Activation of the ciliary neurotrophic factor (CNTF) signalling pathway in cortical neurons of multiple sclerosis patients
1Department of Neurosciences, Lerner Research Institute, Cleveland, 2Mellen Center for Multiple Sclerosis Treatment and Research, Cleveland Clinic Foundation, OH, USA and 3Department of Psychiatry, Kennedy Center for Human Development, Vanderbilt University, Nashville, TN, USA
Correspondence to: Bruce D. Trapp, PhD, Department of Neurosciences/NC30, Lerner Research Institute, Cleveland Clinic, 9500 Euclid Ave, Cleveland, OH 44195, USA E-mail: trappb{at}ccf.org
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Neuronal and axonal degeneration results in irreversible neurological disability in multiple sclerosis (MS) patients. A number of adaptive or neuroprotective mechanisms are thought to repress neurodegeneration and neurological disability in MS patients. To investigate possible neuroprotective pathways in the cerebral cortex of MS patients, we compared gene transcripts in cortices of six control and six MS patients. Out of 67 transcripts increased in MS cortex nine were related to the signalling mediated by the neurotrophin ciliary neurotrophic factor (CNTF). Therefore, we quantified and localized transcriptional (RT-PCR, in situ hybridization) and translational (western, immunohistochemistry) products of CNTF-related genes. CNTF-receptor complex members, CNTFR
, LIFRß and GP130, were increased in MS cortical neurons. CNTF was increased and also expressed by neurons. Phosphorylated STAT3 and the anti-apoptotic molecule, Bcl2, known down stream products of CNTF signalling were also increased in MS cortical neurons. We hypothesize that in response to the chronic insults or stress of the pathogenesis of multiple sclerosis, cortical neurons up regulate a CNTF-mediated neuroprotective signalling pathway. Induction of CNTF signalling and the anti-apoptotic molecule, Bcl2, thus represents a compensatory response to disease pathogenesis and a potential therapeutic target in MS patients.
Key Words: multiple sclerosis; gene expression; CNTF; neuroprotection
Abbreviations: MS, Multiple sclerosis; CNTF, ciliary neurotrophic factor; LIF, leukaemia inhibitory factor; FDR, false discovery rate
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Received March 16, 2007. Revised July 20, 2007. Accepted August 6, 2007.
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Introduction |
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Multiple sclerosis (MS) affects more than 1.5 million people worldwide and is the major cause of non-traumatic neurological disability in young adults in North America and Europe (Weinshenker, 1996
; Noseworthy et al., 2000). Approximately 85% of MS patients initially have a relapsing-remitting disease course (RR-MS) characterized by alternating periods of neurological disability and recovery (Weinshenker, 1996; Noseworthy et al., 2000). Within 25 years a large majority of RR-MS patients exhibit a secondary-progressive disease course (SP-MS) characterized by steadily increasing and irreversible neurological disability (Weinshenker et al., 1989; Noseworthy et al., 2000). Relapses are caused by inflammatory demyelination of white matter, while permanent disability results from neurodegeneration. Axons are transected during inflammatory demyelination (Ferguson et al., 1997; Trapp et al., 1998), but this axonal loss often remains clinically silent due to the ability of the brain to compensate for neuronal loss. Continuous neurological decline during secondary progressive MS (SP-MS) is thought to occur when the brain can no longer compensate for additional axonal or neuronal loss (Trapp et al., 1998). Activation of new cortical areas (Reddy et al., 2000; Rocca et al., 2003) compensate for damage caused by inflammatory demyelination of white matter (Pantano et al., 2002). Other compensatory mechanisms include remyelination (Lassmann et al., 1997), redistribution of sodium channels on demyelinated axons (Waxman, 2006), and expression of neurotrophic factors by immune and CNS resident cells (Martino, 2004).
Ciliary neurotrophic factor (CNTF) is a member of the four alpha-helical cytokine family including interleukin 6 (IL-6), leukaemia inhibitory factor (LIF), interleukin 11 (IL-11), oncostatin M (OSM), cardiotrophin-1 (CT-1) and cardiotrophin-like cytokine (CLC) (Weisenhorn et al., 1999). CNTF plays an important role in brain development and injury (Stockli et al., 1991; Winter et al., 1995; Lee et al., 1997; Dallner et al., 2002) and enhances survival of dorsal root ganglia, hippocampal (Ip et al., 1991), striatal (de Almeida et al., 2001) and retinal neurons (Peterson et al., 2000). CNTF null mice display progressive loss of motor neurons and reduced muscular strength (Masu et al., 1993), while CNTFR null mice die within 24 h of birth because of motor neuron deficits (DeChiara et al., 1995).
CNTF signals through the tripartite complex of CNTFR, LIFRß and GP130 and activates several downstream signalling cascades including JAK/STAT and NF
B (Taga et al., 1989; Taga and Kishimoto, 1997; Heinrich et al., 1998; Middleton et al., 2000). Activation of STATs and NFB, in turn, increases transcription of neuroprotective molecules including FGF, EGF and IGF1. Two to three percent of the human population is CNTF deficient due to a frameshift mutation in exon 2 (Takahashi et al., 1994; Thome et al., 1997). MS patients carrying this null mutation in the CNTF gene show earlier disease onset (Giess et al., 2002b) and faster neurological decline than MS patients with CNTF. CNTF is also increased in CSF from MS patients as they recover from acute exacerbations (Massaro et al., 1997; Massaro, 1998) and studies support a role for CNTF in oligodendrocyte survival and immune suppression in MS animal models (Linker et al., 2002; Kuhlmann et al., 2006). It is possible, therefore, that CNTF delays neurological decline in MS patients. To investigate the molecular mechanisms that contribute to neuronal survival in the cerebral cortex of MS patients, we compared gene transcripts in motor cortex of MS and control patients. We recently quantified and localized gene transcripts which were reduced in MS cortex (Dutta et al., 2006). In this report, we describe significant increases in mRNA's encoding proteins associated with CNTF signalling. Furthermore, these mRNAs and their translational products are enriched in cortical neurons. We propose that a CNTF-mediated neuroprotective response is active in MS cortex and this response is part of the endogenous defense mechanisms mounted by the brain to combat the progressive neurological disability observed in MS patients.
