CXCR2 antagonism promotes oligodendrocyte precursor cell differentiation and enhances remyelination in a mouse model of multiple sclerosis

Journal Pre-proof CXCR2 antagonism promotes oligodendrocyte precursor cell differentiation and enhances remyelination in a mouse model of multiple sclerosis

Lu Wang, Hanyu Yang, Caixia Zang, Yi Dong, Junmei Shang, Jiajing Chen, Yue Wang, Hui Liu, Zihong Zhang, Heng Xu, Xiuqi Bao, Dan Zhang PII:

S0969-9961(19)30305-5

DOI:

https://doi.org/10.1016/j.nbd.2019.104630

Reference:

YNBDI 104630

To appear in:

Neurobiology of Disease

Received date:

31 May 2019

Revised date:

30 August 2019

Accepted date:

1 October 2019

Please cite this article as: L. Wang, H. Yang, C. Zang, et al., CXCR2 antagonism promotes oligodendrocyte precursor cell differentiation and enhances remyelination in a mouse model of multiple sclerosis, Neurobiology of Disease(2019), https://doi.org/10.1016/ j.nbd.2019.104630

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© 2019 Published by Elsevier.

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CXCR2 antagonism promotes oligodendrocyte precursor cell differentiation and enhances remyelination in a mouse model of multiple sclerosis Lu Wang, Hanyu Yang, Caixia Zang, Yi Dong, Junmei Shang, Jiajing Chen, Yue Wang, Hui Liu, Zihong Zhang, Heng Xu* [email protected], Xiuqi Bao* [email protected], Dan Zhang* [email protected]

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State Key Laboratory of Bioactive Substrate and Function of Natural Medicine, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union

Corresponding authors at: State Key Laboratory of Bioactive Substrate and

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*

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Medical College, 1 Xian Nong Tan Street, Beijing 100050, China

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Function of Natural Medicine, Institute of Materia Medica, Chinese Academy of

Abstract

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Beijing 100050, China.

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Medical Sciences and Peking Union Medical College, 1 Xian Nong Tan Street,

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Multiple sclerosis (MS) is a chronic autoimmune demyelinating disease characterized by the autoimmune attack of oligodendrocytes, leading to demyelination and progressive functional deficits. CXC chemokine receptor 2 (CXCR2) is recently reported to orchestrate the migration, proliferation and differentiation of oligodendrocyte precursor cells (OPCs), which implies its possible involvement in the demyelinating process. Here, we used a CXCR2 antagonist, compound 2, as a tool to investigate the role of CXCR2 in demyelination and the underlying mechanism. The primary cultured oligodendrocytes and cuprizone (CPZ)-intoxicated mice were applied in the present study. The results showed that compound 2 significantly

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promoted OPC proliferation and differentiation. In the demyelinated lesions of CPZ-intoxicated mice, vigorous OPC proliferation and myelin repair was observed after compound 2 treatment. Subsequent investigation of the underlying mechanisms identified that upon inhibition of CXCR2, compound 2 treatment upregulated Ki67, transcription factor 2 (Olig2) and Caspr expression, activated PI3K/AKT/mTOR

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signaling, ultimately promoted OPCs differentiation and enhanced remyelination. In conclusion, our results demonstrated that CXCR2 antagonism efficiently promoted

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OPC differentiation and enhanced remyelination in CPZ-intoxicated mice, supporting

diseases such as MS. demyelination;

differentiation

chemokine

receptor

2;

oligodendrocytes;

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Introduction

CXC

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Keywords:

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CXCR2 as a promising therapeutic target for the treatment of chronic demyelinating

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Multiple sclerosis (MS) is a chronic autoimmune demyelination disease of the central nervous system (CNS) characterized by sensory deficits and deteriorated motor coordination. The pathological hallmarks of MS are inflammation, immunization, demyelination and axonal injury, which appear throughout the CNS and are most easily recognized in white matter[1]. Although the etiology of MS remains unclear, myelin destruction is an acknowledged consequence of disease progression that results from the progressive impairment and ultimate death of oligodendrocytes[2, 3]. Current drugs that modify the course of MS, such as interferon beta, glatiramer acetate, mitoxantrone and fingolimod, can only improve symptoms after the attack[4],

Journal Pre-proof but failed as treatments against the progressive neurological deterioration of MS[5, 6]. Oligodendrocytes are myelin-forming cells in the CNS that play critical roles in brain development, neuron function and axon survival[7, 8]. In the early stage of MS, oligodendrocytes are recruited to injured areas, where they engage axons, form myelin and contribute to remyelination[9]. However, this process frequently stops or

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fails in lesions during the later period of MS[10]. Therefore, regulating oligodendrocyte precursor cell (OPC) proliferation and differentiation is crucial for demyelinating

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disorders. Drugs targeting OPC proliferation and differentiation have been identified

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as important therapeutic strategies and might ameliorate the devastating consequences

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of persistent demyelination[11].

