Axonal Regulation of Myelin Myelinating Schwann Cells Protein

The Journal of Neuroscience, September 1988, 8(9): 35153521

Axonal Regulation of Myelin Protein mRNA Levels Actively Myelinating Schwann Cells

Bruce D. Trapp, Peter Hauer, and Greg Lemkel The Johns Hopkins University School of Medicine, Department of Neurology, Neuromuscular Division, Baltimore, Maryland 21205, and ‘Molecular Neurobiology Laboratory, The Salk Institute, San Diego, California 92138

Upon transection of a peripheral nerve, axons distal to the the major myelin proteins are dramatically reduced (Mirsky et transection degenerate. As a consequence of this axonal al., 1980; Politis et al., 1982; Poduslo et al., 1984; Willison et degeneration, myelin-forming Schwann cells cease biosyn- al., 1988). These and related data have been interpreted as thesis of new myelin membrane, contribute to phagocytosis indicating that axons induce and regulate myelin gene expres- of previously formed myelin, and markedly down-regulate sion during formation and maintenanceof the myelin sheath. expression of myelin-specific markers. Among the most We have tested this hypothesis directly. Rat sciatic nerves prominent of these down-regulated markers are the major were transected during a period of active myelination, and the structural proteins of peripheral myelin, P, and myelin basic level and distribution of P, and MBP mRNAs were determined protein (MBP). We have used slot blot and in situ hybridiza- in denervated regions distal to the site of transection. We find tion techniques to demonstrate that for actively myelinating a 40-fold decreasein the level of these messagesat 5 d post- Schwann cells, down-regulation of the P, and MBP genes transection. In situ hybridization studiesdemonstrate that this occurs primarily at the level of mRNA expression. Together decreaseoccurs simultaneously along the entire distal stump. with other recent data, these findings strongly argue for ax- These data indicate that axons regulate myelination at the level onal modulation of P, and MBP gene transcription during of Schwann cell mRNA expression,most probably through di- active myelination. rect modulation of transcription.

