Journal of Neuroscience Research 32507-515 (1992)
Myelin Protein Expression in Dissociated Superior Cervical Ganglia and Dorsal Root Ganglia Cultures K.R. Brunden, Y. Ding, and B.S. Hennington Gliatech, Inc. and Department of Neuroscience, Case Western Reserve University, Cleveland, Ohio (K.R.B.); Department of Biochemistry, University of Mississippi Medical Center, Jackson (Y.D., B.S.H.)
Schwann cells of the adult rat superior cervical ganglia (SCG) synthesize negligible levels of the major myelin glycoprotein, Po, in vivo. This suggests that the sympathetic axons of the SCG are deficient in one of more components involved in the regulation of myelin protein expression. Here we have compared the ability of neurites from neonatal rat SCG and embryonic rat dorsal root ganglia (DRG) to induce Schwann cell expression of myelin proteins after growth in culture using a serum-free medium. Steady-state Po mRNA levels in the SCG and DRG culture paradigms were determined with a sensitive polymerase chain reaction (PCR) assay that amplified cDNA produced by reverse transcription of mRNA. This semiquantitative assay showed a linear response to increasing amounts of Po and actin mRNA and required substantially less cellular RNA than typical hybridization techniques. Using the PCR assay, we found that SCG cultures contained significantly lower amounts of Po mRNA than did DRG cultures. To further confirm that SCG cultures have negligible expression of myelin proteins, immunoblot analyses were done to examine the steady-state levels of both Po and myelin basic protein. While nonmyelinating DRG cultures had readily detectable amounts of these myelin-specific proteins, neither could be demonstrated in the SCG cultures. The data indicate that SCG neurites lack one or more signals needed to induce myelin protein expression. Employing SCG and DRG cultures in comparative biochemical studies should prove useful in identifying the axonal molecule(s) involved in the regulation of myelin protein expression. 0 1992 Wiley-Liss, Inc. Key words: myelin, protein expression, axons, cell culture
onal degeneration due to nerve injury leads to consequent changes in Schwann cell metabolism, including an increase of Schwann cell proliferation (Salzer and Bunge, 1980), a cessation of myelination, and a severe reduction in the expression of myelin-specific proteins (LeBlanc et al., 1987; LeBlanc and Poduslo, 1990; Poduslo et al., 1985; Trapp et al., 1988) and glycolipids (Yao and Poduslo, 1988; Yao et al., 1990). When axons reenter a denervated region, Schwann cells respond by differentiating to a stage characterized by the synthesis and assembly of myelin membranes. While certain peripheral axons clearly regulate myelination, many mature axons do not trigger myelin formation by Schwann cells. This is seen in certain sympathetic nerves, including the cervical sympathetic trunk (CST) where axons are >99% nonmyelinated (Aguayo et al., 1976a). The Schwann cells of the CST express negligible levels of the major myelin glycoprotein, Po, as assayed by immunofluorescence (Jessen et al., 198.5), immunoblotting (Brunden et al., 1990), or metabolic labeling (Brunden et al., 1990). Similarly, the Schwann cells of the superior cervical ganglia (SCG) contain negligible levels of myelin protein (Inuzuka et al., 1988). These data suggest that CST and SCG Schwann cells do not receive the axonal information necessary to up-regulate myelin protein expression. Further evidence of this is provided by the observation that CST Schwann cells appear to be capable of myelination after anastomosis to another nerve (Aguayo et al., 1976a,b; Weinberg and Spencer, 1976) or after interaction with dorsal root ganglia neurites in vitro (Brunden et al., 1990). Although axons often contain information that affects the ability of Schwann cells to synthesize myelin membrane, it appears that axons alone are not sufficient to induce myelination. Studies of myelin formation in
INTRODUCTION
Received July 23, 1991; revised February 25, 1992; accepted March 2, 1992.
Axons profoundly infhence the phenotype of Schwann cells within the peripheral nervous system. Ax-
Address reprint requests to Kurt R. Brunden, Ph.D., Gliatech, Inc., 23420 Commerce Park Rd., Cleveland, OH 44122.
0 1992 Wiley-Liss, Inc.
508
Brunden et al.
culture reveal that Schwann cells must be able to assemble a basement membrane, as well as associate with neurites, before myelination can commence (Bunge et al., 1986; Carey and Todd, 1987; Eldridge et al., 1987). Recent work from this laboratory (Brunden and Brown, 1990; Brunden et al., 1990) indicates that Schwann cells require signaling from only axons, and not basement membrane, for the initiation of Po mRNA and protein expression. This observation has subsequently been confirmed by other investigators (Morrison et al., 1991). Thus, the role of basal lamina in myelination is probably not in the regulation of myelin protein synthesis, and it has been suggested that this extracellular matrix may provide the Schwann cells with a necessary sidedness required for myelin assembly (Bunge et al., 1986). Understanding the mechanism of axonal regulation of myelin protein levels is likely to be of importance in ultimately treating dysmyelinating peripheral nerve diseases. The observation of axonal influence on myelin protein expression by oligodendrocytes (Kidd et al. , 1990) suggests that there may be similarities in the mechanism of axon control within the central and peripheral nervous systems. While there is evidence that axons generally reach a minimal caliber before myelination proceeds (Friede, 1972; Friede and Samorajski, 1968; Griffiths et al., 1991), it is not clear that small axons remain unmyelinated strictly because of physical diameter. Instead, such axons may not express one or more molecules needed to trigger Schwann cell synthesis of myelin proteins and lipids. One approach to examining axon molecules important to the process of myelination is to study the ability of neurites to influence Schwann cell expression of myelin components under defined in vitro conditions. Here we compare the effects of SCG and dorsal root ganglia (DRG) neurites on Schwann cell expression of myelin proteins in cultures grown under conditions that prevent both basal lamina formation and myelination. The data indicate that SCG neurites, unlike those of DRG, are incapable of triggering significant expression of myelin-specific mRNA and proteins, The inability of SCG neurites to up-regulate myelin protein expression suggests that comparative studies with DRG and SCG neuron cultures may shed light onto the mechanism whereby axons regulate myelin protein expression.
MATERIALS AND METHODS Tissue Culture Cultures were grown in 35 mm polystyrene dishes (Corning Glassware) that were coated with rat tail collagen as described (Brunden et al., 1990). Cells were derived from either rat DRG or SCG. DRG were removed from the spinal cord of E l 5 Sprague-Dawley rat
pups and dissociated with 0.25% trypsin in Hanks’ balanced salt solution (HBSS; Gibco) for 35 min at 37°C. Aliquots of the dissociated cells were placed onto the dishes and allowed to attach in serum-containing medium overnight. After attachment, cells were subsequently grown in either serum-free or serum-containing medium for 15 days at 37°C in 5% C02:95% air. Media was changed every 2-3 days. SCG were removed from 4-6 day old neonatal Sprague-Dawley rats, dissociated for 45 min at 37°C in 0.25% trypsin plus 0.03% collagenase in HBSS, and plated onto dishes as above. After overnight attachment of cells, the SCG cultures were switched to serum-free medium and allowed to grow for 15-16 days. Cell counts of random fields selected from photographs of the DRG and SCG cultures (i.e., see Fig. 1) revealed no statistically significant differences in the ratio of Schwann cells to neurons in the two culture paradigms.
Media Serum-free medium (nonmyelinating) was composed of Eagle’s minimal essential medium supplemented with Earle’s salts (Gibco), 1.4 mM L-glutamine, 0.6% glucose, and 100 ng/ml 7 s nerve growth factor (Collaborative Research). Serum-containing medium (myelinating) was as above with the addition of 15% calf serum (HyClone) and 50 Fg/ml ascorbic acid. RNA Preparations Cultured cells were scraped from dishes in 0.5-1 .O ml of 1% SDS/25 mM EDTA in phosphate-buffered saline using sterile, disposable cell scrapers. Generally, the material from four to eight dishes was pooled for a single RNA preparation. An aliquot of the cell homogenate was removed for subsequent determination of cell number (see below), and the remainder of the sample was used for total cellular RNA isolation by the hot phenol method (Brown et al., 1985). We have previously demonstrated that RNA isolated by this technique is intact and of high quality (Brunden and Brown, 1990). In summary, the cell homogenate was mixed with an equal volume of water-saturated phenol for 5 min at 55°C. A known amount of 32P-labeled bovine rRNA was added to the phenol mixture. This rRNA (Sigma Chemical Co.), which was used to follow the recovery of total cellular RNA in subsequent steps, was 5’-labeled by treatment with bacterial alkaline phosphatase and addition of [gamrr~a-~~P]ATP with polynucleotide kinase. The phenol-aqueous mix was centrifuged briefly to separate the phases, and a majority of the upper aqueous phase was recovered. The remainder of the mix was back-extracted for 5 min at 55°C with an equal volume of 0.3 M sodium acetate, pH 5.5. The aqueous phase from the back-extraction was combined with the first aqueous phase, and
Axonal Regulation of Myelin Protein Expression DRG
509
SCG
Fig. 1. Phase-contrast photomicrographs of dissociated DRG and SCG cultures grown in serum-free medium that prevents basal lamina formation. Neurons of both cultures (DRGleft; SCG-right) extend neuritic processes that are populated by Schwann cells that do not form myelin. the sample was treated with 2.5-fold volume excess of ethanol at -20°C overnight. RNA was collected by centrifugation for 60 min at 8,50Og, resuspended in TE buffer (Sambrook et al., 1989) containing 0.3 M sodium acetate, and reprecipitated as above. The final RNA pellet was resuspended in approximately 0.3 ml H,O and an aliquot was counted in a scintillation counter to determine the recovery of the added 32P-rRNA. The fraction of recovered exogenous 32P-rRNA was used to obtain a corrected value for cell-equivalents of total RNA (see below).
culture samples were divided by 6.6 pg, the amount of DNA per diploid rat cell (Lewin, 1980), to calculate the number of cells that were used in each RNA preparation. The fraction of the added ”P-rRNA that was recovered in each RNA sample (see above) was multiplied by the total number of cells used in the preparation to obtain a corrected value of the number of cell equivalents of RNA in the sample. This value served as the basis for determining aliquot sizes to be used in Po and actin mRNA assays.
