CNS interfaces

www.elsevier.com/locate/ymcne Mol. Cell. Neurosci. 28 (2005) 18 – 29

The role of proteoglycans in Schwann cell/astrocyte interactions and in regeneration failure at PNS/CNS interfaces Barbara Grimpe,a Yelena Pressman,b Michael David Lupa,a Kevin Paul Horn,a Mary Bartlett Bunge,b and Jerry Silver a,* a

Department of Neurosciences, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, United States The Miami Project to Cure Paralysis, School of Medicine, University of Miami, Miami, FL 33136, United States

b

Received 13 January 2004; revised 15 June 2004; accepted 15 June 2004 Available online 17 August 2004

In the dorsal root entry zone (DREZ) peripheral sensory axons fail to regenerate past the peripheral nervous system/central nervous system (PNS/CNS) interface. Additionally, in the spinal cord, central fibers that regenerate into Schwann cell (SC) bridges can enter but do not exit at the distal Schwann cell/astrocyte (AC) boundary. At both interfaces where limited mixing of the two cell types occurs, one can observe an up-regulation of inhibitory chondroitin sulfate proteoglycans (CSPGs). We treated confrontation Schwann cell/astrocyte cultures with the following: (1) a deoxyribonucleic acid (DNA) enzyme against the glycosaminoglycan (GAG)-chain-initiating enzyme, xylosyltransferase-1 (XT-1), (2) a control DNA enzyme, and (3) chondroitinase ABC (Ch’ase ABC) to degrade the GAG chains. Both techniques for reducing CSPGs allowed Schwann cells to penetrate deeply into the territory of the astrocytes. After adding sensory neurons to the assay, the axons showed different growth behaviors depending upon the glial cell type that they first encountered during regeneration. Our results help to explain why regeneration fails at PNS/CNS glial boundaries. D 2004 Elsevier Inc. All rights reserved.

Introduction The site of peripheral sensory axon entry into the central nervous system (CNS), the dorsal root entry zone (DREZ), is a unique region where centrally directed regeneration of severed fibers in transit between the peripheral nervous system (PNS) and Abbreviations: AC, astrocyte; Ch’ase ABC, chondroitinase ABC; CNS, central nervous system; CSPG, chondroitin sulfate proteoglycan; DNA, deoxyribonucleic acid; DREZ, dorsal root entry zone; DRG, dorsal root ganglion; ECM, extracellular matrix; GAG, glycosaminoglycan; IgG, immunoglobulin G; PNS, peripheral nervous system; SC, Schwann cell; XT-1, xylosyltransferase-1. * Corresponding author. Department of Neurosciences, School of Medicine, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106. Fax: +1 216 368 4650. E-mail address: [email protected] (J. Silver). Available online on ScienceDirect (www.sciencedirect.com). 1044-7431/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.mcn.2004.06.010

CNS abruptly ceases (Cajal, 1928; Carlstedt, 1985, 1997; Chong et al., 1999; Kliot et al., 1990; Perkins et al., 1980; Reier et al., 1983; Stensaas et al., 1987; Siegal et al., 1990). Whereas the rate of regeneration of afferent fibers in the dorsal root is more sluggish than that of their counterparts in the periphery, the growth response to axotomy of the central process can be augmented by a so-called conditioning injury to the peripheral axon (Chong et al., 1994, 1996; Lu and Richardson, 1991; Lund et al., 2002; Pan et al., 2003; Richardson and Issa, 1984; Richardson and Verge, 1987). Even following peripheral conditioning, however, the number of fibers in the root that manage to regrow past the DREZ is still relatively minimal (Chong et al., 1999). Indeed, the extent of sensory fiber regeneration past the DREZ following a conditioning lesion is less than that which has been documented to occur following conditioning within the territory of the forming glial scar in the dorsal columns (Chong et al., 1994, 1999; Neumann and Woolf, 1999). Thus, the DREZ is a remarkably impenetrable barrier to the passage of axons, at least under the usual circumstances that transpire following root injury. A situation similar to that at the DREZ exists at the distal junction of regeneration-promoting peripheral nerve or Schwann cell (SC) bridges inserted into the parenchyma of the CNS. Here, regeneration is again severely limited as the axons attempt to pass from the peripheral milieu into the central compartment (Perkins et al., 1980; Plant et al., 2001). What are the cellular interactions and molecular mechanisms at these entrances to the CNS that help to create such refractory boundaries to the movement of regrowing axons? At the DREZ and at the ends of PNS conduits, glial ensheathment of axons changes from that provided by Schwann cells to astrocytes (ACs) and oligodendrocytes. The astrocytes, in particular, are believed to play a leading role in the creation of a stop mechanism to axon elongation (Berthold and Carlstedt, 1977a,b; Liuzzi and Lasek, 1987; Pindzola et al., 1993; Zhang et al., 2001). Thus, following dorsal root injury, astrocytes become reactive and extend long processes, unlike stationery oligodendrocytes, into Schwann cell territory (Nomura et al., 2002; Siegal et al., 1990). This cellular organization ensures that reactive astrocytic processes are the first CNS elements that are encountered by regenerating axons and it is

