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Transcript profiling of different types of multiple sclerosis lesions yields FGF1 as a promoter of remyelination.

Mohan H, Friese A, Albrecht S, Krumbholz M, Elliott CL, Arthur A, Menon R, Farina C, Junker A, Stadelmann C, Barnett SC, Huitinga I, Wekerle H, Hohlfeld R, Lassmann H, Kuhlmann T, Linington C, Meinl E - Acta Neuropathol Commun (2014)

Bottom Line: We dissected remyelinated, demyelinated active, and demyelinated inactive white matter MS lesions, and compared transcript levels of myelination and inflammation-related genes using quantitative PCR on customized TaqMan Low Density Arrays.In remyelinated lesions, fibroblast growth factor (FGF) 1 was the most abundant of all analyzed myelination-regulating factors, showed a trend towards higher expression as compared to demyelinated lesions and was significantly higher than in control white matter.Two MS tissue blocks comprised lesions with adjacent de- and remyelinated areas and FGF1 expression was higher in the remyelinated rim compared to the demyelinated lesion core.

View Article: PubMed Central - PubMed

Affiliation: Institute of Clinical Neuroimmunology, Ludwig Maximilian University Munich, Marchioninistraße 15, D-81377, Munich, Germany. hema.mohan@ukmuenster.de.

ABSTRACT
Chronic demyelination is a pathological hallmark of multiple sclerosis (MS). Only a minority of MS lesions remyelinates completely. Enhancing remyelination is, therefore, a major aim of future MS therapies. Here we took a novel approach to identify factors that may inhibit or support endogenous remyelination in MS. We dissected remyelinated, demyelinated active, and demyelinated inactive white matter MS lesions, and compared transcript levels of myelination and inflammation-related genes using quantitative PCR on customized TaqMan Low Density Arrays. In remyelinated lesions, fibroblast growth factor (FGF) 1 was the most abundant of all analyzed myelination-regulating factors, showed a trend towards higher expression as compared to demyelinated lesions and was significantly higher than in control white matter. Two MS tissue blocks comprised lesions with adjacent de- and remyelinated areas and FGF1 expression was higher in the remyelinated rim compared to the demyelinated lesion core. In functional experiments, FGF1 accelerated developmental myelination in dissociated mixed cultures and promoted remyelination in slice cultures, whereas it decelerated differentiation of purified primary oligodendrocytes, suggesting that promotion of remyelination by FGF1 is based on an indirect mechanism. The analysis of human astrocyte responses to FGF1 by genome wide expression profiling showed that FGF1 induced the expression of the chemokine CXCL8 and leukemia inhibitory factor, two factors implicated in recruitment of oligodendrocytes and promotion of remyelination. Together, this study presents a transcript profiling of remyelinated MS lesions and identified FGF1 as a promoter of remyelination. Modulation of FGF family members might improve myelin repair in MS.