Calculation of Cell-Equivalents of RNA Aliquots of the cell homogenates used for RNA preparations were tested for DNA content using a fluorometric assay (Labarca and Paigen, 1980) to obtain a value of total DNA in the original culture homogenates. The samples were extracted with an equal volume of neutral phenol/chloroform (Sambrook et al., 1989) and ethanol-precipitated prior to assaying. Standard curves were constructed using calf thymus DNA that was mixed with 1%sodium dodecyl sulfate (SDS)/25 mM EDTA in phosphate-buffered saline (see RNA preparations) and subsequently extracted with phenol/chloroform and ethanol-precipitated. Such treatment of the standards normalizes any quenching artifacts that might arise due to the SDS extraction. The DNA values obtained for the
Po and actin mRNA levels were determined using a semiquantitative PCR assay (Fuqua et al., 1990). Aliquots of the RNA samples were used for the generation of cDNA and subsequent amplification with a “GeneAmp RNA PCR Kit” (Perkin Elmer Cetus). For Po mRNA determinations, a synthesized oligonucleotide compleprimer (5’-TTGGTGCTTCGGCTGTGGTC-3’) mentary to a sequence within exon 6 of Po mRNA (Lemke et al., 1988) was used at a concentration of 1.5 p M in 20 (~.1of the commercial mix which contained cloned Moloney Murine Leukemia Virus reverse transcriptase (2.5 U/p1) and deoxyribonucleotide triphosphates (1 mM). The reverse transcription proceeded for 45 min at 42°C in a thermocycler (Perkin Elmer Cetus or Thermolyne). After a 5 min denaturation of the reverse
Po and Actin mRNA Determinations
510
Brunden et al.
transcriptase at 99°C 80 pl of a commercial solution was bound antibody, the blots were treated with goat antiadded that contained 2.0 U AmpliTaq DNA polymerase rabbit IgG or goat anti-mouse IgG that were conjugated (Perkin Elmer Cetus) and an “upstream” oligonucle- with alkaline phosphatase (Fisher Scientific). Followotide primer (0.38 pM; 5’-TGTTGCTGCTGTTGC- ing removal of unbound second antibody, staining of TCTTC-3’) complementary to a region of exon 4 of Po the immunoreactive species was performed with nitrotetcDNA (Lemke et al., 1988). After overlaying 50 p1 of razolium blue/5-bromo-4-chloro-3-indoyl phosphate mineral oil onto each sample, the Po cDNA was ampli- (Promega). fied through 30 cycles of the following incubation steps: 94°C for 1 min, 60°C for 2 min, and 72°C for 2 min with 5 sec increases each cycle. Actin mRNA was also as- RESULTS sessed in the total RNA preparations as a control. Ali- Analysis of Po mRNA Levels in SCG quots of RNA were treated with reverse transcriptase as and DRG Cultures There is evidence suggesting that CST and SCG described above, except that randem hexamer primers (0.75 p M ; Perkin Elmer Cetus) were used to initiate axons do not trigger myelin protein synthesis in vivo cDNA synthesis. Following cDNA formation, PCR am- (Brunden et al., 1990; Inuzuka et al., 1988; Jessen et al., plification was carried out as described for Po using an 1985). To further characterize the properties of these actin downstream primer (5’-CAGGTCCAGACGAG- sympathetic axons, we examined whether cultures of disGATGGCAT-3’; 0.25 pM) and upstream primer (5’- sociated SCG were lacking in the ability to increase the CGACATGGAGAAAATCTGCACC-3’; 0.25 FM). expression of the myelin-specific glycoprotein Po. SCG These primers to human p-actin were kindly provided by were removed from neonatal rat pups, and the dissociDrs. S. Younkin and T. Golde (Case Western Reserve ated ganglion cells were grown for 15-16 days in culture University), and have been shown to result in amplified using a serum-free medium that prevents basal lamina actin DNA of 300 base pairs in both human and rat formation (Carey and Todd, 1987; Eldridge et al., 1987). (Golde, 1991). After completion of amplification, ali- For comparison, rat DRG cultures were also grown in quots of the mixtures were analyzed by electrophoresis this medium for 15 days. These DRG cultures have been on 4% agarose gels (low molecular weight grade; Fisher shown to express appreciable amounts of mRNA encodScientific) and stained with ethidium bromide. DNA ing Po (Brunden and Brown, 1990) even though they lack standards were generally included on gels. In one exper- basement membrane and consequently cannot form myiment, aliquots of the Po amplification product from a elin. Representative phase-contrast photomicrographs of DRG culture grown in serum-free medium were digested the two culture paradigms are shown in Figure 1. with approximately 5 units of the restriction enzymes Schwann cells can be seen aligning and interacting with NcoI or PstI in the buffers supplied by the manufacturer the neurites of the DRG and SCG cultures, but these cells (BRL). These samples were then analyzed by agarose gel do not form myelin. Neurites of SCG cultures grown in serum-free medium have been shown to be predomielectrophoresis as above. nantly axonal in nature, with few dendritic processes Immunoblot Analyses (Bruckenstein and Higgens, 1988). To evaluate the steady-state levels of Po mRNA in Cultured tissues were excised from dishes along with the collagen substratum and immediately frozen the SCG and DRG cultures, we employed a semi-quanwith liquid N,. The frozen samples were homogenized titative polymerase chain reaction (PCR) assay which is with 1.25% SDS in water and sonicated in a bath-type significantly more sensitive than typical filter hybridizasonicator for 30 min. Insoluble material was removed by tion techniques. This enhanced sensitivity is particularly centrifugation as described (Bmnden et al., 1990). The important when the amount of cellular RNA is limiting, protein concentrations of the samples were determined as is generally the case with nervous system primary using a BCA assay (Pierce Chemicals). Aliquots of the cultures. Total RNA preparations from a defined number homogenates were separated by SDS-polyacrylamide gel of cells were treated with reverse transcriptase to generelectrophoresis (PAGE) using 15% polyacrylamide gels, ate cDNA which could subsequently be amplified by and the proteins were electrophoretically transferred to PCR (Fuqua et a1., 1990). Oligonucleotide primers were nitrocellulose overnight at 4°C (Yao and Poduslo, 1985). used that would result in the generation of a 181 baseThe nitrocellulose was treated with either Po antibody pair segment of DNA corresponding to a region spanning developed in rabbits against HPLC-purified protein exons 4 through 6 of the Po transcripts (Lemke et al., (Brunden et al., 1987) or with a mouse monoclonal an- 1988) and a 300 base-pair region of actin (Golde, 1991). tibody that reacts with a region of exon 6 of myelin basic As seen in Figure 2, the Po and actin assays demonstrated protein (Boehringer Mannheim Biochemicals). After elevated responses to increasing amounts of RNA from thoroughly washing the nitrocellulose to remove non- 15 day old nonmyelinating DRG cultures, with the am-
Axonal Regulation of Myelin Protein Expression A.
511
Po
Std.
Ct.
Ncol
Pstl
Fig. 3 . Restriction enzyme digests of DNA from the PCR assay of Po mRNA. RNA from DRG cultures grown in serumfree medium was amplified as described in Materials and Methods, and equal-sized aliquots were either digested with NcoI, PstI, or left untreated (Ct). The samples and standard restriction fragments (Std) were subsequentlyanalyzed by agarose gel electrophoresis, with the standards indicated in basepairs (BP).