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precisely at this point where regenerating peripheral axons abort their growth or turn away. Regeneration failure occurs in the same vicinity at the ends of bridging peripheral nerve or Schwann cell grafts (Bignami et al., 1984; Chau et al., 2004; Golding et al., 1999; Kozlova et al., 1995; Liuzzi and Lasek, 1987; Siegal et al., 1990; Xu et al., 1997; Zhang et al., 2001). At the boundary created by such contact interactions between Schwann cells and astroglia, there occurs a rapid and intense up regulation of an extracellular matrix rich in chondroitin sulfate proteoglycans (CSPGs) and other molecules that are believed to play a role in segregating the two glial populations (Ghirnikar and Eng, 1995; Lakatos et al., 2000; Lal et al., 1996; Plant et al., 2001; Wilby et al., 1999). CSPG also appears in the DREZ in vivo during normal development and up-regulates following injuries to the roots or DREZ that are severe enough to open the blood brain barrier (Fitch et al., 1999; Pindzola et al., 1993; Zhang et al., 2000). It is well established that such PG-rich matrices can negatively regulate the outgrowth of regenerating axons (McKeon et al., 1995; Snow et al., 1990, 2002), and it has been suggested that the inhibitory nature of the PG component of the extracellular matrix (ECM) plays a major role in repelling regenerating sensory axons away from the DREZ or disallowing regeneration past the ends of PNS milieu containing scaffolds (Plant et al., 2001). In the present in vitro study, we have analyzed the role of this inhibitory component of the ECM not only as part of the mechanism that disallows regrowth of axons between Schwann cell and astroglial territories, but also as part of the mechanism that might generate the partitioning of CNS/PNS glia in the first place (Lakatos et al., 2000; Lal et al., 1996). We have found that although CSPG digestion or reduction does, in fact, lead to Schwann cell invasion of astroglial territory in our in vitro model (the reverse migration is not as robust), regenerating axons that initiate their growth on Schwann cells, unlike those initiated on astrocytes, tend to remain in close association with the PNS glial surface even when inhibitory CSPGs in the ECM are diminished and the two glial cell types are highly intermingled.

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weeks in vitro (Ghirnikar and Eng, 1995; Lakatos et al., 2000). Instead, they congregate into relatively discrete territories composed of one cell type or the other. At the interface between the CNS and PNS glia, there is an up-regulation of CSPGs (Ghirnikar and Eng, 1995; Lakatos et al., 2000, also see Figs. 2C and D). Some limited cellular intermixing can occur (as it does in vivo), but this is typically manifested as small, isolated fingers of either glial cell type inserted up to a few cell bodies in length into the territory of the other. Is the increased production of PG-enriched ECM at the PNS/CNS glial cell interface involved mechanistically in the cell segregation process in vitro? Two methods of altering CSPGs were utilized to answer this question. The first was the administration of chondroitinase ABC, which digests the N-acetyl glucosamine and glucouronic acid units, the GAG chains of PG. However, some undigested carbohydrate residues, called bstubs,Q remain on the protein core (Sorrell et al., 1990). To confirm the efficacy of chondroitinase ABC in digesting CSPGs in our glial cultures, we used one of the bstubQ antibodies (2B6); positive staining demonstrates that chondroitinase ABC enzyme digestion has occurred (Figs. 1A and B). The second method of reducing GAG chains on PGs is to interfere with their synthesis by the administration of a deoxyribonucleic acid (DNA) enzyme. DNA enzymes are taken up into cells within seconds (Grimpe, 2004). Once inside the cell, they bind to and cut the mRNA of the target protein, xylosyltransferase-1 (XT-1), thereby reducing translation to the final protein (Grimpe and Silver, 2004). XT-1 initiates GAG chain elongation and synthesis of CSPGs in the ER or Golgi compartment (Hoffmann et al., 1984). To investigate the functionality of our DNA enzyme, we examined enzyme uptake into the cells and PG reduction in our glial cultures. With the use of a biotin-labeled DNA enzyme, we were able to monitor uptake and localization in the cytoplasm of astrocytes and Schwann cells (Fig. 2A). The control DNA enzyme-treated cultures as well as untreated confrontation assay cultures show the same high level of staining (Figs. 2C and D). In DNA enzyme-treated confrontation assays, there is a strong down-regulation of the GAG chains in most cells (Fig. 2B).

Results Glycosaminoglycan (GAG) chains are diminished by chondroitinase ABC (Ch’ase ABC) or DNA enzyme against XT-1 shown by immunostaining In confrontation culture assays, astrocytes of the CNS and Schwann cells of the PNS do not readily intermingle, even up to 2

GAG chains are reduced by DNA enzyme treatment as shown by dot blot analysis To investigate the amount of reduction in GAG chains on CSPGs after DNA enzyme or control DNA enzyme treatment, we loaded a defined aliquot of total protein from each group on a dot blot (Fig. 3A). Additionally, as a positive control, we used the

Fig. 1. Confrontation assays treated with chondroitinase ABC and immunostained with an antibody (2B6) for the bstubQ antigen. Panel A shows this staining associated with astrocytes; Panel B with Schwann cells. Scale bars = 10 Am.

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Fig. 2. (A) Both astrocytes (AC + arrows) and Schwann cells (SC + arrow) accumulate labeled DNA enzyme in their cytoplasm. In confrontation assays after (B) DNA enzyme treatment against the XT-1, a marked reduction in GAG chain staining was observed compared to (C) control DNA enzyme-treated or (D) untreated cultures. Scale bars = 10 Am.

same protein amount from JAR cells, which are known to have a high expression rate of XT (Kuhn et al., 2001), as well as the dot blot buffer as a background control. To quantify our result, we did a triple dot blot for each treated or untreated group. The absorbance unit and standard deviation of these triple dot blots are shown below each dot. After staining the nitrocellulose

membrane with the CS-56 antibody, we continued the staining process with an HRP-conjugated secondary antibody. After ECL development, the dot of the DNA enzyme-treated confrontation assays (lane 1), compared with the various control lanes (lanes 2, 3, and 4), shows a strong reduction in GAG chain staining. In order to document that equal amounts of total protein were loaded onto each spot, the nitrocellulose membranes were stripped and restained with GFAP antibody. The JAR cells as well as the dot blot buffer showed no GFAP staining. However, the other three dots contained equal amounts of GFAP protein (Fig. 3B). The results indicate that the DNA enzyme against XT-1 specifically reduces GAG chain incorporation into CSPG core proteins, substantiating the immunostaining data. Schwann cells enter astrocyte regions following GAG chain reduction

