Elsevier

Experimental Neurology

Volume 182, Issue 2, August 2003, Pages 399-411
Experimental Neurology

Regular article
The chondroitin sulfate proteoglycans neurocan, brevican, phosphacan, and versican are differentially regulated following spinal cord injury

https://doi.org/10.1016/S0014-4886(03)00087-6Get rights and content

Abstract

Chondroitin sulfate proteoglycans (CSPGs) are extracellular matrix (ECM) molecules that are widely expressed throughout the developing and adult CNS. In vitro studies demonstrate their potential to restrict neurite outgrowth, and it is believed that CSPGs also inhibit axonal regeneration after CNS injury in vivo. Previous studies demonstrated that CSPGs are generally upregulated after spinal cord injury, and more recent reports have begun to identify individual proteoglycans that may play dominant roles in limiting axonal regeneration. The current study systematically examined the extended deposition patterns after CNS injury of four putatively inhibitory CSPGs that have not been extensively investigated previously in vivo: neurocan, brevican, phosphacan, and versican. After spinal cord injury, neurocan, brevican, and versican immunolabeling increased within days in injured spinal cord parenchyma surrounding the lesion site and peaked at 2 weeks. Neurocan and versican were persistently elevated for 4 weeks postinjury, and brevican expression persisted for at least 2 months. On the other hand, phosphacan immunolabeling decreased in the same region immediately following injury but later recovered and then peaked after 2 months. Combined glial fibrillary acidic protein (GFAP) immunohistochemistry and in situ hybridization demonstrated that GFAP astrocytes constituted a source of neurocan production after spinal cord injury. Thus, the production of several CSPG family members is differentially affected by spinal cord injury, overall establishing a CSPG-rich matrix that persists for up to 2 months following injury. Optimization of strategies to reduce CSPG expression to enhance regeneration may need to target several different family members over an extended period following injury.

Introduction

Axonal regeneration is limited in part by inhibitory molecules that create a nonpermissive environment for axonal growth. Among these molecules is the family of chondroitin sulfate proteoglycans (CSPGs), a class of putatively inhibitory extracellular matrix (ECM) molecules. CSPGs are expressed throughout the developing and adult CNS and are associated with several extracellular substrates, including hyaluronan, tenascin-R and N-CAM (for review see Yamaguchi, 2000). General markers of CSPGs are also reexpressed after a number of CNS injuries Fitch and Silver 1997, Lemons et al 1999, Moon et al 2002, Pasterkamp et al 2001, Plant et al 2001, Stichel et al 1999.

CSPGs are proteoglycans that consist primarily of six core proteins, each of which have varying degrees of associated chondroitin sulfate glycosaminoglycans (for review see Yamaguchi, 2000). Neurocan, brevican, versican, and aggrecan are grouped together as lecticans/hyalecticans; they share similar N-terminal hyaluronan-binding domains and C-terminal globular domains that have a unique lectin domain. NG2 and phosphacan have individual structures and are distinct from the lecticans/hyalecticans. NG2 is a transmembrane CSPG that exhibits no significant homologies to other proteins (Nishiyama et al., 1991), and phosphacan represents the extracellular domain of the transmembrane receptor-type protein tyrosine phosphatase (RPTPβ) (Maurel et al., 1994). In general, when identified by a promiscuous label that detects several different types of CSPG molecules, production of this class of ECM molecules is increased following cortical injury (Fitch and Silver, 1997), fornix lesions (Stichel et al., 1999), nigrostriatal axotomy (Moon et al., 2002), and after spinal cord injury Fitch and Silver 1997, Lemons et al 1999, Pasterkamp et al 2001, Plant et al 2001, Stichel et al 1999. This upregulation of general CSPG markers also correlates with aborted axonal growth (Davies et al., 1997). In vitro studies demonstrate the ability of this class of proteoglycans to limit neurite outgrowth from various classes of cultured neurons and indicate that inhibitory properties attributed to the CSPGs are associated with the attached chondroitin side chains Braunewell et al 1995, Fidler et al 1999, Yamada et al 1997. Additional reports have also shown that the core proteins themselves restrict neurite outgrowth Dou and Levine 1994, Milev et al 1994, Schmalfeldt et al 2000, Ughrin et al 2003.

Several studies have utilized different lesion paradigms to focus on the regulation of single CSPG family members after CNS injury. Cortical wounds enhance NG2 (Levine, 1994), neurocan Asher et al 2000, McKeon et al 1999, brevican (Jaworski et al., 1999), and versican (Asher et al., 2002) expression. Entorhinal lesions upregulate neurocan (Haas et al., 1999) and brevican (Thon et al., 2000) expression. Transection of the nigrastriatal pathway increases immunoreactivity of NG2, neurocan, phosphacan, and versican (Moon et al., 2002). Kainic acid-induced excitotoxic lesions of the hippocampus Bu et al 2001, Ong and Levine 1999 and demyelinating lesions Keirstead et al 1998, Redwine and Armstrong 1998 increase NG2 levels. Spinal cord injury triggers upregulation of NG2 Jones et al 2002, McTigue et al 2001, Plant et al 2001, neurocan (Jones et al., 2002), brevican (Jones et al., 2002), phosphacan Jones et al 2002, Plant et al 2001, and versican (Jones et al., 2002). Spinal cord injury studies have also reported that aggrecan (Lemons et al., 1999) and phosphacan (Jones et al., 2002) are downregulated following injury. While these studies demonstrate increased expression of individual CSPG molecules at select time points across a variety of CNS injuries, no study to date has systematically examined and compared the patterns of long-term regulation of several family members after a common CNS injury.