Cells
Fig. 2 . Linearity of response of the PCR assay used to measure Po and actin mRNA. RNA preparations (0-540 cell-equivalents) from DRG cultures grown in serum-free medium were treated as described in Materials and Methods to generate amplified Po (A) and actin (B) DNA. Equal-sized aliquots of the resulting samples were analyzed by agarose gel electrophoresis, as were DNA standards (Std). The sizes of the standards are listed in base-pairs (BP).
plified products being of the predicted sizes. The expanded region of the Po cDNA contains unique NcoI and PstI restriction enzyme sites (Lemke and Axel, 1985) and treatment of the amplified DNA with these enzymes resulted in the predicted cleavage products (Fig. 3), further verifying that the 181 base-pair product is derived from Po mRNA. It should be noted that, although purified RNA preparations were used here, the Po primers can presumably be used with samples containing total cellular nucleic acids since amplified product derived from genomic DNA will be substantially larger than that from Po cDNA due to the presence of two introns.
We utilized the PCR methodology with RNA from DRG cultures that were grown in serum-free medium (nonmyelinating) or in medium containing serum and ascorbic acid (myelinating) to confirm that the assay accurately measures Po mRNA in the culture preparations. A previous study (Brunden and Brown, 1990) revealed that there are comparable levels of Po mRNA in these two types of DRG cultures, and the nonrnyelinating and myelinating cultures both yielded similar amounts of PCR-amplified DNA (Fig. 4). The amount of 181 basepair DNA from each sample increased with increasing cell number (Fig. 4), as would be expected if the assay were in the linear range of sensitivity. If SCG axons are indeed deficient or reduced in one or more factors needed to induce Schwann cell expression of myelin proteins, it would be expected that SCG cultures should give little or no amplified Po DNA in the PCR assay. This is in fact the case, as shown in Figure 5 where the positive Po signal seen from a DRG culture sample is absent from an SCG RNA preparation. These SCG and DRG samples contained comparable amounts of actin mRNA, as demonstrated in Figure 5. Thus, the lack of Po signal is not due to a general absence of mRNA in the SCG sample. If larger amounts of SCG sample are used in the PCR assay a faint Po signal appears, but the amount of this amplified product is always substantially lower than that of DRG culture samples.
512
Brunden et al. +Myelin
-Myelin
Po
A. 0P 1000 700
500 400
300 200
100
50.
Sld
20 100 20 100
SCG
DUG
20
100
20
B.
Actin
100 0P
Cells 1000
Fig. 4. Analysis of Po mRNA from myelinating and nonmyelinating DRG cultures. RNA samples corresponding to 20 and 100 cell-equivalents of both serum-containing DRG cultures ( + myelin) and serum-free DRG cultures (-myelin) were treated as described in Materials and Methods to generate PCRamplified DNA. Equal-sized aliquots were then analyzed by agarose gel electrophoresis.
Immunoblot Analyses of Po and Myelin Basic Protein in SCG and DRG Cultures The very low level of Po mRNA in the SCG samples would imply that these cultures also express negligible amounts of Po glycoprotein, whereas the DRG cultures should contain this myelin protein. An immunoblot analysis was performed with Po antibody to test these predictions. DRG culture homogenate gave a distinct immunoreactive band of approximately 28 kD and a slightly smaller degradation product, while the SCG culture sample yielded no detectable reaction product when equal-sized aliquots were assayed (Fig. 6). The immunoblot data confirmed the lack of Po expression in the SCG cultures, but it was not known whether these cultures have a general deficit in the synthesis of myelin proteins. Moreover, it has not been demonstrated that the nonmyelinating DRG cultures express measurable myelin basic protein (MBP) in addition to Po. To address these points, immunoblot analyses were done with a monoclonal antibody to MBP. This antibody recognizes a peptide sequence encoded in exon 6 of MBP, and thus reacts with three of the five differentially spliced forms of this group of myelin proteins (Newman et al., 1987). As documented in Figure 7A, comparable
700
500400300-
200
100-
50-
Sld
20
100
DRG
20
100
SIX
Fig. 5. Comparison of Po mRNA levels in DRG and SCG cultures. RNA samples corresponding to 20 and 100 cellequivalents from serum-free DRG and SCG culture preparations were assayed for Po (A) and actin (B) mRNA using the PCR methodology. Equal-sized aliquots were subsequently analyzed by agarose gel electrophoresis. DNA standards (Std), whose sizes are indicated in base-pairs (BP), were also included on the gel.
immunoreactive signals were obtained when 20 p g samples of nonmyelinating and myelinating DRG homogenates were examined, and a strong signal was evident when 50 p g of homogenate from the former was assayed. This is further proof that the neurites of DRG cultures contain the necessary information to trigger general myelin protein synthesis in the absence of basal lamina and active myelination. In contrast to DRG cultures, SCG cultures had little or no immunoreactive MBP (Fig. 7B). Thus, the SCG cultures seem to have a general reduction of myelin protein expression.
Axonal Regulation of Myelin Protein Expression SCG
DRG
513
DRG
A.
-Myelin
+Myelin --36.0 -29.0 --24.0
45.036.0-
29.024.0-
50ug
20ug
20ug 1oug
B. -36.0
30ug
60ug
30 ug
-29.0 -24.0
60 ug
Fig. 6. Immunoblot analysis of Po mRNA levels in homogenates from DRG and SCG cultures grown in serum-free medium. Aliquots (30 pg and 60 pg) from DRG and SCG culture homogenates were separated by SDS-PAGE and immunoblotted to assay for steady-state Po as described in Materials and Methods. The migrations of protein standards are indicated (in W).
DISCUSSION The synthesis of myelin proteins by Schwann cells is dependent on axons, as the disruption of axonSchwann cell interaction via nerve transection results in a drastic down-regulation of myelin protein expression at the mRNA (LeBlanc et al., 1987; LeBlanc and Poduslo, 1990; Trapp et al., 1988) and protein levels (Brunden and Poduslo, 1987; Poduslo et al., 1985). Likewise, Schwann cells from transected nerve demonstrate a decrease in the synthesis of the myelin glycolipid, galactocerebroside (Yao and Poduslo, 1988; Yao et al., 1990). Although the synthesis of myelin components by oligodendrocytes of the central nervous system does not appear to be as strictly dependent on axons (Rome et al., 1986; Zeller et al., 1985), there is evidence suggesting that oligodendrocytes reduce the expression of myelin proteins following nerve transection (Kidd et al., 1990). Given the importance of axons in regulating the synthesis of myelin molecules, surprisingly little is actually known about the mechanisms of this control. Several studies (Friede, 1972; Friede and Samorajski, 1968; Griffiths et al., 1991) point to a correlation of increasing axon di-
DRG
SCG
Fig. 7. Immunoblot analysis of MBP levels in DRG and SCG cultures. A: Nonmyelinating (- myelin) and myelinating ( + myelin) DRG cultures homogenates (10,20, or 50 pg) were assayed for steady-state MBP by SDS-PAGE and immunoblotting as described in Materials and Methods. The migrations of protein standards are indicated (in kD). B: Aliquots (50 pg) of homogenates from DRG and SCG cultures grown in serumfree medium were separated by SDS-PAGE and assayed for MBP by immunoblotting. The migrations of protein standards are shown.
ameter and increased myelination. Models of myelin regulation based solely on physical parameters such as axon caliber or axon surface area are probably simplistic. For example, nonmyelinating Schwann cells in vivo often surround many small diameter axons (Webster et al., 1973), resulting in a large axonal contact area but no myelin formation. Furthermore, other studies have noted myelinated axons with diameters as small as 0.6 pm (Hahn et al., 1987). It is likely that the lack of myelination of smaller axons is not a direct result of their size, but is instead caused by the lack of expression of one or more neuronal factors involved in the regulation of syn-
514
Brunden et al.