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CXC chemokine receptor 2 (CXCR2), which is expressed by both inflammatory

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myeloid cells and oligodendrocytes in the CNS[12, 13], is a member of the chemokine receptor family and primarily modulates immune coordination and inflammation.

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CXCR2 also affects the intracellular processes that modulate a number of cellular events of oligodendrocytes, including migration, proliferation, and differentiation[14-16]. In addition, recent evidence has shown that a high level of CXCR2 is present in the chronic active lesions of MS patients[17], indicating that CXCR2 might be involved in MS pathogenesis. It has been reported that inhibition of CXCR2 could reduce regional recruitment of leukocytes to the spinal cord and attenuate inflammation and immune response in experimental autoimmune encephalomyelitis (EAE) and cuprizone (CPZ) -intoxicated mice[18][19]. Despite all the above-mentioned reports, the precise mechanisms of how CXCR2 antagonism contributes to MS treatment is still

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not fully elucidated and needs further investigation. To date, over a dozen distinct classes of small molecules were identified as potent CXCR2 antagonists and several were advanced into clinical trials mainly toward developing new therapeutics for neutrophil-mediated inflammatory diseases[20]. However, most of these studies were focused on inhibition of CXCR2 in periphery. Recently, the GSK research team reported novel biarylurea-based brain penetrant [21, 22]

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CXCR2 antagonists for the potential treatment of CNS diseases

. Among them,

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compound 2 exhibited nanomolar CXCR2 activity and was able to penetrate into the

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brain (brain/blood ratio = 0.62)[22]. In the present study, we synthesized compound 2

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and profiled it in a β-arrestin assay with an IC50 value of 8.5nM against CXCR2

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(Fig. S1). Therefore, we employed compound 2 as a CXCR2 antagonist and designed

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a set of experiments to elaborate the role of CXCR2 on demyelination and the underlying mechanisms. We found that CXCR2 antagonism significantly improved

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motor deficits and remyelination by promoting OPC proliferation and differentiation. Mechanistic studies illustrated that the remyelination promoted by CXCR2 antagonism was closely related to the activation of the PI3K/AKT/mTOR signaling pathway. Our results clearly showed by directly affecting myelin repair and remyelination, that CXCR2 is a novel therapeutic target for the treatment of demyelination disease such as MS. Materials and Methods 1. Reagents Compound 2 was synthesized in-house according to a published procedure

[21]

.

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Briefly,

2-(tert-butyl)-6-chlorobenzo[d]oxazole-7-sulfonyl

chloride

successively

underwent substitution by using 2-bromopropane under base conditions, oxidation by m-CPBA

and

hydrolysis

by

concentrated

H2SO4

to

generate

6-amino-3-chloro-2-(isopropylsulfonyl)phenol, and followed by a reaction with 1,2-dichloro-3-isocyanatobenzene to afford the target compound 2 as a white solid. 1H NMR (400 MHz, CDCl3) δ 11.00 (s, 1H), 8.35 (d, J = 8.8 Hz, 1H), 8.06 (dd, J =

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7.2, 2.6 Hz, 1H), 7.35 (s, 1H), 7.22 – 7.17 (m, 2H), 7.14 (s, 1H), 7.03 (d, J = 8.8 Hz,

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1H), 3.89 (dt, J = 13.7, 6.9 Hz, 1H), 1.37 (d, J = 6.8 Hz, 6H); HRMS (ESI): m/z (M +

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H+) calcd for C16H16O4N2Cl3S, 436.9891, found: 436.9890; Purity > 95%.

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2. Primary oligodendrocytes culture and treatments

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Mixed glial cultures were prepared from the cortex of postnatal day 1 Sprague

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Dawley rats (Hua Fu Kang Bioscience, China). The brain tissue was cleared of the meninges and blood vessels and dissociated using mechanical trituration. Then, the

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tissue was digested with 0.05 mg/mL trypsin, pipetted into PBS, and filtered through a 70 μm filter screen. Glia were plated on poly-lysine (Sigma, USA)-coated 75 cm2 flasks and cultured in DMEM (HyClone, USA) supplemented with 20% fetal bovine serum (Gibco), 100 U/mL penicillin and 100 μg/mL streptomycin (Pulilai, China) at 37°C in a humidified 5% CO2/95% air incubator. After 9 days of mixed culture, OPCs were sandwiched between microglia and astrocytes. The flasks were incubated on a shaker for 1 h at 200 r.p.m. at 37℃ to remove microglial cells. Then, the flasks were incubated with agitation overnight (18–20 h), and the cell suspension was collected to obtain OPCs. For all in vitro experiments, only cultures with ≥ 95% purity were used.

