# PHYSIOLOGY OF MYELIN FORMING CELLS, FROM MYELINATION TO NEURAL MODULATORS

EDITED BY : Mauricio Antonio Retamal, Fernando C. Ortiz and Marion Baraban PUBLISHED IN : Frontiers in Cellular Neuroscience

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ISSN 1664-8714 ISBN 978-2-88963-274-9 DOI 10.3389/978-2-88963-274-9

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# PHYSIOLOGY OF MYELIN FORMING CELLS, FROM MYELINATION TO NEURAL MODULATORS

Topic Editors:

Mauricio Antonio Retamal, Universidad del Desarrollo, Chile Fernando C. Ortiz, Universidad Autónoma de Chile, Chile Marion Baraban, University of Edinburgh, United Kingdom

Citation: Retamal, M. A., Ortiz, F. C., Baraban, M., eds. (2019). Physiology of Myelin Forming Cells, from Myelination to Neural Modulators. Lausanne: Frontiers Media SA. doi: 10.3389/978-2-88963-274-9

# Table of Contents

*05 Editorial: Physiology of Myelin Forming Cells, From Myelination to Neural Modulators*

Marion Baraban, Mauricio A. Retamal and Fernando C. Ortiz

Tanmoyita Nayak, Jacqueline Trotter and Dominik Sakry

Yousra Laouarem and Elisabeth Traiffort

*58 Galectin-3-Mediated Glial Crosstalk Drives Oligodendrocyte Differentiation and (Re)myelination*

Laura Thomas and Laura Andrea Pasquini

*74 Motor Exit Point (MEP) Glia: Novel Myelinating Glia That Bridge CNS and PNS Myelin*

Laura Fontenas and Sarah Kucenas

Zoe J. Looser, Matthew J. P. Barrett, Johannes Hirrlinger, Bruno Weber and Aiman S. Saab

Domiziana Ortolani, Blandine Manot-Saillet, David Orduz, Fernando C. Ortiz and Maria Cecilia Angulo

*124 Adenosine Actions on Oligodendroglia and Myelination in Autism Spectrum Disorder*

Hai-Ying Shen, Nanxin Huang, Jesica Reemmer and Lan Xiao

*136 Hypomyelination and Oligodendroglial Alterations in a Mouse Model of Autism Spectrum Disorder*

Mariana Graciarena, Araceli Seiffe, Brahim Nait-Oumesmar and Amaicha M. Depino

*147 Connexin and Pannexin-Based Channels in Oligodendrocytes: Implications in Brain Health and Disease*

Sebastián Vejar, Juan E. Oyarzún, Mauricio A. Retamal, Fernando C. Ortiz and Juan A. Orellana

*157 Role of Connexin-Based Gap Junction Channels in Communication of Myelin Sheath in Schwann Cells*

Bruno A. Cisterna, Pablo Arroyo and Carlos Puebla

*165 Pericytes Favor Oligodendrocyte Fate Choice in Adult Neural Stem Cells* Maria Elena Silva, Simona Lange, Bryan Hinrichsen, Amber R. Philp, Carolina R. Reyes, Diego Halabi, Josselyne B. Mansilla, Peter Rotheneichner, Alerie Guzman de la Fuente, Sebastien Couillard-Despres, Luis F. Bátiz, Robin J. M. Franklin, Ludwig Aigner and Francisco J. Rivera

# Editorial: Physiology of Myelin Forming Cells, From Myelination to Neural Modulators

Marion Baraban1 , Mauricio A. Retamal 2,3 and Fernando C. Ortiz 4 \*

1 Mechanics of Neuronal Development Laboratory, Institut de Biologie Paris-Seine—Developmental Biology Laboratory, CNRS UMR7622, Sorbonne Université, UPMC Univ Paris 06, Paris, France, 2 Universidad del Desarrollo, Centro de Fisiologia Celular e Integrativa, Facultad de Medicina Clinica Alemana, Santiago, Chile, 3 Department of Cell Physiology and Molecular Biophysics, Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, TX, United States, 4 Mechanisms of Myelin Formation and Repair Laboratory, Instituto de Ciencias Biomédicas, Facultad de Ciencias de la Salud, Universidad Autónoma de Chile, Santiago, Chile

Keywords: oligodendrocyte, Schwann cell, oligodendrocyte precursor cell, myelin, myelin repair, axon physiology

#### **Editorial on the Research Topic**

#### **Physiology of Myelin Forming Cells, From Myelination to Neural Modulators**

Myelin is a specialized glial-derived membrane that enwraps axons enabling them for saltatory conduction of action potentials. It is organized in sheaths along the axonal compartment, separated by regions enriched in voltage-gated sodium channels—called nodes of Ranvier—where the action potential is regenerated. For many years myelin was considered a static structure, however, the idea of myelin coating as a rigid insulator of axons has been challenged in the last decades. Indeed, both central and peripheral myelin forming cells, namely oligodendrocytes and Schwann cells, interact actively with the surrounding neuronal and non-neuronal cells supporting many processes such as neuronal activity and metabolic supply. In addition, evidences provided by several research groups indicate that myelin synthesis is not only restricted to development, but it is also contributing to the remodeling in adulthood and the repair under pathological conditions. The aim of the present Research Topic is to group articles, reviews and technical reports exploring different aspects of the myelinating cells function in health and disease.