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FGF1-induced gene expression in human primary astrocytes. (a-c) Gene expression was analysed after 8 h (a,b) and 24 h (a,c) with and without FGF1 (10 ng/ml). In all experiments, FGF1 was applied together with heparin (5 U/ml); control cultures contained heparin only. Only differentially expressed probes (corrected p-value <0.05, fold-change > 1.4-fold up or down, and normalized mean expression intensity ≥ 100 in any one of the two groups) are shown. The number of differentially expressed probes is shown in (a). CXCL8 and LIF were among the genes upregulated at both time points (see also Additional file 5: Table S3). (d) For validation, the gene expression of CXCL8, LIF, and the positive control HMOX1 were analysed by qPCR after 8 h and 24 h with FGF1 (10 ng/ml). (e) Secreted CXCL8 and LIF protein were measured by ELISA in the supernatant after 8 and 24 h with and without 10 ng/ml FGF1. In (d,e), the fold-change compared to unstimulated control cultures is displayed. Black bars and boxes indicate medians and 1st/3rd quartiles, respectively. Whiskers extend to the most extreme samples up to 1.5× of the IQR. Red bars indicate means. Fold-changes were analysed by two-sided one-sample tests against the control samples (μ = 1; t-test for FGF1 mRNA, U test for FGF1 protein because of non-normal distribution). *: p < 0.05, **: p < 0.01.
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Fig8: FGF1-induced gene expression in human primary astrocytes. (a-c) Gene expression was analysed after 8 h (a,b) and 24 h (a,c) with and without FGF1 (10 ng/ml). In all experiments, FGF1 was applied together with heparin (5 U/ml); control cultures contained heparin only. Only differentially expressed probes (corrected p-value <0.05, fold-change > 1.4-fold up or down, and normalized mean expression intensity ≥ 100 in any one of the two groups) are shown. The number of differentially expressed probes is shown in (a). CXCL8 and LIF were among the genes upregulated at both time points (see also Additional file 5: Table S3). (d) For validation, the gene expression of CXCL8, LIF, and the positive control HMOX1 were analysed by qPCR after 8 h and 24 h with FGF1 (10 ng/ml). (e) Secreted CXCL8 and LIF protein were measured by ELISA in the supernatant after 8 and 24 h with and without 10 ng/ml FGF1. In (d,e), the fold-change compared to unstimulated control cultures is displayed. Black bars and boxes indicate medians and 1st/3rd quartiles, respectively. Whiskers extend to the most extreme samples up to 1.5× of the IQR. Red bars indicate means. Fold-changes were analysed by two-sided one-sample tests against the control samples (μ = 1; t-test for FGF1 mRNA, U test for FGF1 protein because of non-normal distribution). *: p < 0.05, **: p < 0.01.

Mentions: Since FGF1 is highly abundant in MS lesions (Figure 1) and astrocytes are known to express FGFRs [55,56], we analyzed transcriptional responses of human astrocytes to FGF1. First, we established the response of our cultured human astrocytes to FGF1 by analyzing the induction of HMOX1, which has been reported to be induced in rodent astrocytes by FGF1 [56]. The interaction of FGF1 with its receptors is stabilized and stimulation efficacy is increased by binding to heparan sulfate [57-60]. Experiments to establish appropriate stimulation conditions showed that 5 U/ml heparin and 10 ng/ml FGF1 caused a reliable up-regulation of HMOX1, while heparin alone had no effect (Figure 8 and data not shown).Figure 8


Transcript profiling of different types of multiple sclerosis lesions yields FGF1 as a promoter of remyelination.

Mohan H, Friese A, Albrecht S, Krumbholz M, Elliott CL, Arthur A, Menon R, Farina C, Junker A, Stadelmann C, Barnett SC, Huitinga I, Wekerle H, Hohlfeld R, Lassmann H, Kuhlmann T, Linington C, Meinl E - Acta Neuropathol Commun (2014)