Journal Pre-proof OPCs were plated on poly-L-ornithine (Sigma, USA)-coated 75 cm2 flasks (2*104 cells/cm2). OPCs were first grown in proliferation media consisting of DMEM, 10 ng/mL platelet-derived growth factor AA (PDGFAA, Peprotech, USA), 10 ng/mL fibroblast growth factor (bFGF, Peprotech, USA), 1% fetal bovine serum, and N2 supplement (Invitrogen, USA). After two days of OPCs culture, the proliferation

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medium was replaced with the differentiation medium, which did not contain PDGFAA and bFGF. Additionally, OPCs were treated for 5 days with compound 2 at

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concentrations of 0.1 μM and 1 μM, and triiodothyronine (T3, Sigma, USA) at 10 μM

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was used as a positive control.

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3. Western blots

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Corpus callosum tissue, hippocampus tissue and primary cultured OLs were lysed

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in lysis buffer (Applygen, China). Samples containing 30 μg of protein per lane were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–

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PAGE) and transferred to PVDF membranes. The membranes were blocked in 5% skim milk-TBST (20 mM Tris–HCl at pH 7.5, 500 mM NaCl, 0.1% Tween 20) for 2 h. Then, the GAPDH, PDGFRα, Caspr, Ki67 (Abclonal, China), Olig2, mammalian target of rapamycin (mTOR), p-mTOR (Abcam, USA), MBP (BD Bioscience, USA), phosphoinositide 3-kinase (PI3K), p-PI3K, AKT and p-AKT (Santa Cruz, USA) primary antibodies were added in the same milk and incubated overnight at 4°C. Next, the blots were incubated with horseradish peroxidase-conjugated secondary antibody (Abclonal, China) in TBST for 2 h at room temperature. The blots were developed with an LAS3000 chemiluminescence system (Fujifilm, Tokyo, Japan), and the

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densities of the bands were determined using Gel-Pro Analyzer 4.0 software. 4. Immunofluorescence The cells were fixed with 4% paraformaldehyde for 30 min and penetrated with 0.1% Triton X-100 for 10 min. Then, they were incubated with blocking solution containing 3% normal goat serum for 2 h. Samples were incubated overnight at 4℃

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with Caspr, Ki67 (Abclonal, China), CXCR2, Olig2 (Abcam, USA), MBP and A2B5 (BD Bioscience, USA) antibodies (1:50). Afterward, the cells were washed three

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times in PBS and incubated for 1 h with FITC- or rhodamine-labeled secondary

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antibodies (Abcam, USA) (1:500). The cultures were washed in PBS and mounted

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with DAPI-Fluoromount-G (Gentihold, China) for 5 min to stain the nuclei. All

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washing and incubation procedures were performed at room temperature, unless

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stated otherwise, with 24-well plates and gentle shaking. Images were collected using a Leica DM5000B microscope (Leica, USA) and Leica Applications Suite software.

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5. Animals and treatments

Male C57/BL mice of 6-8 weeks old and weighing 22–25 g were supplied by Hua Fu Kang Bioscience. Mice were maintained in a 12-h light/dark cycle at 24°C in a room with a 60% relative humidity and received food and water ad libitum. Mice were adapted for 1 week to the conditions described above before experimentation. Mouse demyelination was induced by adding 0.2% CPZ (Sigma, USA) to the standard rodent chow for 6 weeks, with 10 mice in each group. Then, the mice were orally administered compound 2 at doses of 25, 50, and 100 mg/kg or the positive control drug cyclosporin A (CyA, Neocyspin, China) 70 mg/kg twice per day for 9

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consecutive days without CPZ. Control and CPZ-treated mice were administered 0.5% carboxymethylcellulose (CMC)-Na. All experimental procedures were performed in accordance with the guidelines of the Beijing Municipal Ethics Committee for the care and use of laboratory animals. 6. Assessment of neurological deficits

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Neurological deficits were evaluated by a classical 5-degree scale: score 0, mouse tail is tilted upward at an angle greater than 45 degrees above the ground; score 0.5,