Schwann cells of the PNS can be divided into 2 distinct cell Materials and Methods types, thosethat ensheathemultiple axons and thosethat tightly Tissues. Sixteen35d-old Sprague-Dawley rats were used for morpho- and repeatedly wrap a singleaxon, resulting in formation of the logical studies. Under appropriate anesthetic, the right sciatic nerve was transected above the sciatic notch in 8 of the rats. The proximal stump myelin sheath (Raine, 1984; Webster and Favilla, 1984). Al- and attached L4 and L5 roots were removed to prevent reinnervation though all Schwanncells have the potential to form this sheath, of the distal stump which remained in its normal orientation so that they will do so only upon induction by appropriate peripheral various regions could be analyzed. At 2, 5, 10, and 20 d posttransection, axons (Aguayo et al., 1976; Weinberg and Spencer, 1976; Bray 2 normal rats and 2 rats having right sciatic nerve transections were et al., 1981). A key feature of this axon-mediated induction is perfused with 4% paraformaldehyde in 0.08 M phosphate buffer. The distal stumps and the left sciatic nerves were removed from transected the activation and high-level expressionof the genesencoding animals, and the right sciatic nerve was removed from control animals. the major myelin proteins P, and myelin basic protein (MBP) These nerves were dissected into 3 equal segments (proximal, mid, and (Lemke, 1986). distal), placed in fixative overnight and then processed to paraffin by P, and MBP accumulate to very high levels during postnatal standard procedures. Two paraffin blocks were produced for each time point. Both contained the transected distal stump, the untransected left development, with a time coursethat closely parallels the rate sciatic nerve, and the right sciatic nerve from a control animal. Sections of myelin production in peripheral nerve (Wood and Engel, were cut at a thickness of 6 pm, placed on glass slides, deparaffinized, 1976; Lemke and Axel, 1985; Willison et al., 1987). Schwann and rehydrated prior to use. In situ hybridization and immunohisto- cells that do not elaborate myelin, however, do not express chemistry were performed separately on adjacently cut sections. significant amountsofthese proteins (Brockeset al., 1979;Trapp Fifty 35-d-old Sprague-Dawley rats were used for biochemical studies. Under appropriate anesthetic, both sciatic nerves were transected above et al., 1981, 1987; Willison et al., 1988). Previous studieshave the sciatic notch in 25 of the animals. The proximal end of each distal also indicated that maintenance of the myelin sheath depends stump was sutured into the musculature at the inferior aspect of the on survival of myelinated axons. When myelinated axons de- thigh. At 1, 2, 5, 10, and 20 d after transection, the distal stumps from generate, for example, both myelin synthesisand expressionof 5 animals and the sciatic nerves from 5 control animals were removed and stored in liquid nitrogen prior to RNA isolation. In situ hybridization. Sections on slides were prehybridized and hybridized as described previously (Trapp et al., 1987). Probes were Received Dec. 10, 1987; revised Jan. 29, 1988; accepted Feb. 2, 1988. YS-labeled nick-translated PO and MBP cDNAs. Autoradiography was We thank Dr. John Griffin for critical review of the manuscriot. Cindi Cootauco performed by standard procedures. Following development of the emul- for technical assistance, and Rod Graham for typing and edit& the manuscript. sion, sections were stained with hematoxylin, dehydrated, and overlaid This work was supported by Grants NS 22849 (B.D.T.) and NS 23896 (G.L.) from the NIH and Grant RG 1844 (G.L.) from the National Multiple Sclerosis Society. with coverslips. Sections were photographed witha Zeiss Axiophat mi- B.D.T. is a Harry Weaver Nemosciences Scholar of the National Multipie croscone under both bright and dark fields. The P, and MBP cDNA Sclerosis Society; G.L. is an awardee of the Pew Scholars Program in the Biomed- clones-used have been characterized and described elsewhere (Roach et ical Sciences. al., 1983; Lemke and Axel, 1985). Correspondence should be addressed to Bruce D. Trapp, The Johns Hopkins Zmmunohistochemistry. Sections were stained with a well-character- University School of Medicine, 600 North Wolfe Street, Meyer 6- 18 1, Baltimore, ized P, antiserum(Trapp et al., 1979) by the peroxidase-antiperoxidase MD 21205. procedure as described previously (Trapp et al., 1981). Copyright 0 1988 Society for Neuroscience 0270-6474/88/093515-07$02.00/O RNA isolation and analysis. Total RNA was extracted from frozen 3516 Trapp et al. - Axons Regulate Myelin mRNA Levels

I PO mRNA MBP mRNA c T 1 c T 2- 5

0 10 20 3b DAYS POST TRANSECTION DAYS POST TRANSECTION Figure 1. Slot blot determination of major myelin RNA levels in control and transected rat sciatic nerves. Total cellular RNA (1 pg) from control (C) or transected (7’) rat sciatic nerves and from rat liver (L) was immobilized to a nylon support and hybridized at high stringency to 32P-radiolabeled P, (left) and MBP (right) cDNA clones. Numbers on blots refer to days posttransection; a day 10 posttransection control point was not taken. Exposure time for the PO blot was 12 hr with an intensifying screen; for the MBP blot, 24 hr with an intensifying screen. Each blot autoradiogram was scanned with a densitometer, and percentage control values were calculated by dividing the integrated hybridization signal from each experimental (transected) point by the average of the hybridization signal for the 4 control points. Autoradiography was performed at -70°C using preflashed X-ray film. tissue by homogenization in guanidine thiocyanate and subsequent pre- and immobilized to GeneScreen (New England Nuclear) using a slot cipitation with lithium chloride as described by Cathala et al. (1983). blotter (Schleicher and Schuell). Slot blots were hybridized and washed Then, 0.4, 1, and 2 fig of total RNA from transected and control sciatic at high stringency using nick-translated 32P-labeled cDNA (P, and MBP) nerves and from liver were denatured in 6 x SSC/7.4% formaldehyde probes as described previously (Lemke and Axel, 1985).

