Ciliary Neurotrophic Factor Role in Myelin Oligodendrocyte Glycoprotein Expression in Cuprizone-Induced Multiple Sclerosis Mice

Abstract

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system that leads to loss of myelin and oligodendrocytes and damage to axons. Myelin oligodendrocyte glycoprotein (MOG) is a minor component of the myelin sheath, but is an important autoantigen linked to the pathogenesis of MS. Ciliary neurotrophic factor (CNTF) has been shown to enhance the generation, maturation, and survival of oligodendrocytes in culture medium. The aim of this study was to demonstrate the role of CNTF on MOG expression in the cerebral cortex of Cuprizone-induced MS mice. The mice were treated by Cuprizone for five weeks in order to induce MS. The mice were then divided into 3 groups. The first group was injected subcutaneously (SC) by CNTF in the amount of 250 μg/kg BW per day. The second group (SHAM) was injected SC by normal saline and the third group was left without injection as the control group. After four weeks the mice were killed and the cerebral cortex was harvested and the expression of MOG was studied by Western blotting. The data from this study show that the MOG expression was significantly increased in the CNTF-injected group as compared to the other groups. It is concluded that CNTF increases the MOG expression and may be important in the pathophysiology of MS. It is also concluded that CNTF may play a role in the process of remyelination by inducing the MOG expression.

Introduction

The myelin sheath is a multilamellar membrane structure that envelopes large-diameter axons. Most of the central nervous system (CNS) myelin is made up of lipid (up to 70 % by dry weight). In the CNS the main proteins of myelin are proteolipid protein (PLP), myelin basic protein (MBP), and myelin oligodendrocyte glycoprotein (MOG). MOG is a minor component of the myelin sheath, but it is an important autoantigen linked to the pathogenesis of multiple sclerosis (MS) (Johns and Bernard 1999). It has been shown that MOG is a potent encephalitogen that triggers strong T-cell and B-cell responses (Genain et al. 1995).

Multiple sclerosis (MS) is a chronic disease of the CNS characterized by the presence of inflammatory infiltrates containing few autoreactive T cells and a multitude of pathogenic non-specific mononuclear cells in areas of demyelination, severe glial scarring, and axonal loss (Owens et al. 2003). Inflammatory mechanisms contribute to demyelination; autoimmune cells were also shown to have neuroprotective properties by the release of growth and neurotrophic factors (Hohlfeld et al. 2000).

In the CNS, oligodendrocytes synthesize large amounts of membranes that wrap around axons and compact to form myelin (Ferguson et al. 1997). Spontaneous remyelination occurs in MS, but the extent of myelin repair remains insufficient to prevent the progression of disability. Failure to remyelinate takes place despite the survival of oligodendrocytes (Lucchinetti et al. 1999) and/or recruitment of oligodendrocyte precursor cells (OPCs) around and within MS lesions (Maeda et al. 2001). This suggests that failure of remyelination may be the result of an incapability of OPCs to mature into myelin-forming cells or of differentiated oligodendrocytes to achieve their normal function. After injury and disease, astrocytes become reactive and prevent regeneration; however, it has also been suggested that astrocytes can become activated and promote regeneration. Thus, it is hypothesized that astrocytes have an important role in modulating CNS repair (Nash et al. 2011). Although there is growing knowledge about the factors involved in the induction and specification of oligodendrocyte lineage, as well as about survival, differentiation, and proliferation of oligodendrocytes, little is known about the molecular control of the myelination itself. Possible reason for the abortive remyelination includes astrogliosis and accumulation of extracellular matrix (ECM), lack of myelinating cells, or unfavorable combination of growth factors (Maier et al. 2005).

Growth factor plays important roles in the brain development and function (Mashayekhi and Gholizadeh 2011; Mashayekhi et al. 2011). Growth factor expression could participate in the repair process of the demyelinating disease by modulating the activity of microglia/macrophages, by inducing the expression of other factors that can affect myelin regeneration or degeneration, and also by directly stimulating the localized proliferation and/or regeneration of oligodendrocytes within lesion areas (Rosenberg et al. 2006). Among growth factors, ciliary neurotrophic factor (CNTF) is a cytokine that has been demonstrated to stimulate myelination in vitro (Stankoff et al. 2002). CNTF is the only cytokine found exclusively in the nervous system and variety of neural cells (Eckenstein et al. 1990). CNTF signaling is mediated by a tripartite complex of CNTF receptor-α (CNTFR-α), LIF receptor (LIFR), and gp130. CNTFR- α is expressed only in the nervous system and skeletal muscle (Davis et al. 1991). As with the other cytokines, it is the dimerization of gp130 with LIFR that results in signal transduction (Stahl and Yancopoulos 1994). Increased concentrations of LIF and brain-derived neurotrophic factor (BDNF) in the cerebrospinal fluid of patients with MS has been reported (Mashayekhi and Salehi 2011; Mashayekhi et al. 2012).