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mouse tail is tilted upward at an angle less than 45 degrees above the ground; score 1,

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mouse tail is parallel to the ground; score 1.5, the root of the mouse tail is parallel to

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the ground and the tip of the tail hangs down; and score 2, the root of the mouse tail

design. 7. Rotarod test

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hang down. All evaluations were done by a researcher blind to the experimental

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The rotarod test, which requires mice to balance and walk on a rotating cylinder, was used to measure coordinated motor skills. The rotarod test was performed as previously described[23]. Briefly, mice were positioned on the rotarod, which then revolved at 30 rpm for up to 120 sec. The rotarod automatically recorded the time point when the animal fell off the rod, which was designated as the latency. The mice were tested three times, and the latency was recorded for each test. Between each trial, the animals rested for 1 h. All evaluations were done by a researcher blind to the experimental design. 8. Luxol fast blue (LFB) staining

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Mice were anesthetized with pentobarbital and transcardially perfused with saline and then with cold 4% paraformaldehyde in 0.1 M phosphate buffer at pH 7.4. After the brains were fixed overnight, the brains were frozen, coronally sectioned to 8 μm thickness and mounted onto slides. The extent of the demyelination of the corpus callosum was evaluated using Luxol fast blue (LFB) staining. Briefly, silane-coated

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slides were rehydrated in a graded series of alcohol to 95% and incubated in LFB solution (0.01%) overnight at 60°C. Thereafter, sections were differentiated in a

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lithium carbonate solution (0.05%). Afterward, the sections stained with LFB were

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observed using a light microscope (NIKON 600, Japan).

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9. Statistical analyses

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Data are expressed as the mean ± S. D. and were analyzed by one-way ANOVA

statistically significant.

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Results

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followed by Dunnett’s post hoc test with SPSS 18.0. P < 0.05 was considered

1. Compound 2 treatment promoted primary cultured OPC differentiation. In chronic demyelinating diseases, the failure of remyelination is usually caused by a decrease in the efficiency of OPC differentiation. PDGFRα is a receptor located on the surface of OPCs in the precursor stage, making it a negative marker of remyelination[9]. A2B5 is another marker of immature oligodendrocytes. In the present experiment, the incubation of compound 2 with primary cultured OPCs remarkably decreased the expression of PDGFRα in a dose-dependent manner (Fig. 1A, B). We then employed confocal microscopy to further validate the effects of compound 2 on OPC differentiation by observing A2B5 expression. Consistent with

Journal Pre-proof the above findings, the proportion of A2B5+ cells dramatically decreased with the presence of compound 2 (Fig. 1D). MBP is a protein that expresses only in mature oligodendrocytes and is frequently used as a marker for OPC maturation. The Western blot results showed that compound 2 treatment significantly increased the expression of MBP by approximately 1.5-fold relative to the control cells (Fig. 1A, C). The

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proportion of MBP+ cells was also increased, which was detected by immunofluorescence. Consistently, compound 2 treatment induced an increased cell

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body size, and denser networks of MBP+ cells compared with control cells (Fig. 1E).

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The promotion of OPC differentiation induced by compound 2 was equivalent to that

25]

. It promotes the expression of differentiation gene and

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differentiation[24,

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of T3, an agent that was required in vitro for the normal timing of oligodendrocyte

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myelination of primary cultured OPCs [26]. Therefore, T3 has become the most widely used positive control in oligodendrocyte differentiation research in vitro. These

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findings suggested that CXCR2 antagonism could promote the differentiation of OPCs to oligodendrocytes.

2. Compound 2 treatment upregulated Ki67, Olig2 and Caspr expression in primary cultured oligodendrocytes. There are many proteins that reported to regulate the proliferation and differentiation of OPCs, of which Ki67[27], Olig2[9] and Caspr[28] are known as critical regulators. We therefore investigated whether these proteins contributed to OPC maturation induced by CXCR2 antagonism. As expected, treatment with compound 2 markedly upregulated Ki67, Olig2 and Caspr expression detected by Western blot and

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immunofluorescence in primary cultured oligodendrocytes (Fig. 2). These data provided evidences that inhibition of CXCR2 activity could promote the proliferation and differentiation of OPCs, and the formation of myelin. 3. Compound 2 treatment improved motor behavior and alleviated the neurological impairment of CPZ-intoxicated mice.