Figure 2. Comparison of the distribution of P, mRNA in longitudinally oriented parathn sections of aged-matched control (A, C, E) and 2 (B), 5 (O), and 20 d Q transected rat sciatic nerves. Clusters of P, mRNA were readily detectable in all control sections. The general distribution of PO mRNA 2 d after transection was similar to controls. P, mRNA clusters were reduced dramatically 5 d after transection and were not detected in 20 d transected nerves. Dark-field optics. Scale bar, 75 pm. The Journal of Neuroscience, September 1988, 8(9) 3517

Figure 3. Bright-field analysis of sections in Figure 2. Clusters of silver grains representing PO mRNA were concentrated around Schwann cell nuclei in control sections (A, C, E). Smaller P, mRNA clusters containing few silver grains surrounded Schwann cell nuclei 2 d after transection (B). Perinuclear concentrations of P, mRNA were rarely found 5 d after transcription (0, arrowhead)and were not detected in sections from 20 d posttransected nerves (F). A dramatic increase in the number of cell nuclei per unit area of nerve occurred between 5 and 20 d after transection. Scale bar, 30 pm.

tribution of POand MBP mRNAs in tissuesections of 2, 5, 10, Results and 20 d transected distal stumps and compared this distribu- Myelin RNA levelsin transectedsciatic nerves tion to that found in age-matchedcontrol nerves. For eachtime We extracted total cellular RNA from normal sciatic nervesand point, sectionsof control and transectednerves were hybridized from the distal stumps of transectednerves at 1, 2, 5, 10, and on the sameslide. The relative intensities of hybridization sig- 20 d posttransection. The relative levels of POand MBP tran- nals in normal and transected nerves were thereby internally scripts present in each of these RNA preparations were then controlled. The general distribution of PO and MBP mRNAs determined by Northern and slot blot hybridizations. Slot blots was analyzed in dark-field images(Figs. 2 and 4); more precise were performed using 3 different amounts of RNA in order to localization of thesemRNAs wasobtained in bright-field images quantitate changesin myelin transcript levels accurately. The photographed at higher magnification (Figs. 3 and 5). results of thesestudies (Fig. 1) indicate that transection causes POmessage was readily detected in control nerves at all ages a dramatic reduction in the steady-statelevel of PO and MBP studied (Fig. 2). Silver grains resulting from cDNA-mRNA hy- RNAs: These transcripts were eachreduced approximately 40- bridization appearedin clusters that were distributed through- fold by 5 d posttransection. Although the kinetics of this re- out these sections. The general distribution of P, mRNA in duction were similar for the 2 genes,MBP messagelevels fell sectionsof 2 d distal stumps wassimilar to that found in control slightly faster than those for PO. Both the P, and MBP RNAs nerves. In sectionsfrom 5 d distal stumps,however, POmessage are reduced by 1 d posttransection, with maximal reduction clusterswere rare and lessintense than those found in control observed by 5 d after the operation. Both messagesalso showed nerves. These messageclusters were not detected in 10 and 20 a very slight but consistent increasebetween 5 and 20 d post- d distal stumps (Fig. 2). transection. Analysis of Northern blot hybridizations indicated When analyzed under bright-field optics, silver grains rep- no changein the size of PO or MBP transcripts following tran- resenting PO messagewere concentrated around Schwann cell section (data not shown). nuclei in sectionsfrom control nerves(Fig. 3). The hybridization apparent in dark-field imagesof 2 d transected nerves wasalso In situ hybridization locatedaround Schwanncell nuclei. Comparedwith age-matched In order to assessaxonal effects on glial gene expressionat the control nerves, the number of silver grains in the 2 d transected cellular level, we used in situ hybridization to examine the dis- nerves was significantly reduced. Only occasionalnuclei were 3518 Trapp et al. l Axons Regulate Myelin mRNA Levels

Figure 4. Comparison of the distribution of MBP mRNA in longitudinally oriented paraffin sections of age-matched control (A, C, E) and 2 (B), 5 (D), and 20 d Q transected rat sciatic nerves. MBP mRNA was distributed diffusely throughout all control sections and was localized in small clusters 2 d after transection. MBP message was not detected in sections from 5 and 20 d transected nerves. Dark-field images. Scale bar, 75 pm.