Intercellular communication between myelinating glial cells and the rest of the cellular components of the nervous tissue is essential to maintain CNS homeostasis. This function is mainly based on the ability of glial cells to be metabolic and electrically coupled between themselves and neurons, participating in trophic support and synchronization of the glial network. This communication is established by devoted channels constituted by connexins or pannexins which are permeables to ions and small molecules. In this regard, Cisterna et al. summarize the organization and the functional role of gap junction channels constituted by connexins in Schwann cells. They further report the consequences of the mutation of genes encoding connexin 32, leading to Charcot Marie Tooth X-linked form 1 disease (Cisterna et al.). Vejar et al. review the functional evidence of electrical and metabolic coupling through connexin- and pannexin-based channels within and between oligodendrocytes in the central nervous system. Afterward, the authors focus on the interaction and function of oligodendrocytes/astrocytes coupling, a well-known process that provides metabolic support to axons.

Subsequently, two technical reports illustrate the use of advanced techniques based on optogenetics and optical sensors, applied to the study of oligodendroglia physiology (Looser et al.; Ortolani et al.). Looser et al. optimize the combination of electrophysiological recordings and imaging of an ATP biosensor in the fully myelinated optic nerve. The ATP sensor was delivered

Edited and reviewed by: Arianna Maffei, Stony Brook University, United States

> \*Correspondence: Fernando C. Ortiz<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Cellular Neurophysiology, a section of the journal Frontiers in Cellular Neuroscience

> Received: 30 July 2019 Accepted: 08 October 2019 Published: 22 October 2019

#### Citation:

Baraban M, Retamal MA and Ortiz FC (2019) Editorial: Physiology of Myelin Forming Cells, From Myelination to Neural Modulators. Front. Cell. Neurosci. 13:475. doi: 10.3389/fncel.2019.00475

**5**

by reliable intravitreal injections of adeno-associated virus vectors which make this protocol adaptable to any genetically encoded sensor in any mutated mice. This method will permit to gain knowledge on the metabolism and homeostasis of this myelinated nerve in both physiological and pathological conditions. Moreover, Ortolani et al. established a protocol to perform photo-stimulation of GABAergic neurons in vivo, in the awake mouse pups. The authors confirmed that GABAergic synaptic inputs have no effect on OPC proliferation of the somatosensory cortex during development. Remarkably, this framework was optimized at an early post-natal stage, during the critical period of intense myelination. This might be readily adapted to investigate activity-dependent myelination occurring in other brain areas of the mouse.

The next section recapitulates some of the key aspects of myelination in the central nervous system. While it is known that oligodendrocytes myelinate specific axons, the complex rules governing the correct targeting of CNS myelination only begin to be understood. Almeida highlights the molecular and cellular codes that possibly target the myelination of the appropriate axon. After the initial step of targeted myelination, one can ask how the CNS myelination pattern changes during adulthood. Williamson and Lyons provide an overview of the recent findings on myelination dynamics and remodeling, specially they discuss the interesting results obtained by recent longitudinal imaging studies, concerning the apparent stability of the myelination patterns along the axon over lifetime.

Many human neurologic conditions have been associated with glial cells dysfunction or destruction in the CNS. Among them multiple sclerosis (MS) represents the archetypical example of a demyelinating disease, being the second cause of disabilities for the young adult population and more than 2 million people having the condition worldwide. By using the experimental autoimmune encephalomyelitis (EAE) MS model, Osorio-Barrios et al. examine the contribution of dopamine receptor D5 pathway on T-Cells, knowing to play a key role on the early stages of the demyelination insult in MS. It is known that after this initial demyelinated stage there is a spontaneous remyelination process that normally remains incomplete. This has led to the develop of many studies trying to shed light on the remyelination mechanisms, aiming to improve this spontaneous repair process. One strategy to increase the efficiency of remyelination might be to produce additional myelinating cells with the help of neighboring cells. In this regard, Silva et al. demonstrate that pericytes, central components of the vascular niche, influence neural stem cells toward the generation of oligodendrocytes. In this same line, to identify molecular factors involved in adult oligodendrogenesis is central in the search of myelin regeneration strategies. Laouarem and Traiffort highlight the importance of the neural morphogen hedgehog and its pathway during myelin repair, as well as providing an overview on the role of this signaling molecule during oligodendrogenesis at early developmental stages. Recent studies have pointed out oligodendroglia abnormalities as a contributing factor on complex pathologies such as the autistic spectrum disorder (ASD). To investigate in further details those abnormalities, Graciarena et al. characterize myelination and oligodendroglia population of different brain areas in a murine ASD model induced by prenatal exposition to valproic acid. In addition, Shen et al. summarize the contribution of adenosine signaling to both oligodendrocyte homeostasis and myelination in the context of the ASD.

Finally, a collection of reviews and articles concentrates on summarizing the miscellaneous functions of myelinating glial cells. Couve and Schmachtenberg describe the dental pulp system in human, focused on its innervation, the myelinating and the non-myelinating Schwann cells. Interestingly, they review the striking plasticity of those two types of Schwann cells preserving the dental pulp innervation, after an acute nerve injury caused by caries as well as the long-term changes related to aging. Fontenas and Kucenas introduce and depict an exceptional novel population of glial cells called motor exit point (MEP) glia. MEP glial cells are located at the transition zone in the interface between the peripheral and the central nervous systems, being able to myelinate motor neurons at that particular axonal region. The authors report the main characteristics of this recently described glia population based on their in vivo studies in zebrafish, emphasizing the importance of in vivo experimental models to study cellular dynamics (Fontenas and Kucenas). The next article examines one particular aspect of the microglia/oligodendroglia interaction: Thomas and Pasquini concentrate on the role of microglia derived-galectin-3 in oligodendrocyte differentiation and how this signaling molecule can foster remyelination. Finally, Nayak et al. bring our attention to the intracellular domain (ICD) of the NG2 proteoglycan, a molecular hallmark of oligodendrocyte progenitors, by showing that this domain induce mRNA translation mainly through the mTOR pathway. They suggest that the intracellular domain of NG2 could accelerate cell cycle kinetic in OPC and hypothesize that NG2 ICD might play a role also in tumor progression.