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Material and methods |
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Tissue and microarray analysis
Six MS and six control brains were obtained from Cleveland Clinic and CNMD Brain Bank, University of Pittsburgh, and have been characterized previously (Mirnics et al., 2000
; Dutta et al., 2006). Brains were removed according to a rapid autopsy protocol, sliced into 1-cm thick slices, and either fixed in 4% paraformaldehyde for morphological studies or rapidly frozen for biochemical analysis. Sections (14-µm thick) from frozen blocks of the motor cortex were cut and immunostained for myelin proteolipid protein (PLP) & MHC Class II as described previously (Trapp et al., 1998). Blocks without demyelination or other pathology indicted by increased activation of MHC class II positive cells were sectioned as described later for preparation of RNA and protein. Fixed blocks from the contralateral motor cortex were similarly characterized for demyelination and other frank pathology and used for immunocytochemical and in situ hybridization studies. Demographics of the MS and control patients were described previously and the tissue analysed here have been used to describe decreases in mitochondrial and GABA gene transcripts in MS cortex (Dutta et al., 2006). Details of RNA isolation, labelling and microarray analysis have been described previously (Dutta et al., 2006). Briefly, non-lesion motor cortex samples from six MS patients and six individuals without neurological disease were compared on Affymetrix U133A and U133B microarrays. Among 555 significantly altered transcripts, 488 were decreased and 67 were increased in the MS samples (P < 0.05, 1.5-fold). We used a permutation-based false discovery rate (FDR) analysis to determine whether transcript changes were due to inherent biological differences between control and MS tissue or by ‘chance’. The FDR was only 5.9% (Dutta et al., 2006).
Quantitative RT-PCR
Cortical gray matter was separated from subcortical white matter by scoring the block at the white matter/gray matter juncture with a scalpel prior to cutting multiple 60-µm thick sections in a cryostat. RNA was isolated from the cortical sections as described and used for microarray and RT PCR experiments (Dutta et al., 2006). RNA from the six MS samples and six control samples was reverse transcribed as described previously (Dutta et al., 2006). Gene-specific PCR was performed using SYBR Green I kit and a Roche Lightcycler (Roche Diagnostics, Indianapolis, IN) and standardized to 18S RNA (Ambion Inc., Austin, TX). Primer sequences are appended (Supplemental Data I). Each sample was run in triplicate with melting curve analysis to detect primer dimer artefacts. Normal distribution of the data was confirmed using a Shapiro–Wilk test (Analyse-It Software, Leeds, UK). Delta Ct values were subsequently used to determine relative expression changes. The data was compared by the Student's t-test.
Western blots
For protein analysis, 60 µm thick frozen cortical sections from five MS and five control brains were homogenized in protein extraction buffer, separated on 4–12% NuPage Bis–Tris gels (Invitrogen, Carlsbad, CA) and transferred to PVDF membranes (Invitrogen Inc., Carlsbad CA) as described previously (Dutta et al., 2006). After blocking (5% non-fat milk), membranes were placed in primary antibodies overnight, incubated in horseradish peroxidase-tagged secondary antibodies, treated with Supersignal Pico detection reagent (Pierce Inc., Rockford, IL) and then exposed to Kodak X-Omat film (Kodak Inc., Rochester, NY). Membranes were stripped and subsequently re-probed with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The average intensity for each band was quantified with Image J (NIH, Bethesda, MD). Differences between control and MS samples were compared by the Student's t-test.
Antibodies
The antibodies used in the study are commercially available, well characterized and include CNTF, GAPDH, MAP2 and PLP (Chemicon Inc., Temecula, CA), CNTFR (R&D Biosystems, Minneapolis, MN), SOCS3, LIFRß and GP130 (Santa Cruz Biotechnologies, Santa Cruz, CA), STAT3 and pSTAT3 (Cell Signaling Technology, Danvers, MA), Bcl2 (AbCam Inc., Cambridge, MA), GFAP and MHC Class II (Dako, Carpentenia, CA).
Immunocytochemistry
Free-floating sections (30-µm thick) were cut from paraformaldehyde-fixed blocks, rinsed, boiled for antigen retrieval and immunostained for CNTF (1:100), SOCS3 (1:200), LIFR (1:250), GP130 (1:250) and STAT3 (1:100) as described previously (Trapp et al., 1998) using the avidin–biotin (ABC) procedure and diaminobenzidine (DAB). Frozen (14-µm thick) sections were air dried for 30–60 min, fixed for 10 min in 95% ethanol and used to localize CNTFR (1:200), pSTAT3 (1:50) and Bcl2 (1:200). For double-labelling, free-floating sections were pretreated as described earlier, incubated with primary antibodies for 5 days and then incubated with Texas Red- and FITC-conjugated secondary antibodies for 2 h. Sections were rinsed, mounted with vectashield (Vector Labs, Burlingame, CA) and examined in a Leica confocal microscope (Leica Microsystems, Exton, PA).
