Mohan et al. Acta Neuropathologica Communications (2014) 2:168 DOI 10.1186/s40478-014-0168-9 RESEARCH ARTICLE Open Access Transcript profiling of different types of multiple sclerosis lesions yields FGF1 as a promoter of remyelination Hema Mohan1,10†, Anita Friese1†, Stefanie Albrecht2†, Markus Krumbholz1†, Christina L Elliott3, Ariel Arthur3, Ramesh Menon4, Cinthia Farina4, Andreas Junker5, Christine Stadelmann5, Susan C Barnett3, Inge Huitinga6, Hartmut Wekerle7, Reinhard Hohlfeld1,8, Hans Lassmann9, Tanja Kuhlmann2, Chris Linington3 and Edgar Meinl1* 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. Keywords: Multiple sclerosis, Remyelination, Demyelination, Fibroblast growth factor Introduction but also compromises axonal survival by enhancing sus- The adult central nervous system contains a large pool of ceptibility to damage by inflammatory mediators [4], and mitotic oligodendroglial progenitor cells (OPCs) which by disrupting trophic support provided by myelinating rapidly differentiate and remyelinate lesions in models of oligodendrocytes [5-7]. The frequently observed failure of toxin or immune mediated demyelination [1]. In MS, remyelination in MS does not appear to be due to an however, this endogenous repair mechanism frequently intrinsic, absolute defect of myelination, because de- and fails, resulting in the formation of chronic demyelinated remyelinated MS lesions are frequently found side by side plaques with glial scars, the pathological hallmark of the in the majority of patients. Furthermore, early MS lesions disease [2,3]. This has profound functional consequences often show signs of remyelination [8-12]. as demyelination not only disrupts saltatory conduction, It is unclear why many MS lesions fail to remyelinate. The presence of OPCs and premyelinating oligodendro- * Correspondence: [email protected] cytes in many demyelinated lesions [13,14] suggests that †Equal contributors this could be caused by failure of OPC to differentiate into 1 Institute of Clinical Neuroimmunology, Ludwig Maximilian University myelinating oligodendrocytes. Overcoming this differenti- Munich, Marchioninistraße 15, D-81377 Munich, Germany Full list of author information is available at the end of the article ation block to enhance remyelination by endogenous OPC © 2014 Mohan et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Mohan et al. Acta Neuropathologica Communications (2014) 2:168 Page 2 of 18 is considered a logical strategy to restore saltatory conduc- contained 12 white matter lesions, two of them with a tion and reduce accumulation of disability due to axonal demyelinated core and a remyelinated rim. As control, we pathology [15,16]. Achieving this goal, however, is compli- used 6 control tissue blocks from 4 healthy subjects with- cated as a multitude of cellular and molecular changes out clinical or histological evidence of CNS disease (details within the MS lesions can influence OPC migration, sur- in Additional file 1: Table S1). vival or differentiation [17-19]. While experimental studies For immunostaining of FGF1 we used 10 formalin demonstrate that many factors can individually influence fixed paraffin embedded (FFPE) tissue blocks from 7 MS OPC migration and/or differentiation [20-32], their rela- patients containing remyelinated, demyelinated inactive tive expression within MS lesions and their significance in and demyelinated active areas as well as and 3 control modulating remyelination remains unclear. Expression tissue blocks from 3 healthy subjects. Most of the patients profiling of MS lesions can provide new insight into had secondary progressive MS. The mean disease dur- pathomechanisms [33-35], but this approach has not been ation was 24 years, the sex (f/m) ratio was 2:1 (details applied to remyelinated lesions yet. in Additional file 1: Table S1). To identify endogenous pathways that might be MS lesions were classified according to defined criteria: exploited to enhance remyelination we analyzed white Active lesions contained abundant macrophages with de- matter lesions and used qPCR to focus on the expression graded myelin products visualized by luxol fast blue (LFB) of genes regulating oligodendrocytes. We compared dis- or oil red O staining. Inactive demyelinated lesions were sected remyelinated lesions, actively demyelinating lesions, sharply demarcated from the normal appearing white inactive demyelinated lesions and control white matter. matter and largely devoid of macrophages. Remyelinated This revealed an important role of the FGF family in the lesions were sharply demarcated from the normal appear- regulation of remyelination. The FGF family is known to ing white matter and identified by a fainter LFB staining. regulate oligodendrocyte biology and myelin thickness Tissue was collected from donors from whom a writ- [36-40], but its role in MS lesion development is un- ten informed consent was provided for brain autopsy clear. We found that among the analyzed myelination- and the use of the material and clinical information for regulating genes FGF1 was the most abundant transcript research purposes. in remyelinated lesions. In two lesions that contained a demyelinated core and a remyelinated rim, the FGF1 Dissection of brain specimens, RNA extraction, cDNA transcript levels were higher in the remyelinated parts synthesis, and quantitative PCR of brain tissue suggesting that the increased availability of FGF1 may Selected areas from tissue blocks were obtained as follows: support remyelination. Cryosections (20 μm) were mounted on PEN slides (P.A. FGF1 has been reported to promote proliferation of glial L.M. Microlaser, Bernried, Germany). To identify demyeli- precursors [41], but had so far not been linked to remyeli- nated, remyelinated, control white matter, and grey matter nation or its failure in MS. We employed a dissociated areas every 6th section (30 μm) was stained with LFB. The myelinating culture system [42-44], a remyelinating slice unstained sections were superimposed on LFB stained culture model [45,46], a pure oligodendrocyte culture, and sections and the lesion areas were marked and macrodis- genome wide expression profiling of astrocytes to deduce sected manually. In total 200–300 μm of each block was the functional relevance. This revealed that FGF1 promotes used for transcript analysis. To check the precision of myelination as well as remyelination, presumably via an dissection, the macrodissected sections were stained indirect mechanism. In astrocytes, FGF1 induced leukemia with LFB. The control tissue samples used for qPCR inhibitory factor (LIF) and the chemokine CXCL8,both exclusively contained white matter. Two MS tissue blocks implicated in the recruitment of oligodendrocytes and the with adjacent de-and remyelinated lesions were analyzed promotion of remyelination. This suggests that also in vivo individually. FGF1 stimulates astrocytes to release factors promoting RNA was obtained from the dissected tissue specimens remyelination and that selective modulation of FGF signal- by guanidinium thiocyanate-phenol-chloroform extraction ing pathways could provide a novel strategy to enhance (TRI® Reagent, SIGMA, Munich, Germany), and cDNA remyelination in MS and other demyelinating diseases. was synthesized using random hexamers (High Capacity cDNA Reverse Transcription kit, Applied Biosystems Materials and methods (ABI,