surrounded by concentrations of silver grains in sectionsfrom 5 d transected nerves. The number of these grains was dra- Immunocytochemistry matically reduced compared with age-matchedcontrols, con- Myelin sheaths in control nerves from all time points were sistent with the data we obtained from slot blot hybridizations. stainedintensely by POantiserum. The distribution of POprotein The distribution of Schwanncell nuclei in 2 and 5 d transected in sectionsof 2 d distal stumps was similar to the distribution nerves did not differ significantly from controls, although, as found in control nerves. However, in sections of 5 d distal expected, a significant increasein the density of nuclei was ob- stumps, PO antiserum stained discontinuous ovoids, which are served in 10 and 20 d transected nerves. While silver grains a hallmark of the early stagesof Wallerian degeneration. The were diffusely scattered throughout these 10 and 20 d sections size and number of thesePO-positive ovoids were decreasedin at a low level, concentrations of grains were never observed sectionsof 10 d distal stumps. Since our Northern and slot blot around cell nuclei. analysisshowed a small increasein PO and MBP mRNA levels MBP mRNA was also readily detected in sectionsof control between 5 and 20 d posttransection, PO-stainedsections of 20 nerves at all agesstudied (Fig. 4). In contrast to PO message, d distal stumps were examined for evidence of remyelination however, MBP mRNA was distributed more diffusely over my- (Fig. 6). Although PO immunoreactivity was present in these elinated fibers. This diffuse distribution was reduced by 2 d sections, it was restricted to myelin debris that had not been posttransection: occasional,randomly distributed foci of MBP removed. No evidence of remyelination was found. hybridization were observed. MBP messagewas not detectable in dark-field imagesof sectionsfrom 5, 10, and 20 d transected Discussion nerves. When analyzed with bright-field optics, the majority of Schwann cells exhibit remarkable phenotypic plasticity, which silver grains correspondingto MBP mRNA were diffusely scat- is largely controlled through their associationwith various types tered throughout myelinated fibers, although occasionalclusters of axons (Aguayo et al., 1976; Spenceret al., 1979; Mirsky et of grains were apparent. These clusterswere smaller and much al., 1980; Politis et al., 1982). The primary metabolic level at less intense than those found in sections hybridized with PO which this control is exercised, however, has not been firmly probes and were not consistently associatedwith Schwann cell ertablished. Recent transection studies, performed with adult nuclei (Fig. 5). Few silver grains were observed over sections nerves in which myelination is essentiallycomplete, have been from 5, 10, and 20 d transected nerves. taken to suggestthat axonal influences may be mediamd at The Journal of Neuroscience, September 1988, 8(9) 3519

Figure 5. Bright-field analysis of sections in Figure 4. Silver grains representing MBP message are distributed diffusely in control sections (A. C, E). Small clusters of silver grains,not alwaysassociated with cell nuclei(arrowheads) werealso present in control and 2 d transectednerves (B). Fewsilver grainswere present at 5 (0) and 20 (0 d after transection.Scale bar, 30 pm.