Summarizing, this Research Topic present original articles and updating reviews on the cellular and molecular mechanisms of the classical function associated with myelin forming cells, namely myelin formation and repair processes. But more interestingly, the topic also recapitulates evidence on, possibly unexpected, new functions (i.e., preserving dental pulp cytoarchitecture); new cellular interactions, such as the role of pericytes on remyelination; and even a new population of myelinating glia (i.e., MEP glia). By organizing this topic, we expect to provide a unique and broad overview on myelinating cells function in health and disease, encouraging to the community to develop more investigation on this very active field.

# AUTHOR CONTRIBUTIONS

MB and FO conceived the major ideas developed in the manuscript. MB wrote the original manuscript which was edited and corrected by all the authors. MB, MR, and FO approved the final manuscript.

#### ACKNOWLEDGMENTS

The editors are grateful to all the authors for their contributions to this topic. We also thank the reviewers of all the articles who kindly helped us to improve the content of this compelling Research Topic. We thank Rodrigo Varas for his helpful comments on the manuscript.

**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Baraban, Retamal and Ortiz. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Dopamine Receptor D5 Signaling Plays a Dual Role in Experimental Autoimmune Encephalomyelitis Potentiating Th17-Mediated Immunity and Favoring Suppressive Activity of Regulatory T-Cells

#### Francisco Osorio-Barrios 1 , Carolina Prado1 , Francisco Contreras 1 and Rodrigo Pacheco1,2 \*

1Laboratorio de Neuroinmunología, Fundación Ciencia & Vida, Santiago, Chile, 2Departamento de Ciencias Biológicas, Facultad de Ciencias de la Vida, Universidad Andres Bello, Santiago, Chile

#### Edited by:

Mauricio Antonio Retamal, Universidad del Desarrollo, Chile

#### Reviewed by:

Robert Weissert, University of Regensburg, Germany Marcella Reale, Università degli Studi G. d'Annunzio Chieti e Pescara, Italy

> \*Correspondence: Rodrigo Pacheco<EMAIL_ADDRESS><EMAIL_ADDRESS>
Received: 22 February 2018 Accepted: 14 June 2018 Published: 10 July 2018

#### Citation:

Osorio-Barrios F, Prado C, Contreras F and Pacheco R (2018) Dopamine Receptor D5 Signaling Plays a Dual Role in Experimental Autoimmune Encephalomyelitis Potentiating Th17-Mediated Immunity and Favoring Suppressive Activity of Regulatory T-Cells. Front. Cell. Neurosci. 12:192. doi: 10.3389/fncel.2018.00192 A number of studies have shown pharmacologic evidence indicating that stimulation of type I dopamine receptor (DR), favors T-helper-17 (Th17)-mediated immunity involved in experimental autoimmune encephalomyelitis (EAE) and in some other inflammatory disorders. Nevertheless, the lack of drugs that might discriminate between DRD1 and DRD5 has made the pharmacological distinction between the two receptors difficult. We have previously shown genetic evidence demonstrating a relevant role of DRD5-signaling in dendritic cells (DCs) favoring the CD4+ T-cell-driven inflammation in EAE. However, the role of DRD5-signaling confined to CD4+ T-cells in the development of EAE is still unknown. Here, we analyzed the functional role of DRD5-signaling in CD4+ T-cellmediated responses and its relevance in EAE by using a genetic approach. Our results show that DRD5-signaling confined to naive CD4+ T-cells exerts a pro-inflammatory effect promoting the development of EAE with a stronger disease severity. This pro-inflammatory effect observed for DRD5-signaling in naive CD4+ T-cells was related with an exacerbated proliferation in response to T-cell activation and to an increased ability to differentiate toward the Th17 inflammatory phenotype. On the other hand, quite unexpected, our results show that DRD5-signaling confined to Tregs strengthens their suppressive activity, thereby dampening the development of EAE manifestation. This anti-inflammatory effect of DRD5-signaling in Tregs was associated with a selective increase in the expression of glucocorticoid-induced tumor necrosis factor receptorrelated protein (GITR), which has been described to play a critical role in the expansion

**Abbreviations:** Ab, antibody; CFSE, carboxyfluorescein succinimidyl ester; CNS, central nervous system; CDn, cluster of differentiation n; CFA, complete Freund's adjuvant; CTLA4, cytotoxic T-lymphocyte antigen 4; DCs, dendritic cells; DR, Dopamine Receptor; DRDn, DR Dn; DRDnKO, DRDn Knockout; EAE, Experimental Autoimmune Encephalomyelitis; IFN-n, interferon n; IL-n, interleukin n; mAb, monoclonal antibody; MFI, mean fluorescence intensity; MOG, myelin oligodendrocyte glicoprotein; OVA, ovalbumin; pOT-II, peptide OVA323–339; pMOG, peptide MOG35–55; PE, phycoerythrin; PMA, phorbol 12-myristate 13-acetate; RORγt, RAR-related orphan receptor gamma; STAT3, signal transducer and activator of transcription 3; TGF-β, transforming growth factor β; Tregs, regulatory T-cells; Tbet, T-box transcrption factor encoded by TBX21; Thn, T helper n; WT, wild-type.

of Tregs. Our findings here indicate a complex role for DRD5-signaling in CD4+ T-cellsdriven responses potentiating early inflammation mediated by effector T-cells in EAE, but exacerbating suppressive activity in Tregs and thereby dampening disease manifestation in late EAE stages.