In situ hybridization
Paraffin-embedded sections from control and MS motor cortex were hybridized with riboprobes specific to CNTFR, Bcl2, CNTF and proteolipid protein as described previously (Dutta et al., 2006). Riboprobes were generated from PCR products using gene-specific primers for CNTFR, Bcl2 and a combination of primers for CNTF (all sequences appended in Supplementary Data 1). For cellular resolution of mRNA expression, sections were dipped in autoradiographic NTB2 emulsion (Kodak, Rochester, NY), counterstained with hematoxylin-and-eosin and examined by dark-field and bright-field microscopy. Bright-field images were used to quantify mRNA abundance. Grains per neuronal perikaryal area were quantified for at least 30–35 upper motor neurons in at least six sections from two control and two MS motor cortices. Normal distribution of the data was confirmed using a Shapiro–Wilk test (Analyse-It Software, Leeds, UK). Statistical significance (P < 0.05) was determined by the Student's t-test.
Tissue sections were processed for the combined localization of CNTF mRNA and the astrocytic marker, glial fibrillary acidic protein (GFAP) immunoreactivity. Paraffin-embedded sections were processed overnight at 4°C in rabbit anti-GFAP (1:250), rinsed in RNAse-free PBS, incubated in biotinylated goat anti-rabbit IgG, and processed for the localization of antibody binding with the avidin–biotin technique and diaminobenzidine (DAB) as the chromogen. Sections were subsequently dried and processed for CNTF cRNA hybridization as described earlier. The relative localizations of silver grains and immunoprecipitate of GFAP were assessed by bright-field microscopy.
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Results |
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Myelinated, non-lesion motor cortex samples from six MS patients and six individuals without neurological disease were obtained at autopsy and compared on Affymetrix U133A and U133B microarrays (Dutta et al., 2006
). Statistical significance was determined by permutation-based FDR analysis. Among the 555 significantly altered transcripts, 488 were decreased and 67 were increased in the MS samples (1.5-fold, P < 0.05) (Dutta et al., 2006). Nine of the 67 increased gene transcripts were related to CNTF signalling (Fig. 1A). CNTF-related genes increased in MS cortical microarrays were also increased when measured by RT-PCR. Computer generated hierarchical clustering of transcripts, gene accession numbers, P-values and fold changes of microarray and RT-PCR measurements are shown in Fig. 1A. Location of these transcripts with regards to CNTF signalling pathways is shown schematically in Fig. 1B.
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CNTF receptors
CNTF signals through the tripartite receptor complex which includes CNTFR
, GP130 and LIFßR. We compared transcriptional and translational products of these three genes in control and MS motor cortex. There was a significant increase in levels of CNTFR mRNA (1.82-fold by microarray, P < 0.05; 1.90-fold by RT-PCR, P < 0.05, Fig. 2A) and CNTFR protein (1.52-fold; P < 0.0008, Fig. 2B) were significantly increased in MS cortex. mRNAs encoding LIFßR (+1.6-fold P = 0.067) and GP130 (+1.2-fold, P = 0.61) were also increased in microarrays but did not reach statistical significance (Supplemental Data, Fig. A). However, when measured by RT-PCR, LIFRß and GP130 mRNAs were increased by 2.14-fold (P < 0.05) and 2.65-fold (P < 0.005), respectively, in MS cortex (Fig. 2A). Translational products of these transcripts were also significantly increased (LIFßR 1.31-fold, P < 0.005; GP130 1.40-fold, P < 0.0001) when measured by western blots (Fig. 2B). We next determined the immunocytochemical distribution of CNTF-receptor subunits. CNTFR (Fig. 2C and D), LIFßR (Fig. 2E and F) and GP130 (Fig. 2G and H) were highly enriched in neurons in both control (Fig. 2C, E and G) and MS cortex (Fig. 2D, F and H). Upon activation, LIFßRs are translocated from the nucleus to the cytoplasm (Gouin et al., 1999; Gardiner et al., 2002). In contrast to control sections where most LIFßR immunoreactivity localized to neuronal nuclei (Fig. 2E inset), significant LIFßR was detected in the perinuclear cytoplasm and proximal dendrites of neurons in MS cortex (Fig. 2F inset). CNTFR mRNA was localized and quantified by in situ hybridization using radiolabelled probes. mRNA encoding CNTFR was highly enriched in neurons in both control (Fig. 3A) and MS (Fig. 3B and C) brains and grain densities were significantly increased (1.80-fold; P < 0.05) in neurons in MS cortex when compared to control cortex (Fig. 3D). Silver grain densities for myelin proteolipid protein (PLP) mRNA were similar in control (Fig. 3E) and MS (Fig. 3F) cortex. CNTFR mRNA was below detection limits in microarrays of both control white matter (n = 13) and white matter lesions (n = 12) (Supplemental Data, Fig. B). This was consistent with our immunohistochemical staining, which failed to detect CNTFR-positive glial cells in sections of control or MS brains. In addition, we did not detect any LIFRß and GP130 positive glial cells in control and MS cortex. CNTFR mRNA was readily detected in microarrays of cortical lesions (n = 13) but was not significantly increased compared to control cortex (n = 8) (Supplemental Data, Fig. B). Based on these comparisons, increase in CNTF-receptor expression appears to be a specific response of neurons in non-lesion motor cortex in MS patients.