Fig. 3. Dot blots of confrontation assays 13 days in culture (DNA enzyme, control DNA enzyme, untreated, and JAR cells). (A) Dots after staining for GAG chains of PGs with the CS-56 antibody. Below each dot are the evaluations of the absorption units and standard deviations of the triple experiment for each treatment group. The dot blot buffer is an internal control for background staining. The dot blot shows clearly that the DNA enzyme against XT-1 specifically reduced GAG chain incorporation into CSPG core proteins. (B) The nitrocellulose membrane was stripped and restained with an antibody against GFAP to verify that the same amount of protein was used in each dot.

Chondroitinase ABC treatment After 13 days of no treatment or chondroitinase ABC treatment in confrontation assays, sufficient time had passed to allow the two glial cell types to approach one another along a significant length of their interface. The untreated controls, however, developed only short distance overlapping of the two glial types (Fig. 4B). Only very rarely in control cultures does a Schwann cell come to land well within the astroglial territory. In chondroitinase ABC-treated Schwann cell/astrocyte confrontation cultures, Schwann cells penetrate much more deeply into the territory of the astrocytes (Fig. 4A). These differences in cell mixing are presented in Figs. 4A, AV, B, and BV where pseudocoloring helps to differentiate the two cell types. The chondroitinase ABC-treated coverslips show a marked reduction in CS-56 staining, which further confirmed that GAG chain digestion had occurred (data not shown).

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Fig. 4. Thirteen-day confrontation assays. (A) Photograph and (AV) pseudo-color image of chondroitinase ABC-treated cultures. Note that the Schwann cells (green) grow into the astrocyte layer (red). (AU) Three photographs from one coverslip, which was used to quantify our observation, shows the longitudinal interface of such treated astrocytes and Schwann cells in the confrontation assay. (B) Photograph and (BV) pseudo-color image of untreated cultures, showing that Schwann cells do not intermix with astrocytes, in contrast to A, AV, and AU. (BU) The longitudinal interface from one coverslip, which was used to quantify our observation. (C) Photograph and (CV) pseudo-color image of DNA enzyme-treated cultures. (CW) Three photographs of the interface of the astrocyte/ Schwann cell confrontation assay after treatment with the DNA enzyme to the XT-1. The Schwann cells deeply penetrate the astrocyte layer after GAG chain reduction. (D) Photograph and (DV) pseudo-color image of control DNA enzyme-treated cultures show the same unmixed phenotype as in untreated cultures (B and BV). (DU) Three photographs from one coverslip, which was used to quantify our observation and which shows the longitudinal interface of the confrontation assay. Scale bars = 100 Am.

DNA enzyme XT-1 treatment To investigate whether the use of a newly designed DNA enzyme against XT-1 increases intermixing between Schwann cells and astrocytes in the confrontation assay, we treated the cultures with the enzyme beginning at 2 h after plating and for 13 additional days. We observed many Schwann cells in the astrocyte layer (Fig. 4C). Indeed, many Schwann cells move completely through and emerge beyond the far side of the astroglial cell lane. The control DNA enzyme-treated cultures show an unmixed phenotype identical to untreated control cultures (Fig. 4D). Again pseudo-colors were used to demonstrate the effect more clearly (Figs. 4CVand DV).

Quantification Because Schwann cells move robustly into astrocyte areas but not vice versa, for each coverslip we quantified five evenly spaced strips for the presence of Schwann cells within the territory of the astrocytes. This was done using color-specific pixel intensity of the immunostained cells in three to four treated or control coverslips. For analysis and statistical evaluation by Tina 2.09, each strip was divided into two equal areas (each 3  20 mm), one far and one near to the initial astrocyte/Schwann cell interface (Fig. 6). Quantitative data are presented for confrontation cultures that were treated with chondroitinase ABC, DNA enzyme against XT-1, control

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Table 1 Pixel intensity analysis of Schwann cells in the astrocyte layer of confrontation assays

DNA enzyme, as well as those that were untreated (Table 1). The data in Table 1 confirm our observations that the degradation of CS-GAG chains by chondroitinase ABC or their reduction by the DNA enzyme against XT-1 leads to a striking intermixing of Schwann cells with astrocytes in the confrontation assay. Regeneration of adult dorsal root ganglion (DRG) axons in the confrontation assay of astrocytes and Schwann cells To investigate if the deep integration of Schwann cells into the astrocyte layer by treatment with chondroitinase ABC or DNA enzyme against XT-1 enables adult primary sensory neurons to regenerate across Schwann cell/astrocyte interfaces, we applied approximately 200–300 DRG neurons on either side of the culture midline for 1 day. The DRG neurons were compelled to land on either largely astrocyte or Schwann cell territories but close to the

