Oligodendroglial fibroblast growth factor receptor 1 ...

Brain Pathology ISSN 1015–6305

RESEARCH ARTICLE

Oligodendroglial fibroblast growth factor receptor 1 gene targeting protects mice from experimental autoimmune encephalomyelitis through ERK/AKT phosphorylation squez2*, Christine Stadelmann3, Martin Berghoff1 Ranjithkumar Rajendran1*, Mario Giraldo-Vela 1 2 3

Department of Neurology, University of Giessen, Klinikstrasse 33, Giessen, 35385, Germany. Department of Neurology, Sozialstiftung Bamberg, Buger Strasse 80, Bamberg, 96049, Germany. €ttingen, Robert-Koch-Strasse 40, Go €ttingen, 37099, Germany. Institute of Neuropathology, University of Go

Keywords demyelination, EAE, FGFR1, oligodendrocytes. Corresponding author: Martin Berghoff, MD, Department of Neurology, University of Giessen, 35385 Giessen, Germany (E-mail: [email protected]) Received 24 June 2016 Accepted 18 January 2017 Published Online Article Accepted 24 January 2016 *Authors contributed equally to this work There are no conflicts of interest. doi:10.1111/bpa.12487

Abstract Fibroblast growth factors (FGFs) exert diverse biological effects by binding and activation of specific fibroblast growth factor receptors (FGFRs). FGFs and FGFRs have been implicated in demyelinating pathologies including multiple sclerosis. In vitro activation of the FGF2/ FGFR1 pathway results in downregulation of myelin proteins. FGF1, 2 and 9 have been shown to be involved in the pathology of multiple sclerosis. Recent studies on the function of oligodendroglial FGFR1 in a model of toxic demyelination showed that deletion of FGFR1 led to increased remyelination and preservation of axonal density and an increased number of mature oligodendrocytes. In the present study the in vivo function of oligodendroglial FGFR1 was characterized using an oligodendrocyte-specific genetic approach in the most frequently used model of multiple sclerosis the MOG35-55-induced EAE. Oligodendroglial FGFR1 deficient mice (referred to as Fgfr1ind2/2) showed a significantly ameliorated disease course in MOG35-55-induced EAE. Less myelin and axonal loss, and reduced lymphocyte and macrophage/microglia infiltration were found in Fgfr1ind2/2 mice. The reduction in disease severity in Fgfr1ind2/2 mice was accompanied by ERK/AKT phosphorylation, and increased expression of BDNF and TrkB. Reduced proinflammatory cytokine and chemokine expression was seen in Fgfr1ind2/2 mice compared with control mice. Considering that FGFR inhibitors are used in cancer trials, the oligodendroglial FGFR1 pathway may provide a new target for therapy in multiple sclerosis.

INTRODUCTION Fibroblast growth factors (FGFs) are secreted glycoproteins which exert diverse biological effects by binding and activation of specific fibroblast growth factor receptors (FGFRs) (25, 40). FGF2 is the prototypic ligand of the FGF family with complex paracrine and autocrine modes of action mediated by four receptor tyrosine kinases FGFR1-4 (25, 33, 40). In vitro and in vivo data suggest that FGF play a role in demyelinating pathologies (41, 50, 57). In humans, the most common demyelinating disease is multiple sclerosis (MS). In MS increased expression of FGF1 in remyelinated lesions (50), increased glial expression of FGF9 in active demyelinated lesions (41) and a high number of FGF21 macrophages/activated microglia in active lesions were described (11). Furthermore, high levels of FGF2 protein expression were found during relapse in serum (27) and cerebrospinal fluid (CSF) (59). In a model of toxic demyelination FGF2 k.o. mice showed enhanced oligodendrocyte repopulation of demyelinated lesions (3), enhanced remyelination (2) and reduced axonal damage (64). In rats application of FGF2 into the CSF over 3 days caused disruption of mature Brain Pathology •• (2017) ••–•• C 2017 International Society of Neuropathology V

oligodendrocytes, loss of myelin and axon degeneration (8). In contrast to these findings suggesting a negative role for FGF2, in MOG35–55-induced experimental autoimmune encephalomyelitis (EAE), the most widely used animal model of MS, FGF2 ablation leads to a more severe disease course, increased lymphocyte and macrophage infiltration and decreased remyelination (57). This finding is supported by a FGF2 gene therapy study in EAE resulting in less disease severity, reduced lymphocyte and macrophage infiltration and an increased number of myelin-forming oligodendrocytes (58). In vitro activation of the FGF2/FGFR1 pathway in oligodendrocyte lineage cells resulted in downregulation of myelin proteins (13) and altered patterns of FGF receptor expression (4). These data support an important role of FGF2 in MS and demyelinating models including EAE. The function of the corresponding receptor FGFR1 in MS and EAE is not well-defined. FGFR1 is present on oligodendrocytes, astrocytes and neurons in humans (22) and rodents (56), where it has been shown to be expressed in both oligodendrocyte progenitor cells (OPC) and differentiated oligodendrocytes. In patients with MS FGFR1 is upregulated in OPC in active lesions and around 1