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CNTF
Following confirmation of increased CNTF-receptor complex in neurons, we compared levels and locations of CNTF mRNA in MS and control cortices using radioactive in situ hybridization. Dark-field images contained large and small silver grain aggregates suggesting CNTF mRNA expression by both neurons and glial cells in control (Fig. 4A) and MS cortex (Fig. 4B). Upper motor neurons (layers V–VI) were identified by size and shape in bright-field images (Fig. 4C and D) and silver grains quantified. Silver grain densities were significantly (P < 0.05) increased in MS cortical neurons compared to control (Fig. 4E). To identify the CNTF mRNA-positive glial cells, we combined immunohistochemistry for GFAP and in situ hybridization for CNTF. Silver grains were localized over GFAP positive astrocytes (Fig. 4F). Collectively, these data support increased CNTF mRNA in both neurons and astrocytes in MS cortex. We next compared CNTF levels in western blots and found a 1.71-fold (P < 0.001) increase of CNTF in MS cortex (Fig. 4G). CNTF was detected in cortical neurons in control (Fig. 4H) and MS patients (Fig. 4I) by immunohistochemistry. Interestingly, CNTF-positive astrocytes were not detected in MS cortex (Fig. 4J).
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STAT signalling
The JAK/STAT pathway is one of the primary intracellular signalling cascades activated by CNTF (Rajan et al., 1996
; Taga and Kishimoto, 1997; Heinrich et al.,1998). Signal transducers and activators of transcription (STATs) reside in the cytoplasm in a latent, hypophosphorylated form. Upon activation by CNTF signalling, STAT3 is phosphorylated, dimerized and translocated to the nucleus where it binds to DNA elements and stimulates transcription of downstream genes (Leaman et al., 1996). We quantified STAT3 and pSTAT3 levels in MS and control cortex (Fig. 5A). Total STAT3 levels were similar in control and MS cortical homogenates (Fig. 5A). In !contrast, levels of pSTAT3 were significantly (P < 0.05) increased in MS cortex (Fig. 5A). Immunocytochemically, STAT3 was enriched in neuronal cytoplasm (Fig. 5D and E) while pSTAT3 was enriched in neuronal nuclei in both control (Fig. 5B) and MS (Fig. 5C) cortex. STAT3 activation is associated with increase in levels of its inhibitor SOCS3 (Suppressor of Cytokine 3), thereby initiating an inhibitory feedback (Auernhammer et al., 1999; Naka et al., 1999). SOC3 mRNA and protein was increased in MS cortex (Fig. 5F and G) and SOCS3 is enriched in cortical neurons in control (Fig. 5H) and MS cortex (Fig. 5I). Collectively, these data support increased phosphorylation of STAT3 and activation of JAK/STAT signalling in neurons in MS cortex.
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Neurotrophic factors
Since neuroprotection can also be mediated by nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and glial-derived neurotrophic factor (GDNF) [reviewed in (Thoenen and Sendtner, 2002
; Chao, 2003; Hohlfeld et al., 2006)], we compared by RT-PCR levels of the mRNAs which encode these neurotrophic molecules and their cognate receptors in control and MS cortex (Table 1, Supplementary Data). With the exception of TrkB, the receptor for BDNF, which was increased in MS cortex (2.4-fold, P = 0.037), levels of all the other neurotrophic factors and receptors were similar in control and MS cortex. These results raise the possibility that activation of CNTF signalling is the major neuroprotective pathway induced in cortex. The significance of increased TrkB remains to be determined.
Transcription factors and neuroprotective genes
If CNTF is inducing a neuroprotective signalling cascade, it should result in transcription of neuroprotective molecules. Our microarray comparison detected a 1.9-fold increase in mRNA encoding the anti-apoptotic molecule Bcl2 in MS cortex. This increase in Bcl2 transcripts was confirmed by RT-PCR (2.1-fold; P < 0.05) (Fig. 6A). In situ hybridization studies localized most Bcl2 mRNA to neurons and supports increased neuronal Bcl2 transcripts in MS cortex (Fig. 6C) when compared to control cortex (Fig. 6B). Significant increases in Bcl2 protein (1.43-fold; P < 0.008) were also detected in MS cortex (Fig. 6D). Similar to Bcl2 mRNA, most Bcl2 protein was detected in neurons in both control (Fig. 6E) and MS (Fig. 6F) cortex. We detected significant decrease by RT-PCR (1.98-fold, P = 0.0001) in expression of TGF-ß inducible early growth response transcript (TIEG) mRNA in MS cortex. This result provides further evidence of an anti-apoptotic response in MS cortex as increased expression of TIEG is associated with increased apoptosis (Ribeiro et al., 1999). These data support the induction of a Bcl2-mediated anti-apoptotic pathway in neurons in the motor cortex of MS patients. This anti-apoptotic response was also coupled with increased expression of several neuroprotective molecules including FGF2 (fibroblast growth factor 2) (3.4-fold by RT-PCR, P = 0.050), ACVR2 (Activin receptor 2) (+2.82, P = 0.045), ANG-1 (Angiopoietin-1) (+2.94, P = 0.040) and epidermal growth factor receptor (EGFR) (2.04-fold, P = 0.050) (Alzheimer and Werner, 2002; Reuss and Bohlen und, 2003; Valable et al., 2003) supporting upregulation of a neuroprotective response in MS cortex. Our microarray comparisons and RT-PCR validation detected increase in mRNA encoding three transcription factors, cFOS (1.49-fold, P = 0.021), CEBPß (1.67-fold, P = 0.011) and CEBP
(1.96-fold, P = 0.011) which are known downstream activators of STAT3 signalling (Numata et al., 2005). These data support transcriptional activation of CNTF-mediated signalling as well as an increase in a neuroprotective response in MS cortex.
