Osteopontin is extensively expressed by macrophages following CNS demyelination but has a redundant role in remyelination
Introduction
Remyelination, a regenerative event in which demyelinated axons are reinvested with new myelin sheaths, is a complex process involving interactions between oligodendrocyte precursor cells (OPCs), demyelinated axons and a wide spectrum of secreted or membrane bound proteins (Franklin and Goldman, 2004). There is strong evidence based on in vitro and developmental studies that extracellular matrix (ECM)-integrin interactions are potent regulators of OPC biology (ffrench-Constant and Colognato, 2004). It might therefore be expected that similar interactions will be involved in the regulation of remyelination. In this study we focus on osteopontin (OPN), an Arg-Gly-Asp (RGD) integrin-binding motif-containing phosphoglycoprotein component of the ECM that has many developmental, physiological and immunological roles in a wide range of tissue (Ashkar et al., 2000, Rangaswami et al., 2006, Reinholt et al., 1990, Sodek et al., 2000).
In the normal adult CNS OPN is expressed in neuronal cell bodies in several brain regions. The levels of expression are substantially increased following a wide range of CNS injuries including ischemia (Ellison et al., 1998, Lee et al., 1999, Wang et al., 1998), toxic injury (Choi et al., 2003, Kim et al., 2002), and virus induced encephalomyelitis (Shin and Koh, 2004). In the context of CNS pathology it has been most extensively studied in the primary human demyelinating disease of the CNS, multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE), where it is strongly implicated in the exacerbation of disease activity through the promotion of T-cell survival (Chabas et al., 2001, Hur et al., 2007, Sinclair et al., 2005). OPN has also emerged as a significantly upregulated gene during the remyelination of demyelination induced in the mouse brain by the dietary toxin cuprizone (Selvaraju et al., 2004). A possible role in remyelination is suggested by in vitro studies in which recombinant OPN was shown to increase myelin basic protein (MBP) synthesis and enhance myelin membrane formation in oligodendrocyte precursor cell (OPC) lines (Selvaraju et al., 2004).
In this study we use toxin-models of demyelination, characterised by an inflammatory response predominantly mediated by cells of the innate immune response and in which there is a clear temporal separation between the acute demyelination and subsequent remyelination, to describe the expression pattern of OPN during remyelination and to assess its functional role in remyelination using OPN−/− mice.
Section snippets
Focal demyelination in caudal cerebellar peduncle in rats and mice and spinal cord white matter in mice
For most experiments female Sprague–Dawley rats (Harlan, UK) aged 8–10 weeks (young) and 9–12 months (old) were used and procedures performed in compliance with United Kingdom Home Office regulations. Anaesthesia was induced with isoflurane and maintained with intravenous injection of propofol (10 mg/ml) supplemented with diazepam (0.15 mg/100 g body weight) and buprenorphine (0.003 mg/100 g). Demyelination was induced bilaterally by stereotaxic injection of 4μl of 0.01% ethidium bromide (EB)
Osteopontin is expressed in grey but not white matter in adult CNS
Sections of the normal adult CNS (brain stem at the level of the caudal cerebellar peduncles and spinal cord) were examined by in situ hybridization (ISH) for expression of OPN mRNA. Consistent with previous reports (Shin et al., 1999), OPN mRNA expression in these two locations was confined to neuronal cell bodies of brain stem nuclei (most prominently in the vestibular nuclei) (Fig. 1 A) and spinal cord grey matter, especially in ventral horn motoneurons. Immunohistochemistry (IHC) revealed a
Discussion
OPN has been proposed as a potential regulator of myelination and remyelination based on its increased expression during the remyelination phase of cuprizone-induced demyelination in the mouse brain and it ability to enhance proliferation and expression of a differentiation-associated gene, myelin basic protein (MBP) in an OPC-like cell lines (Selvaraju et al., 2004). A role in remyelination might also be expected since OPN is a ligand for αV integrins, several of which have been shown to
Acknowledgments
This work was supported by grants from the UK Multiple Sclerosis Society. We are very grateful to Michael Peacock for his excellent technical assistance. We are also very grateful to Dr Susan Rittling for the donation of the OPN−/− mice.
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