initial interface so that rapidly growing axons could have access to both cell types. DRG growth beginning on Schwann cells In the confrontation assay, robust glial cell mixing occurs following treatment with either chondroitinase ABC or DNA enzyme against XT-1 as reported above. Nonetheless, when starting from a Schwann cell, the majority (92.3%) of regenerating DRG axons in DNA enzyme-treated cultures remain closely associated with Schwann cell surfaces (Table 2), even when Schwann cells and their associated axons penetrate deeply into and sometimes cross directly over astrocytes. A relatively small number of axons (7.7%), having first begun growth on a Schwann cell, change between the two glial cell types and none of the axons continued on astrocytes. In chondroitinase ABC-treated confrontation assays, more axons could escape from the Schwann cell, although they remain largely faithful (76%) to this cell type. Axons are even capable of following thin Schwann cell processes around tight curves as they travel over astrocyte surfaces (Tables 2 and Figs. 5A and AV). In the z-section of Fig. 5AV, one can observe that the axons (blue) were positioned adjacent to both types of glial cell surfaces at various places along their journey. Although the axons appear capable of accessing both types of glia, just 8% of the axons change allegiance repeatedly between the two glial cell types when growth begins on Schwann cells and 16% of the axons abandon Schwann cells to grow only along astrocytes. In the control DNA enzyme-treated confrontation assays, again, the majority of Schwann-cell-initiated DRG axons (87.1%) regrow only on Schwann cells (Table 2), and a minor number (7.7%) alternate between the two cell types. The lowest percentages (5.2%) of the axons that start growing on a Schwann cell continue their growth only on astrocytes. The untreated confrontation assays show the same tendencies as in the three treatment groups (Table 2). In summary, chondroitinase ABC digestion somewhat increases the number of axons that transit between Schwann cell and astrocyte surfaces. DNA enzyme treatment failed to engender axonal regeneration onto astrocytes when regrowth is initiated on PNS glia. DRG growth beginning on astrocytes The substrate fidelity of regenerating DRG axons observed when the cell body initiates regeneration on the surfaces of Schwann cells changes when the DRG are plated first on astrocytes. Indeed, when DRG are plated on the astrocyte side in untreated control confrontation cultures, only about half of the axons (53.3%) that initiate on astrocytes and then grow among Schwann cells remain faithful to the astrocytes. Having initiated growth on astrocytes, many (37.8%) regenerating axons that come into contact with Schwann cells grow onto but then leave the Schwann cell surface

Table 2 Interaction of DRG with Schwann cells or astrocytes in the confrontation assay

DNA XT-1 Ch’ase ABC DNA control Untreated DNA XT-1 Ch’ase ABC DNA control Untreated

Cells plated on

Axons on both (%)

Axons on SCs only (%)

Axons on ACs only (%)

Total no. of coverslips

Schwann cells Schwann cells Schwann cells Schwann cells Astrocytes Astrocytes Astrocytes Astrocytes

7.7 8.0 7.7 14.5 28.9 42.3 61.7 37.8

92.3 76.0 87.1 81.8 1.2 2.6 2.5 8.9

0.0 16.0 5.2 3.7 69.9 55.1 35.8 53.3

2 3 4 5 5 7 5 4

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Fig. 5. Treated confrontation assays. (A) In chondroitinase ABC-treated cultures, an axon (blue, and follow the arrows) initiating from a soma that came to lie on a Schwann cell (green) remains with the Schwann cell process (green) even though the Schwann cell crosses over several astrocytes (red). For more clarity, each single-colored channel is shown in separate pictures [AV blue, DRG (neurofilament); AU green, Schwann cells (p75); Aj red, astrocytes (GFAP)]. (A) Picture A in a z-section that shows that the axon (blue) contacts both astrocyte (red, right asterisk) and Schwann cell (green, left asterisk) surfaces. (B) A DNA enzyme-treated culture. In this example, the DRG landed on and initiated its axons on the astrocyte layer (red) first. The axons (blue) of this cell crossed between astrocytes (red) and Schwann cells (green) and back again (follow the arrows). Again, for more clarity, each single-colored channel is shown in separate pictures [BVblue, DRG (neurofilament); BU green, Schwann cells (p75); Bj red, astrocytes (GFAP)]. (C) In a control DNA enzyme-treated culture, the DRG cell body (blue) is positioned on an astrocyte. Its axons (blue) grow first on astrocytes (red), but then switch to a Schwann cell (green) and then back to astrocytes (follow the arrows). For more clarity, each single-colored channel is shown in separate pictures [CVblue, DRG (neurofilament); CW green, Schwann cells (p75); Cj red, astrocytes (GFAP)]. Scale bars = 20 Am.

once again. A minority of DRG axons (8.9%) that begin regrowth on an astrocyte, but then stray onto a Schwann cell, continue growing on the Schwann cell surface (Table 2). Control DNA enzyme-treated confrontation assays plated with DRG on the astrocyte side result in

similar observations. Thus, at interfaces where PNS and CNS glia are partially interlaced (i.e., compared to XT-1 modified cultures), 35.8% stay only on the astrocytes whereas 61.7% of astrocyteinitiated axons grow on both cell types. A few DRG axons that first

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extend from a DRG lying on top of an astrocyte but then grow towards a Schwann cell continue elongation only on the Schwann cell (2.5%, Table 2 and Fig. 5C). Neither chondroitinase ABC nor the DNA enzyme against XT1 markedly changes the tendency of the DRG initiating their regeneration on astrocytes to be relatively flexible in their preference for growth. Thus, in both cases, 69.9% (DNA XT-1; Table 2 and Fig. 5B) or 55.1% (chondroitinase ABC; Table 2) of the axons remain on astrocytes after plating on astrocytes. However, 28.9% (DNA XT-1) or 42.3% (chondroitinase ABC) of the axons actually grow freely between astrocytes and Schwann cells. Again, the minority of the axons that started on astrocytes continue growth only on Schwann cells (DNAas XT-1, 1.2%, chondroitinase ABC 2.5%). Finally, we calculated the statistical relevance of the results in Table 2. The evaluation shows that in all of our treatment groups (DNA XT-1, chondroitinase ABC, DNA control, untreated), the results are highly significant (P V 0.0001).