Neuronal differentiation of cortical neural precursor cells was markedly inhibited by CNTF, whereas glial differentiation was markedly potentiated (Bonni et al. 1997). CNTF has also been shown to enhance the generation, maturation, and survival of oligodendrocytes (Mayer et al. 1994).

As CNTF plays a key role in myelin formation in vitro (Stankoff et al. 2002), we examined the in vivo effects of this cytokine on the expression of MOG in the cerebral cortex of Cuprizone-induced MS mice.

Materials and Methods

Antibodies and Reagents

Myelin oligodendrocyte glycoprotein (ab109746) and CNTF (ab9786) were purchased from Abcam, UK; and Cuprizone was purchased from Sigma-Aldrich. The secondary universal antibody was bought from Vector laboratory, UK.

Animals

Balb/c mice were maintained on 12–12 light: dark cycle beginning at 8.00 am. They were kept at a constant temperature in mice boxes with unrestricted access to laboratory food and water. The colony was maintained through random pair mating. Timed mating was carried out by placing a male and female together and checking for the presence of a vaginal plug. The presence of a vaginal plug was taken as gestational day zero (E0) and the day of birth was designated as postnatal day 0 (P0). All animal procedures were carried out in accordance with the Animals (Scientific Procedure) Act, 1986.

Induction of Demyelination and Treatment with CNTF

Demyelination was induced by feeding 8-10-week-old mice a diet containing 0.2 % cuprizone (bis-cyclohexanone oxaldihydrazone, Sigma-Aldrich Inc.) mixed into ground standard rodent chow. Cuprizone is a well-known copper-chelating agent, discovered and described in the early 1950s (Messori et al. 2007). The underlying mechanisms of cuprizone-induced oligodendrocyte cell death are not fully understood. It is well known that feeding of cuprizone causes the formation of mega-mitochondria in the liver. Liver tissue from cuprizone-fed animals was studied with respect to mitochondrial dysfunction. The formation of mega-mitochondria also termed “giant mitochondria” is linked to metabolic injury (Hoppel and Tandler 1973).

The cuprizone diet was administered for 5 weeks for demyelination. The control group received breeder chow without cuprizone admixture. Animals were then put on standard rodent chow without cuprizone to induce remyelination. The mice were then divided into three groups. The first group was injected subcutaneously (SC) by CNTF for 6 weeks in the amount of 250 μg/kg BW per day. The amount of injected CNTF has been administered according to the previous study (Xu et al. 1998). The second group (SHAM) was injected by normal saline and the third group was left without injection as the control group. After four weeks the cerebral cortex was harvested after euthanasia by intraperitoneal injection of an overdose of anesthetic (Sodium pentobarbitone) and the cerebral cortex was removed and processed as described. The total numbers of 36 animals were used in this study (n = 12 for each group).

Cell Extract

Frozen tissue samples (10 mg each) were chopped into tiny pieces and suspended in 0.5 ml of protein lysis buffer [150 mM NaCl, 1.0 % NP40, 20 mM Tris (pH 7.5), 5 mM EDTA, and Complete Mini protease inhibitor cocktail (Roche Diagnostics Ltd., West Sussex, UK)] and then mechanically homogenized by sonication. After centrifugation, the protein extracts were recovered and stored at −70 °C until they were used.

Total Protein Concentration and Western Blotting

The total protein concentration in cerebral cortex extracts was determined by the Bio-Rad protein assay based on the Bradford dye procedure. For Western blot, protein extracts (50 μg/lane) were separated on 10 % SDS–polyacrylamide gel and transferred to a polyvinylidene difluoride membrane (Bio-Rad Laboratories Ltd. Hertfordshire, UK). The membranes were blocked with phosphate buffered saline (PBS) containing 0.05 % Tween 20 and 5 % dry milk and probed either with monoclonal mouse anti-MOG antibody (R&D system, UK; Catalogue No. MAB3228) (1:1,000 dilution) or a mouse monoclonal anti-β-tubulin antibody (Abcam plc, Cambridge, UK) (1:10,000 dilution) and then treated with the appropriate horseradish peroxidase-conjugated secondary antibodies. Immunoreactive protein was visualized using the Enhanced Chemiluminescence Western blotting detection system (Amersham Pharmacia Biotech, Piscataway, NJ). Densitometric analysis was performed by scanning immunoblots and quantitating protein bands using an image analyzer (Metaview Software).

Statistical Analysis

All data presented are expressed as mean ± standard error of the mean (SEM). Statistical analysis was performed using Student’s t test and only values with P ≤ 0.05 were considered as significant.

Results

Total Protein Concentration

The total protein concentration in the brain extracts from CNTF-injected, SHAM, and control groups was determined by the Bio-Rad protein assay based on the Bradford dye mixture. The total protein contents of CNTF-injected, SHAM, and control were 0.94 ± 0.02, 0.93 ± 0.03, and 0.92 ± 0.03 (g/l), respectively. No significant increase in the total protein concentration was seen in the CNTF-injected brain samples compared with those from the SHAM and control groups (P > 0.05) (Fig. 1).