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chronic–active and chronic–inactive lesions (11) and in remyelinated lesions (50). In rats FGFR1 is expressed on macrophages/ activated microglia in the acute phase of EAE (43). FGFR1 deficient mice showed a reduction in myelin sheath thickness and expression of myelin proteins, there was no effect on OPC proliferation or oligodendrocyte differentiation in these k.o. mice (19). In a model of toxic demyelination, conditional FGFR1 deletion in oligodendrocytes results in increased remyelination and axonal density and an increased number of mature oligodendrocytes (69) suggesting that FGFR1 may be an inhibitor of remyelination in this model. Effects of FGFR1 are mediated through ERK/AKT pathways (19, 29). Activation of the (PI3K)/AKT pathway in mice results in enhanced myelination (15, 53) but has no effect on OPC proliferation or the number of mature oligodendrocytes (15). ERK deficient mice show reduced myelin thickness, but there is no effect on the number or differentiation of oligodendrocytes (32). In MOG35–55-EAE ERK1 null knockout mice and AKT32/2 mice present with a more severe disease course, increased inflammation and demyelination (1, 65). Apart from findings in MS or demyelinating disease models, FGFR1 has been shown to play a key role in carcinogenesis (12), and multikinase or selective FGFR inhibitors and monoclonal FGFR antibodies are currently being tested in preclinical and clinical trials (12). The objective of this study was to characterize the role of oligodendroglial FGFR1 in MOG35–55-induced EAE. Based on recent findings on the function of oligodendroglial FGFR1 in toxic demyelination we hypothesized that deletion of FGFR1 would result in a less severe disease course, reduced demyelination and axonal injury, and decreased inflammation. B6.Cg-Tg(PLP1-cre/ERT)3Pop Fgfr1tm5.1Sor mice were administered tamoxifen to induce conditional Fgfr1 deletion in oligodendrocyte lineage cells (referred to as Fgfr1ind2/2 mice). In agreement with the hypothesis a milder disease course, reduced myelin degeneration, increased axonal density, and decreased lymphocyte and macrophage infiltration were found in Fgfr1ind2/2 mice. The underlying mechanisms include increased ERK and AKT phosphorylation, an increase in BDNF and TrkB expression, and decreased expression of LINGO-1. These data suggest that oligodendroglial FGFR1 is a modulator of demyelination and axonal degeneration in MOG35–55-induced EAE.

MATERIALS AND METHODS Generation of Fgfr1 conditional knock-out mice B6;129S4-Fgfr1tm5.1Sor/J, B6.Cg-Tg(Plp1-cre/ERT)3Pop/J and C57BL/6 mice were purchased from The Jackson Laboratories (Bar Harbor, ME, USA). B6;129S4-Fgfr1tm5.1Sor/J mice were crossbred with B6.Cg-Tg(Plp1-cre/ERT)3Pop/J mice and backcrossed to a C57BL/6J background. Genomic DNA was isolated (DirectPCR-Tail, Peqlab, Erlangen, Germany) to identify the deletion of FGFR1 floxed allele and PLP transgene, mice were genotyped by PCR with the following primers: FGFR1 forward primer 50 -GGACTGGGATAGCAAGTCTCTA-30 and reverse primer 50 -GTGGATCTCTGTGAGCCTGAG-30 ; PLP transgene forward 50 -GCGGTCTGGCAGTAAAAACTATC-30 and reverse primer 50 -GTGAAACAGCATTGCTGTCACTT-30 ; CRE internal positive control primer forward 50 -CTAGGCCACAGAATTGAAAGATC 2

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T-30 and reverse primer 50 -GTAGGTGGAAATTCTAGCATC ATCC-30 . The FGFR1 primers in our genotyping assay flank the upstream loxP site in intron 3. Genotyping protocols were followed as provided by The Jackson Laboratories. PCR products were detected by agarose gel electrophoresis (Peqlab, Erlangen, Germany). For induction of Cre recombinase 4-week-old B6.C g-Tg(PLP1-cre/ERT)3Pop Fgfr1tm5.1Sor female mice were treated with 1 mg of tamoxifen (Sigma-Aldrich, Steinheim, Germany) in 100 mL sunflower oil/ethanol i.p. over five consecutive days (referred to as Fgfr1ind2/2) (Figure 1A). B6.Cg-Tg(PLP1cre/ERT)3Pop Fgfr1tm5.1Sor littermate control mice received a sunflower oil/ethanol mixture (referred to as controls) (Figure 1A). Animal experiments were approved by the local state authorities of Hesse, Giessen, Germany (GI 20/23-Nr. 31/2008).

Induction and clinical evaluation of MOG35-55-induced EAE EAE was induced in 8- to 12-week-old female Fgfr1ind2/2 mice and control mice (Figure 1A) by subcutaneous injection of 300 mg myelin oligodendrocyte glycoprotein peptide (MOG35-55; Charite Hospital, Berlin, Germany) emulsified in complete Freund0 s adjuvant (Sigma, Steinheim, Germany) containing 10 mg Mycobacterium tuberculosis (Difco, Michigan, USA). Intraperitoneal injections of 300 ng pertussis toxin (Calbiochem, Darmstadt, Germany) were given at the time of immunization and 48 hours later. The clinical disease course was monitored until day 62 in a blinded manner. Mice were sacrificed at days 18–20 p.i. for the acute phase and at day 62 p.i. for the chronic phase (Figure 1A). Three independent experiments were performed. EAE disease course was evaluated using a scale that ranged from 0 to 5: 0 5 normal, 0.5 5 distal tail weakness, 1 5 complete tail weakness, 1.5 5 mild hind limb weakness, 2 5 ascending hind limb weakness, 2.5 5 severe hind limb weakness, 3 5 hind limb paralysis, 3.5 5 hind limb paralysis and moderate forelimb weakness, 4 5 hind limb paralysis and severe forelimb weakness, 4.5 5 tetraplegia and incontinence, to 5 5 moribund/death.