In addition, few previous studies have examined cell types in the injured CNS that are responsible for altered ECM production. Whereas in vitro studies demonstrate that astrocytes can produce NG2 Fidler et al 1999, Hirsch and Bahr 1999 and keratan sulfate proteoglycans Geisert et al 1996, Geisert et al 1992, Powell and Geller 1999, studies in the adult injured CNS indicate instead that macrophages and oligodendrocyte progenitor cells are sources of NG2 Bu et al 2001, Jones et al 2002, McTigue et al 2001, Zhang et al 2001 and that microglia, macrophages, and oligodendrocyte progenitor cells are sources of keratan sulfate proteoglycans (Jones and Tuszynski, 2002) after injury. Thus, the present study used a spinal cord injury model to evaluate and compare the deposition patterns of neurocan, brevican, phosphacan, and versican over a 2-month period following spinal cord injury, and the cell types in the injured milieu that participate in altered production of CSPGs. Increased, but differential, long-term expression of individual CSPG family members was observed, and astrocytes were identified as contributors to this in vivo response.

Section snippets

Animal subjects and surgery

Adult female Fischer 344 rats (160–200 g) were subjects of this study. NIH guidelines for laboratory animal care and safety were strictly followed. Animals had free access to food and water throughout the study. All surgeries were performed under anesthesia with a combination (2 ml/kg) of ketamine (25 mg/ml), xylazine (1.3 g/ml), and acepromazine (0.25 mg/ml). A total of 28 rats were used in this study at postinjury time points as follows: 1 day (n = 4), 3 days (n = 4), 7 days (n = 4), 9 days

Cellular response to spinal cord injury

Nissl staining was performed to evaluate lesion extent, general cellular staining, and extent of cavitation (Fig. 2A–G). All sections showed consistent lesions of the dorsal white matter and a portion of the underlining gray matter. Cellular accumulation at the lesion interface was evident within 72 h, peaked at 1–2 weeks, and diminished by 4 and 8 weeks postinjury. Over this time period, cystic degeneration of the lesion site was evident.

Neurocan immunolabeling peaks 2 weeks after spinal cord injury

In the intact spinal cord, neurocan was expressed at

Discussion

This report describes the differential and long-term production of several putatively inhibitory CSPG molecules that could significantly affect axonal regeneration after spinal cord injury. Nearly all members of the CSPG family—neurocan, brevican, phosphacan, and versican—are upregulated after spinal cord injury, but with contrasting temporal and spatial patterns. Further, astrocytes are identified as a cellular source of production of neurocan, one CSPG family member that contributes to this

Acknowledgements

This work was supported by the National Institute for Health, the Veterans Administration, the Hollfelder Foundation, and the Heumann foundation. The brevican monoclonal antibody (RB18) was a gift from Dr. Yu Yamaguchi, The Burnham Institute (La Jolla, CA). The versican monoclonal antibody (12C5), the phosphacan monoclonal antibody (3F8), and the neurocan monoclonal antibody (IF6) were obtained from the Developmental Studies Hybridoma Bank. We thank Dana Sajed for excellent technical assistance.

References (45)

  • Y Zhang et al.

    Correlation between putative inhibitory molecules at the dorsal root entry zone and failure of dorsal root axonal regeneration

    Mol. Cell. Neurosci.

    (2001)
  • R.A Asher et al.

    Neurocan is upregulated in injured brain and in cytokine-treated astrocytes

    J. Neurosci.

    (2000)
  • R.A Asher et al.

    Versican is upregulated in CNS injury and is a product of oligodendrocyte lineage cells

    J. Neurosci.

    (2002)
  • E.J Bradbury et al.

    Chondroitinase ABC promotes functional recovery after spinal cord injury

    Nature

    (2002)
  • K.H Braunewell et al.

    Functional involvement of sciatic nerve-derived versican- and decorin-like molecules and other chondroitin sulphate proteoglycans in ECM-mediated cell adhesion and neurite outgrowth

    Eur. J. Neurosci.

    (1995)
  • J Bu et al.

    Transient expression of the NG2 proteoglycan by a subpopulation of activated macrophages in an excitotoxic hippocampal lesion

    Glia

    (2001)
  • S.J Davies et al.

    Regeneration of adult axons in white matter tracts of the central nervous system

    Nature

    (1997)
  • C.L Dou et al.

    Inhibition of neurite growth by the NG2 chondroitin sulfate proteoglycan

    J. Neurosci.

    (1994)
  • P.S Fidler et al.

    Comparing astrocytic cell lines that are inhibitory or permissive for axon growththe major axon-inhibitory proteoglycan is NG2

    J. Neurosci.

    (1999)
  • R Grill et al.

    Cellular delivery of neurotrophin-3 promotes corticospinal axonal growth and partial functional recovery after spinal cord injury

    J. Neurosci.

    (1997)
  • C.A Haas et al.

    Entorhinal cortex lesion in adult rats induces the expression of the neuronal chondroitin sulfate proteoglycan neurocan in reactive astrocytes

    J. Neurosci.

    (1999)
  • S Hirsch et al.

    Immunocytochemical characterization of reactive optic nerve astrocytes and meningeal cells

    Glia

    (1999)
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