Keywords: Th17 response, T-cell activation, regulatory T-cells, dopamine, knockout mice, experimental autoimmune encephalomyelitis, neuroimmunology

#### INTRODUCTION

During last 15 years dopamine has emerged as a major regulator of inflammation. All five dopamine receptors (DRs, DRD1- DRD5) have been found to be expressed in immune cells, including dendritic cells (DCs) and T-cells among others, where they exert a complex regulation of immunity (Pacheco et al., 2014). The outcome of the dopamine effects in the immune response depends in many factors, including differential expression of DRs in the immune cells present in the inflamed tissue, the local levels of dopamine and the signaling coupled to and the affinity of the different DRs involved. Current evidence has indicated that stimulation of low-affinity DRs, for instance DRD1 and DRD2, are coupled to anti-inflammatory mechanisms, thereby dampening inflammation (Shao et al., 2013; Yan et al., 2015). Conversely, signaling triggered by high-affinity DRs, including DRD3 and DRD5, has been found consistently to promote inflammation (Prado et al., 2012, 2018; Contreras et al., 2016).

Multiple sclerosis (MS) is an inflammatory disorder involving a CD4+ T-cells driven response against the myelin sheath in the central nervous system (CNS). Previous evidence suggested that T-helper-1 (Th1) as well as T-helper-17 (Th17) cells are the phenotypes of autoreactive T-cells involved in MS and its mouse model, the experimental autoimmune encephalomyelitis (EAE; Dardalhon et al., 2008; Oukka, 2008). Importantly, DCs by producing interleukin 12 (IL-12) and IL-23, might induce the differentiation of naive CD4+ T-cells into Th1 and Th17 cells respectively (Haines et al., 2013; O'Connor et al., 2013). Consistent with the prominent role of dopamine in regulating adaptive immunity, relevant effects for dopaminergicsignaling have been found in MS (Prado et al., 2012, 2018). In this regard, CD4+ T-cells infiltrate the CNS where they can be exposed to dopamine and other neurotransmitters. In fact, striatal dopamine levels are significantly altered in mouse models of MS (Balkowiec-Iskra et al., 2007). Accordingly, emerging evidence has shown a relevant role of type I DRs (including DRD1 and DRD5) in the regulation of CD4+ T-cellmediated autoimmune response involved in human individuals undergoing MS and in EAE (Prado et al., 2013). In this regard, type I DRs have been described to be expressed in DCs (Nakano et al., 2008; Prado et al., 2012) and CD4+ T-cells from human and mouse origin (Kipnis et al., 2004; Cosentino et al., 2007; Kim et al., 2013; Franz et al., 2015).

The first work addressing the role of DRs in T-cell mediated immunity, showed that antagonizing type I DRs expressed on human DCs altered acquisition of functional phenotype by naive CD4+ T-cells increasing the Th1-to-Th17 ratio in co-cultures of DCs and T-cells in vitro (Nakano et al., 2008). Moreover, the same authors reported later that human DCs contain intracellular vesicles loaded with dopamine, which are released during Ag-presentation to naive CD4+ T-cells in vitro (Nakano et al., 2009). The in vivo relevance of these observations was evaluated by using a pharmacological approach in EAE (Nakano et al., 2008). In that study, the treatment of mice with the systemic administration of a type I DRs antagonist, (R)- (+)-7-Chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (SCH23390), reduced disease severity by impairing the Th17 response. However, this pharmacologic approach does not allow the discrimination between the effects mediated by DRD5 or DRD1 since the drug inhibits both type I DRs with similar affinities (Bourne, 2001). Furthermore, this study could not identify the cell-type responsible for the anti-inflammatory effect of type I DRs antagonism. Importantly, we have contributed to elucidate this mechanism using a genetic approach that allows us to determine the contribution of DRD5-signaling in DCs and its consequences in EAE development. In agreement with previous studies (Nakano et al., 2008), we demonstrate that DRD5-deficient mice exhibited delayed EAE progression with reduced severity compared with normal mice (Prado et al., 2012). In addition, we demonstrated that DRD5-deficiency confined to DCs resulted in exacerbated activation of signal transducer and activator of transcription 3 (STAT3), triggering impaired production of IL-12 and IL-23 with a consequent attenuation in Th1 and Th17 responses and reduced EAE manifestation (Prado et al., 2018). Nevertheless, the extent of attenuation of disease severity was stronger when EAE manifestation was compared between wild-type (WT) and general DRD5 knockout (DRD5KO) mice than when comparing WT animals with mice harboring DRD5-deficiency confined to DCs, suggesting that DRD5 expressed in other cell types different of DCs were also relevant in the regulation of EAE development (Prado et al., 2012).

Addressing the dopaminergic regulation of CD4+ T-cells mediated by type I DRs, pharmacological evidence has suggested that signaling triggered by these receptors in human T-cells in vitro favors the differentiation toward the Th2 phenotype (Nakano et al., 2009). Another study performed in a mouse model of ovalbumin (OVA)-induced acute asthma shows that pharmacologic antagonism of type I DRs impaired Th17 function and thereby ameliorated the allergic response (Gong et al., 2013). In addition, our previous results using a genetic approach have shown that DRD5-stimulation in mouse CD4+ T-cells favors T-cell activation and without detectable effects in Th1 differentiation when activated with Abs to CD3 and CD28 and a Th1-biased mixture of blocking Abs and cytokine milieu in vitro (Franz et al., 2015). Regarding the role of type I DRs on Tregs physiology, two independent groups have shown pharmacological evidence indicating that, by stimulating DRD1/DRD5, dopamine reduces the suppressive function of Tregs (Kipnis et al., 2004; Cosentino et al., 2007). This dopamine-mediated inhibitory mechanism involves a reduction in IL-10 and transforming growth factor β (TGF-β) production and diminished expression of cytotoxic T-lymphocyte antigen 4 (CTLA4), which participate in the cytokine-mediated and contact-mediated suppression exerted by Tregs, respectively. Together, these findings support an important role for type I DRs in the regulation of CD4+ T-cells physiology and reveal a relevant involvement of these receptors in autoimmunity. Nonetheless, the precise contribution of DRD1- and DRD5-signaling in the regulation of the CD4+ T-cell mediated autoimmune response associated to EAE remains unknown.