Histopathology and immunohistochemistry Fgfr1ind2/2 mice and control mice were anesthetized and transcardially perfused with 4% paraformaldehyde in the acute and chronic phases. Spinal cords were removed and embedded in paraffin blocks. A minimum of six spinal cord cross sections was examined per animal. Spinal cord sections were stained for inflammatory infiltrates (hematoxylin and eosin), myelin loss (Luxol fast blue/ periodic acid-Schiff) and axonal degeneration (Bielschowsky silver impregnation). The extent of myelin loss, encompassing primary demyelination and myelin loss caused by axonal damage, was calculated by relating the area of myelin loss to the total white matter area in LFB/PAS-stained sections. Axonal densities were evaluated in Bielschowsky silver impregnated sections as described earlier (68). Counting was performed in the white matter in spinal cord sections comprising of cervical, thoracic and lumbar spinal cord at 10003 magnification. Axons were counted in minimum of 5 white matter lesions with microscopic fields using a 25-point ocular grid eyepiece from Olympus (Hamburg, Germany). The number of points crossing axons was counted as a fraction of the total number of points of the ocular morphometric grid. Axonal density within Brain Pathology •• (2017) ••–•• C 2017 International Society of Neuropathology V

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Figure 1. Tamoxifen-induced, conditional deletion of Fgfr1 in oligodendrocytes followed by EAE induction with the MOG3555 peptide (A). About 100 mL of Tamoxifen (Fgfr1ind2/2) or vehicle (sunflower oil/ ethanol; control) were injected on five consecutive days in 4-week-old female B6.Cg-Tg(PLP1-cre/ERT)3Pop Fgfr1tm5.1Sor mice. EAE was induced at 8–12 weeks of age. Clinical symptoms were monitored until day 62 p.i. Samples were collected on day 18–20 p.i. (acute phase) and on day 62 p.i. (chronic phase). B, Fgfr1ind2/2 mice showed a delayed onset of disease. The maximum EAE score was at day 14 p.i. in control and day 16 p.i. in Fgfr1ind2/2 mice. A milder disease course was found in Fgfr1ind2/2 mice from day 33 p.i. to the end of the chronic phase (with exception of days 47 and 48). In the chronic phase Fgfr1ind2/2 mice presented with a mild paraparesis whereas controls still had a severe paraparesis. n 5 22 for each group, data are presented as mean 6 SEM. C, Fgfr1ind2/2 mice showed improvement in EAE scores compared with control mice. Mean improvement per animal was calculated by subtracting the EAE score at day 62 from the peak EAE score. Fgfr1 expression was less in Fgfr1ind2/2 mice compared with control mice in the acute (D) and chronic phases (E) of EAE.

the lesion was compared with the normal appearing white matter. Axonal density in lesions is given as the percentage of axons compared with the normal appearing white matter. Importantly, MOG35–55-induced EAE in B6 mice is characterized by substantial axonal pathology (16, 37). To differentiate primary demyelination from myelin loss caused by axonal damage and loss, a side-by-side assessment of myelin histochemistry (LFB) and axonal preservation (Bielschowsky silver impregnation) was performed for all lesions. For immunohistochemistry, spinal cord sections were deparaffinized, hydrated and antigen-retrieval was performed by boiling the sections in citrate buffer (10 mM, pH 6). Endogenous peroxidase was blocked for 10 min with 3% hydrogen peroxide. Sections were then incubated with 10% FCS for 1 hour and with primary antibodies for macrophages/activated microglia (Mac 3, clone M3/84, 1:200, Pharmingen, San Diego, CA, USA), activated B cells (B220, clone RA3-6B2, 1:200, Pharmingen, San Diego, CA, USA), T cells (CD3, clone CD3-12, 1:150, Serotec, Oxford, Brain Pathology •• (2017) ••–•• C 2017 International Society of Neuropathology V

UK), Olig2(1) and Nogo-A(1) oligodendrocyte populations (Olig 2, 1:300, IBL, Gunma, Japan; Nogo-A(1), 1:50, Santa Cruz Biotechnology, CA, USA) overnight. Afterward, biotinylated secondary antibodies [goat anti-rat (Mac 3, B220) and goat anti-rabbit (CD3, Olig2, Nogo-A)] were applied and the signal was detected by incubation with an avidin-biotin complex. Microscopic images were captured using a light microscope (Olympus BX51, Hamburg, Germany) and acquired by a digital camera (Olympus DP71, Olympus America Inc., Center Valley PA, USA). For analysis of myelin loss, total white matter areas and areas of myelin loss were determined by manually tracing the regions by using the Image J software (ImageJ 1.47d, National Institute of Health, USA). The percentage of myelin loss per mouse was obtained by dividing the total area of myelin loss by the total area of white matter. The density of axons was counted with an ocular 25-point grid at 10003 magnification (oil immersion). Mac3(1), B220(1) and CD3(1) cells were counted at 4003 magnification with an ocular 3


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morphometric grid. Cellular immunohistological quantifications were performed using an ocular morphometric grid. T cells, B cells and macrophages/activated microglia were done in a minimum of six inflammatory lesions per mouse from the entire spinal cord comprising cervical, thoracic and lumbar spinal cord. Analyses were not restricted to the subpial areas. The average numbers of positive cells were normalized to an area of 1 mm2.

LINGO-1 forward 50 -TCATCAGGTGAGCGAGAGGA-30 and reverse 50 -CAGTACCAGCAGGAGGATGG-30 ; TGFb forward 50 -CTCCTGCTGCTTTCTCCCTC-30 and reverse 50 -GTGGGG TCTCCCAAGGAAAG-30 ; SEMA3A forward 50 -GGATGGG TCCTCATGCTCAC-30 and reverse 50 -TGGTGCTGCAAGTCAG AGCAG-30 ; GAPDH was used as the internal control gene, and the comparative DDCT method was used to evaluate gene expression.