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As CXCR2 was involved in OPC proliferation and differentiation, we therefore further validated whether CXCR2 antagonism could improve the demyelination in a

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CPZ mouse model. Mice challenged with CPZ for 6 weeks exhibited dysfunction of

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motor behavior and neurological deficits (Fig. S2). Based on previous experiments[21],

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compound 2 was intragastrical administered at 25, 50, 100mg/kg to mice for 9

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consecutive days. The motor behavior of the mice was measured at the 5th and 9th day

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of compound 2 treatment. The results showed that the time of mice treated with compound 2 staying on the rod significantly increased in a dose-dependent manner at

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the 5th day (Fig. 3A). Administration of compound 2 to mice for 9 days further alleviated the symptoms of CNS injury, as indicated by markedly improved motor behavior and neurological function (Fig. 3C, D). These in vivo data strongly suggested that CXCR2 antagonism is effective in improving the pathological changes caused by demyelination. 4. Compound 2 treatment promoted the remyelination in the corpus callosum and hippocampus of CPZ-intoxicated mice. We next investigated the remyelination properties of compound 2 in the CPZ-induced remyelination mouse model. The results showed that there was robust

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demyelination in the corpus callosum upon CPZ challenge, detected by the staining of myelin with LFB. When CXCR2 was inhibited by compound 2 treatment, the amount of LFB-stained myelin was significantly increased in the corpus callosum (Fig. 4A, B). The remyelination effects were further evaluated in Western blot assay by analyzing the proliferation and differentiation markers, PDGFRα and MBP. As shown in Figure

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4, PDGFRα expression in the corpus callosum and hippocampus in compound 2 treated mice was significantly reduced in a dose-dependent manner. The same results

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were obtained in detection of MBP expression in the corpus callosum and

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hippocampus. It was worth noting that the capacity of compound 2 to promote

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remyelination was more potent than that of the positive control drug CyA, which is an

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immunosuppressive agent and commonly used for treatment of acute exacerbations in

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early multiple sclerosis[29]. It has also demonstrated inhibition of demyelination in a study of demyelinated animal models

[30]

. Combined with the above data, these

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observations indicated that CXCR2 antagonism could promote remyelination and myelin repair in the mouse model of demyelination. 5. Compound 2 treatment upregulated Ki67, Olig2 and Caspr expression in the corpus callosum and hippocampus of CPZ-intoxicated mice. We also evaluated the effects of compound 2 on the expression of Ki67, Olig2 and Caspr in CPZ-induced mouse demyelination model. Consistent with the in vitro results, mice in compound 2 group exhibited higher expression of Ki67, Olig2 and Caspr in both the corpus callosum and hippocampus (Fig. 5). Similarly, the efficacy of compound 2 on Ki67, Olig2 and Caspr expression was better than that of the positive

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drug CyA. These results suggested that CXCR2 antagonism effectively promoted the proliferation and differentiation of OPCs in CPZ mice. 6. Compound 2 treatment affected PI3K/AKT gene transcription in CPZ-intoxicated mice. To clarify the mechanisms of CXCR2 antagonism in remyelination, we performed a

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transcriptomics study to examine the differentially expressed genes after CPZ intoxication and compound 2 treatment. As shown in Figure 6A, red represents high

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expression of the gene and green represents low expression of the gene. Comparing

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the color of each group on the same plane could reflect the expression of the same

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gene under different treatments. The results showed that compared to control mice,

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the expression of each gene was greatly impacted by CPZ challenge, which was partly

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reversed by compound 2 treatment. The results of the heat map might shed a light on the mechanism of compound 2 on CPZ-intoxicated mice. The Venn diagram

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illustrated that 39 genes were affected by both CPZ challenge and compound 2 treatment (Fig. 6B). The transcriptomics data were further analyzed and revealed that the PI3K/AKT signaling was one of the most affected pathways after CPZ challenge and compound 2 treatment (Fig. 6C, D). 7. Compound 2 treatment promoted remyelination by activating the PI3K/AKT/mTOR signaling pathway. We then validated the effect of compound 2 on the activity of PI3K and AKT in primary cultured oligodendrocytes and CPZ-intoxicated mice. Treatment with compound 2 significantly increased the phosphorylation of PI3K and AKT both in

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vitro and in vivo, indicating that the PI3K/AKT signaling pathway was activated by CXCR2 antagonism (Fig 7A, B, D, E, G, H). In addition, we examined the activation of mTOR, a critical downstream signaling of AKT activation and is closely related to cell proliferation and differentiation. As shown in Figure 7 C, F and I, treatment with compound 2 significantly increased the phosphorylation of mTOR in primary cultured

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oligodendrocytes, as well as in the corpus callosum and the hippocampus of CPZ-intoxicated mice, indicating that mTOR was activated upon inhibition of

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CXCR2. These findings suggested that the CXCR2 antagonism could activate the

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PI3K/AKT/mTOR signaling pathway. This pathway may be involved in the OPC

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proliferation and differentiation induced by CXCR2 antagonism.