transcriptional, posttranscriptional, translational, and/or pro- tion, for example, lose POmRNA expressionover a time course tein processinglevels (Poduslo et al., 1985; Brunden and Po- similar to that of cells located more distally. duslo, 1987; LeBlanc et al., 1987). Our data clearly indicate that MBP mRNA was distributed diffusely over myelinated fibers during active myelination, axons regulate Schwann cell phe- in control sections. This diffuse distribution reflects the trans- notype primarily at the level of mRNA expression.For reasons location of MBP mRNA from Schwanncell perinuclear regions discussedbelow, this regulation is likely to be mediateddirectly to cytoplasmic domains along the myelin internode and is con- through changesin gene transcription. sistent with translation of this messagenear the sites of MBP Our quantitative slot blot analyses(Fig. 1) demonstrate that insertion into compact myelin (Trapp et al., 1987). This distri- P, and MBP mRNA levels in distal stumps are reduced by bution is markedly different from the perinuclear localization similar degreesat 1 d (3-fold), 2 d (9-fold), and 5 d (40-fold) of PO mRNA. This difference almost certainly reflects the dif- after nerve transection. These data indicate that major myelin ferent translational modesused for these2 messages:PO mRNA messagelevels are reduced by axon withdrawal, just asthey are encodesan integral membrane protein that is translated on induced during postnatal development (Lemke and Axel, 1985; membrane-bound polysomes (rough ER) localized near the Lemke, 1986). Our in situ hybridization studies confirm the Schwann cell nucleus (Trapp et al., 1987), while MBP mRNA extent and time course of the reduction of these mRNAs and encodesa soluble,extrinsic membraneprotein that is translated provide additional information about their cellular and subcel- on free ribosomes(Colman et al., 1982). lular distribution. For example, although reduced by 90%, the The distribution of MBP mRNA in 2 d distal stumpsdiffered general distribution of P, messagein sectionsfrom 2 d distal from control nerves in that silver grains often appearedin clus- stumps did not differ significantly from control nerves: The ters that were evenly distributed along the length of the distal number of Schwanncells expressingdetectable levels of mRNA stump. This hybridization pattern is suggestiveof a general was similar, although the number of silver grains surrounding down-regulation of MBP messagelevels as well as altered MBP individual cells was substantially reduced. Theseresults suggest mRNA transport along each myelin internode. In contrast to that P, messagelevels are down-regulated generally in all my- P, mRNA, the MBP messageclusters in 2 d transected nerves elin-forming Schwanncells, rather than differentially in a select often had no apparent associationwith Schwann cell nuclei. subpopulation. Those cells situated close to the site of transec- Significant levels of MBP mRNA could not be detected by in 3520 Trapp et al. l Axons Regulate Myelin mRNA Levels