ERK/AKT and BDNF/TrkB receptor protein quantification

Statistics

In the acute and chronic phase spinal cords were separately homogenized in lysis buffer with Tissue ruptor (Qiagen Instruments, Hombrechtikon, Switzerland). The amount of protein was quantified (PierceV BCA Protein Assay Kit, Thermo Scientific, IL, USA) and normalized. Equal amounts of protein (60 mg) were loaded and separated by 10% SDS-PAGE, transferred (Trans Blot, Semi dry Transfer cell, BioRad) to a nitrocellulose membrane (GE Healthcare, AmershamTM Hybond ECL, Buckinghamshire, UK) and blocked with 5% BSA for 1 hour. The membranes were incubated overnight at 4 C with the primary antibodies against pERK and pAKT (1:1000; Cell Signaling, MA, USA), BDNF and TrkB receptor (1:500; Santa Cruz Biotechnology, CA, USA), Membranes were then incubated for 1 hour with goat anti-rabbit secondary antibody (1:1000, Santa Cruz Biotechnology, CA, USA) and developed using SuperSignal West Pico Chemiluminescent Substrate (Thermo, Pierce Biotechnology, Rockford, IL, USA) using a Fusion Fx7 chemiluminescence system (Peqlab, Erlangen, Germany). GAPDH (Santa Cruz Biotechnology, CA, USA) was used as a loading control; proteins were analyzed by use of the Fusion BioID software (Peqlab, Erlangen, Germany). R

Reverse transcription-PCR In the acute and chronic phase spinal cords were removed, snap frozen in liquid nitrogen and homogenized with Tissue ruptor (Qiagen Instruments, Hombrechtikon, Switzerland). Total RNA was isolated (NucleoSpin RNA II, Macherey-Nagel, D€uren, Germany). cDNA was synthesized from 1 mg of total RNA using the QuantiTectV Reverse Transcription Kit (Qiagen GmbH, Hilden, Germany). Quantitative PCR was performed for FGFR1, proinflammatory cytokines, CX3CL1, CX3CR1, FGF2, LINGO-1, SEMA3A and TGFb using the iTaqTM Universal SYBRV Green qPCR Master Mix (Bio-Rad, CA, USA) by 40 cycles at an annealing temperature of 60 C using the StepOneV Real-Time PCR system (Applied Biosystems, Darmstadt, Germany). In our mice model exon 4 of the Fgfr1 gene is flanked with lox sites. Upon tamoxifen injection Cre mediated deletion of the Frs2/3-binding site on Fgfr1 occurs in oligodendrocytes and the primers will pair on exon 4. Quantification of target genes was performed using the following primers: FGFR1, forward 50 -CAGATGCACTCCCA TCCTCG-30 and reverse 50 -GGGAGCTACAGGGTTTGGTT-30 ; TNFa, forward 50 -CGGTCCCCAAAGGGATGAGAAGT-30 and reverse 50 -ACGACGTGGGCTACAGGCTT-30 ; IL1b forward 50 -TACCTGTGGCCTTGGGCCTCAA-30 and reverse 50 -GCTT GGGATCCACACTCTCCAGCT-30 ; IL6 forward 50 -CTCTG CAAGAGACTTCCA-30 and reverse 50 -AGTCTCCTCTCCGG ACTT-30 ; FGF2 forward 50 -GGC TGC TGG CTT CTA AGT G T-30 and reverse 50 -ACT GGA GTA TTT CCG TGA CCG-30 ; R

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All analyses were performed blinded to the genotype. All mice and samples were included into the analyses. Positively labeled cells in lesions of randomly selected spinal cord sections were counted. Each category analyzed included five or more spinal cord sections per mouse and three mice per condition. Specific numbers of animals per sample are noted in the figure legends. Statistical analysis for differences between the genotypes in the clinical course and histological data was performed using the Mann–Whitney U test. A Ttest was used to analyze reverse transcription-PCR and protein data. Statistical significance was set at P 0.05. Values are expressed as mean 6 standard error of mean. * indicates P 0.05, ** indicates P < 0.01, *** indicates P < 0.001. Statistical analysis was performed using Systat 12 software (Systat, San Jose, CA, USA). Graphs were prepared using the Sigmaplot 10 software (Systat, San Jose, CA, USA).

RESULTS Fgfr1ind2/2 mice show a milder EAE disease course Mice with oligodendroglial Fgfr1 deletion (Fgfr1ind2/2) were analyzed for their susceptibility to MOG35-55 peptide EAE induction (Figure 1B). EAE was induced in 22 eight- to twelve-week-old female Fgfr1ind2/2 mice and compared with 22 age- and sexmatched littermate controls. The onset of clinical symptoms was on day 9.7 6 0.3 in controls and on day 10.9 6 0.2 in Fgfr1ind2/2 mice (P < 0.001). From day 33 p.i. on (with exception of days 47 and 48) Fgfr1ind2/2 mice showed a milder disease course than controls (P 0.05). In this disease phase, Fgfr1ind2/2 mice presented with a mild paraparesis whereas controls still suffered from a severe paraparesis. There was a marked improvement in function in Fgfr1ind2/2 mice (P 5 0.01, Figure 1C). Disease incidence in both genotypes was 100%. No effect of gene deletion on mortality was observed. There were no differences in body weight between Fgfr1ind2/2 and control mice. Fgfr1 mRNA expression was less in Fgfr1ind2/2 mice in the acute (P 5 0.013, Figure 1D) and chronic (P 5 0.017, Figure 1E) phase of EAE.

Reduced myelin loss and increased axonal density in Fgfr1ind2/2 mice The differences in the clinical course were most evident in the chronic phase of EAE. As the next step, Fgfr1ind2/2 mice were assessed for their susceptibility to myelin loss and axonal density in spinal cord white matter. Myelin loss was evaluated by Luxol fast blue staining and axonal density by Bielschowsky staining. In the acute phase of EAE no difference in the extent of myelin loss (Figure 2D–F) was observed between Fgfr1ind2/2 mice and controls. Brain Pathology •• (2017) ••–•• C 2017 International Society of Neuropathology V

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Figure 2. Inflammation, myelin loss and axonal density in spinal cord sections in acute (A–I) and chronic (J–R) EAE in controls and Fgfr1ind2/2 mice. Representative images of spinal cord sections are shown. The inflammatory index (A–C) and myelin loss (D–F) were not different between Fgfr1ind2/2 mice and controls in the acute phase.