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Discussion

Currently, the therapeutic strategies for MS mainly focus on modifying the disease

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course by anti-inflammatory and immune-based therapies. Due to the critical role of

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demyelination in the pathogenesis of MS, more researchers are trying to find new treatments which lead to potential remyelination. In the present study, we found that CXCR2 antagonism could promote the proliferation and differentiation of OPCs. In addition, CXCR2 antagonism significantly improved motor behavior and neurological function in CPZ-induced demyelinated mouse model through myelin repair and remyelination. CXCR2 is a receptor of CXC chemokines and belongs to the G-protein-coupled receptor (GPCR) superfamily. Studies have shown that the expression of CXCR2 increases in the brains of rodents and humans with chronic demyelinating disease, indicating that CXCR2 is somehow related to the pathology of demyelination. It was

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reported that mice lacking CXCR2 had impaired autoimmune system and were resistant

to

demyelination[12,31].

In

focal

lysophosphatidylcholine-induced

demyelinating lesions, the localized inhibition of CXCR2 was found to reduce the release of cytokines and enhance remyelination[16]. Although the important role that CXCR2 plays on remyelination have been revealed by several reports, current studies

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did not fully elucidate the precise mechanisms of how CXCR2 antagonism promotes remyelination. Compound 2 is a CNS penetrant CXCR2 antagonist, making it a good

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tool to study the influence of CXCR2 on the demyelination in CNS. And in our

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experiments, we found that compound 2 can inhibit the expression of CXCR2 both in

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vivo and in vitro (Fig. S3). Our present study gave evidences that CXCR2 antagonism

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by compound 2 treatment markedly promoted the maturation of primary cultured

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OPCs to oligodendrocytes and thus enhanced myelin repair in CPZ-intoxicated mouse model, further verifying that CXCR2 antagonism contributes to remyelination. We

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also clarified the underlying mechanism that PI3K/AKT/mTOR pathway plays a critical role in the proliferation and differentiation of OPCs following CXCR2 antagonism.

In the present study, we employed primary cultured OPCs and demyelinating mouse model to investigate the remyelination effect of CXCR2. OPCs, a subtype of glial cells in the CNS, are precursors to oligodendrocytes. They proliferate and migrate throughout the CNS during late embryonic development, and then differentiate into mature myelinating oligodendrocytes[32]. Oligodendrocytes are well known as myelin-forming cells with crucial role in facilitating the rapid conduction of

Journal Pre-proof neuronal action potentials[33] and supporting axonal survival[34]. Primary culture of OPCs provides powerful means to characterize their differentiation, properties and potential for myelin repair. Our present study showed that compound 2 treatment could effectively promote the differentiation and mature of primary cultured OPCs to oligodendrocytes. The CPZ mouse model is a well-characterized model of demyelination that closely resembles the chronic and progressive clinical form of

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demyelination disease[35]. CPZ-challenged mice exhibit several clinical deficits, such

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as progressive impaired motor coordination, paralysis associated with axonal loss and

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demyelination[36]. CPZ could also induce oxidative/nitrative stress, causing

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mitochondrial impairment, inflammation, oligodendrocytes death and myelin loss.

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Our in vivo data demonstrated that compound 2 treatment significantly ameliorated

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the clinical symptoms and repaired nerve damage of CPZ-intoxicated mice. As CXCR2 is closely correlated with immunity and inflammation, we also examined the

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capacity of compound 2 to suppress neuroinflammation in CPZ-intoxicated mice. We found that compound 2 treatment significantly inhibited the activation of microglia and astrocytes, indicating its potent anti-neuroinflammatory capacity (Fig. S4). Our results indicated that inhibition of CXCR2 not only promoted OPC differentiation, but also attenuated inflammation and immune response, which might further have an influence on OPC differentiation. To further validate the effect of CXCR2 antagonism on remyelination, we investigated the change of proteins including Ki67, Olig2 and Caspr both in vitro and in vivo. In MS lesions, myelin repair requires the proliferation of OPCs to provide

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enough cells to form myelin. Ki67 is a nuclear protein associated with cellular proliferation[27]. Olig2 is a transcription factor that is well known for determining motor neuron differentiation[28, 37]. Caspr is a membrane protein found in the neuronal membrane and is mainly expressed in the paranodal section of the axon during myelination[28]. Therefore, promoting the expression of Ki67, Olig2 and Caspr is very

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important in remyelination. The present data illustrated that compound 2 treatment upregulated the expression of these proteins both in vivo and in vitro, which ensured

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that CXCR2 antagonism could promote the regeneration of myelin.

