondary antibody was used at 1:75 dilution. Phalloidin staining and SLI counts. Two fixed sciatic nerves from two ⫹/shi mice were incubated in fluorescein-conjugated phalloidin (Sigma Chemical Co., St. Louis, MO) (1:70 dilution in PBS) overnight at 4°C and examined by confocal fluorescence microscopy. A total of 209 myelinated axons were sampled, and the
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number of SLI was determined as described in Gould et al. (1995). Sciatic nerves from wt and shi/shi mice were incubated with Texas red-conjugated phalloidin (Sigma Chemical Co., St. Louis, MO) (1:70 dilution in PBS) overnight at 4°C. Tissue preparation. The detergent-independent isolation of sciatic nerves and liver membranous fraction was performed according to the method of Hertzberg (Hertzberg, 1984). Protein determination. Protein content was determined by the method of Lowry (Lowry et al., 1951) using the BioRad DC Protein Assay kit (Bio-Rad Laboratories, Hercules, CA). Western blot. Protein samples were run on 8 –16% polyacrylamide gradient gels (Novex, San Diego, CA) according to the method of Laemmli (1970) and transferred to supported nitrocellulose according to the method of Towbin (Towbin et al., 1979). Immunodetection was performed with antibodies to myelin components and horseradish peroxidase-conjugated secondary antibodies. Antibodies were used at the following dilutions: mouse anti-MAG (1/1000), rabbit anti-P0 (1/ 3000), rabbit anti-PMP22 (1/2000), mouse anti-MBP (1/ 1000), mouse anti-Cx32 (1/1000), horseradish-conjugated donkey anti-rabbit (1/1000), and horseradishconjugated sheep anti-mouse (1/1000). Enhanced chemiluminescence reagents (Supersubstrate kit, Pierce Chemical Co., Rockford, IL) were used to detect immune complexes. Immuno-dot blot and quantitative analysis. Solubilized membrane fractions from 90-day-old wt, ⫹/shi, and shi/shi sciatic nerves having the same protein concentration were serially diluted and blotted in duplicate directly onto supported nitrocellulose. Immunodetection was performed as for Western blot. The immune complexes visualized on X-ray films were photographed with a digital camera, and densitometry and quantification were done using the Molecular Dynamics ImageQuaNT analysis system (Molecular Dynamics, Sunnyvale, CA). The intensity levels of P0, PMP22, MBP, MAG, and Cx32 proteins were measured in wt, ⫹/shi, and shi/shi samples, and those in wt samples were set arbitrarily to 1. The amounts of the proteins from ⫹/shi and shi/shi were expressed relative to the wt values. Protein levels were determined in at least five independent experiments for wt and shi/shi samples and in at least three independent experiments for ⫹/shi samples. Each sample consisted of two sciatic nerves from the same animal. The mean and standard deviation were calculated for each protein and a paired T test was performed using Microsoft Excel (Microsoft Corp.).