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Axonal density was increased in Fgfr1ind2/2 mice (G–I). In the chronic phase Fgfr1ind2/2 mice showed a reduced inflammatory index compared with control mice (J–L). The degree of myelin loss was less in Fgfr1ind2/2 mice (M–O). The axonal density was increased in Fgfr1ind2/2 mice (P–R). n 5 3. Bar: 200 mm, 20 mm (insert).

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Figure 3. Mac3(1) cells (macrophages/activated microglia), B220(1) B cells and CD3(1) T cells in white matter lesions of spinal cord sections in the acute (A–I) and chronic phases (J–R) of EAE in controls and Fgfr1ind2/2 mice. Representative images of spinal cord sections are presented. The number of Mac3(1) cells per mm2 was reduced in Fgfr1ind2/2 mice in both the acute (A–C) and chronic (J–L)

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phase of EAE. B220(1) B cell numbers per mm2 were significantly reduced in Fgfr1ind2/2 mice in both the acute (D–F) and chronic (M–O) phase of EAE. The number of CD3(1) T cells per mm2 was not different between Fgfr1ind2/2 mice and controls in the acute phase (G–I), whereas in the chronic phase the number of CD3 (1) T cells was reduced significantly (P–R). n 5 3. Bar: 200 mm, 20 mm (insert).


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Figure 4. Number of Olig2(1) and NogoA(1) oligodendrocytes in spinal cord sections in controls and Fgfr1ind2/2 mice. Representative images of spinal cord sections in the acute phase (A, B, D, E) and the chronic phase (G, H, J, K) are shown. There were no differences

in the number of Olig2(1) oligodendrocytes in the acute (C) or chronic phase (I). A trend toward an increased number of NogoA(1) oligodendrocytes in Fgfr1ind2/2 mice was seen at the acute phase (F) and the chronic phase (L). n 5 3. Bar: 200 mm.

At this time point, the axonal density was higher in Fgfr1ind2/2 mice than in controls (P 5 0.05) (Figure 2G–I). In the chronic phase of EAE (Figure 2M–R) Fgfr1ind2/2 mice showed less myelin loss (P 5 0.046) and a higher axonal density (P 5 0.05).

L). With regard to cellular infiltration the number of CD3(1) T cells was not affected by FGFR1 deletion in the acute phase of EAE (P 5 0.275, Figure 3G–I). However, the number of Mac3(1) cells (P 5 0.05) and B220(1) B cells (P 5 0.05) was reduced in Fgfr1ind2/2 mice compared with controls (Figure 3A–F). In the chronic phase a reduction of Mac3(1) cells (P 5 0.05), B220(1) B cells (P 5 0.05) and CD3(1) T cells (P 5 0.05) was observed in Fgfr1ind-/- mice compared with controls (Figure 3J–R).

Inflammation in Fgfr1ind2/2 mice is characterized by reduced numbers of lymphocytes and macrophages/microglia To study the underlying cellular mechanisms that drive myelin loss and axonal damage, the degree of inflammation was analyzed in spinal cord white matter lesions on H&E stained sections of Fgfr1ind2/ 2 mice and littermate controls. In the acute phase the inflammatory index was not significantly different between Fgfr1ind2/2 mice and controls (Figure 2A–C), whereas in the chronic phase the inflammatory index was reduced in Fgfr1ind2/2 mice (P 5 0.05; Figure 2J– Brain Pathology •• (2017) ••–•• C 2017 International Society of Neuropathology V

Deletion of Fgfr1 does not affect numbers of oligodendrocyte lineage cells In the mouse CNS Olig2 (36) and Nogo-A (38) are expressed by oligodendrocyte lineage cells. To answer the question whether deletion of oligodendroglial FGFR1 affects oligodendrocyte lineage cells in EAE the number of Olig2(1) and NogoA(1) 7


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Increased phosphorylation of ERK1/2 and AKT in Fgfr1ind2/2 mice To study the underlying mechanisms associated with less myelin loss and an increase of axonal density in the Fgfr1ind2/2 mice we examined ERK1/2 and AKT phosphorylation in the spinal cord of the acute and chronic phases of EAE. No differences in ERK1/2 and AKT phosphorylation were seen in the acute phase of EAE (Figure 5A,B). In the chronic phase of EAE, however, ERK1/2 (P 5 0.016) and AKT (P 5 0.024) phosphorylation was increased in Fgfr1ind2/2 mice compared with controls (Figure 5E,F).

Deletion of FGFR1 causes changes in BDNF and TrkB receptor expression To investigate whether less myelin loss and increased axonal density in the spinal cord was accompanied by changes in BDNF synthesis, the effect of oligodendroglial ablation of FGFR1 on the neurotrophin BDNF and its receptor TrkB was studied on protein level in the acute and chronic phase. In the acute phase BDNF and TrkB receptor protein expression was similar between Fgfr1ind2/2 mice and controls (Figure 5C,D). In the chronic phase BDNF (P 5 0.010) and TrkB receptor protein (P 5 0.003) was increased in Fgfr1ind2/2 mice when compared with controls (Figure 5G,H).