In this study, we analyzed the precise role of DRD5-signaling in the CD4+ T-cell response in vivo using a genetic approach. For this purpose, we dissected the role of DRD5 expressed in naive CD4+ T-cells and Tregs from that of DRD5 expressed in other hematopoietic cells in EAE. Afterward, the role of DRD5 expressed in CD4+ T-cells in inflammation was validated in other in vivo paradigms. Our results indicate that DRD5-signaling in CD4+ T-cells favors T-cell activation and contributes significantly to the differentiation toward the Th17-inflammatory phenotype in vivo. Furthermore, our data show unexpectedly that DRD5-signaling potentiates the suppressive activity of Tregs in vivo and in vitro. Thus, our findings indicate that DRD5-signaling in CD4+ T-cells plays a dual role in EAE development favoring early to the Th17-mediated inflammation and later contributing to the suppressive activity of Tregs.

# MATERIALS AND METHODS

#### Animals

C57BL/6 WT (cd45.2+/+) mice and B6.SJL-Ptprca (cd45.1+/+) mice in the C57BL/6 background were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). DRD5KO mice were kindly donated by Dr. David Sibley (Hollon et al., 2002), which were back-crossed for at least ten-generations in the C57BL/6 (cd45.2+/+) genetic background. OVA-specific OT-II transgenic mice expressing specific T-cell receptors (TCRs) for I-Ab /OVA323–339 in the C57BL/6 (cd45.2+/+) genetic background were gently donated by Dr. María Rosa Bono (Ureta et al., 2007). DRD5KO/OT-II mice were generated by crossing parental DRD5KO and OT-II mice. Six-to-10 weeks old mice were used in all experiments. All mice were maintained and manipulated according to institutional guidelines at the pathogen-free facility of the Fundación Ciencia and Vida.

#### Reagents

Monoclonal antibodies (mAbs) for flow cytometry: anti-FoxP3 (clone FJK-16S) conjugated to Phycoerythrin (PE)-Cyanine 7 (Cy7) and Allophycocyanin (APC), and anti-IFN-γ (clone XMG1.2) conjugated to PE-Cy7 were obtained from eBioscience (San Diego, CA, USA). Anti-CD4 (clone GK1.5) conjugated to APC and APC-Cy7; anti-CD25 (clone PC61) conjugated to Fluorescein isothiocyanate (FITC); anti-CD44 (clone IM7) conjugated to PE; anti-CD62L (clone MEL14) conjugated to APC-Cy7; anti-IL-17A (clone TC11-181710.1) conjugated to APC; anti-CD45.2 (clone 104) conjugated to PE-Cy7; anti-CD45.1 (clone A20) conjugated to Brilliant Violet (Bv)421; anti-CTLA4 (clone UC10-4139) conjugated to Bv421; anti-ICOS (clone C398.4A) conjugated to PE-Cy7; anti-CD39 (clone Duha59) conjugated to PE; anti-CD73 (clone Ty/11.8) conjugated to Peridin-Chlorophyll (PerCP); anti-CCR6 (clone 29-2L17) conjugated to APC; anti-glucocorticoidinduced tumor necrosis factor receptor-related protein (GITR; clone YGITR756) conjugated to PE-Cy7; anti-CD104 (clone 2E7) conjugated to Pacific Blue; anti-B220 (clone RA3-6B2) conjugated to PerCP; anti-TCRVα2 (clone B20.1) conjugated to PE and TCRVβ5 (clone MR9-4) conjugated to APC were purchased from Biolegend (San Diego, CA, USA). mAbs for Cell Culture: the followings mAbs low in endotoxins and azida free (LEAF) were purchased from Biolegend: anti-IL-4 (clone 11B11), anti-CD28 (clone 37.51), anti-CD3ε (clone 145-2C11), anti-IFN-γ (clone AN-18) and anti-IL-2 (clone JES6-1A12). Carrier-Free cytokines TGF-β3, TGF-β1, IL-6, IL-12, IL-23, IL-1β and IL-2 were purchased from Biolegend. Zombie AquaTM Fixable Viability dye detectable by flow cytometry was purchased from Biolegend. Phorbol 12-myristate 13-acetate (PMA) and ionomycin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cell Trace Carboxyfluorescein succinimidyl ester (CFSE), Brefeldin A and Fetal Bovine Serum (FBS) were obtained from Life Technologies (Carlsbad, CA, USA). The peptide derived from myelin oligodendrocyte glycoprotein (pMOG35–55) was purchased from GeneTel Laboratories (Madison, WI, USA). The peptide derived from the chicken ovalbumin (OVA323–339; OT-II peptide or pOT-II) was purchased from Genescript (Piscataway, NJ, USA). Freund's Complete Adjuvant (CFA) was purchased from Thermo Scientific. Bovine Serum Albumin (BSA) was purchased to Rockland (Limerick, PA, USA).

#### Generation of Bone Marrow Chimeras

Five-to-six weeks old B6.SJL-Ptprca (cd45.1+/+) mice were lethally irradiated with two doses of 550 rad each (source of Co60) separated for 3 h. One-day later, irradiated mice received the i.v. transference of bone marrow precursors (107 cells per mouse) obtained from WT (cd45.2+/+) or DRD5KO (cd45.2+/+) donor mice and maintained for 8 weeks with antibiotics (Trimethroprim and Sulfadoxine; Gorban 0.1% purchased from Merck Animal Health, Madison, NJ, USA) in the drinking water. The percentage of chimerism was determined as the percentage of CD45.2+ cells from the CD45+ leukocytes in peripheral blood, typically fluctuating between 80% and 85% (see Supplementary Figure S1). Bone marrow precursors were obtained from femurs and tibias as described before (Inaba et al., 1992).