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Discussion |
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Neurodegeneration is a major cause of permanent neurological disability in chronic MS patients. Remyelination, redistribution of sodium channels along demyelinated axons and activation of new cortical areas help delay progressive neurological disability in MS patients. Microarray comparisons of MS and control motor cortex detected significant increases in CNTF-related gene transcripts in MS motor cortex. Based upon this unbiased gene transcript search (Fig. 1) and reports that MS patients with CNTF-null mutations have earlier onset and more aggressive disease (Giess et al., 2002b
), we quantified and localized translational and transcriptional products of genes associated with CNTF-mediated neuroprotection in control and MS cortices. The tripartite CNTF-receptor complex (CNTFR, LIFR ß and GP130), CNTF, pSTAT3 and the anti-apoptotic molecule, Bcl2, were significantly increased in MS motor cortex and highly enriched in neurons. These data support induction of CNTF-mediated signalling pathways, which have the potential to inhibit neuronal death and delay neurological disability in MS patients.
Although the precise stoichiometry of CNTF-receptor subunit assembly is unknown (Schuster et al., 2003), it is reasonable to assume that all three members should increase in concert to facilitate assembly of the tripartite complex and intracellular signalling. mRNA and protein for all three receptor subunits were increased in MS cortex when measured by RT-PCR and western blotting. Immunocytochemistry established that the complex members were highly enriched in neurons and quantitative in situ hybridization detected a significant increase in CNTFR mRNA in MS cortical neurons. Interestingly, CNTFR mRNA and protein were not detected in oligodendrocytes in either gray or white matter from control and MS patients. LIFRß was enriched in neuronal nuclei in control cortex, indicating low levels of LIFRß activity (Fig. 2E inset) (Gouin et al., 1999; Gardiner et al., 2002). In contrast, LIFRß was highly enriched in perinuclear neuronal cytoplasm in MS cortex (Fig. 2F inset). This translocation is essential for formation of the tripartite complex and mandatory for CNTF signalling. LIFRß translocation from nucleus to cytoplasm provides anatomical support for increased CNTF signalling in MS cortical neurons. Increased CNTF-receptor subunit expression and translocation of LIFRß subunits to the neuronal cytoplasm may be the result of increased CNTF expression as transcriptional and translational products of CNTF were significantly increased in MS motor cortex and detected in cortical neurons. Detection of CNTF mRNA in astrocytes and cortical neurons of MS patients raises the possibility of both an autocrine and a paracrine mode of CNTF action in MS cortex.
Activation of the CNTF tripartite receptor complex is known to activate several signalling pathways including JAK/STAT, NFB and MAP kinase [Reviewed in (Segal and Greenberg, 1996)]. In stable non-differentiating cells, CNTF preferentially activates STAT3 compared to other members of the STAT family (Stahl et al., 1995). We provide evidence for STAT3 activation in neurons in the motor cortex of MS patients. While STAT3 levels were similar in homogenates from control and MS cortex, its phosphorylation state was significantly increased in MS samples. We further demonstrate that STAT3 is highly enriched in neurons and more importantly that pSTAT3 is enriched in neuronal nuclei in MS cortex. One of the primary targets associated with activation of STAT3 is its inhibitor SOCS3 (Auernhammer et al., 1999; Naka et al., 1999). To provide further evidence of STAT3 activation, we quantified levels of SOCS3 in MS cortex. Increased levels of SOCS3 protein was detected in MS cortical neurons. Our data, therefore, provides evidence that known downstream components of STAT3 and CNTF signalling are activated in MS cortex. It should be stressed, however, that these data are correlative in nature and we recognize the possibility that STAT3 could be activated by multiple signalling pathways.
We also quantified mRNAs encoding NGF, BDNF, NT-3 and GDNF. CNTF was the only neurotrophin increased in MS cortex. Using RT-PCR, mRNA encoding TrkB, the receptor for BDNF, was increased MS cortex. TrkB was previously detected in neurons close to MS plaques (Kerschensteiner et al., 1999) and BDNF expression was shown to be increased in inflammatory white matter lesions in MS brains (Kerschensteiner et al., 1999). Since our samples were collected from non-inflamed regions of MS cortex, it was not surprising that BDNF levels were not increased. While the relevance of increased TrkB expression and identification of a possible source of its ligand remains to be determined, it is possible that TrkB signalling may also act in parallel with CNTF signalling in MS cortical neurons.
Essential to a CNTF-mediated neuroprotective response is the expression of neuroprotective molecules. Transcripts encoding three transcription factors, cFOS (Kuroda et al., 2001; Kelly et al., 2004) CEBPß and CEBP (Cantwell et al., 1998;Weihua et al., 2000) that are activated during STAT3 signalling (Numata et al., 2005) were increased in microarrays of MS cortex. More importantly, transcripts encoding three neuroprotective molecules Bcl2, FGF2 and ANG-1 were also significantly increased in MS cortical microarrays. Because of its anti-apoptotic function, we quantified and localized Bcl2 transcriptional and translational products. Bcl2 protein and mRNA were significantly increased in homogenates from MS cortex and was shown to be highly enriched in neurons by immunocytochemistry and in situ hybridization. Quantification of silver grain densities detected a significant increase of Bcl2 transcripts in MS cortical neurons compared to control cortical neurons. We previously reported an increase in apoptotic neurons in lesions in MS cortex (Peterson et al., 2001). Induction of a Bcl2-mediated anti-apoptotic pathway may inhibit neuronal apoptosis in non-lesion cortex in MS patients.