Altered pattern of cytokine and chemokine expression in Fgfr1ind2/2 mice

Figure 5. Protein quantification of ERK1/2 and AKT phosphorylation, and brain-derived neurotrophic factor (BDNF)/TrkB receptor expression in spinal cord lysates of controls and Fgfr1ind2/2 mice. Representative Western blots are shown for the acute and chronic phases of EAE. In the acute phase no effect of Fgfr1 deletion on ERK1/2 and AKT phosphorylation (A,B) and BDNF/TrkB receptor protein expression (C,D) was seen. In the chronic phase ERK1/2 and AKT phosphorylation (E,F), and BDNF and TrkB receptor protein expression (G,H) were upregulated in Fgfr1ind2/2 mice. n 5 3.

oligodendrocytes was studied in spinal cord white matter lesions in the acute and chronic phase. There was no effect of FGFR1 deletion on the number of Olig2(1) oligodendrocytes at both time points (Figure 4A–C for acute phase, Figure 4G–I for chronic phase). A trend toward an increased number of NogoA(1) oligodendrocytes in Fgfr1ind2/2 mice was found both in the acute phase (P 5 0.124, Figure 4D–F) and the chronic phase (P 5 0.118, Figure 4J–L). 8

To characterize the underlying molecular mechanisms of cellular infiltration, proinflammatory cytokine gene expression was analyzed in the spinal cord in the acute and chronic phase (Figure 6). In the acute phase Fgfr1ind2/2 mice showed decreased expression of TNFa (P < 0.001), IL1b (P 5 0.019) and IL6 (P < 0.001) compared with controls (Figure 6A–C). In the chronic phase no differences in proinflammatory cytokine expression for TNFa, IL1b, IL6 were observed (Figure 6F–H). Expression of inducible NOS (iNOS) and IL12 was not changed in Fgfr1ind2/2 mice in both phases of EAE. In addition, to define further molecular mechanisms of cellular infiltration, the expression of the chemokine CX3CL1 and its receptor CX3CR1 in the spinal cord were studied in the acute and chronic phase. In the acute phase no effect of FGFR1 deletion on gene expression of CX3CL1 or CXC3R1 was seen (Figure 6D,E). In the chronic phase CX3CL1 (P 5 0.020) and CX3CR1 (P 5 0.028) expression was decreased in Fgfr1ind2/2 mice compared with controls (Figure 6I,J).

Ablation of FGFR1 affects remyelination inhibitor expression To study whether differences in myelin damage in the spinal cord were accompanied by changes in inhibition of remyelination, the effect of FGFR1 on remyelination inhibitors such as FGF2, LINGO-1, SEMA3A and TGFb were studied on mRNA level (Figure 7). In the acute phase no differences for FGF2, LINGO-1, SEMA3A and TGFb, expression were seen between Fgfr1ind2/2 mice and controls (A-D). In the chronic phase gene expression of LINGO-1 was decreased in Fgfr1ind2/2 mice (P 5 0.018, Figure 7F), a trend toward reduced expression of FGF2 was seen in Fgfr1ind2/2 mice (P 5 0.071, Figure 7E). No differences were found for SEMA3A and TGFb expression (Figure 7G,H). Brain Pathology •• (2017) ••–•• C 2017 International Society of Neuropathology V

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macrophages and activated microglia (43). In the present study the in vivo function of FGFR1 was characterized using an oligodendrocyte-specific genetic approach, which allowed defining the function of this receptor in a cell-specific and inducible fashion. Impaired signaling via oligodendroglial FGFR1 resulted in a milder disease course. The underlying neuropathology included less myelin loss and axon degeneration, and reduced cellular infiltration. Furthermore, impaired signaling via oligodendroglial FGFR1 was associated with phosphorylation of the ERK/AKT pathway, enhanced expression of the neurotrophin BDNF and decreased expression of LINGO-1, and downregulation of proinflammatory cytokines and chemokines. The key clinical finding of our study is that Fgfr1ind2/2 mice showed a milder disease course in the chronic phase of EAE. Furthermore, Fgfr1ind2/2 mice presented with a delay in onset of symptoms. These clinical findings are in contrast to those obtained

Figure 6. Gene expression of proinflammatory cytokine and chemokine/receptor levels in the spinal cord of controls and Fgfr1ind2/ 2 mice. Findings in the acute phase (A–E) and the chronic phase (F– J) are shown. In the acute phase decreased mRNA expression of TNFa, IL1b and IL6 was observed in Fgfr1ind2/2 mice compared with controls (A–C). In the chronic phase TNFa, IL1b, IL6 were not different between Fgfr1ind2/2 mice and controls (F–H). There was no difference in the expression of the chemokine CX3CL1 and its receptor CX3CR1 in Fgfr1ind2/2 mice and controls in the acute phase (D,E) whereas in the chronic phase CX3CL1 and CX3CR1 were reduced in Fgfr1ind2/2 mice (I,J). n 5 6.

DISCUSSION Recent studies suggest that FGFR1 plays a role in MS and a model of toxic demyelination (69). FGFR1 is upregulated on oligodendrocyte precursor cells within active and around chronic MS brain lesions (11). In EAE it is expressed on effector cells such as Brain Pathology •• (2017) ••–•• C 2017 International Society of Neuropathology V

Figure 7. Gene expression of the remyelination inhibitors FGF2, LINGO-1, SEMA3A and TGFb in the spinal cord of controls and Fgfr1ind2/2 mice. Findings in the acute phase (A–D) and the chronic phase (E–H) are shown. In the acute phase FGF2, LINGO-1, SEMA3A and TGFb expression was not different between Fgfr1ind2/2 mice and controls. In the chronic phase LINGO-1 was reduced in Fgfr1ind2/2 mice, furthermore a trend toward a reduced expression of FGF2 was seen. n 5 6.