thesis of myelin macromolecules. The extent of synthesis of such putative axonal factors may be correlated with axon diameter. The identification of axonal components involved in the control of myelination would be greatly aided by the characterization of neuron populations deficient in one or more of these critical factors. Earlier studies suggested that Schwann cells of the CST do not receive the necessary information to form myelin membrane (Aguayo et al., 1976a,b; Weinberg and Spencer, 1976). More recent work demonstrated that CST and SCG Schwann cells synthesize a very low level of myelin protein (Brunden et al., 1990; Inuzuka et al., 1988). Together, the data suggest that the axons of these sympathetic neurons are not signaling Schwann cells to produce myelin proteins. In this study, we have compared the ability of SCG and DRG neurites to induce myelin protein expression by Schwann cells in vitro. Cultures were grown in a defined medium that prevents basal lamina formation and myelination, since neurites are known to be capable of up-regulating the synthesis of the myelin glycoprotein, Po, under these growth conditions (Brunden and Brown, 1990; Brunden et al., 1990; Morrison et al., 1991). When the steady-state levels of Po mRNA were examined with a sensitive PCR assay it was found that SCG cultures had little mRNA encoding this myelin protein. In contrast, nonmyelinating DRG cultures contained Po mRNA in amounts comparable to those found in myelinating DRG cultures grown with serum and ascorbic acid. The low level of Po mRNA in the SCG cultures is presumably not a consequence of an atypical Schwann cell population since nonmyelinating Schwann cells of the CST can respond normally to DRG neurites by synthesizing Po (Brunden et al., 1990). Examination of the steady-state levels of myelin proteins further demonstrates that SCG cultures differ from their DRG counterparts. While nonmyelinating DRG cultures contained MBP at levels comparable to myelinating DRG cultures, little or no MBP was found in SCG homogenates. Similarly, immunoblots performed with Po antibody revealed immunoreactive protein in nonmyelinating DRG cultures but none in SCG samples. The mRNA and protein measurements convincingly demonstrate that SCG Schwann cells receive little of no exposure to the axonal signal(s) needed for the expression of myelin proteins. During the preparation of this manuscript, a report was published indicating that SCG cultures grown in the presence of serum contain approximately 50% as much Po mRNA as myelinating DRG cultures (Morrison et al., 1991). This result would appear to differ from the data presented here, although the discrepency might be explained by the fact that our cultures were grown in serum-free medium. Such an expla-
nation would imply that serum contains one or more factors that increase myelin protein expression. The neuronal factor controlling the expression of myelin proteins may exist as an axonal membrane component that interacts with a Schwann cell surface receptor. This interaction could subsequently initiate an intracellular cascade that affected gene expression. While the possibility of axons secreting a soluble factor involved in the control of myelin protein synthesis cannot be eliminated, the close proximity of nonmyelinated and myelinated fibers in nerve fascicles would seem to preclude such a mechanism of regulation. The reduction or absence within SCG axons of one or more “myelination factors” should prove useful in obtaining an understanding of this signaling process. Dissociated SCG and DRG can be readily grown in culture under conditions that eliminate Schwann cells and fibroblasts, leaving a pure population of neuronsheurites that can be analyzed for differences in expression of macromolecules. Identification of molecules synthesized by DRG neurons that are absent from SCG neurons should provide candidates that might be involved in the axonal regulation of myelin protein expression.
ACKNOWLEDGMENTS This work was supported by NIH grant NS-27587. The authors thank J. Anthony Warrington for technical assistance, and Drs. S. Younkin and T. Golde for providing actin primers.
REFERENCES Aguayo AJ, Bray GM, Terry LC, Sweezy E (1976a): Three dimensional analysis of unmyelinated fibers in normal and pathologic autonomic nerves. J Neuropathol Exp Neurol 35: 136-157. Aguayo AJ, Charron L, Bray GM (1976b): Potential of Schwann cells from unmyelinated nerve to produce myelin: A quantitative ultrastructural and radiographic study. Neurocytology 5565573. Brown DT, Wellman SE, Sittman DB (1985): Changes in the level of three different classed of histone mRNA during murine erythroleukemia cell differentiation, Mol Cell Biol 5:2879-2886. Bruckenstein DA, Higgens D (1988): Morphological differentiation of embryonic rat sympathetic neurons in tissue culture. I. Conditions under which neurons form axons but not dendrites. Dev Biol 128:324-336. Brunden KR, Poduslo JF (1987): Lysosomal delivery of the major myelin glycoprotein in the absence of myelin assembly: posttranslational regulation of the level of expression by Schwann cells. J Cell Biol 104:661-669. Brunden KR, Brown DT (1990): Po mRNA expression in cultures of Schwann cells and neurons that lack basal lamina and myelin. J Neurosci Res 27:159-168. Brunden KR, Berg CT, Poduslo JF (1987): Isolation of an integral membrane glycoprotein by chloroform-methanol extraction and C,-reversed-phase high-performance liquid chromatography. Anal Biochem 164:474-481.
Axonal Regulation of Myelin Protein Expression Brunden KR, Windebank AJ, Poduslo JF (1990): Role of axons in the regulation of Po biosynthesis by Schwann cells. J Neurosci Res 26:135-143. Bunge RP, Bunge MB, Eldridge CF (1986): Linkage between axonal ensheathment and basal lamina production by Schwann cells. Annu Rev Neurosci 9:95-102. Carey DJ, Todd MS (1987): Schwann cell myelination in a chemically defined medium: demonstration of a requirement for additives that promote Schwann cell extracellular matrix formation. Dev Brain Res 32:95-102. Eldridge CF, Bunge MB, Bunge RB, Wood PM (1987): Differentiation of axon-related Schwann cells in vitro. I. Ascorbic acid regulates basal lamina assembly and myelin formation. J Cell Biol 105:1023-1034. Friede RL (1972): Control of myelin formation by axon caliber. J Comp Neurol 144:233-252. Friede RL, Samorajski T (1968): Myelin formation in the sciatic nerve of the rat. J Neuropathol Exp Neurol 27:546-57 1. Fuqua SAW, Fitzgerald SD, McGuire WL (1990): A simple polymerase chain reaction method for detection and cloning of lowabundance transcripts. BioTechniques 9:206-210. Golde T (1991): Ph.D Thesis, Case Western Reserve University, Cleveland, Ohio. Griffiths IR, McCulloch MC, Barrie JA, Kyriakides E (1991): Expression of Po mRNA in myelinating Schwann cells is related to fibre size. J Neurocytol 20:396-403. Hahn AF, Chang Y, Webster HdeF (1987): Development of myelinated nerve fibers in the sixth cranial nerve of the rat: a quantitative electron microscopic study. J Comp Neurol 260:491500. Inuzuka T, Quarles RH, Trapp BD, Heath JW (1988): Analysis of myelin proteins in sympathetic peripheral nerve of adult rats. Dev Brain Res 38:191-199. Jessen KR, Morgan L, Brammer J, Mirsky R (1985): Galactocerebroside is expressed by non-myelin-forming Schwann cells in situ. J Cell Biol 101:1135-1143. Kidd GJ, Hauer PE, Trapp BD (1990): Axons modulate myelin protein messenger RNA levels during central nervous system myelination in vivo. J Neurosci Res 26:409-418. Labarca C, Paigen K (1980): A simple, rapid, and sensitive DNA assay procedure. Anal Biochem 102:344-352. LeBlanc AC, Poduslo JF (1990): Axonal modulation of myelin gene expression in the peripheral nerve. J Neurosci Res 26:31-326. LeBlanc AC, Poduslo JF, Mezei C (1987): Gene expression in the presence or absence of myelin assembly. Mol Brain Res 2: 57-67. Lemke G, Axel R (1985): Isolation and sequence analysis of cDNA encoding the major structural protein of peripheral myelin. Cell 40:501-508.
515
Lemke G , Lamar E, Patterson J (1988): Isolation and analysis of the gene encoding peripheral myelin protein zero. Neuron 1:7383. Lewin B (1980): “Gene Expression,” 2nd Ed. New York: John Wiley and Sons, pp 962-963. Momson S, Mitchell LS, Ecob-Prince MS, Griffiths IR, Thomson CE, Barrie JA, Kirkham D (1991): Po gene expression in cultured Schwann cells. J Neurocytol 20:769-780. Newman S, Kitamura K, Campagnoni AT (1987): Identification of a cDNA coding for a fifth form of myelin basic protein in mouse. Proc Natl Acad Sci USA 845386-890. Poduslo JF, Dyck PJ, Berg CT (1985): Regulation of myelination: Schwann cell transition from a myelin-maintaining state to a quiescent state after permanent nerve transection. J Neurochem 44:388-400. Rome LH, Bullock PN, Chiappelli F, Cardwell M, Adinolfi AM, Swanson D (1986): Synthesis of a myelin-like membrane by oligodendrocytes in culture. J Neurosci Res 15:49-65. Salzer JL, Bunge RP (1980): Studies of Schwann cell proliferation. I. An analysis in tissue culture of proliferation during development, Wallerian degeneration, and direct injury. J Cell Biol 841739-752. Sambrook J, Fritsch EF, Maniatis T (1989): “Molecular Cloning. A Laboratory Manual.” Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. Trapp BD, Hauer P, Lemke G (1988): Axonal regulation of myelin protein mRNA levels in actively myelinating Schwann cells. J Neurosci 81:3515-3521. Webster HdeF, Martin JR, O’Connell MF (1973): The relationships between interphase Schwann cells and axons before myelination: a quantitative electron microscopic study. Dev Biol 32: 40 1-4 16. Weinberg HJ, Spencer PS (1976): Studies on the control of myelinogenesis. 11. Evidence for neuronal regulation of myelin production. Brain Res 113:363-378. Yao JK, Poduslo JF (1985): Association and release of the major intrinsic membrane glycoprotein from peripheral nerve myelin. Biochem J 228:43-54. Yao JK, Poduslo JF (1988): Biosynthesis of neutral glucocerebroside homologues in the absence of myelin assembly after nerve transection. J Neurochem 50:630-638. Yao JK, Windebank AJ, Poduslo JF, Yoshino JE (1990): Axonal regulation of Schwann cell glycolipid synthesis. Neurochem Res 15:279-282. Zeller NK, Behar TN, Dubois-Dalcq ME, Lazzarini RA (1985): The timely expression of myelin basic protein gene in cultured rat brain oligodendrocytes is independent of continuous neuronal influences. J Neurosci 5:2955-2962.