FGF1-induced gene expression in human primary astrocytes. (a-c) Gene expression was analysed after 8 h (a,b) and 24 h (a,c) with and without FGF1 (10 ng/ml). In all experiments, FGF1 was applied together with heparin (5 U/ml); control cultures contained heparin only. Only differentially expressed probes (corrected p-value <0.05, fold-change > 1.4-fold up or down, and normalized mean expression intensity ≥ 100 in any one of the two groups) are shown. The number of differentially expressed probes is shown in (a). CXCL8 and LIF were among the genes upregulated at both time points (see also Additional file 5: Table S3). (d) For validation, the gene expression of CXCL8, LIF, and the positive control HMOX1 were analysed by qPCR after 8 h and 24 h with FGF1 (10 ng/ml). (e) Secreted CXCL8 and LIF protein were measured by ELISA in the supernatant after 8 and 24 h with and without 10 ng/ml FGF1. In (d,e), the fold-change compared to unstimulated control cultures is displayed. Black bars and boxes indicate medians and 1st/3rd quartiles, respectively. Whiskers extend to the most extreme samples up to 1.5× of the IQR. Red bars indicate means. Fold-changes were analysed by two-sided one-sample tests against the control samples (μ = 1; t-test for FGF1 mRNA, U test for FGF1 protein because of non-normal distribution). *: p < 0.05, **: p < 0.01.
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Fig8: FGF1-induced gene expression in human primary astrocytes. (a-c) Gene expression was analysed after 8 h (a,b) and 24 h (a,c) with and without FGF1 (10 ng/ml). In all experiments, FGF1 was applied together with heparin (5 U/ml); control cultures contained heparin only. Only differentially expressed probes (corrected p-value <0.05, fold-change > 1.4-fold up or down, and normalized mean expression intensity ≥ 100 in any one of the two groups) are shown. The number of differentially expressed probes is shown in (a). CXCL8 and LIF were among the genes upregulated at both time points (see also Additional file 5: Table S3). (d) For validation, the gene expression of CXCL8, LIF, and the positive control HMOX1 were analysed by qPCR after 8 h and 24 h with FGF1 (10 ng/ml). (e) Secreted CXCL8 and LIF protein were measured by ELISA in the supernatant after 8 and 24 h with and without 10 ng/ml FGF1. In (d,e), the fold-change compared to unstimulated control cultures is displayed. Black bars and boxes indicate medians and 1st/3rd quartiles, respectively. Whiskers extend to the most extreme samples up to 1.5× of the IQR. Red bars indicate means. Fold-changes were analysed by two-sided one-sample tests against the control samples (μ = 1; t-test for FGF1 mRNA, U test for FGF1 protein because of non-normal distribution). *: p < 0.05, **: p < 0.01.
Mentions: Since FGF1 is highly abundant in MS lesions (Figure 1) and astrocytes are known to express FGFRs [55,56], we analyzed transcriptional responses of human astrocytes to FGF1. First, we established the response of our cultured human astrocytes to FGF1 by analyzing the induction of HMOX1, which has been reported to be induced in rodent astrocytes by FGF1 [56]. The interaction of FGF1 with its receptors is stabilized and stimulation efficacy is increased by binding to heparan sulfate [57-60]. Experiments to establish appropriate stimulation conditions showed that 5 U/ml heparin and 10 ng/ml FGF1 caused a reliable up-regulation of HMOX1, while heparin alone had no effect (Figure 8 and data not shown).Figure 8

Bottom Line: We dissected remyelinated, demyelinated active, and demyelinated inactive white matter MS lesions, and compared transcript levels of myelination and inflammation-related genes using quantitative PCR on customized TaqMan Low Density Arrays.In remyelinated lesions, fibroblast growth factor (FGF) 1 was the most abundant of all analyzed myelination-regulating factors, showed a trend towards higher expression as compared to demyelinated lesions and was significantly higher than in control white matter.Two MS tissue blocks comprised lesions with adjacent de- and remyelinated areas and FGF1 expression was higher in the remyelinated rim compared to the demyelinated lesion core.

View Article: PubMed Central - PubMed

Affiliation: Institute of Clinical Neuroimmunology, Ludwig Maximilian University Munich, Marchioninistraße 15, D-81377, Munich, Germany. hema.mohan@ukmuenster.de.

ABSTRACT
Chronic demyelination is a pathological hallmark of multiple sclerosis (MS). Only a minority of MS lesions remyelinates completely. Enhancing remyelination is, therefore, a major aim of future MS therapies. Here we took a novel approach to identify factors that may inhibit or support endogenous remyelination in MS. We dissected remyelinated, demyelinated active, and demyelinated inactive white matter MS lesions, and compared transcript levels of myelination and inflammation-related genes using quantitative PCR on customized TaqMan Low Density Arrays. In remyelinated lesions, fibroblast growth factor (FGF) 1 was the most abundant of all analyzed myelination-regulating factors, showed a trend towards higher expression as compared to demyelinated lesions and was significantly higher than in control white matter. Two MS tissue blocks comprised lesions with adjacent de- and remyelinated areas and FGF1 expression was higher in the remyelinated rim compared to the demyelinated lesion core. In functional experiments, FGF1 accelerated developmental myelination in dissociated mixed cultures and promoted remyelination in slice cultures, whereas it decelerated differentiation of purified primary oligodendrocytes, suggesting that promotion of remyelination by FGF1 is based on an indirect mechanism. The analysis of human astrocyte responses to FGF1 by genome wide expression profiling showed that FGF1 induced the expression of the chemokine CXCL8 and leukemia inhibitory factor, two factors implicated in recruitment of oligodendrocytes and promotion of remyelination. Together, this study presents a transcript profiling of remyelinated MS lesions and identified FGF1 as a promoter of remyelination. Modulation of FGF family members might improve myelin repair in MS.

Show MeSH
Related in: MedlinePlus