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after systemic deletion of its key ligand FGF2. FGF22/2 mice are more severely affected in the chronic phase of EAE (57), a finding, which is supported by data from a FGF2 gene therapy study (58). There was no effect of systemic FGF2 deletion in the acute phase of EAE. Intrathecal injection of a HSV multigene vector engineered with the human FGF2 gene at day 20 after EAE induction also resulted in a milder disease course 5 days later to the end of the study (58). Using cuprizone toxic demyelination in FGF2 null k.o. mice and in oligodendroglial FGFR1 conditional knockout mice, evaluation revealed improved motor function, less demyelination and axon degeneration as in our study (48). In human brain tissue from patients with MS, demyelination in active/chronic lesion areas was associated with an increase of FGF2 and FGFR1 (11) suggesting a role of the FGF/FGFR pathway in neurorepair and neuroprotection. Differences in findings between studies may be explained by (a) alternative binding of other ligands to FGFR1 or activation of other FGFRs by FGF2, (b) up- or downregulation of members of the FGF family during demyelination, (c) different k.o. approaches including systemic deletion of a ligand and conditional k.o. of a receptor in specific cells, (d) differences in proteins used to induce EAE and (e) various demyelinating disease models. In vitro data suggest that the deleterious effects of FGFR1 activation by FGF2 on differentiated oligodendrocytes are associated with a downregulation of myelin proteins (17). Myelin sheaths play an important role in protecting axons from degeneration (30). Our findings suggest that impaired signaling via oligodendroglial FGFR1 results in less myelin loss and in enhanced axonal density. Similar effects of oligodendroglial FGFR1 were also observed in toxic demyelination, where remyelination and axonal integrity is enhanced (69). The degree of myelin loss in Fgfr1ind2/2 mice in our study is in agreement with findings in FGF2 null mice in toxic demyelination, in which enhanced remyelination (2) and reduced axonopathy were found (64). In contrast, in a PLP-EAE study in FGF2 null mice increased myelin degeneration and axonal damage was seen in the chronic disease phase (57). Oligodendroglial FGFR1 did not affect the number of oligodendrocyte lineage cells in our model. In agreement, in the model of toxic demyelination reduced oligodendroglial FGFR1 expression did not have an effect on OPC numbers (69). We observed increased phosphorylation of ERK1/2 and AKT in the chronic phase of Fgfr1ind2/2 mice. Both ERK1/2 and AKT3 are predominantly expressed in the brain and play a role in brain development and neurodegeneration. In the hypomyelinated CNS of Fgfr1/Fgfr2 double mutant mice ERK1/2MAPK activity was reduced, these mice showed reduced myelin protein gene expression and myelin thickness (19). In contrast to these findings, we observed enhanced ERK1/2 activation in the chronic phase of EAE. The difference between the studies may be caused by the additional k.o. of FGFR2 expressed in mature oligodendrocytes. It is known that active ERK1/2 and AKT phosphorylation increases myelination in the CNS (15, 53). ERK1/2 activation is capable of reinitiating myelin growth by preexisting oligodendrocytes, even after active myelination is terminated (31). Growth factors associated with an activation of the ERK1/2 and AKT pathway include FGF and BDNF (66). In vitro inactivation of ERK1/2 in oligodendrocytes results in reduced extension of oligodendrocyte processes (32). ERK1/2 k.o. mice have normal numbers of oligodendrocytes, delayed myelination and a failure to upregulate key myelin proteins (15, 20, 21). In vivo data suggest that activation of 10

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AKT causes enhanced myelination (15) and increases myelin thickness (24, 28). Inhibition of the downstream target of AKT mTOR, results in reduced myelin thickness (53). In agreement with these findings on the function of ERK1/2 and AKT, upregulation of ERK1/2 and AKT in our study was associated with reduced myelin degeneration without an alteration in the number of oligodendrocytes. In the chronic phase of EAE we found increased BDNF and TrkB expression in Fgfr1ind2/2 mice presenting with less axonal degeneration. In vitro FGF2 has been suggested to promote the role of BDNF through upregulation of the respective receptor TrkB expression (7). BDNF is a neurotrophic factor involved in neuronal survival and differentiation as well as axonal growth (10). Interactions of FGFR1 with other growth factor receptors are not well studied. FGF upregulate BDNF and TrkB in retinal ganglion cells by activating the ERK signaling pathway (5, 61), the TNFa/TNFR pathway (13), expression of Nrf2 (67), VEGF/VEGFR2 (51, 60) and the PDGF receptor (52). In MS immune cells are a major source of BDNF (35). More BDNF(1) cells are found in active demyelinating lesions compared with inactive lesions (62). Furthermore, neurons around active lesions and reactive astrocytes within lesions express high levels of its high affinity receptor gp145trkB (62). In EAE (39, 42) and other lesion models (45) BDNF is expressed by activated immune cells and it is also associated with axonal protection (42). Activation of TrkB by an agonist reduces the severity of EAE, reduces myelin loss, protects against axonal degeneration and attenuates inflammation (44). Activation of TrkB induces the Akt/STAT3 pathway associated with neuronal survival and myelination (44). In agreement, we observed an increase in BDNF/TrKB expression and AKT phosphorylation in Fgfr1ind2/2 mice. In both acute and chronic phases of EAE, impaired signaling via FGFR1 was associated with a decreased number of macrophages/ activated microglia and B220(1) B cells in the spinal cord. Activated macrophages and microglia release a number of soluble factors such as nitric oxide (NO), excitotoxins and proteases, which cause structural damage in the CNS (26). In Fgfr1ind2/2 mice a significant reduction of CD3(1) T cells was observed in the chronic phase of EAE, there was no difference in CD3(1) T cells number in the acute phase of EAE as described for AKT32/2 mice, where an increase of T cells was detected (65). We found increased AKT phosphorylation in the chronic phase of EAE suggesting that oligodendroglial FGFR1 mediates inflammation through the AKT pathway. In contrast to our findings, in FGF22/2 mice the number of macrophages and T cells was increased in acute EAE (57). As discussed above, differences between studies may be explained by alternative binding of other ligands to FGFR1, up- or downregulation of members of the FGF family or different k.o. approaches. Data on oligodendrocyte-specific FGFR1 ablation and immune cell infiltration into the CNS are not available from the toxic demyelination model (52). In brain tissue of patients with MS, FGF2 has been detected in inflammatory cells such as microglia and infiltrating T cells and B cells in chronic active lesions (11). There are only few data on the interaction of FGF/FGFR and inflammation. In patients with rheumatoid arthritis FGF1 activated FGFR1 expressing T cells are increased in the peripheral blood (9) suggesting that immune-growth factor networks contibute to multiple interactions that maintain chronic inflammation. Brain Pathology •• (2017) ••–•• C 2017 International Society of Neuropathology V