Myelin Protein Expression in Dissociated Superior Cervical Ganglia and Dorsal Root Ganglia Cultures K.R. Brunden, Y. Ding, and B.S. Hennington Gliatech, Inc. and Department of Neuroscience, Case Western Reserve University, Cleveland, Ohio (K.R.B.); Department of Biochemistry, University of Mississippi Medical Center, Jackson (Y.D., B.S.H.)
Schwann cells of the adult rat superior cervical ganglia (SCG) synthesize negligible levels of the major myelin glycoprotein, Po, in vivo. This suggests that the sympathetic axons of the SCG are deficient in one of more components involved in the regulation of myelin protein expression. Here we have compared the ability of neurites from neonatal rat SCG and embryonic rat dorsal root ganglia (DRG) to induce Schwann cell expression of myelin proteins after growth in culture using a serum-free medium. Steady-state Po mRNA levels in the SCG and DRG culture paradigms were determined with a sensitive polymerase chain reaction (PCR) assay that amplified cDNA produced by reverse transcription of mRNA. This semiquantitative assay showed a linear response to increasing amounts of Po and actin mRNA and required substantially less cellular RNA than typical hybridization techniques. Using the PCR assay, we found that SCG cultures contained significantly lower amounts of Po mRNA than did DRG cultures. To further confirm that SCG cultures have negligible expression of myelin proteins, immunoblot analyses were done to examine the steady-state levels of both Po and myelin basic protein. While nonmyelinating DRG cultures had readily detectable amounts of these myelin-specific proteins, neither could be demonstrated in the SCG cultures. The data indicate that SCG neurites lack one or more signals needed to induce myelin protein expression. Employing SCG and DRG cultures in comparative biochemical studies should prove useful in identifying the axonal molecule(s) involved in the regulation of myelin protein expression. 0 1992 Wiley-Liss, Inc. Key words: myelin, protein expression, axons, cell culture
onal degeneration due to nerve injury leads to consequent changes in Schwann cell metabolism, including an increase of Schwann cell proliferation (Salzer and Bunge, 1980), a cessation of myelination, and a severe reduction in the expression of myelin-specific proteins (LeBlanc et al., 1987; LeBlanc and Poduslo, 1990; Poduslo et al., 1985; Trapp et al., 1988) and glycolipids (Yao and Poduslo, 1988; Yao et al., 1990). When axons reenter a denervated region, Schwann cells respond by differentiating to a stage characterized by the synthesis and assembly of myelin membranes. While certain peripheral axons clearly regulate myelination, many mature axons do not trigger myelin formation by Schwann cells. This is seen in certain sympathetic nerves, including the cervical sympathetic trunk (CST) where axons are >99% nonmyelinated (Aguayo et al., 1976a). The Schwann cells of the CST express negligible levels of the major myelin glycoprotein, Po, as assayed by immunofluorescence (Jessen et al., 198.5), immunoblotting (Brunden et al., 1990), or metabolic labeling (Brunden et al., 1990). Similarly, the Schwann cells of the superior cervical ganglia (SCG) contain negligible levels of myelin protein (Inuzuka et al., 1988). These data suggest that CST and SCG Schwann cells do not receive the axonal information necessary to up-regulate myelin protein expression. Further evidence of this is provided by the observation that CST Schwann cells appear to be capable of myelination after anastomosis to another nerve (Aguayo et al., 1976a,b; Weinberg and Spencer, 1976) or after interaction with dorsal root ganglia neurites in vitro (Brunden et al., 1990). Although axons often contain information that affects the ability of Schwann cells to synthesize myelin membrane, it appears that axons alone are not sufficient to induce myelination. Studies of myelin formation in
INTRODUCTION
Received July 23, 1991; revised February 25, 1992; accepted March 2, 1992.
Axons profoundly infhence the phenotype of Schwann cells within the peripheral nervous system. Ax-
Address reprint requests to Kurt R. Brunden, Ph.D., Gliatech, Inc., 23420 Commerce Park Rd., Cleveland, OH 44122.
0 1992 Wiley-Liss, Inc.
508
Brunden et al.
culture reveal that Schwann cells must be able to assemble a basement membrane, as well as associate with neurites, before myelination can commence (Bunge et al., 1986; Carey and Todd, 1987; Eldridge et al., 1987). Recent work from this laboratory (Brunden and Brown, 1990; Brunden et al., 1990) indicates that Schwann cells require signaling from only axons, and not basement membrane, for the initiation of Po mRNA and protein expression. This observation has subsequently been confirmed by other investigators (Morrison et al., 1991). Thus, the role of basal lamina in myelination is probably not in the regulation of myelin protein synthesis, and it has been suggested that this extracellular matrix may provide the Schwann cells with a necessary sidedness required for myelin assembly (Bunge et al., 1986). Understanding the mechanism of axonal regulation of myelin protein levels is likely to be of importance in ultimately treating dysmyelinating peripheral nerve diseases. The observation of axonal influence on myelin protein expression by oligodendrocytes (Kidd et al. , 1990) suggests that there may be similarities in the mechanism of axon control within the central and peripheral nervous systems. While there is evidence that axons generally reach a minimal caliber before myelination proceeds (Friede, 1972; Friede and Samorajski, 1968; Griffiths et al., 1991), it is not clear that small axons remain unmyelinated strictly because of physical diameter. Instead, such axons may not express one or more molecules needed to trigger Schwann cell synthesis of myelin proteins and lipids. One approach to examining axon molecules important to the process of myelination is to study the ability of neurites to influence Schwann cell expression of myelin components under defined in vitro conditions. Here we compare the effects of SCG and dorsal root ganglia (DRG) neurites on Schwann cell expression of myelin proteins in cultures grown under conditions that prevent both basal lamina formation and myelination. The data indicate that SCG neurites, unlike those of DRG, are incapable of triggering significant expression of myelin-specific mRNA and proteins, The inability of SCG neurites to up-regulate myelin protein expression suggests that comparative studies with DRG and SCG neuron cultures may shed light onto the mechanism whereby axons regulate myelin protein expression.
MATERIALS AND METHODS Tissue Culture Cultures were grown in 35 mm polystyrene dishes (Corning Glassware) that were coated with rat tail collagen as described (Brunden et al., 1990). Cells were derived from either rat DRG or SCG. DRG were removed from the spinal cord of E l 5 Sprague-Dawley rat
pups and dissociated with 0.25% trypsin in Hanks’ balanced salt solution (HBSS; Gibco) for 35 min at 37°C. Aliquots of the dissociated cells were placed onto the dishes and allowed to attach in serum-containing medium overnight. After attachment, cells were subsequently grown in either serum-free or serum-containing medium for 15 days at 37°C in 5% C02:95% air. Media was changed every 2-3 days. SCG were removed from 4-6 day old neonatal Sprague-Dawley rats, dissociated for 45 min at 37°C in 0.25% trypsin plus 0.03% collagenase in HBSS, and plated onto dishes as above. After overnight attachment of cells, the SCG cultures were switched to serum-free medium and allowed to grow for 15-16 days. Cell counts of random fields selected from photographs of the DRG and SCG cultures (i.e., see Fig. 1) revealed no statistically significant differences in the ratio of Schwann cells to neurons in the two culture paradigms.
Media Serum-free medium (nonmyelinating) was composed of Eagle’s minimal essential medium supplemented with Earle’s salts (Gibco), 1.4 mM L-glutamine, 0.6% glucose, and 100 ng/ml 7 s nerve growth factor (Collaborative Research). Serum-containing medium (myelinating) was as above with the addition of 15% calf serum (HyClone) and 50 Fg/ml ascorbic acid. RNA Preparations Cultured cells were scraped from dishes in 0.5-1 .O ml of 1% SDS/25 mM EDTA in phosphate-buffered saline using sterile, disposable cell scrapers. Generally, the material from four to eight dishes was pooled for a single RNA preparation. An aliquot of the cell homogenate was removed for subsequent determination of cell number (see below), and the remainder of the sample was used for total cellular RNA isolation by the hot phenol method (Brown et al., 1985). We have previously demonstrated that RNA isolated by this technique is intact and of high quality (Brunden and Brown, 1990). In summary, the cell homogenate was mixed with an equal volume of water-saturated phenol for 5 min at 55°C. A known amount of 32P-labeled bovine rRNA was added to the phenol mixture. This rRNA (Sigma Chemical Co.), which was used to follow the recovery of total cellular RNA in subsequent steps, was 5’-labeled by treatment with bacterial alkaline phosphatase and addition of [gamrr~a-~~P]ATP with polynucleotide kinase. The phenol-aqueous mix was centrifuged briefly to separate the phases, and a majority of the upper aqueous phase was recovered. The remainder of the mix was back-extracted for 5 min at 55°C with an equal volume of 0.3 M sodium acetate, pH 5.5. The aqueous phase from the back-extraction was combined with the first aqueous phase, and
Axonal Regulation of Myelin Protein Expression DRG
509
SCG
Fig. 1. Phase-contrast photomicrographs of dissociated DRG and SCG cultures grown in serum-free medium that prevents basal lamina formation. Neurons of both cultures (DRGleft; SCG-right) extend neuritic processes that are populated by Schwann cells that do not form myelin. the sample was treated with 2.5-fold volume excess of ethanol at -20°C overnight. RNA was collected by centrifugation for 60 min at 8,50Og, resuspended in TE buffer (Sambrook et al., 1989) containing 0.3 M sodium acetate, and reprecipitated as above. The final RNA pellet was resuspended in approximately 0.3 ml H,O and an aliquot was counted in a scintillation counter to determine the recovery of the added 32P-rRNA. The fraction of recovered exogenous 32P-rRNA was used to obtain a corrected value for cell-equivalents of total RNA (see below).
culture samples were divided by 6.6 pg, the amount of DNA per diploid rat cell (Lewin, 1980), to calculate the number of cells that were used in each RNA preparation. The fraction of the added ”P-rRNA that was recovered in each RNA sample (see above) was multiplied by the total number of cells used in the preparation to obtain a corrected value of the number of cell equivalents of RNA in the sample. This value served as the basis for determining aliquot sizes to be used in Po and actin mRNA assays.