Discussion A few days after birth in rodents (Carlstedt, 1985), changes occur in the glial organization of the DREZ that, following injury, are thought to play a critical role in disallowing reentry of regenerating primary sensory axons into the spinal cord. The extension of astrocyte processes into the dorsal roots is not only thought to confer tensile strength to the damaged DREZ (Livesey and Fraher, 1992), but also increases the surface area of direct contact between Schwann cells and astrocytes, generating a unique blend of CNS and PNS glial environments that are lacking in oligodendrocytes and therefore their purported axon growth inhibitors (Chen et al., 2000; Qiu et al., 2002; Wang et al., 2002). It has been proposed that reactive astrocyte ECM may play a pivotal role in regeneration failure at the DREZ (Pindzola et al., 1993). Importantly, it has been recently reported that chondroitinase ABC degradation of CSPGs in vivo at the ends of peripheral nerve bridges (Coomes and Houle, 2002; Mayes and Houle, 2003), as well as Schwann cell filled guidance channels (Chau et al., 2004), lead to enhanced glial cell mixing in the tube (Chau et al., 2004) and regeneration of a relatively small number (about 20%; Houle, personal communication) of axons into the spinal cord beyond the end of the Schwann cells within the bridge (Chau et al., 2004; Coomes and Houle, 2002). The results of our in vitro experiments with chondroitinase ABC digestion (Fig. 4), as well as the aforementioned in vivo experiments, suggest that whereas the increase in CSPGs at PNS–CNS glial interfaces does appear to play a role in creating a regeneration boundary, it is not the only potential mechanism that tends to restrict axon regeneration to the PNS compartment. One possibility is that the incompletely digested carbohydrate stubs of the PG core protein that remain following chondroitinase ABC treatment are inhibitory to axon outgrowth (Lemons et al., 2004). Another possibility is that after PG modulation, some other type of inhibitory molecule (e.g., Tenascin) remains at the interface between or upon the surfaces of the two glial cell types and influences axon substrate preferences depending on the collection of receptors that the growth cone produces for the inhibitor when in contact with one or the other glial cell type. While CSPG-mediated inhibition may not be the only factor in disallowing sensory axon entrance into the CNS, it does appear to

be a very important part of the mechanism that normally constrains migration of Schwann cells into astroglial territory, at least in vitro. There is a vast literature showing that high concentrations of naturally occurring as well as synthetic (Petersen et al., 1996) PGs positioned at interfaces between differing cell types can create a potent barrier to axonal regeneration as well as to cellular intermixing (Pindzola et al., 1993; Plant et al., 2001; Snow et al., 1990, 2002). In many instances, inhibition is conferred by the GAG component of the PG. The lack of intermixing between Schwann cells and astroglia across their own self-assembled PGrich interface is yet another example of this phenomenon. It is unclear why, after CSPG digestion (chondroitinase ABC) or GAG chain reduction (DNA enzyme to XT-1), the movement of Schwann cells was so far ranging whereas astrocytes remained relatively stationary. It may be that the substrate conditions in our in vitro model bind astroglia exceptionally tightly or that astroglia are relatively less motile intrinsically than are Schwann cells. Another possibility could be that the normal production of CSPGs by the Schwann cells is especially critical in keeping these glial cells together. When CSPGs are digested (via chondroitinase ABC) or inhibited in their expression (via DNA enzyme), the Schwann cells in particular may become more motile, which leads to their deep growth into the astrocyte layer. Also, preliminary BrdU experiments showed that there were no differences in the percentages of dividing cells in the treated versus the untreated cultures. Our results do suggest that the boundary forming function of CSPGs may be one component of the mechanism that keeps the robust migratory potential of peripheral glia in check and thereby disallows them from moving substantially past astroglia in the injured adult as well as during development (Golding et al., 1996, 1999). Additionally, our present findings suggest that some type of molecular baddictiveQ behavior between DRG and Schwann cells may also play a role in contributing to the regeneration refractory nature of the PNS/CNS interface. Although the precise mechanism that underlies the preference of adult axons for Schwann cells is unknown, it is likely that this phenomenon involves some form of receptor modulation (like that suggested above) that is also dependent upon the sequence of contact that occurs between mature neuron cell bodies or their axons and the type of glial cell that is seen by the regenerating fiber as it first emerges from the soma. Thus, it may be that the cohort of receptors that is induced when regenerating DRG encounter astrocytes first is relatively nonspecific or quickly malleable. In the situation, where sensory fibers fail to regenerate which occurs at the DREZ or the rostral end of Schwann cell bridges, the dominant receptor(s) may be fixed and/or highly specific for growth-promoting ligands or neurotrophins on Schwann cell surfaces. Their persistent activation could disallow movement of the growth cone off the Schwann cell membrane. Another facet of the baddictionQ phenomenon may involve some critical length of time that an axon spends on one or the other of the two glial environments that they encounter. Thus, it is possible that axons emerging from an astrocytic environment but then quickly switched to a Schwann cell environment for a long period of time may also undergo Schwann cell baddiction.Q The reverse situation (brief period of growth on Schwann cells but longer on astrocytes) may be able to break the axon’s preference for the Schwann cell surface. An underlying premise behind the addiction hypothesis and one with considerable experimental evidence is that the