potential mediator of axonal influences on myelin gene expression in vivo. Recent studiesexamining the effectsof permanenttransection of adult sciatic nerves on Schwann expression of P, protein have been reported to result in a modest (3-fold) decreasein P, mRNA levels (LeBlanc et al., 1987), an alteration of the glycosylation pattern of the P, protein (Poduslo, 1985), and an augmentation of the rate at which this protein is delivered to lysosomes(Brunden and Poduslo, 1987). Thus, these studies have been alternatively interpreted as indicating that axonal effects can be mediated at transcriptional, posttranscriptional, protein processing,and protein sorting steps. Which of these regulatory control points is paramount during normal Schwann Figure 6. Paraffin section of 20 d transected nerve oriented longitu- cell differentiation has proven difficult to assess,largely because dinally and immunostained with PO antibodies. Detectable P, protein in the adult, Schwann cell differentiation and myelination are is restricted to myelin debris (arrowheads). No evidence of remyelina- essentiallycomplete and the major myelin RNAs have conse- tion was found. Scale bar, 40 grn. quently fallen to low relative levels (Lemke and Axel, 1985; LeBlanc et al., 1987). situ hybridization to tissue sectionsof 5 d distal stumps, al- In contrast, the studiesdescribed in this report involve anal- though small clusters of P, messagesurrounding an occasional ysis of axonal regulation in the sciatic nerves of 35-d-old rats, Schwann cell nucleus were still observed (Figs. 2-5). Our in- a time at which the rate of myelin deposition is just below the ability to detect MBP messageby in situ hybridization to these peak levels observed around postnatal day 2 1. Similarly, the in sectionsprobably resultsfrom both the lower absolute concen- vitro studiesof Lemke and Chao (1988) involve analysisof cells tration of this mRNA (relative to PO)and its diffuse distribution dissociated from 2-3 d sciatic nerve, a time at which the rate within Schwann cells. The density of Schwann cells increased of myelin formation is also very high. The congruence of the dramatically between 5 and 20 d posttransection. Although this data obtained from these 2 independent approaches strongly increasein cell density was accompaniedby a slight increasein suggeststhat axons regulate Schwann differentiation primarily major myelin messagelevels as determined by slot blot hybrid- at the level of gene expression,probably through direct mod- ization (Fig. l), neither POnor MBP mRNA was detected by in ulation of transcription. It should be emphasizedthat this mod- situ hybridization to sectionsfrom 10 and 20 d nerves. These ulation is not always positive, however. Nerve growth factor observations support the hypothesis that in 10 and 20 d distal receptors (Taniuchi et al., 1986) and NGF receptor mRNA stumpsmajor myelin mRNAs are present at low concentrations (Lemke and Chao, 1988) have recently been shown to increase in many cells rather than at high concentrations in restricted dramatically upon withdrawal of axons, a finding that indicates populations. that axons are capableof both positive and negative regulation Taken together, our data clearly demonstrate that during a of Schwann cell gene expression. The availability of cloned period of active myelination, axonal regulation of Schwanncell probes for the major myelin and NGF receptor genesshould phenotype is primarily effected through modulation of Schwann now allow us to investigate this regulatory capability directly. cell mRNA levels. This hypothesis is supported by a seriesof experiments recently performed with purified neonatalSchwann References cells cultured in vitro in the absenceof axons. When dissociated Aguayo, A. J., L. Charron, and G. M. Bray (1976) Potential of Schwann from the neonatal nerve at a time of rapid myelination and cells from unmyelinated nerves to produce myelin: A quantitative ultrastructural and autoradiographic study. J. Neurocytol. 5: 565- placed into culture, thesecells ceasemyelin synthesisand mark- 573. edly down-regulate expression of the major myelin proteins Bray, G. M., M. Rasminsky, and A. J. Aguayo (1981) Interactions (Mirsky et al., 1980). Lemke and Chao (1988) have observed between axons and their sheath cells. Annu. Rev. Neurosci. 4: 127- that this “dedifferentiation” is accompaniedby a corresponding 162. 40-fold down-regulation in the level of PO and MBP mRNAs Brockes, J. P., M. C. Raff, D. J. Nishiguchi, and J. Winter (1979) Studies on cultured rat Schwann cells. III. Assays for peripheral my- over 5 d in culture. The magnitude and time course of this elin proteins. J. Neurocytol. 9: 67-77. reduction are in remarkably good agreementwith the in vivo Brunden, K. R., and J. F. Poduslo (1987) Lysosomal delivery of the data describedabove. That thesechanges directly reflect mod- major myelin glycoprotein in the absence of myelin assembly. J. Cell ulation of myelin gene transcription can be inferred from 2 Biol. 104: 661-669. Cathala, G., J.-F. Savouret, B. Mendez, B. L. West, M. Karin, J. A. additional observations. Lemke and Chao (1988) have shown Martial, and J. D. Baxter (1983) A method for isolation of intact, that the low levels of POand MBP mRNAs expressedby Schwann translationally active ribonucleic acid. DNA 2: 329-335. cells cultured in the absenceof axons can be markedly elevated Colman, D. R., G. Kreibich, A. B. Frey, and D. D. Sabatini (1982) by forskolin-induced increasesin intracellular cyclic AMP. This Synthesis and incorporation of myelin polypeptides into CNS myelin. second messengerhas, in turn, been demonstrated to strongly J. Cell Biol. 95: 598-608. LeBlanc, A. C., J. F. Poduslo, and C. Mezei (1987) Gene expression potentiate the transcriptional activity of the cloned promoter in the presence or absence of myelin assembly. Mol. Brain Res. 2: region of the rat PO gene: This promoter, when linked to a 57-67. heterologous reporter enzyme and transfected into cultured Lemke, G. (1986) Molecular biology of the major myelin genes. Trends Schwann cells, is active only when thesecells are grown in the Neurosci. 9: 266-270. Lemke, G., and R. Axe1 (1985) Isolation and sequence of a cDNA presenceof an agent that elevates intracellular CAMP (Lemke encoding the major structural protein of peripheral myelin. Cell 40: et al., 1988). Cyclic AMP, therefore, should be consideredas a 501-508. The Journal of Neuroscience, September 1988, 8(9) 3521

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