Calculation of Cell-Equivalents of RNA Aliquots of the cell homogenates used for RNA preparations were tested for DNA content using a fluorometric assay (Labarca and Paigen, 1980) to obtain a value of total DNA in the original culture homogenates. The samples were extracted with an equal volume of neutral phenol/chloroform (Sambrook et al., 1989) and ethanol-precipitated prior to assaying. Standard curves were constructed using calf thymus DNA that was mixed with 1%sodium dodecyl sulfate (SDS)/25 mM EDTA in phosphate-buffered saline (see RNA preparations) and subsequently extracted with phenol/chloroform and ethanol-precipitated. Such treatment of the standards normalizes any quenching artifacts that might arise due to the SDS extraction. The DNA values obtained for the
Po and actin mRNA levels were determined using a semiquantitative PCR assay (Fuqua et al., 1990). Aliquots of the RNA samples were used for the generation of cDNA and subsequent amplification with a “GeneAmp RNA PCR Kit” (Perkin Elmer Cetus). For Po mRNA determinations, a synthesized oligonucleotide compleprimer (5’-TTGGTGCTTCGGCTGTGGTC-3’) mentary to a sequence within exon 6 of Po mRNA (Lemke et al., 1988) was used at a concentration of 1.5 p M in 20 (~.1of the commercial mix which contained cloned Moloney Murine Leukemia Virus reverse transcriptase (2.5 U/p1) and deoxyribonucleotide triphosphates (1 mM). The reverse transcription proceeded for 45 min at 42°C in a thermocycler (Perkin Elmer Cetus or Thermolyne). After a 5 min denaturation of the reverse
Po and Actin mRNA Determinations
510
Brunden et al.
transcriptase at 99°C 80 pl of a commercial solution was bound antibody, the blots were treated with goat antiadded that contained 2.0 U AmpliTaq DNA polymerase rabbit IgG or goat anti-mouse IgG that were conjugated (Perkin Elmer Cetus) and an “upstream” oligonucle- with alkaline phosphatase (Fisher Scientific). Followotide primer (0.38 pM; 5’-TGTTGCTGCTGTTGC- ing removal of unbound second antibody, staining of TCTTC-3’) complementary to a region of exon 4 of Po the immunoreactive species was performed with nitrotetcDNA (Lemke et al., 1988). After overlaying 50 p1 of razolium blue/5-bromo-4-chloro-3-indoyl phosphate mineral oil onto each sample, the Po cDNA was ampli- (Promega). fied through 30 cycles of the following incubation steps: 94°C for 1 min, 60°C for 2 min, and 72°C for 2 min with 5 sec increases each cycle. Actin mRNA was also as- RESULTS sessed in the total RNA preparations as a control. Ali- Analysis of Po mRNA Levels in SCG quots of RNA were treated with reverse transcriptase as and DRG Cultures There is evidence suggesting that CST and SCG described above, except that randem hexamer primers (0.75 p M ; Perkin Elmer Cetus) were used to initiate axons do not trigger myelin protein synthesis in vivo cDNA synthesis. Following cDNA formation, PCR am- (Brunden et al., 1990; Inuzuka et al., 1988; Jessen et al., plification was carried out as described for Po using an 1985). To further characterize the properties of these actin downstream primer (5’-CAGGTCCAGACGAG- sympathetic axons, we examined whether cultures of disGATGGCAT-3’; 0.25 pM) and upstream primer (5’- sociated SCG were lacking in the ability to increase the CGACATGGAGAAAATCTGCACC-3’; 0.25 FM). expression of the myelin-specific glycoprotein Po. SCG These primers to human p-actin were kindly provided by were removed from neonatal rat pups, and the dissociDrs. S. Younkin and T. Golde (Case Western Reserve ated ganglion cells were grown for 15-16 days in culture University), and have been shown to result in amplified using a serum-free medium that prevents basal lamina actin DNA of 300 base pairs in both human and rat formation (Carey and Todd, 1987; Eldridge et al., 1987). (Golde, 1991). After completion of amplification, ali- For comparison, rat DRG cultures were also grown in quots of the mixtures were analyzed by electrophoresis this medium for 15 days. These DRG cultures have been on 4% agarose gels (low molecular weight grade; Fisher shown to express appreciable amounts of mRNA encodScientific) and stained with ethidium bromide. DNA ing Po (Brunden and Brown, 1990) even though they lack standards were generally included on gels. In one exper- basement membrane and consequently cannot form myiment, aliquots of the Po amplification product from a elin. Representative phase-contrast photomicrographs of DRG culture grown in serum-free medium were digested the two culture paradigms are shown in Figure 1. with approximately 5 units of the restriction enzymes Schwann cells can be seen aligning and interacting with NcoI or PstI in the buffers supplied by the manufacturer the neurites of the DRG and SCG cultures, but these cells (BRL). These samples were then analyzed by agarose gel do not form myelin. Neurites of SCG cultures grown in serum-free medium have been shown to be predomielectrophoresis as above. nantly axonal in nature, with few dendritic processes Immunoblot Analyses (Bruckenstein and Higgens, 1988). To evaluate the steady-state levels of Po mRNA in Cultured tissues were excised from dishes along with the collagen substratum and immediately frozen the SCG and DRG cultures, we employed a semi-quanwith liquid N,. The frozen samples were homogenized titative polymerase chain reaction (PCR) assay which is with 1.25% SDS in water and sonicated in a bath-type significantly more sensitive than typical filter hybridizasonicator for 30 min. Insoluble material was removed by tion techniques. This enhanced sensitivity is particularly centrifugation as described (Bmnden et al., 1990). The important when the amount of cellular RNA is limiting, protein concentrations of the samples were determined as is generally the case with nervous system primary using a BCA assay (Pierce Chemicals). Aliquots of the cultures. Total RNA preparations from a defined number homogenates were separated by SDS-polyacrylamide gel of cells were treated with reverse transcriptase to generelectrophoresis (PAGE) using 15% polyacrylamide gels, ate cDNA which could subsequently be amplified by and the proteins were electrophoretically transferred to PCR (Fuqua et a1., 1990). Oligonucleotide primers were nitrocellulose overnight at 4°C (Yao and Poduslo, 1985). used that would result in the generation of a 181 baseThe nitrocellulose was treated with either Po antibody pair segment of DNA corresponding to a region spanning developed in rabbits against HPLC-purified protein exons 4 through 6 of the Po transcripts (Lemke et al., (Brunden et al., 1987) or with a mouse monoclonal an- 1988) and a 300 base-pair region of actin (Golde, 1991). tibody that reacts with a region of exon 6 of myelin basic As seen in Figure 2, the Po and actin assays demonstrated protein (Boehringer Mannheim Biochemicals). After elevated responses to increasing amounts of RNA from thoroughly washing the nitrocellulose to remove non- 15 day old nonmyelinating DRG cultures, with the am-
Axonal Regulation of Myelin Protein Expression A.
511
Po
Std.
Ct.
Ncol
Pstl
Fig. 3 . Restriction enzyme digests of DNA from the PCR assay of Po mRNA. RNA from DRG cultures grown in serumfree medium was amplified as described in Materials and Methods, and equal-sized aliquots were either digested with NcoI, PstI, or left untreated (Ct). The samples and standard restriction fragments (Std) were subsequentlyanalyzed by agarose gel electrophoresis, with the standards indicated in basepairs (BP).