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To elucidate how CXCR2 antagonism affected proliferation and differentiation

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of OPCs, we performed a transcriptomic analysis in CPZ-challenged mice and found

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that the PI3K/AKT signaling was the most affected pathway by compound 2

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treatment. The transcriptomic results were further validated in oligodendrocytes and CPZ-intoxicated mice. The PI3K/AKT pathway could target many signaling

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components that control almost every aspect of physiological and pathological cellular functions. mTOR is a direct substrate of the AKT kinase that promotes protein translation, growth, angiogenesis, and metabolism. In developing oligodendrocytes, mTOR signaling is required for radial sorting of axons, lipid biosynthesis, and myelin growth, which is activated before the onset of myelination and declines as myelination starts[38]. Consistent with these studies, the present data demonstrated that mTOR activation contributed to OPC maturation upon inhibition of CXCR2. Therefore, our in vivo and in vitro investigation further validated PI3K/AKT/mTOR signaling as a functionally important intracellular signaling cascade that regulates

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OPCs proliferation and differentiation and promotes CNS remyelination after CXCR2 antagonism[39, 40]. In conclusion, the present study demonstrated that the CXCR2 antagonism could improve the motor behavior and alleviate neurological impairment in CPZ-intoxicated mice. This protective effect may be related to its ability to promote OPC proliferation

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and differentiation. Further mechanistic studies showed that CXCR2 antagonism promoted remyelination through the activation of PI3K/AKT/mTOR signaling

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pathway. Collectively, our data support CXCR2 as a promising therapeutic target for

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chronic demyelinating diseases by directly targeting OPC maturation.

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Competing interests

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The authors declare that there are no competing interests. Ethical approval

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All experimental procedures were performed in accordance with the guidelines

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of the Beijing Municipal Ethics Committee for the care and use of laboratory animals. Abbreviations

MS, Multiple sclerosis; CXCR2, CXC chemokine receptor 2; OPCs, oligodendrocyte precursor cells; CPZ, cuprizone; PDGFRα, platelet-derived growth factor receptor α; MBP, myelin basic protein; Olig2, transcription factor 2; CNS, central nervous system; EAE, experimental autoimmune encephalomyelitis; LPC, lysophosphatidylcholine;

mTOR,

mammalian

target

of

rapamycin;

PI3K,

phosphoinositide 3-kinase; PDGFAA, platelet-derived growth factor AA; bFGF, fibroblast growth factor; CyA, cyclosporin A; CMC, carboxymethylcellulose; LFB, Luxol fast blue; GPCR, G-protein-coupled receptor.

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Acknowledgements This work was supported by grants from National Sciences Foundation of China (81630097, 81773718, 21772235), CAMS Innovation Fund for Medical Sciences (No. 2016-I2M-3-011), National Major Scientific and Technological Special Project for “Significant New Drugs Development” during the Thirteen Five-year Plan Period

CAMS

The

Fundamental

Research

Funds

for

the

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(2018RC350002).

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(2018ZX09711001-008-005, 2018ZX09711001-003-005, 2018ZX09711001-003-020),

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lP

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Figures

Central

Universities

Journal Pre-proof Figure 1. Effects of CXCR2 antagonism on the expression of PDGFRα, A2B5 and MBP in primary cultured oligodendrocytes. Primary cultured OPCs were incubated with Compound 2 at 0.1, 1 μM or T3 at 10 μM for five consecutive days. (A)Western blot bands image of PDGFRα and MBP. (B) Statistical analysis of PDGFRα expression. (C) Statistical analysis of MBP expression. A representative immunoblot

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from four experiments was shown. (D）Confocal analysis of A2B5 expression. (E) Confocal analysis of MBP expression. A representative image from four experiments

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was shown. A2B5 (green), MBP (green). DAPI (blue). Results were expressed as

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mean ± SD. *P < 0.05, **P < 0.01, *** P < 0.001 vs. Control.