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In the CNS T cells are reactivated resulting in the release of proinflammatory cytokines and chemokines (14). In the acute phase of EAE, impaired signaling via FGFR1 was associated with decreased secretion of proinflammatory cytokines such as TNFa, IL1b and IL6. There is evidence for a deleterious role of the CX3CL1/CX3CR1 pathway in MS. CX3CR1 is present on astrocytes and oligodendrocytes in active and silent MS lesions (54). Increased expression of serum CX3CL1 is observed in patients with MS (34). Data from a genetic study suggest that the CX3CR1 I249T280 haplotype may protect from switch to secondary progressive MS (63). The role of the CX3CL1/CX3CR1 pathway appears to be controversial. We observed that impaired signaling via oligodendroglial FGFR1 is associated with downregulation of the CX3CL1/CX3CR1 pathway and decreased immune cell infiltration in the chronic phase of EAE. This deleterious role of the pathway is supported by a study where treatment with an anti-CX3CL1 neutralizing antibody resulted in reduced clinical disease severity and less lymphocyte infiltration in EAE (49) and activation of FGFR1 in mammary tumor cells promotes the recruitment of macrophages in a CX3CL1/R1 dependent manner in vitro and in vivo (6, 55). In contrast to these findings, CX3CR1-deficient mice show a more severe disease course and increased IFN-g expression (23). Further, since we observed this positive effect during chronic phase, it is possible that the difference in the microenvironment of the acute and chronic lesions may play a role in governing the outcome of oligodendroglial Fgfr1 loss, such as astrocyte derived FGF2 which persists in the chronic phase. Apart from findings on FGF2, other FGF have been shown to play a role in demyelinating lesions of patients with MS. In vivo, FGF1 was localized on astrocytes, neurons, oligodendrocytes, as well as microglia and infiltrating T cells and B cells. FGFR1 and FGFR2 were significantly higher expressed in remyelinated lesions compared with control white matter (50). In vitro, FGF1 induced the leukemia inhibitory factor and chemokine CXCL8 in astrocytes, which caused recruitment of oligodendrocytes and promotion of remyelination (50). Furthermore, in vitro downregulation of myelin basic protein, myelin oligodendrocyte glycoprotein and myelin associated glycoprotein after exposure to FGF1 was observed. FGF9 was found in active demyelinating lesions in patients with MS, and was made responsible for tissue damage, lesion formation through activating proinflammatory cytokine and chemokine production in the CNS. In vitro FGF9 inhibited myelination and remyelination (41). The nogo receptor-interacting protein (LINGO-1) is a key inhibitor of oligodendrocyte precursor cell differentiation and myelination in vitro (47) and in vivo (46). Deletion of LINGO-1 causes a less severe EAE disease course, improved axonal integrity and promoted axon remyelination (46). An anti-LINGO-1 antagonist applied before and after onset of symptoms results in less disease activity in acute EAE (46). LINGO-1 can interact with the TrkB receptor to inhibit phosphorylation induced by BDNF thereby suppressing oligodendrocyte differentiation (18). In our study impaired signaling via oligodendroglial FGFR1 was associated with downregulated LINGO-1 expression in the chronic phase of EAE suggesting that this inhibitor is controlled by oligodendroglial FGFR1 as well. The finding that signaling via oligodendroglial FGFR1 modulates clinical disease activity and inflammation gives the opportunity for a targeted molecular therapy. Selective and multikinase Brain Pathology •• (2017) ••–•• C 2017 International Society of Neuropathology V


FGFR inhibitors are currently beeing tested in patients with genetically selected tumors with promising efficacy in some of these trials (12). Tyrosine kinase inhibitors are active against kinases such as VEGFR or PDGFR. Luzitanib, a VEGFR/FGFR inhibitor has been applied successfully in FGFR1-amplified breast tumors (12). In addition, monoclonal antibodies such as FP-1039 binding to the extracellular domain of FGFRs are used in clinical testings (12). Signaling via oligodendroglial FGFR1 also modulates LINGO-1 expression suggesting that LINGO-1 inhibition could be a promising approach to decrease the effects of the receptor. A LINGO-1 antagonist has been used succesfully to promote remyelination in EAE (46) and inhibition of LINGO-1 is has been tested in a Phase I clinical MS trial (NCT01244139). In summary, impaired signaling via oligodendroglial FGFR1 has numerous beneficial effects on MOG35–55-induced EAE. Ablation of oligodendroglial FGFR1 resulted in a less severe clinical disease course, less myelin loss and axonal degeneration, and reduced cellular inflammation. These changes are accompanied by an increase of ERK/AKT phosphorylation as well as increased expression of BDNF and TrkB. Furthermore, there is an association between oligodendroglial FGFR1 and the expression of LINGO-1. Considering that FGFR inhibitors are used in cancer trials, the oligodendroglial FGFR1 pathway may provide a new target for therapy in multiple sclerosis.

ACKNOWLEDGMENTS The study was funded by Merck Serono GmbH, Darmstadt, Germany. We thank Salar Kamali and Liza Mittal for help with animal experiments.

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