Cells
Fig. 2 . Linearity of response of the PCR assay used to measure Po and actin mRNA. RNA preparations (0-540 cell-equivalents) from DRG cultures grown in serum-free medium were treated as described in Materials and Methods to generate amplified Po (A) and actin (B) DNA. Equal-sized aliquots of the resulting samples were analyzed by agarose gel electrophoresis, as were DNA standards (Std). The sizes of the standards are listed in base-pairs (BP).
plified products being of the predicted sizes. The expanded region of the Po cDNA contains unique NcoI and PstI restriction enzyme sites (Lemke and Axel, 1985) and treatment of the amplified DNA with these enzymes resulted in the predicted cleavage products (Fig. 3), further verifying that the 181 base-pair product is derived from Po mRNA. It should be noted that, although purified RNA preparations were used here, the Po primers can presumably be used with samples containing total cellular nucleic acids since amplified product derived from genomic DNA will be substantially larger than that from Po cDNA due to the presence of two introns.
We utilized the PCR methodology with RNA from DRG cultures that were grown in serum-free medium (nonmyelinating) or in medium containing serum and ascorbic acid (myelinating) to confirm that the assay accurately measures Po mRNA in the culture preparations. A previous study (Brunden and Brown, 1990) revealed that there are comparable levels of Po mRNA in these two types of DRG cultures, and the nonrnyelinating and myelinating cultures both yielded similar amounts of PCR-amplified DNA (Fig. 4). The amount of 181 basepair DNA from each sample increased with increasing cell number (Fig. 4), as would be expected if the assay were in the linear range of sensitivity. If SCG axons are indeed deficient or reduced in one or more factors needed to induce Schwann cell expression of myelin proteins, it would be expected that SCG cultures should give little or no amplified Po DNA in the PCR assay. This is in fact the case, as shown in Figure 5 where the positive Po signal seen from a DRG culture sample is absent from an SCG RNA preparation. These SCG and DRG samples contained comparable amounts of actin mRNA, as demonstrated in Figure 5. Thus, the lack of Po signal is not due to a general absence of mRNA in the SCG sample. If larger amounts of SCG sample are used in the PCR assay a faint Po signal appears, but the amount of this amplified product is always substantially lower than that of DRG culture samples.
512
Brunden et al. +Myelin
-Myelin
Po
A. 0P 1000 700
500 400
300 200
100
50.
Sld
20 100 20 100
SCG
DUG
20
100
20
B.
Actin
100 0P
Cells 1000
Fig. 4. Analysis of Po mRNA from myelinating and nonmyelinating DRG cultures. RNA samples corresponding to 20 and 100 cell-equivalents of both serum-containing DRG cultures ( + myelin) and serum-free DRG cultures (-myelin) were treated as described in Materials and Methods to generate PCRamplified DNA. Equal-sized aliquots were then analyzed by agarose gel electrophoresis.
Immunoblot Analyses of Po and Myelin Basic Protein in SCG and DRG Cultures The very low level of Po mRNA in the SCG samples would imply that these cultures also express negligible amounts of Po glycoprotein, whereas the DRG cultures should contain this myelin protein. An immunoblot analysis was performed with Po antibody to test these predictions. DRG culture homogenate gave a distinct immunoreactive band of approximately 28 kD and a slightly smaller degradation product, while the SCG culture sample yielded no detectable reaction product when equal-sized aliquots were assayed (Fig. 6). The immunoblot data confirmed the lack of Po expression in the SCG cultures, but it was not known whether these cultures have a general deficit in the synthesis of myelin proteins. Moreover, it has not been demonstrated that the nonmyelinating DRG cultures express measurable myelin basic protein (MBP) in addition to Po. To address these points, immunoblot analyses were done with a monoclonal antibody to MBP. This antibody recognizes a peptide sequence encoded in exon 6 of MBP, and thus reacts with three of the five differentially spliced forms of this group of myelin proteins (Newman et al., 1987). As documented in Figure 7A, comparable
700
500400300-
200
100-
50-
Sld
20
100
DRG
20
100
SIX
Fig. 5. Comparison of Po mRNA levels in DRG and SCG cultures. RNA samples corresponding to 20 and 100 cellequivalents from serum-free DRG and SCG culture preparations were assayed for Po (A) and actin (B) mRNA using the PCR methodology. Equal-sized aliquots were subsequently analyzed by agarose gel electrophoresis. DNA standards (Std), whose sizes are indicated in base-pairs (BP), were also included on the gel.
immunoreactive signals were obtained when 20 p g samples of nonmyelinating and myelinating DRG homogenates were examined, and a strong signal was evident when 50 p g of homogenate from the former was assayed. This is further proof that the neurites of DRG cultures contain the necessary information to trigger general myelin protein synthesis in the absence of basal lamina and active myelination. In contrast to DRG cultures, SCG cultures had little or no immunoreactive MBP (Fig. 7B). Thus, the SCG cultures seem to have a general reduction of myelin protein expression.
Axonal Regulation of Myelin Protein Expression SCG
DRG
513
DRG
A.
-Myelin
+Myelin --36.0 -29.0 --24.0
45.036.0-
29.024.0-
50ug
20ug
20ug 1oug
B. -36.0
30ug
60ug
30 ug
-29.0 -24.0
60 ug
Fig. 6. Immunoblot analysis of Po mRNA levels in homogenates from DRG and SCG cultures grown in serum-free medium. Aliquots (30 pg and 60 pg) from DRG and SCG culture homogenates were separated by SDS-PAGE and immunoblotted to assay for steady-state Po as described in Materials and Methods. The migrations of protein standards are indicated (in W).
DISCUSSION The synthesis of myelin proteins by Schwann cells is dependent on axons, as the disruption of axonSchwann cell interaction via nerve transection results in a drastic down-regulation of myelin protein expression at the mRNA (LeBlanc et al., 1987; LeBlanc and Poduslo, 1990; Trapp et al., 1988) and protein levels (Brunden and Poduslo, 1987; Poduslo et al., 1985). Likewise, Schwann cells from transected nerve demonstrate a decrease in the synthesis of the myelin glycolipid, galactocerebroside (Yao and Poduslo, 1988; Yao et al., 1990). Although the synthesis of myelin components by oligodendrocytes of the central nervous system does not appear to be as strictly dependent on axons (Rome et al., 1986; Zeller et al., 1985), there is evidence suggesting that oligodendrocytes reduce the expression of myelin proteins following nerve transection (Kidd et al., 1990). Given the importance of axons in regulating the synthesis of myelin molecules, surprisingly little is actually known about the mechanisms of this control. Several studies (Friede, 1972; Friede and Samorajski, 1968; Griffiths et al., 1991) point to a correlation of increasing axon di-
DRG
SCG
Fig. 7. Immunoblot analysis of MBP levels in DRG and SCG cultures. A: Nonmyelinating (- myelin) and myelinating ( + myelin) DRG cultures homogenates (10,20, or 50 pg) were assayed for steady-state MBP by SDS-PAGE and immunoblotting as described in Materials and Methods. The migrations of protein standards are indicated (in kD). B: Aliquots (50 pg) of homogenates from DRG and SCG cultures grown in serumfree medium were separated by SDS-PAGE and assayed for MBP by immunoblotting. The migrations of protein standards are shown.
ameter and increased myelination. Models of myelin regulation based solely on physical parameters such as axon caliber or axon surface area are probably simplistic. For example, nonmyelinating Schwann cells in vivo often surround many small diameter axons (Webster et al., 1973), resulting in a large axonal contact area but no myelin formation. Furthermore, other studies have noted myelinated axons with diameters as small as 0.6 pm (Hahn et al., 1987). It is likely that the lack of myelination of smaller axons is not a direct result of their size, but is instead caused by the lack of expression of one or more neuronal factors involved in the regulation of syn-
514
Brunden et al.
