Elsevier

Experimental Neurology

Volume 184, Issue 2, December 2003, Pages 981-990
Experimental Neurology

Intact aggrecan and chondroitin sulfate-depleted aggrecan core glycoprotein inhibit axon growth in the adult rat spinal cord

https://doi.org/10.1016/S0014-4886(03)00383-2Get rights and content

Abstract

Aggrecan is a chondroitin sulfate (CS)/keratan sulfate (KS)-substituted proteoglycan (PG) abundant in cartilage which is also present within the mammalian embryonic, adult, and injured adult central nervous system (CNS). Although its role within the CNS is not clear, cell culture studies show that when substituted with CS, aggrecan inhibits neurite extension. To better understand the inhibitory effect of aggrecan on injured adult axons in vivo, we developed a model to independently test intact aggrecan and CS-depleted aggrecan core glycoprotein. Acute rat spinal cord hemisection cavities were filled with a growth-promoting matrix, Matrigel, and severed dorsal rootlets were placed into this matrix. This created an assay in which axons readily grew. The extent of ingrowth in this baseline assay was compared to the ingrowth in Matrigel loaded with intact aggrecan or the purified core glycoprotein of aggrecan. Our results show that both intact aggrecan and equivalent concentrations of the core glycoprotein component significantly inhibit axonal growth in this model system. These results confirm that aggrecan can inhibit the growth of adult axons in vivo and suggest that the inhibitory effects of aggrecan may be mediated, at least in part, by structures located on the core glycoprotein in the absence of the bulk of the CS chains.

Introduction

Intact aggrecan core protein and core fragments are present in immature, adult, and injured adult rat spinal cord (Lemons et al., 2001). Aggrecan core protein is also present in immature and adult rat brain Milev et al., 1998, Yamaguchi, 2000 as well as immature chick brain Schwartz and Shattil, 2000, Schwartz et al., 1993. Although the nature and degree of glycosaminoglycan (GAG) and oligosaccharide substitution of aggrecan core protein in the adult central nervous system (CNS) is largely unknown, several lines of research suggest that, if it is substituted with chondroitin sulfate (CS), then aggrecan has the potential for influencing axon growth. Studies of CS-substituted proteoglycans (PGs) support, indirectly, a role for CS-substituted aggrecan in inhibition of axon growth in the developing CNS Oakley and Tosney, 1991, Snow et al., 1991, Snow et al., 2001, Snow and Letourneau, 1992. The local abundance of CS-substituted PGs is also correlated with regenerative failure in the adult CNS Davies et al., 1997, Fitch and Silver, 1997, Gates et al., 1996a, Gates et al., 1996b, Lemons et al., 1999, McKeon et al., 1995, although, the exact identity of the PGs and degree of CS substitution were not established. In vitro McKeon et al., 1995, Snow et al., 1990, Snow et al., 2001, Zuo et al., 1998 and in vivo in both the embryo (Chung et al., 2000) and adult Bradbury et al., 2002, Moon et al., 2001, Yick et al., 2000, the inhibitory effects of CS-substituted PGs are diminished by treatment with chondroitinase ABC. Such treatment degrades CS chains in situ since only treated samples exhibit high reactivity with MAbs 2B6 and/or 3B3 which detect the protein-bound “stubs” of chondroitin-4-sulfate and chondroitin-6-sulfate, respectively (Couchman et al., 1984). However, the effect of chondroitinase ABC treatment on other molecules in vivo, such as hyaluronan (HA), has not been determined. This is important because chondroitinase ABC is a potent hyaluronidase (Yamagata et al., 1968) and depolymerization of HA may result in significant loss or disorganization of HA-binding proteins (such as the lecticans aggrecan, brevican, versican, and neurocan). Thus, it is possible that chondroitinase ABC treatment may result, indirectly, in the loss or disorganization of intact lecticans and/or their core glycoproteins.

To begin to understand the possible independent effects of the CS and core glycoproteins of the CSPGs on adult axon growth, we tested the separate effects of intact aggrecan and CS depleted-aggrecan (referred to as aggrecan core glycoprotein). By filling an acute spinal hemisection cavity with Matrigel, a permissive terrain for axonal growth was created. In separate groups of rats, Matrigel was used alone, loaded with intact aggrecan or aggrecan core glycoprotein. This assay builds on previous in vitro studies because it permits adult neurons to remain in vivo while their axons are presented with a controlled culture-like environment, within which axon growth can be assessed. Our results show that both CS-substituted aggrecan and aggrecan core glycoprotein-alone significantly inhibit the growth of adult rat axons into the Matrigel. These results suggest that the inhibitory effects of CS-substituted PGs may be mediated, at least in part, by core glycoprotein components, such as specific protein domains, N-linked or O-linked oligosaccharides, and/or the linkage regions of CS chains which remain after chondroitinase digestion.

Section snippets

Methods

For all surgical procedures, adult Long Evans rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (35 mg/kg for females and 40 mg/kg for males). All animals were given the antibiotic, penicillin G procaine (Phoenix Pharmaceutical Inc., St. Joseph, MO), subcutaneously for 7 days, beginning on the day of surgery [0.1 cm3 (30,000 U)/250 g]. All surgical procedures were carried out under aseptic conditions and on a warming pad. Rats recovered in veterinary intensive

Results

The goals of this study were to use a novel in vivo model to determine if a structurally defined aggrecan preparation could inhibit axonal growth of adult neurons in vivo and secondly to determine to what extent any inhibitory affect was dependent on the presence of intact aggrecan. Our results show that, as expected, native CS-substituted aggrecan profoundly inhibits axonal growth of adult neurons in vivo. Interestingly, a similarly marked inhibitory effect was also seen with the CS-depleted

Discussion

Our results show that aggrecan loading of Matrigel can inhibit the ingrowth of adult axons that normally innervate the spinal cord. Using an in vivo assay, in which Matrigel is introduced into a spinal hemisection lesion, very clear inhibition of axonal growth by both intact aggrecan and aggrecan core glycoprotein was achieved. These findings, together with the identification and characterization of aggrecan core protein species in adult spinal cord (Lemons et al., 2001), suggest that aggrecan

Acknowledgements

This study was supported by the Department of Veterans Affairs, the State of Florida Brain and Spinal Cord Injury Rehabilitation Trust Fund, the C.M. and K.E. Overstreet Endowment and the Shriners of North America. We thank Vivian Thompson for biochemical analysis and G. Othel O'Steen and Wilbur O'Steen for technical assistance during surgery and with animal care.

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