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repertoire or concentrations of growth-promoting or neurotrophic (Eddlestone and Mucke, 1993) versus inhibitory molecules (Krekoski et al., 2002) made by Schwann cells and reactive astrocytes are quite different. What these critical differences may be whether the addiction phenomenon is peculiar to the DRG and precisely how these differences in substrate characteristics manifest their effects on the regenerating growth cone are now subjects of investigation. An explanation for the presence of P75+ cells in the astrocyte territory following DRG addition to the cultures is the possibility of contaminating satellite cells that closely resemble Schwann cells and might migrate from the DRG cell bodies into the astrocyte layer. These cells are also P75+, and thus they could be mistaken for Schwann cells. Given the robust capacity for Schwann cell or astrocyte mixing following CSPG alteration and the relatively small number of satellite glia in our DRG cultures, it is unlikely, but cannot be ruled out completely (without prelabeling the Schwann cells), that all P75+ cells were satellite glia. Nonetheless, the presence of contaminating satellite glia cannot explain the behavior of DRG axons when their cell bodies first come to lie upon one glial cell type or the other (Fig. 5 and Table 2). Transplanting Schwann cells or segments of pre-degenerated peripheral nerves in the form of bridges between severed ends of the spinal cord has been and continues to be a classic strategy for promoting axonal regeneration in the adult (Perkins et al., 1980; Richardson et al., 1980; Takami et al., 2002; Xu et al., 1997). Schwann cells are a prime cell type widely documented to promote axon regrowth from a variety of CNS neurons since they produce growth stimulating molecules such as neurotrophins, laminin, and L1 (Bunge and Kleitman, 1999; Jasmin et al., 2000; Taniuchi et al., 1988). Furthermore, they have the potential to remyelinate the axons that they guide (Lankford et al., 2002; Xu et al., 1997). Schwann cells also have the unfortunate tendency to disallow axon passage beyond their distal interface into the CNS. Nonetheless, even strong barriers to regeneration are the product of a balance between growth inhibitory versus stimulatory factors both intrinsic and extrinsic to the neuron (Chen et al., 2000; Dergham et al., 2002; McKeon et al., 1995; Neumann et al., 2002; Pindzola et al., 1993; Qiu et al., 2002; Wong et al., 2002). Regeneration failure at the DREZ or at experimentally created PNS/CNS interfaces is a clear example of this principle since vigorous, albeit short distance, regeneration centrally past the DREZ or past experimentally constructed PNS/CNS boundaries has been achieved with the use of highly growth supportive immature astroglial bridges (Kliot et al., 1990) or olfactory ensheathing glia (Ramon-Cueto and Nieto-Sampedro, 1994; Ramon-Cueto et al., 1998) or the use of potent neurotrophins produced in the dorsal horn (Chau et al., 2004; Menei et al., 1998; Oudega and Hagg, 1999; Ramer et al., 2002; Romero et al., 2001; Tuszynski et al., 2003). Once near the end of the PNS part of their journey, regenerating axons may be maximally enticed to leave the Schwann cell surface when a strategy of modifying the intrinsic growth potential of the axon (Benn et al., 2002; Borisoff et al., 2003; Cai et al., 2002; Chong et al., 1994; Tropea et al., 2003) is offered in combination with a means for decreasing inhibitors (Chau et al., 2004; Coomes and Houle, 2002) and simultaneously increasing neurotrophic support in the synaptic target (Bamber et al., 2001; Li and Raisman, 1997; Oudega et al., 2001; Plant et al., 2001; Stichel et al., 1996; Tropea et al., 2003).

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Experimental methods Preparation and culture of astrocytes, Schwann cells, DRG neurons, and JAR cells Astrocyte (AC) cultures were prepared from cerebral cortices of newborn (P0) Sprague–Dawley rats and cultured for 14 days in Dulbecco’s modified Eagle’s medium-F12 (DMEM, GibcoLife Sciences, Maryland) supplemented with 10% fetal calf serum (FCS, Gibco-Life Sciences) and 10 ml/l penicillin/ streptomycin on poly-l-lysine (PLL; 10 Ag/ml)-coated Nunc flasks at 378C, 95% humidity, and 5% CO2. The astrocytes used in the experiments have always been in culture for 14 days. The Schwann cells (SC, passage 2) were generated in the Miami Project from sciatic nerves of adult Fischer 344 rats (Harlan, Indianapolis, IN) as described elsewhere (Meijs et al., in press; Plant et al., 2001). After mincing, the explants were grown in DMEM/10% FCS and manipulated for 2 weeks to remove fibroblasts (Morrissey et al., 1991). Explants were dissociated and the cells plated in medium with three mitogens, forskolin (0.8 Ag/ml, Sigma, Missouri), heregulin (20 ng/ml, Genetech, California), and pituitary extract (2 mg/ml Invitrogen Corp., California). Growth to confluency and transfer to new dishes were performed twice. Their purity (95–98%) was determined (Takami et al., 2002) and then frozen for shipment. Schwann cells were thawed upon arrival and cultured again in the mitogencontaining medium, growth to confluency, and then frozen and thawed as needed to use in the assay after 14 days of incubation. Living Schwann cell cultures were not used beyond three further months. The confrontation assay experiments were similar in design to those described in Lakatos et al. (2000) with a few modifications. Coverslips were coated with PLL (0.2 mg/ml) and laminin (5Ag/ ml). Suspensions of each glial cell type were positioned on these coverslips by pipetting the cells in just one strip of each type of cell. Each strip (a total of two stripes per coverslip) were approximately 1.5 cm in length and 0.8 cm in width. At the time of plating, the culture medium was changed to DMEM/FCS/ penicillin/streptomycin without additional supplements. The strips contained 106 astrocytes or 106 Schwann cells. After 13 days, the cultures were fixed in 2% paraformaldehyde (PFA) phosphatebuffered saline (PBS, 1 mM KH2PO4, 10 mM Na2HPO4, 0.137 M NaCl, 2.7 mM KCl, pH 7.4). The adult dorsal root ganglia (DRG) were harvested from adult Sprague–Dawley rats using a technique described by Davies et al. (1999). On the 13th day of glial coculture, the DRG neurons were seeded at a concentration of 150 cells/Al. Plated on either the astrocyte or the Schwann cell side adjacent to the midline was1.5 Al. After 10 min to allow the cells to adhere, the cultures were moved to the incubator where they remained for 1 day. This was followed by fixing in 2% PFA for 30 min at 378C. JAR cells were obtained from Neal Rote (Metrohealth, Cleveland, OH) and cultivated in the recommended medium (RPMI 1640 Gibco, 10% FCS), 2 mM glutamine, 1.5 g/l NaHCO3, 4.5 g/l glucose, 10 mM Hepes, 1 mM Na-Pyruvate, and 10 ml/l penicillin/streptomycin. The JAR cell is a fetal placental choriocarcinoma cell line that serves as a positive control for several of the XT-1 experiments because these cells express high amounts of xylosyltransferase and PGs (Gotting et al., 2000).