A role for CNTF-mediated neuroprotection in MS patients was previously proposed based upon earlier disease onset and more severe symptoms in MS patients with CNTF null mutations (Giess et al., 2002b). Hoffman and colleagues (Hoffmann and Hardt, 2002), however, failed to detect differences in disease onset or progression in MS patients with CNTF-null mutations and suggested that other neurotrophic factors may compensate for loss of CNTF. Our data describing upregulation of endogenous CNTF and CNTF signalling components in MS cortical neurons provide additional support for CNTF-mediated neuroprotection in chronic MS patients. Significant increases in CSF levels of CNTF in MS patients recovering from relapses suggest that this neuroprotective pathway is operational during early stages of MS. mRNA encoding CNTF have been reported increased following spinal cord injury (Widenfalk et al., 2001). As in MS, ALS patients with CNTF null mutations have earlier onset and a more aggressive disease (Giess et al., 2002a). Exogenous delivery of CNTF has also been associated with decreased degeneration of striatal neurons and cortical-striatal circuits in animal models of Parkinson's and Huntington's disease (Emerich et al., 1997). CNTF, therefore, appears to have a neuroprotective role in several neurodegenerative diseases.
While the therapeutic potential for CNTF in CNS diseases has been clear for sometime, systemic delivery of CNTF as a therapeutic is complicated by its short half-life and its inability to readily pass the blood–brain barrier. In an ALS clinical trial, systemic delivery of CNTF at relatively high doses (>5 µg/kg of body weight) caused a number of side effects including aseptic meningitis, respiratory failure and hepatic infections (Miller et al., 1996). It was recently shown that at the concentrations used in the ALS trial, CNTF can activate the IL-6R (Schuster et al., 2003), raising the possibility that these side effects were not due to interaction of CNTF with its cognate receptor. Development of human CNTF variants that interact specifically with the cognate CNTF-receptor complex may provide a more effective mechanism for testing the efficacy of systemic CNTF therapies. The data described here raises the possibility that therapeutic enhancement of endogenous CNTF signalling pathways as an additional therapeutic approach for increasing neuronal survival in MS and other neurodegenerative diseases.
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Supplementary material |
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Supplementary material is available at Brain online.
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Footnotes |
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*Present address: Oak Clinic for MS Research at Kent State University, Kent, OH, USA
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Acknowledgements |
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This work was supported by the NIH (NINDS) grants RO1 NS35058 and PO1 NS38667 (B.D.T). Authors would like to thank Thalia Torres for her excellent technical help with the in situ hybridization.
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References |
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Alzheimer C, Werner S. Fibroblast growth factors and neuroprotection. Adv Exp Med Biol (2002) 513:335–51.[Web of Science][Medline]
Auernhammer CJ, Bousquet C, Melmed S. Autoregulation of pituitary corticotroph SOCS-3 expression: characterization of the murine SOCS-3 promoter. Proc Natl Acad Sci USA (1999) 96:6964–9.
Cantwell CA, Sterneck E, Johnson PF. Interleukin-6-specific activation of the C/EBPdelta gene in hepatocytes is mediated by Stat3 and Sp1. Mol Cell Biol (1998) 18:2108–17.
Chao MV. Neurotrophins and their receptors: a convergence point for many signalling pathways. Nat Rev Neurosci (2003) 4:299–309.[CrossRef][Web of Science][Medline]
Dallner C, Woods AG, Deller T, Kirsch M, Hofmann HD. CNTF and CNTF receptor alpha are constitutively expressed by astrocytes in the mouse brain. Glia (2002) 37:374–8.[CrossRef][Web of Science][Medline]
de Almeida LP, Zala D, Aebischer P, Deglon N. Neuroprotective effect of a CNTF-expressing lentiviral vector in the quinolinic acid rat model of Huntington's disease. Neurobiol Dis (2001) 8:433–46.[CrossRef][Web of Science][Medline]
DeChiara TM, Vejsada R, Poueymirou WT, et al. Mice lacking the CNTF receptor, unlike mice lacking CNTF, exhibit profound motor neuron deficits at birth. Cell (1995) 83:313–22.[CrossRef][Web of Science][Medline]
Dutta R, McDonough J, Yin X, et al. Mitochondrial dysfunction as a cause of axonal degeneration in multiple sclerosis patients. Ann Neurol (2006) 59:478–89.[CrossRef][Web of Science][Medline]
Emerich DF, Winn SR, Hantraye PM, et al. Protective effect of encapsulated cells producing neurotrophic factor CNTF in a monkey model of Huntington's disease. Nature (1997) 386:395–9.[CrossRef][Medline]
Ferguson B, Matyszak MK, Esiri MM, Perry VH. Axonal damage in acute multiple sclerosis lesions. Brain (1997) 120:393–9.
Gardiner NJ, Cafferty WB, Slack SE, Thompson SW. Expression of gp130 and leukaemia inhibitory factor receptor subunits in adult rat sensory neurones: regulation by nerve injury. J Neurochem (2002) 83:100–9.[CrossRef][Web of Science][Medline]
Giess R, Holtmann B, Braga M, et al. Early onset of severe familial amyotrophic lateral sclerosis with a SOD-1 mutation: potential impact of CNTF as a candidate modifier gene. Am J Hum Genet (2002a) 70:1277–86.[CrossRef][Web of Science][Medline]
Giess R, Maurer M, Linker R, et al. Association of a null mutation in the CNTF gene with early onset of multiple sclerosis. Arch Neurol (2002b) 59:407–9.