#### EAE Induction and Evaluation

Mice were injected s.c. with 50 µg pMOG (Genetel Laboratories, Madison, WI, USA) emulsified in complete Freund's adjuvant (CFA; Invitrogen) supplemented with heat-inactivated Mycobacterium tuberculosis H37 RA (Difco Laboratories, Detroit, MI, USA). In addition, mice received 500 ng pertussis toxin (Calbiochem, La Jolla, CA, USA) i.p. on days 0 and 2. Clinical signs were assessed daily according to the following scoring criteria: 0, no detectable signs; 1, flaccid tail; 2, hind limb weakness or abnormal gait; 3, complete hind limb paralysis; 4, paralysis of fore and hind limbs; and 5, moribund or death. In some EAE experiments, total splenic CD4+ T-cells were purified by negative selection using magnetic beads-based kit (MACSr, Miltenyi Biotec, Bergisch Gladbach, Germany) and i.v. injected (5 × 106 cells per mouse) 24 h before EAE induction. In other EAE experiments, splenic naive (CD25−CD62L+CD44−) CD4+ T-cells were purified by cell-sorting and i.v. injected (3 × 106 cells per mouse) 24 h before EAE induction. In other experiments splenic Tregs (CD4+ CD25high T-cells) were purified by cell-sorting and i.v. injected (7 × 105 cells per mouse) 24 h before EAE induction.

### CD4+ T-Cell Isolation and Activation in Vitro

Total CD4+ T-cells were obtained by negative selection of splenocytes (MACSr). Naive (CD4+ CD25−) T-cell isolation was achieved by cell sorting using a FACS Aria II (BD, Franklin Lakes, NJ, USA), obtaining purities over 98%. Splenic DCs were purified using a CD11c+ magnetic selection kit (MACSr). All in vitro experiments were performed using complete IMDM medium (Life Technologies) 10% FBS. To assess proliferation, naive T-cells from OT-II mice were stained with CFSE (10 µM as indicated in figure legends) and cultured on a 5:1 (T-cells:DCs) ratio on U-bottom 96-well plates in the presence of OT-II peptide (OVA323–339, pOT-II; 200 ng/ml) for 2 or 3 days. T-cell activation was determined as IL-2 secretion in the co-culture supernatant by ELISA as previously described (González et al., 2013). The extent of T-cell proliferation was determined as the percentage of dilution of CFSE-associated fluorescence by flow cytometry.

# CD4+ T-Cell Differentiation in Vitro

Naive (CD4+CD62L+CD44−CD25−) T-cell were isolated by cell-sorting and stimulated with 50 ng/well of plate-bound anti-CD3 and 2 µg/mL soluble anti-CD28 Abs on flat-bottom 96-well plates (Thermo Scientific) for 5 days. To force the differentiation of the different Th phenotypes, naïve CD4+ T-cells were incubated in the conditions indicated above, in addition to a mixture of cytokines and blocking antibodies: Th1: 20 ng/mL IL-12, 10 ng/mL IL-2 and 5 µg/mL anti-IL-4; non-pathogenic Th17 (Th17np): during the first 2 days with 25 ng/mL IL-6, 5 ng/mL TGF-β1, 20 ng/mL IL-1β, 5 µg/mL anti-IL-4, 5 µg/mL anti-IL-2 and 5 µg/mL anti-IFN-γ and during the last 3 days with 25 ng/mL IL-6, 5 ng/mL TGF-β1; pathogenic Th17 (Th17p): during the first 2 days with 25 ng/mL IL-6, 5 ng/mL TGF-β3, 20 ng/mL IL-1β, 5 µg/mL anti-IL-4, 5 µg/mL anti-IL-2 and 5 µg/mL anti-IFN-γ and during the last 3 days with 25 ng/mL IL-6, 20 ng/mL IL-1β and 20 ng/mL IL-23. At different incubation times, cells were assessed for gene expression real time RT-PCR.

#### Quantitative RT-PCR

Total RNA extracted from cells using the Total RNA EZNA kit (Omega Bio-Tek, Norcross, GA, USA), was DNasedigested using the TURBO DNA-free kit (Ambion, Thermo Fisher Scientific) and 1 µg of RNA was used to synthesize cDNA utilizing M-MLV reverse transcriptase, according to manufacturer's instructions (Life Technologies). Quantitative gene expression analysis was performed using Brilliant II SYBR Green QPCR Master Mix (Agilent Technologies, Santa Clara, CA, USA), according to manufacturer's recommendations. Primers were used at a concentration of 0.5 µM. We used 40 PCR cycles as follows: denaturation 10 s at 95◦C, annealing 20 s at 60◦C and extension 20 s at 72◦C. Expression of target genes was normalized to Gapdh. The sequences of the primers used are indicated in **Table 1**.