Fig. 1.

Total protein concentration in the cerebral cortical tissues extracts from CNTF-injected, SHAM, and control groups (g/l). No significant change was seen in total protein concentration between the groups. In each of the experimental groups the number of animals investigated was n = 12

Analysis of MOG Expression by Western blotting

Western blot analysis was performed to quantitatively evaluate MOG expression in the cerebral cortical extracts. A Western blot analysis using anti-MOG antibody as a probe confirmed the presence of MOG in all the extracts (Fig. 2). An image analyzer was used to determine the intensities of the band in the respective lanes. Quantification of the Western blot bands from repeated experiments (n = 12) showed that the amount of MOG was significantly increased in the CNTF-injected cerebral cortical extracts when compared with SHAM and control groups (P < 0.0001) (Fig. 3).

Fig. 2.

MOG expression in the cerebral cortex extracts from CNTF-injected (lane 2), SHAM (lane 1), and control (lane 3) groups. β-tubulin (50-kDa) expression was determined as a protein loading control. In each of the experimental groups the number of animals investigated was n = 12

Fig. 3.

Signal intensities from MOG expression in the CNTF-injected, SHAM, and control cerebral cortex immunoblotting experiments were determined by densitometric analysis. In each of the experimental groups the number of animals investigated was n = 12. Significant increase in the MOG expression was seen in CNTF-injected group when compared with SHAM and control groups (P < 0.001)

Discussion

MS is characterized by focal myelin damage, oligodendrocyte loss, and infiltration of macrophages and T lymphocytes (Barnett and Prineas 2004). During early stage of MS some repair is possible, presumably associated with partial remyelination, initiated by either surviving oligodendrocytes or newly formed oligodendrocytes generated from oligodendrocyte progenitor cells (OPCs) (Prineas et al. 1984; Blackmore and keirstead, Blakemore and Keirstead 1999). OPCs are known to persist in the adult state in MS patients and rodents (Nishiyama et al. 1997). In animal models of demyelination, OPCs proliferate and migrate into demyeliated areas (Redwine and Armstrong 1998).

Acute demyelination of the CNS in MS is initially followed by a process of remyelination (Barnett and Sutton 2006). This repair process is guaranteed by the extensive proliferation of OPCs in response to demyelination. It has been demonstrated that growth factor expression could be important in the repair process of this demyelinating disease by inducing the expression of other factors that can affect myelin regeneration and also by directly stimulating the localized proliferation and/or regeneration of oligodendrocytes within lesion areas (Rosenberg et al. 2006). Among growth factors, CNTF has been shown to promote oligodendrocyte survival (Barres et al. 1993). It was shown that exogenous CNTF was protective in rat experimental autoimmune encephalomyelitis (EAE) optic neuritis (Maier et al. 2004). CNTF was previously shown to stimulate myelination when added to mature cortical cocultures (Stankoff et al. 2002).

The pathogenic process to MS is currently unknown, but there are a couple of theories based on current research. One of the current leading theories is antibody-mediated demyelination, where the immune system is attacking the body: specifically the central nervous system, leading to demyelination. In this theory, target antigens mark the body for antibodies to attack. T cell and B cells have been widely implicated in the MS pathogenesis through an antibody-mediated demyelination (Tomassini et al. 2007). Two suspected antigens involved in pathogenesis of MS are myelin basic protein (MBP), which has already been shown to have many antibodies present against it in early MS (Berger et al. 2003) and the other is myelin oligodendrocyte glycoprotein (MOG). Both have been identified as targets of the immune response. These antibodies that result from the immune response might be factors that contribute to the development of multiple sclerosis (Tomassini et al. 2007).

MOG is a glycoprotein believed to be important in the process of myelinization of nerves in the CNS (Pham-Dinh et al. 1995). It is a transmembrane protein expressed on the surface of oligodendrocyte cell and on the outermost surface of myelin sheaths and speculated to serve as a necessary adhesion molecule to provide structural integrity to the myelin sheath and is known to develop late on the oligodendrocyte (Berger and Reindl 2007).

Interest in MOG has centered on its role in demyelinating diseases such as MS. It is a target antigen that leads to autoimmune-mediated demyelination. MOG has received much of its laboratory attention in studies dealing with MS. Several studies have shown a role for antibodies against MOG in the pathogenesis of MS (Egg et al. 2001).

Several studies have shown a role for antibodies against MOG in the pathogenesis of MS. Here we show that daily administration of murine CNTF, a neurotrophic factor that has been described as a survival and differentiation factor for neurons and oligodendrocyte, significantly increases MOG expression in cuprizone-induced mice cerebral cortex. As CNTF increases MOG expression, it is concluded that this growth factor may be important in the process of remyelination. It is also concluded that CNTF may play a key role in the pathogenesis of MS.