Gouin F, Couillaud S, Cottrel M, Godard A, Passuti N, Heymann D. Presence of leukaemia inhibitory factor (LIF) and LIF-receptor chain (gp190) in osteoclast-like cells cultured from human giant cell tumour of bone. Ultrastructural Distribution Cytokine (1999) 11:282–9.
Heinrich PC, Behrmann I, Muller-Newen G, Schaper F, Graeve L. Interleukin-6-type cytokine signalling through the gp130/Jak/STAT pathway. Biochem J (1998) 334:297–314.[Web of Science][Medline]
Hoffmann V, Hardt C. A null mutation in the CNTF gene is not associated with early onset of multiple sclerosis. Arch Neurol (2002) 59:1974–5.
Hohlfeld R, Kerschensteiner M, Stadelmann C, Lassmann H, Wekerle H. The neuroprotective effect of inflammation: implications for the therapy of multiple sclerosis. Neurol Sci (2006) 27(Suppl 1):S1–7.[CrossRef][Web of Science][Medline]
Ip NY, Li YP, van de Stadt I, Panayotatos N, Alderson RF, Lindsay RM. Ciliary neurotrophic factor enhances neuronal survival in embryonic rat hippocampal cultures. J Neurosci (1991) 11:3124–34.[Abstract]
Kelly JF, Elias CF, Lee CE, et al. Ciliary neurotrophic factor and leptin induce distinct patterns of immediate early gene expression in the brain. Diabetes (2004) 53:911–20.
Kerschensteiner M, Gallmeier E, Behrens L, et al. Activated human T cells, B cells, and monocytes produce brain-derived neurotrophic factor in vitro and in inflammatory brain lesions: a neuroprotective role of inflammation? J Exp Med (1999) 189:865–70.
Kuhlmann T, Remington L, Cognet I, et al. Continued administration of ciliary neurotrophic factor protects mice from inflammatory pathology in experimental autoimmune encephalomyelitis. Am J Pathol (2006) 169:584–98.
Kuroda H, Sugimoto T, Horii Y, Sawada T. Signaling pathway of ciliary neurotrophic factor in neuroblastoma cell lines. Med Pediatr Oncol (2001) 36:118–21.[CrossRef][Web of Science][Medline]
Lassmann H, Bruck W, Lucchinetti C, Rodriguez M. Remyelination in multiple sclerosis. Mult Scler (1997) 3:133–6.
Leaman DW, Leung S, Li X, Stark GR. Regulation of STAT-dependent pathways by growth factors and cytokines. FASEB J (1996) 10:1578–88.[Abstract]
Lee MY, Deller T, Kirsch M, Frotscher M, Hofmann HD. Differential regulation of ciliary neurotrophic factor (CNTF) and CNTF receptor alpha expression in astrocytes and neurons of the fascia dentata after entorhinal cortex lesion. J Neurosci (1997) 17:1137–46.
Linker RA, Maurer M, Gaupp S, et al. CNTF is a major protective factor in demyelinating CNS disease: a neurotrophic cytokine as modulator in neuroinflammation. Nat Med (2002) 8:620–4.[CrossRef][Web of Science][Medline]
Martino G. How the brain repairs itself: new therapeutic strategies in inflammatory and degenerative CNS disorders. Lancet Neurol (2004) 3:372–8.[CrossRef][Web of Science][Medline]
Massaro AR. Are there indicators of remyelination in blood or CSF of multiple sclerosis patients? Mult Scler (1998) 4:228–31.
Massaro AR, Soranzo C, Carnevale A. Cerebrospinal-fluid ciliary neurotrophic factor in neurological patients. Eur Neurol (1997) 37:243–6.[Web of Science][Medline]
Masu Y, Wolf E, Holtmann B, Sendtner M, Brem G, Thoenen H. Disruption of the CNTF gene results in motor neuron degeneration. Nature (1993) 365:27–32.[CrossRef][Medline]
Middleton G, Hamanoue M, Enokido Y, et al. Cytokine-induced nuclear factor kappa B activation promotes the survival of developing neurons. J Cell Biol (2000) 148:325–32.
Miller RG, Bryan WW, Dietz MA, et al. Toxicity and tolerability of recombinant human ciliary neurotrophic factor in patients with amyotrophic lateral sclerosis. Neurology (1996) 47:1329–31.
Mirnics K, Middleton FA, Marquez A, Lewis DA, Levitt P. Molecular characterization of schizophrenia viewed by microarray analysis of gene expression in prefrontal cortex. Neuron (2000) 28:53–67.[CrossRef][Web of Science][Medline]
Naka T, Fujimoto M, Kishimoto T. Negative regulation of cytokine signaling: STAT-induced STAT inhibitor. Trends Biochem Sci (1999) 24:394–8.[CrossRef][Web of Science][Medline]
Noseworthy JH, Lucchinetti C, Rodriguez M, Weinshenker BG. Multiple sclerosis. N Engl J Med (2000) 343:938–52.
Numata A, Shimoda K, Kamezaki K, et al. Signal transducers and activators of transcription 3 augments the transcriptional activity of CCAAT/enhancer-binding protein alpha in granulocyte colony-stimulating factor signaling pathway. J Biol Chem (2005) 280:12621–9.
Pantano P, Iannetti GD, Caramia F, et al. Cortical motor reorganization after a single clinical attack of multiple sclerosis. Brain (2002) 125:1607–15.