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Figure 2. CXCR2 antagonism up-regulated the expression of Ki67, Olig2 and Caspr in primary cultured oligodendrocytes. Primary cultured OPCs were incubated with Compound 2 at 0.1 and 1 μM or T3 at 10 μM for five consecutive days. (A) Western blot analysis of Ki67 expression. (B) Confocal analysis of Ki67 expression. (C) Western blot analysis of Olig2 expression. (D) Confocal analysis of Olig2 expression. (E) Western blot analysis of Caspr expression. (F) Confocal analysis of Caspr expression. A representative image from four experiments was shown. MBP (green). Ki67 (red), Olig2 (red), Caspr (red). DAPI (blue). Results were expressed as mean ±

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SD. *P < 0.05, **P < 0.01, *** P < 0.001 vs. Control.

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Figure 3. CXCR2 antagonism treatment improved motor behavior and alleviated neurological impairment in MS mice. Mice were intoxicated with CPZ for 6 weeks

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and then administrated with Compound 2 for 9 days. (A) Motor behavior of mice treated with Compound 2 for 5 days. (B) Neurological function score of mice treated with Compound 2 for 5 days. (C) Motor behavior of mice treated with Compound 2 for 9 days. (D) Neurological function score of mice treated with Compound 2 for 9 days. Results were expressed as mean ± SD. n = 10. *

###

P < 0.001 vs. Control mice.

P < 0.05, **P < 0.01, ***P < 0.001 vs. CPZ-intoxicated mice.

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Figure 4. CXCR2 antagonism promoted remyelination in CPZ intoxicated mice. Mice were intoxicated with CPZ for 6 weeks, then treated with Compound 2 at doses of 25, 50 and 100 mg/kg for 9 consecutive days. (A) LFB staining of corpus callosum. Top, bar=125 μm, Bottom, bar=50 μm. (B) Quantification of myelinated area percentage in corpus callosum. (C, D, E) Western blot bands and analysis of PDGFRα and MBP expression in the corpus callosum of mice. (F, G, H) Western blot bands and analysis of PDGFRα and MBP expression in the hippocampus of mice. A representative image from four experiments was shown. Results were expressed as mean ± SD. # P < 0.05,

###

P < 0.001 vs. Control mice. *P < 0.05,

CPZ-intoxicated mice.

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P < 0.01,

***

P < 0.001 vs.

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Figure 5. CXCR2 antagonism up-regulated the expression of Ki67, Olig2 and Caspr

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expression in corpus callosum and hippocampus of CPZ-intoxicated mice. Mice received CPZ intoxication for 6 weeks and were administrated with 25, 50 and 100

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mg/kg compound 2 or 70mg/kg CyA for 9 consecutive days.

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(A, B, C) Western blot assay of Ki67, Olig2, Caspr expression in corpus callosum of mice. (D, E, F) Western blot assay of Ki67, Olig2, Caspr expression in hippocampus of mice. A representative image from four experiments was shown. Results were expressed as mean ± SD. # P < 0.05, ###P < 0.001 vs. Control mice. *P < 0.05, **P < 0.01 vs. CPZ-intoxicated mice.

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Figure 6. Transcriptomics analysis of CPZ intoxicated mice after CXCR2 antagonism

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treatment. Mice received CPZ intoxication for 6 weeks and then treated with

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100mg/kg compound 2 for 9 consecutive days. (A) Heat map of control group mice, CPZ-intoxicated mice and compound 2 treated mice. (B) Venn diagram of CPZ-intoxicated mice vs. control mice and CPZ-intoxicated mice vs. Compound 2-treated mice. (C) Changed signaling pathways between CPZ-intoxicated mice and control mice. (D) Changed signaling pathways between CPZ-intoxicated mice and Compound 2-treated mice.

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Figure 7. CXCR2 antagonism activated PI3K/AKT/mTOR signaling pathway both in vitro and in vivo. Primary cultured OPCs were incubated with compound 2 at 0.1 and 1 μM for 5 consecutive days. (A, B, C) Western blot analysis of p-PI3K, p-AKT, p-mTOR expression. Mice were intoxicated by CPZ for 6 weeks and treated with 25, 50 and 100 mg/kg compound 2 or 70mg/kg CyA for 9 consecutive days. (D, E, F) Western blot analysis of p-PI3K, p-AKT, p-mTOR expression in corpus callosum of

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mice. (G, H, I) Western blot analysis of p-PI3K, p-AKT, p-mTOR expression in hippocampus of mice. A representative image from four experiments was shown. Results were expressed as mean ± SD. #P < 0.05, ##P < 0.01 vs. Control mice. *P < 0.05, *** P < 0.001 vs. CPZ-intoxicated mice.

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Highlights 1 Compound 2 promotes OPCs proliferation and differentiation. 2 Compound 2 enhances remyelination in a MS mouse model.

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3 Inhibition of CXCR2 activates PI3K/AKT/mTOR signaling pathway.