thesis of myelin macromolecules. The extent of synthesis of such putative axonal factors may be correlated with axon diameter. The identification of axonal components involved in the control of myelination would be greatly aided by the characterization of neuron populations deficient in one or more of these critical factors. Earlier studies suggested that Schwann cells of the CST do not receive the necessary information to form myelin membrane (Aguayo et al., 1976a,b; Weinberg and Spencer, 1976). More recent work demonstrated that CST and SCG Schwann cells synthesize a very low level of myelin protein (Brunden et al., 1990; Inuzuka et al., 1988). Together, the data suggest that the axons of these sympathetic neurons are not signaling Schwann cells to produce myelin proteins. In this study, we have compared the ability of SCG and DRG neurites to induce myelin protein expression by Schwann cells in vitro. Cultures were grown in a defined medium that prevents basal lamina formation and myelination, since neurites are known to be capable of up-regulating the synthesis of the myelin glycoprotein, Po, under these growth conditions (Brunden and Brown, 1990; Brunden et al., 1990; Morrison et al., 1991). When the steady-state levels of Po mRNA were examined with a sensitive PCR assay it was found that SCG cultures had little mRNA encoding this myelin protein. In contrast, nonmyelinating DRG cultures contained Po mRNA in amounts comparable to those found in myelinating DRG cultures grown with serum and ascorbic acid. The low level of Po mRNA in the SCG cultures is presumably not a consequence of an atypical Schwann cell population since nonmyelinating Schwann cells of the CST can respond normally to DRG neurites by synthesizing Po (Brunden et al., 1990). Examination of the steady-state levels of myelin proteins further demonstrates that SCG cultures differ from their DRG counterparts. While nonmyelinating DRG cultures contained MBP at levels comparable to myelinating DRG cultures, little or no MBP was found in SCG homogenates. Similarly, immunoblots performed with Po antibody revealed immunoreactive protein in nonmyelinating DRG cultures but none in SCG samples. The mRNA and protein measurements convincingly demonstrate that SCG Schwann cells receive little of no exposure to the axonal signal(s) needed for the expression of myelin proteins. During the preparation of this manuscript, a report was published indicating that SCG cultures grown in the presence of serum contain approximately 50% as much Po mRNA as myelinating DRG cultures (Morrison et al., 1991). This result would appear to differ from the data presented here, although the discrepency might be explained by the fact that our cultures were grown in serum-free medium. Such an expla-
nation would imply that serum contains one or more factors that increase myelin protein expression. The neuronal factor controlling the expression of myelin proteins may exist as an axonal membrane component that interacts with a Schwann cell surface receptor. This interaction could subsequently initiate an intracellular cascade that affected gene expression. While the possibility of axons secreting a soluble factor involved in the control of myelin protein synthesis cannot be eliminated, the close proximity of nonmyelinated and myelinated fibers in nerve fascicles would seem to preclude such a mechanism of regulation. The reduction or absence within SCG axons of one or more “myelination factors” should prove useful in obtaining an understanding of this signaling process. Dissociated SCG and DRG can be readily grown in culture under conditions that eliminate Schwann cells and fibroblasts, leaving a pure population of neuronsheurites that can be analyzed for differences in expression of macromolecules. Identification of molecules synthesized by DRG neurons that are absent from SCG neurons should provide candidates that might be involved in the axonal regulation of myelin protein expression.
ACKNOWLEDGMENTS This work was supported by NIH grant NS-27587. The authors thank J. Anthony Warrington for technical assistance, and Drs. S. Younkin and T. Golde for providing actin primers.
REFERENCES Aguayo AJ, Bray GM, Terry LC, Sweezy E (1976a): Three dimensional analysis of unmyelinated fibers in normal and pathologic autonomic nerves. J Neuropathol Exp Neurol 35: 136-157. Aguayo AJ, Charron L, Bray GM (1976b): Potential of Schwann cells from unmyelinated nerve to produce myelin: A quantitative ultrastructural and radiographic study. Neurocytology 5565573. Brown DT, Wellman SE, Sittman DB (1985): Changes in the level of three different classed of histone mRNA during murine erythroleukemia cell differentiation, Mol Cell Biol 5:2879-2886. Bruckenstein DA, Higgens D (1988): Morphological differentiation of embryonic rat sympathetic neurons in tissue culture. I. Conditions under which neurons form axons but not dendrites. Dev Biol 128:324-336. Brunden KR, Poduslo JF (1987): Lysosomal delivery of the major myelin glycoprotein in the absence of myelin assembly: posttranslational regulation of the level of expression by Schwann cells. J Cell Biol 104:661-669. Brunden KR, Brown DT (1990): Po mRNA expression in cultures of Schwann cells and neurons that lack basal lamina and myelin. J Neurosci Res 27:159-168. Brunden KR, Berg CT, Poduslo JF (1987): Isolation of an integral membrane glycoprotein by chloroform-methanol extraction and C,-reversed-phase high-performance liquid chromatography. Anal Biochem 164:474-481.
Axonal Regulation of Myelin Protein Expression Brunden KR, Windebank AJ, Poduslo JF (1990): Role of axons in the regulation of Po biosynthesis by Schwann cells. J Neurosci Res 26:135-143. Bunge RP, Bunge MB, Eldridge CF (1986): Linkage between axonal ensheathment and basal lamina production by Schwann cells. Annu Rev Neurosci 9:95-102. Carey DJ, Todd MS (1987): Schwann cell myelination in a chemically defined medium: demonstration of a requirement for additives that promote Schwann cell extracellular matrix formation. Dev Brain Res 32:95-102. Eldridge CF, Bunge MB, Bunge RB, Wood PM (1987): Differentiation of axon-related Schwann cells in vitro. I. Ascorbic acid regulates basal lamina assembly and myelin formation. J Cell Biol 105:1023-1034. Friede RL (1972): Control of myelin formation by axon caliber. J Comp Neurol 144:233-252. Friede RL, Samorajski T (1968): Myelin formation in the sciatic nerve of the rat. J Neuropathol Exp Neurol 27:546-57 1. Fuqua SAW, Fitzgerald SD, McGuire WL (1990): A simple polymerase chain reaction method for detection and cloning of lowabundance transcripts. BioTechniques 9:206-210. Golde T (1991): Ph.D Thesis, Case Western Reserve University, Cleveland, Ohio. Griffiths IR, McCulloch MC, Barrie JA, Kyriakides E (1991): Expression of Po mRNA in myelinating Schwann cells is related to fibre size. J Neurocytol 20:396-403. Hahn AF, Chang Y, Webster HdeF (1987): Development of myelinated nerve fibers in the sixth cranial nerve of the rat: a quantitative electron microscopic study. J Comp Neurol 260:491500. Inuzuka T, Quarles RH, Trapp BD, Heath JW (1988): Analysis of myelin proteins in sympathetic peripheral nerve of adult rats. Dev Brain Res 38:191-199. Jessen KR, Morgan L, Brammer J, Mirsky R (1985): Galactocerebroside is expressed by non-myelin-forming Schwann cells in situ. J Cell Biol 101:1135-1143. Kidd GJ, Hauer PE, Trapp BD (1990): Axons modulate myelin protein messenger RNA levels during central nervous system myelination in vivo. J Neurosci Res 26:409-418. Labarca C, Paigen K (1980): A simple, rapid, and sensitive DNA assay procedure. Anal Biochem 102:344-352. LeBlanc AC, Poduslo JF (1990): Axonal modulation of myelin gene expression in the peripheral nerve. J Neurosci Res 26:31-326. LeBlanc AC, Poduslo JF, Mezei C (1987): Gene expression in the presence or absence of myelin assembly. Mol Brain Res 2: 57-67. Lemke G, Axel R (1985): Isolation and sequence analysis of cDNA encoding the major structural protein of peripheral myelin. Cell 40:501-508.
515
Lemke G , Lamar E, Patterson J (1988): Isolation and analysis of the gene encoding peripheral myelin protein zero. Neuron 1:7383. Lewin B (1980): “Gene Expression,” 2nd Ed. New York: John Wiley and Sons, pp 962-963. Momson S, Mitchell LS, Ecob-Prince MS, Griffiths IR, Thomson CE, Barrie JA, Kirkham D (1991): Po gene expression in cultured Schwann cells. J Neurocytol 20:769-780. Newman S, Kitamura K, Campagnoni AT (1987): Identification of a cDNA coding for a fifth form of myelin basic protein in mouse. Proc Natl Acad Sci USA 845386-890. Poduslo JF, Dyck PJ, Berg CT (1985): Regulation of myelination: Schwann cell transition from a myelin-maintaining state to a quiescent state after permanent nerve transection. J Neurochem 44:388-400. Rome LH, Bullock PN, Chiappelli F, Cardwell M, Adinolfi AM, Swanson D (1986): Synthesis of a myelin-like membrane by oligodendrocytes in culture. J Neurosci Res 15:49-65. Salzer JL, Bunge RP (1980): Studies of Schwann cell proliferation. I. An analysis in tissue culture of proliferation during development, Wallerian degeneration, and direct injury. J Cell Biol 841739-752. Sambrook J, Fritsch EF, Maniatis T (1989): “Molecular Cloning. A Laboratory Manual.” Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. Trapp BD, Hauer P, Lemke G (1988): Axonal regulation of myelin protein mRNA levels in actively myelinating Schwann cells. J Neurosci 81:3515-3521. Webster HdeF, Martin JR, O’Connell MF (1973): The relationships between interphase Schwann cells and axons before myelination: a quantitative electron microscopic study. Dev Biol 32: 40 1-4 16. Weinberg HJ, Spencer PS (1976): Studies on the control of myelinogenesis. 11. Evidence for neuronal regulation of myelin production. Brain Res 113:363-378. Yao JK, Poduslo JF (1985): Association and release of the major intrinsic membrane glycoprotein from peripheral nerve myelin. Biochem J 228:43-54. Yao JK, Poduslo JF (1988): Biosynthesis of neutral glucocerebroside homologues in the absence of myelin assembly after nerve transection. J Neurochem 50:630-638. Yao JK, Windebank AJ, Poduslo JF, Yoshino JE (1990): Axonal regulation of Schwann cell glycolipid synthesis. Neurochem Res 15:279-282. Zeller NK, Behar TN, Dubois-Dalcq ME, Lazzarini RA (1985): The timely expression of myelin basic protein gene in cultured rat brain oligodendrocytes is independent of continuous neuronal influences. J Neurosci 5:2955-2962.