# CD4+ T-Cell Proliferation and Differentiation in Vitro

To analyze T-cell proliferation in vivo, splenic CD4+ T-cells were isolated from OT-II mice, stained with 10 µM CFSE and then i.v. transferred (12.5 × 106 cells/mouse) into WT recipient mice. One-day later mice were i.p. immunized with

25 µg OVA or just injected with vehicle (PBS). Three-days after immunization mice were sacrificed and spleen cells were isolated, immunostained with fluorochrome-conjugated antibodies for B220, CD4, TCR-Vα2 and TCR-Vβ5 and then CFSE-associated fluorescence was analyzed in the B220− CD4+ TCR-Vα2 + TCR-Vβ5 + gate. The extent of T-cell proliferation was determined as the percentage of cells displaying dilution of CFSE-associated fluorescence or by the decrease of MFI in CFSE associated fluorescence in the CD4+ T-cell population expressing the transgenic OT-II TCR (TCR composed by Vα2 and Vβ5 chains) by flow cytometry. To determine the acquisition of inflammatory phenotypes by CD4+ T-cells in vivo, naive CD4+ CD25− CD44− CD62L+ T-cells were isolated from OT-II/cd45.2+/+ mice by cell-sorting and then i.v. transferred (105 cells/mice) into WT/cd45.1+/+ recipient mice. One-day later, mice were s.c. immunized with 100 µg of pOT-II in CFA and the inflammatory phenotype of CD4+ T-cells in the draining lymph nodes (inguinal lymph nodes) was analyzed 7 and 14 days after immunization by intracellular cytokine staining of IL-17 and IFN-γ .

#### Tregs Suppression Assay

Splenic naive CD4+ CD25− T-cells were isolated from OT-II mice by cell-sorting, stained with 5 µM CFSE and then co-cultured with splenic DCs on a 5:1 (T-cells:DCs) ratio on U-bottom 96-well plates in the presence of OVA (5 µM) for 5 days. Splenic CD4+ CD25high T-cells (nTregs) were isolated from OT-II mice by cell-sorting and added to the co-cultures at indicated ratios. The extent of proliferation of naive T-cells (effector T cells; Teffs) was determined as the percentage of cells displaying dilution of the CFSE-associated fluorescence. One-hundred percent proliferation corresponded to the extent of proliferation of Teffs in the absence of nTregs (positive control). % Suppression was calculated as 100% proliferation (from positive control) minus % proliferation observed for each sample.

# Flow Cytometry

For analysis of cytokine production, cells were restimulated with 1 µg/mL ionomycin and 50 ng/mL PMA in the presence of 5 µg/mL brefeldin A for 4 h. Cell surface staining was carried out in PBS with 2% FBS. For intracellular staining, cells were first stained with Zombie Aqua Fixable Viability kit (Biolegend), followed by staining for cell-surface markers and then resuspended in fixation/permeabilization solution (3% BSA and 0.5% saponin in PBS). Samples including the analysis of Foxp3 transcription factor were resuspended in fixation/permeabilization solution (Foxp3 Fixation/Permeabilization; eBioscience) according to the manufacturer instructions. To determine absolute number of cells, samples were analyzed in the presence of 123count eBeads (eBioscience) according to manufacturer's guidelines. Data were collected with a Canto II (BD) and results were analyzed with FACSDiva (BD) and FlowJo software (Tree Star, Ashlan, OR, USA).

# Statistical Analysis

Differences in means between two groups (different genotype or different conditions) were analyzed by unpaired 2-tailed Student's t-test. Progression of EAE severity curves were compared with a non-parametric Mann–Whitney rank sums two-tailed U test. P value ≤ 0.05 was considered significant. Analyses were performed with GraphPad Prism 6 software.

# Ethics Statement

This study was carried out in accordance with the recommendations of the institutional guidelines of Fundación Ciencia & Vida. The protocol was approved by the Bioethics and Biosecurity committee of the Fundación Ciencia & Vida.

# RESULTS

### DRD5-Signaling in Naive CD4+ T-Cells Favors the Development of the Inflammatory Response Associated to EAE

Since we previously appreciated a difference in the severity of EAE manifestation between animals deficient in DRD5 confined to DCs and animals displaying a global deficiency of DRD5 (Prado et al., 2012), we wondered whether DRD5-signaling in other immune cells was relevant in the regulation of the inflammatory response involved in EAE. To address this question, we first compared how was the development of EAE in animals displaying a global deficiency of DRD5 with animals bearing DRD5-deficiency confined to the hematopoietic compartment. For this purpose, we generated chimeric animals by killing bone marrow of WT recipient animals with a myeloablative dose of γ-irradiation and then transferring WT or DRD5-deficient bone marrow. Eight weeks after bone marrow transplantation, we obtained chimeric animals bearing WT or DRD5-deficient blood cells and WT genetic background outside the hematopoietic compartment (Supplementary Figure S1). When EAE was induced in these chimeric animals we observed that DRD5-deficiency confined to bone marrow resulted in attenuated EAE manifestation (**Figure 1A**), although the reduction in EAE severity was less marked than attenuation observed between WT and DRD5KO mice (**Figure 1B**). Of note, this lower difference in EAE manifestation observed between chimeric animals bearing WT or DRD5-deficient bone marrow may be due to the fact that the percentage of chimerism was around 80%–85% but not complete (Supplementary Figure S1). For this reason and also considering that autoimmune response in EAE is driven by CD4+ T-cells, which have been described to express DRD5 (Franz et al., 2015), we next carried out gaining of function experiments by performing adoptive transfer of CD4+ T-cells. In this regard, we hypothesized that if DRD5-signaling in CD4+ T-cells is relevant in contributing to the inflammation associated to EAE, we should rescue the phenotype of DRD5KO mice in EAE upon transfer of WT CD4+ T-cells. Accordingly, our results show that when DRD5KO mice received the transference of WT CD4+ T-cells, EAE severity was significantly stronger than DRD5KO mice, although still in a lesser extent than EAE severity observed in WT mice (**Figure 1C**). Since