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Design of the DNA enzyme and control DNA enzyme against Xylosyltransferase (XT)-1 The DNA enzyme (Grimpe and Silver, 2004) was designed as an end-capped phosphorothioate oligodeoxyribonucleotide (ODNPS) with two nucleotides on each end. It was obtained from a commercial supplier (MWG Biotech, South Carolina). The sequence corresponded to the 3V end of the published xylosyltransferase-1 mRNA sequence (Accession No.: RNO295748; Gotting et al., 2000). It had no homology to other mammalian sequences registered in the GenBank databases of the NIH (Altshul et al., 1997). For the catalytic digestion of the targeted mRNA, the DNA enzyme contained a loop structure (Santoro and Joyce, 1997). This control DNA enzyme could not digest target message. The control DNA enzyme was designed not to bind to any mRNA and served as a control for the presence of exogenous single stranded DNA in a cell.

PBS and incubating with Texas red-labeled streptavidin (1:200, Molecular Probes) for 2 h at room temperature on a shaker. Additionally, the coverslips were rinsed again four times in PBS and mounted in Citifluor. Astrocyte, Schwann cell, and DRG staining Astrocytes The fixed confrontation assay coverslips were rinsed in PBS four times for 10–15 min. The cultures were stained with the first antibody, a monoclonal mouse IgG antibody against GFAP (1:400, Chemicon) with 5% NGS in PBS plus 0.1% BSA and 0.1% Triton (Sigma) and incubated overnight at 48C on a shaker. The next day, the cultures were rinsed four times for 10–15 min in PBS, followed by incubation with the second antibody, Alexa 594-labeled goat antibody specific for a mouse IgG (1:200, Molecular Probes) for 2 h at room temperature, and rinsed again four times, for 5–10 min in PBS and mounted.

Treatment of the confrontation cultures The Schwann cell and astrocyte confrontation cultures were treated 2–3 h after initial plating for 13 further days with 8 AM of the DNA enzyme or control DNA enzyme every second day. Untreated control cultures received no addition. Other confrontation cultures were treated with 0.2 U/Al of chondroitinase ABC (Seikagaku Kogyo Co., LTD Tokyo, Japan) every second day at the same time the medium was changed. Finally, after 13 days, the cultures were prepared for immunocytochemistry or dot blot experiments. CSPG staining The fixed coverslips from the confrontation assays were incubated for 30 min at 378C and rinsed in PBS four times for 10–15 min followed by incubation with the first antibody, the mouse monoclonal CS-56 IgM antibody (1:200, Sigma) with 5% normal goat serum (NGS, Gibco) diluted in PBS and 0.1% bovine serum albumin (BSA, Sigma) overnight at 48C on a shaker. The following day, the coverslips were rinsed again four times for 10– 15 min in PBS and incubated for 2 h at room temperature with the centrifuged (5000–7000 rpm) second antibody, a biotin-labeled goat anti-mouse IgM antibody (1:200, Chemicon, California), on a shaker. Coverslips were rinsed four times 10–15 min in PBS and incubated with centrifuged (5000–7000 rpm) Texas red-labeled streptavidin (1:200, Molecular Probes, Oregon) for 2 h at room temperature on a shaker. This was followed by rinsing the coverslips four times in PBS and mounting them in Citifluor (Ted Pella, California). In a triple staining with astrocyte and Schwann cells (see later), the last step contained an Alexa 350labeled streptavidin (1:200, Molecular Probes). After chondroitinase ABC digestion of confrontation cultures, they were stained with an antibody that recognizes the undigested carbohydrates (bstubQ-antigen) at the protein core of PGs. This stub antibody (2B6, Seikagaku Kogyo Co., LTD), a mouse immunoglobulin G (IgG), was used at a concentration of 1:100 diluted in PBS, 0.1% BSA, 5% NGS, and incubated overnight at 48C on a shaker. The next day, the cultures were rinsed four times for 10–15 min in PBS and then incubated with the second antibody, a biotinlabeled goat anti-mouse IgG (1:200, Chemicon) in PBS, 0.1% BSA, and 5% NGS on a shaker for 2 h at room temperature. This was followed by rinsing the coverslips four times for 10–15 min in

