The role of proteoglycans in Schwann cell/astrocyte interactions and in regeneration failure at PNS/CNS interfaces

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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.

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 CNS abruptly ceases (Cajal, 1928, Carlstedt, 1985, Carlstedt, 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, Chong et al., 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, Chong et al., 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, Berthold and Carlstedt, 1977b, 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 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, Snow et al., 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.

Section snippets

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 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

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,

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, Gibco-Life Sciences, Maryland) supplemented with 10% fetal calf serum (FCS, Gibco-Life Sciences) and 10 ml/l penicillin/streptomycin on poly-l-lysine (PLL; 10 μg/ml)-coated Nunc flasks at 37°C, 95% humidity, and 5% CO2. The astrocytes used in the experiments have always been in culture for 14 days. The Schwann cells (SC,

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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