Peterson JW, Bo L, Mork S, Chang A, Trapp BD. Transected neurites, apoptotic neurons and reduced inflammation in cortical MS lesions. Ann Neurol (2001) 50:389–400.[CrossRef][Web of Science][Medline]
Peterson WM, Wang Q, Tzekova R, Wiegand SJ. Ciliary neurotrophic factor and stress stimuli activate the Jak-STAT pathway in retinal neurons and glia. J Neurosci (2000) 20:4081–90.
Rajan P, Symes AJ, Fink JS. STAT proteins are activated by ciliary neurotrophic factor in cells of central nervous system origin. J Neurosci Res (1996) 43:403–11.[CrossRef][Web of Science][Medline]
Reddy H, Narayanan S, Arnoutelis R, et al. Evidence for adaptive functional changes in the cerebral cortex with axonal injury from multiple sclerosis. Brain (2000) 123:2314–320.
Reuss B, Bohlen und HO. Fibroblast growth factors and their receptors in the central nervous system. Cell Tissue Res (2003) 313:139–57.[CrossRef][Web of Science][Medline]
Ribeiro A, Bronk SF, Roberts PJ, Urrutia R, Gores GJ. The transforming growth factor beta(1)-inducible transcription factor TIEG1, mediates apoptosis through oxidative stress. Hepatology (1999) 30:1490–7.[CrossRef][Web of Science][Medline]
Rocca MA, Mezzapesa DM, Falini A, et al. Evidence for axonal pathology and adaptive cortical reorganization in patients at presentation with clinically isolated syndromes suggestive of multiple sclerosis. Neuroimage (2003) 18:847–55.[CrossRef][Web of Science][Medline]
Schuster B, Kovaleva M, Sun Y, et al. Signaling of human ciliary neurotrophic factor (CNTF) revisited. The interleukin-6 receptor can serve as an alpha-receptor for CTNF. J.Biol Chem (2003) 278:9528–35.
Segal RA, Greenberg ME. Intracellular signaling pathways activated by neurotrophic factors. Annu Rev Neurosci (1996) 19:463–89.[Web of Science][Medline]
Stahl N, Farruggella TJ, Boulton TG, Zhong Z, Darnell JE Jr, Yancopoulos GD. Choice of STATs and other substrates specified by modular tyrosine-based motifs in cytokine receptors. Science (1995) 267:1349–53.
Stockli KA, Lillien LE, Naher-Noe M, et al. Regional distribution, developmental changes, and cellular localization of CNTF-mRNA and protein in the rat brain. J Cell Biol (1991) 115:447–59.
Taga T, Hibi M, Hirata Y, et al. Interleukin-6 triggers the association of its receptor with a possible signal transducer, gp130. Cell (1989) 58:573–81.[CrossRef][Web of Science][Medline]
Taga T, Kishimoto T. Gp130 and the interleukin-6 family of cytokines. Annu Rev Immunol (1997) 15:797–819.[CrossRef][Web of Science][Medline]
Takahashi R, Yokoji H, Misawa H, Hayashi M, Hu J, Deguchi T. A null mutation in the human CNTF gene is not causally related to neurological diseases. Nat Genet (1994) 7:79–84.[CrossRef][Web of Science][Medline]
Thoenen H, Sendtner M. Neurotrophins: from enthusiastic expectations through sobering experiences to rational therapeutic approaches. Nat Neurosci (2002) 5(Suppl):1046–50.[CrossRef][Web of Science][Medline]
Thome J, Nara K, Foley P, et al. Ciliary neurotrophic factor (CNTF) genotypes: influence on choline acetyltransferase (ChAT) and acetylcholine esterase (AChE) activities and neurotrophin 3 (NT3) concentration in human post mortem brain tissue. J Hirnforsch (1997) 38:443–51.[Web of Science][Medline]
Trapp BD, Peterson J, Ransohoff RM, Rudick R, Mork S, Bo L. Axonal transection in the lesions of multiple sclerosis. N Engl J Med (1998) 338:278–85.
Valable S, Bellail A, Lesne S, et al. Angiopoietin-1-induced PI3-kinase activation prevents neuronal apoptosis. FASEB J (2003) 17:443–5.
Waxman SG. Ions, energy and axonal injury: towards a molecular neurology of multiple sclerosis. Trends Mol Med (2006) 12:192–5.[CrossRef][Web of Science][Medline]
Weihua X, Hu J, Roy SK, Mannino SB, Kalvakolanu DV. Interleukin-6 modulates interferon-regulated gene expression by inducing the ISGF3 gamma gene using CCAAT/enhancer binding protein-beta(C/EBP-beta). Biochim Biophys Acta (2000) 1492:163–71.[Medline]
Weinshenker BG. Epidemiology of multiple sclerosis. Neurol Clin (1996) 14:291–308.[CrossRef][Web of Science][Medline]
Weinshenker BG, Bass B, Rice GP, et al. The natural history of multiple sclerosis: a geographically based study. I. Clinical course and disability. Brain (1989) 112:133–46.
Weisenhorn DM, Roback J, Young AN, Wainer BH. Cellular aspects of trophic actions in the nervous system. Int Rev Cytol (1999) 189:177–265.[Web of Science][Medline]
Widenfalk J, Lundstromer K, Jubran M, Brene S, Olson L. Neurotrophic factors and receptors in the immature and adult spinal cord after mechanical injury or kainic acid. J Neurosci (2001) 21:3457–75.
Winter CG, Saotome Y, Levison SW, Hirsh D. A role for ciliary neurotrophic factor as an inducer of reactive gliosis, the glial response to central nervous system injury. Proc Natl Acad Sci USA (1995) 92:5865–9.
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