351 RNA isolation. Total RNA was isolated from sciatic nerves with Tri Reagent (Sigma BioSciences, St. Louis, MO) according to the manufacturer’s instructions. Each preparation consisted of two sciatic nerves crushed in liquid nitrogen and suspended in 1 ml of Tri Reagent. The yield of RNA averaged around 1 g per sciatic nerve. RNA samples were stored at ⫺75°C until use. Ribonuclease protection assay (RPA). RPA was performed using the Hybspeed RPA kit from Ambion (Ambion, Austin, TX) according to the manufacturer’s instructions. The protected fragments were separated by electrophoresis on 6% TBE-Urea gels (Novex, San Diego, CA) and detected by autoradiography. Films were photographed with a digital camera, and quantification by densitometry was done using Molecular Dynamics ImageQuaNT, Version 4.2 (Molecular Dynamics, Sunnyvale, CA). At least three independent RPA determinations were done for each mRNA in wt and shi/shi sciatic nerve samples. The mean and standard deviation of the ratios of myelin mRNA to control mRNA intensity values were calculated and a paired T test was performed using Microsoft Excel (Microsoft Corp.). cDNA templates. The following cloned DNAs were used to prepare the antisense RNA probes: a 515-bp rat PMP22 cDNA inserted in the HindIII site of pRc/CMV (D’Urso, personal communication); a 2,049-bp rat LMAG cDNA inserted in the ApaI site of Bluescript KS⫹ (Salzer et al., 1987); and a 1458-bp rat Cx32 cDNA inserted in the KpnI site of pGEM3Zf⫹ (Werner et al., 1991). Antisense RNA probes. Template for cyclophilin was obtained from Ambion (Ambion Inc., Austin, TX). The cyclophilin antisense probe was 165 nucleotides long and protected a 103-nucleotide fragment. The PMP22 containing plasmid was cut with XmnI and SP6 polymerase was used to make a 424 nucleotide probe, which protected a 315-nucleotide fragment. The Cx32 containing plasmid cut with KpnI and T7 polymerase was used to make a 281-nucleotide probe, which protected a 264-nucleotide fragment. The L-MAG containing plasmid cut with EcoRI and T3 polymerase was used to make a 361-nucleotide probe, which protected a 341-nucleotide fragment of L-MAG and a 303-nucleotide fragment of S-MAG. These two fragments could not be resolved in the gel system used. In vitro transcription of [␣- 32P]UTP (800 Ci/mmol) (DuPont NEN, Boston, MA) labeled antisense RNA probes and RNA markers was performed with the Maxiscript In Vitro Transcription kit from Ambion (Ambion Inc., Austin, TX), according to the manufacturer’s instructions. Labeled probes were gel purified
352 using 6% TBE-Urea gels (Novex, San Diego, CA). The RNA Century Marker Plus Template set was from Ambion. Antibodies, plasmids, and reagents. Mouse antiMAG antibody was a generous gift from Dr. R. Quarles (NIH, Bethesda, MD). Rabbit anti-P0 was a generous gift from Dr. M. Filbin (Hunter College, NY, NY). Rabbit anti-PMP22 was a generous gift from Dr. H. W. Muller (Laboratory for Molecular Neurobiology, Department of Neurology, University of Dusseldorf, Germany). Mouse anti-MBP was purchased from Sternberger Monoclonals Inc. (Baltimore, MD). Mouse antiCx32 (R5.21C) was obtained from The Developmental Studies Hybridoma Bank (University of Iowa, IA). Mouse and rabbit anti-Cx32 were purchased from Zymed Laboratories (San Francisco, CA). An inhibitor peptide for the Cx32 antibodies was also purchased from Zymed Laboratories. Texas red-conjugated goat anti-mouse IgG was purchased from Accurate Chemical & Scientific Corp. (Westbury, NY). Fluorescein-conjugated goat anti-mouse IgG was purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Horseradish peroxidase-conjugated donkey antirabbit, sheep anti-rat, and sheep anti-mouse antibodies were purchased from Amersham Life Sciences (Buckinghamshire, England). Rat PMP22 cDNA was a generous gift from Drs. H. W. Muller and D. D’Urso (Heinrich-Heine-University, Dusseldorf, Germany). Rat L-MAG cDNA was a generous gift from Dr. J. Salzer (New York University School of Medicine, New York, NY). Rat Cx32 cDNA was a generous gift from Dr. R. Werner (University of Miami School of Medicine, Miami, FL). Except where otherwise noted all reagents were from Sigma Chemical Co. (St. Louis, MO).
ACKNOWLEDGMENTS This work was supported by NIH Grant NS19943 (E.B.), and by a graduate fellowship from the U. of CT. Health Ctr. (C.S.-S). We thank Drs. D’Urso, M. Filbin, H. W. Muller, R. Quarles, and J. Salzer for their generous gifts of reagents, and Dr. D. Oliver (U. Conn. Health Ctr. Farmington, CT) for his comments on the manuscript. We also thank Frank Morgan and Maureen Pons for technical assistance. The Cx32 antibody developed by Dr. Daniel Goodenough was obtained form the Developmental Studies Hybridoma Bank maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242, under contract NO1-HD-7-3263 from the NICHD.
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