Schwann cells The Schwann cells were immunostained with the same procedure as used for astrocytes but with P75 antibody, which also stained the DRG neurons albeit at a much lower level, a polyclonal rabbit antibody used at a concentration of 1:500 (Babco, California) with 5% NGS in PBS, 0.1% BSA, and 0.1% Triton overnight at 48C on a shaker. The second antibody, Alexa 488labeled goat anti-rabbit antibody (1:500, Molecular Probes) was used for 2 h at room temperature or overnight at 48C on a shaker. Dorsal root ganglion Staining of DRG with neurofilament antibody was performed after staining for GFAP and P75. The neurofilament antibody, a monoclonal mouse IgG2b, was used at a concentration of 1:400 (Roche, Indiana) with 5% NGS in PBS, 0.1% BSA, and 0.1% Triton overnight at 48C on a shaker. The second antibody, Alexa 350-labeled goat anti-mouse IgG2b (1:200, Molecular Probes), was used for 2 h at room temperature or overnight at 48C on a shaker. Triple-stained images were obtained with a Leitz Orthoplan2 fluorescence microscope connected to a Magnafire color CCD camera (Optronics, California). In each channel, color bleed through was removed by MetaVue v4.6 (Universal Imaging Corp) and the photomicrographs were overlaid and aligned in Photoshop (Adobe). Axons from DRG neurons were distinguished from Schwann cells by staining in the blue (DRG) channel compared to the green (Schwann cell) channel. Dot Blot Schwann cells and astrocytes from five wells of confrontation assays for each group (DNA enzyme and control DNA enzyme treated and untreated) were collected in dot blot buffer [10 mM Tris–HCl (pH 7.6), 150 mM NaCl, 0.2 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM EDTA (pH 8), 1% Triton, and 0.5% nonidet Px-40 (NP-40)]. The protein concentration was measured by using the bicinchoninic acid (BCA) assay (Pierce, Illinois) following the manufacture’s protocol for the microplate assay. Equal amounts of total protein were diluted in Tris-buffered saline (150 mM NaCl, 20 mM Tris, pH 7.5, TBS) at a concentration of 1:100, and 200 Al was loaded into the dot blot apparatus (BioRad, California) on a nitrocellulose membrane (BioRad) in a triple experiment following the manufacture’s protocol. The membrane

B. Grimpe et al. / Mol. Cell. Neurosci. 28 (2005) 18–29

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Each individual rectangle (approximately 800 mm2) was divided into two equal parts (black and white in the schematic Fig. 6). The optical density of Schwann cells in these two areas was measured with TINA 2.09 (Raytest). The average Schwann cell density was calculated for each coverslip. For this and further statistical evaluation (such as percentage and standard deviation shown as error bars), Excel (Microsoft) was used. These results are shown in Table 1. DRG axon quantification

Fig. 6. Schwann cells (light gray) and astrocytes (dark gray) were plated in two adjacent lanes that were given sufficient time for the cells to reach one another. The diagram shows a schematic description of the way our coverslips were quantified. The areas of interest to our quantification are shown in black and white bars. The whole area needed to be divided into two equal parts (approximately 800 mm2). For more details, see Experimental methods.

was blocked in 1 TBS with 3% BSA overnight at 48C on a shaker. On the next day, the membrane was washed in TBS-T (TBS plus 0.05% Tween 20) three times for 20 min and incubated in TBS-3% BSA with CS-56 antibody (1:500) overnight at 48C on a shaker. On the following day, the nitrocellulose blot was rinsed again in TBS-T and incubated with the second antibody conjugated with horseradish peroxidase (HRP) in TBS-3% BSA for 3 h at room temperature on a shaker. This step was followed by the enhanced chemoluminescence (ECL) reaction (Amersham, New Jersey) according to the manufacture’s protocol. The Hyperfilm (Amersham) was developed after 5 s. The film was evaluated by Aida v3.21 (Raytest, Germany) in a transmission mode with absorption units. For further statistical evaluation, Excel (Microsoft) was used. The dot blot was reused by incubating it at 508C two times for 30 min in stripping buffer (100 mM DTT, 2% SDS, 62.5 mM Tris–HCl, pH 6.7), reblocked and restained with a rabbit antibody to GFAP (Accurate, New York) in a 1:1000 dilution overnight at 48C on a shaker. The next day, the blot was rinsed three times in TBS-T and incubated with the second antibody conjugated with HRP in TBS-T-5% dry milk in a 1:1000 dilution for 2 h at room temperature. This was followed by rinsing the dot blot three times for 30 min in TBS-T. The incubation was terminated by using the ECL reaction and the dot blot was developed with Hyperfilm (Amersham). Glial cell mixing quantification To characterize the density of Schwann cells (SC) in the astrocyte (AC) layer under different treatment conditions (chondroitinase ABC, DNA enzyme against XT-1, control DNA enzyme, and untreated), photomicrographs were taken with a Leitz Orthoplan2 fluorescence microscope connected to a Magnafire color CCD camera (Optronics). Five equally sized, rectangularly shaped areas that spanned the intersection between the two lanes of glial cells were randomly chosen for each coverslip that was to be analyzed. Three to four coverslips per group were observed at 10.

We also analyzed coverslips that contained DRG neurons plated on either side of the midline between the Schwann cell or astrocyte lanes. Starting from the cell body, we followed individual axons and their branches and scored the cell type upon which each branch of the axon was associated. It was noted whether the axon remained with the same cell type during growth or shifted from one cell type to another. To quantify the statistical relevance of these results, we used the chi-square test. First, we added the numbers of axons in the group, called baxons growing on both cell typesQ to the group referred to as baxons growing only on astrocytes.Q This new group was compared to the group baxons growing only on Schwann cells.Q This procedure was done for all treatment groups and comparisons were made of the data gathered for DRG axons that started growth on Schwann cells versus those that initiated growth on astrocytes.

Acknowledgments We thank Albert Ries for his outstanding help in protein chemistry and photographic quantification. We also thank Prof. Paul Jones from the Department of Epidemiology and Biostatistics at Case Western Reserve University for his help in the statistical evaluation procedures. This work was supported by NS 25713, NS 09923, the Christopher Reeve Paralysis Foundation, the Daniel Heumann Fund, and the Brumagin Memorial Fund.

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