FIGURE 1 | DRD5-signaling in naive CD4+ T-cells favors the development of the inflammatory response associated to experimental autoimmune encephalomyelitis (EAE). (A) Wild-type (WT) cd45.1+/+ mice were lethally irradiated and then bone marrow (107 cells/per mouse) from WT cd45.2+/+ (black symbols) or DRD5KO cd45.2 +/+ (red symbols) mice were i.v. transferred to reconstitute the hematopoietic compartment. Eight-week later, EAE was induced by immunization with pMOG in freund's complete adjuvant (CFA) followed by Pertussis Toxin injection and disease severity was daily determined throughout the time-course of disease development. Values represent mean ± SEM with n = 5–6 mice per group. ∗∗p < 0.01 by Mann-Whitney U-test. (B) EAE was induced in WT (black symbols) and DRD5KO (red symbols) mice by immunization with pMOG in CFA followed by Pertussis Toxin injection and disease severity was daily determined throughout the time-course of disease development. Values represent mean ± SEM with n = 4–7 mice per group. ∗∗p < 0.01 by Mann-Whitney U-test. (C) Total splenic CD4+ T-cells were isolated from WT mice by negative selection using magnetic beads-based kit (from Miltenyi) and then they were i.v. injected into DRD5KO mice (5 × 106 cells/per mouse; white symbols). One-day later, EAE was induced by immunization with pMOG in CFA followed by Pertussis Toxin injection and disease severity was daily determined throughout the time-course of disease development. EAE was also induced in WT (black symbols) and untransferred DRD5KO (red symbols) mice as control groups. Values represent mean ± SEM with n = 4 mice per group. ∗p < 0.05 and ∗∗∗∗p < 0.0001 WT vs. untransferred DRD5KO mice; ##p < 0.01 and ####p < 0.0001 WT vs. transferred DRD5KO; &&&p < 0.001 transferred vs. untransferred DRD5KO mice by Mann-Whitney U-test. (D) Naive CD4+ T-cells (CD4+ CD25− CD62L+ CD44−) were isolated from WT mice by cell-sorting and then they were i.v. injected into DRD5KO mice (3 × 106 cells/per mouse; white symbols). One-day later, EAE was induced by immunization with pMOG in CFA followed by Pertussis Toxin injection and disease severity was daily determined throughout the time-course of disease development. EAE was also induced in WT (black symbols) and untransferred DRD5KO (red symbols) mice as control groups. Values represent mean ± SEM with n = 3–4 mice per group. ∗p < 0.05 WT vs. untransferred DRD5KO mice; &&p < 0.01 transferred vs. untransferred DRD5KO mice by Mann-Whitney U-test.

Tregs, a subpopulation of CD4+ T-cells, play a relevant role attenuating EAE development in late stages of the disease manifestation (Petermann et al., 2010) and they also express DRD5 (Kipnis et al., 2004; Cosentino et al., 2007), we next wanted to evaluate the effect of DRD5-signaling confined to CD4+

T-cells without the potential contribution of DRD5-signaling from Tregs. For this purpose, we next performed a set of experiments in which we isolate naive CD25− CD4+ CD44− CD62L+ T-cells from WT mice, a subset devoid of Tregs cells (CD25high CD4+), and then these cells were transferred into DRD5KO recipient mice just before EAE induction. The results show that the transfer of DRD5-sufficient naive CD4+ T-cells, in the absence of Tregs, into DRD5-defficient animals results in a complete rescue in EAE manifestation, displaying similar EAE severity than observed for WT mice (**Figure 1D**). Thereby, these results indicate that DRD5-signaling in naive CD4+ T-cells exerts a pro-inflammatory role favoring EAE development.

### DRD5-Signaling in CD4+ T-Cells Potentiates T-Cell Activation

To address the question of how DRD5-signaling favors the inflammatory potential of CD4+ T-cells we next evaluated the role of this receptor in T-cells activation. Accordingly, we first determined the role of DRD5-signaling in the proliferation of CD4+ T-cells in vivo, which is directly related with the potency of T-cell activation. For this purpose, we performed adoptive transfer experiments using CD4+ T-cells from OT-II transgenic mice, which express a transgenic T-cell receptor (TCR) specific for the recognition of a peptide derived from the chicken ovalbumin (OVA323–339; pOT-II). To determine T-cell activation in vivo, we isolate CD4+ T-cells from WT/OT-II or from DRD5KO/OT-II mice, which were loaded with the fluorescent probe CFSE and then transferred into WT recipient mice. Subsequently, transferred mice were immunized with pOT-II in CFA and the potency of proliferation induced in pOT-II-specific T-cells was determined 3 days later in the spleen as the extent of the dilution of CFSE-associated fluorescence as previously described

FIGURE 2 | DRD5-signaling in CD4+ T-cells potentiates T-cell activation. (A,B) CD4+ T-cells were isolated from WT/OT-II or DRD5KO/OT-II mice by negative selection using magnetic beads-based kit (from Miltenyi), stained with 10 µM carboxyfluorescein succinimidyl ester (CFSE) and then i.v. transferred (12.5 × 106 cells/mice) into WT recipient mice. One-day later mice were i.p. immunized with 25 µg ovalbumin (OVA) or just injected with vehicle (PBS). Three-day after immunization mice were sacrificed and spleen cells were isolated, immunostained with fluorochrome-conjugated antibodies for B220, CD4, TCR-Va2 and TCR-Vβ5 and then CFSE-associated fluorescence was analyzed in the B220− CD4+ TCR-Va2+ TCR-Vβ5 + gate. (A) Representative histograms of CFSE-associated fluorescence vs. number of cells (events) are shown. Markers delimiting proliferating cells are shown. Percentages of CD4+ T-cells in the region of proliferating cells are indicated. (B) Percentages of CD4+ T-cells in the proliferating area from three independent experiments are represented in the bars graphs. Values represent mean ± SEM. ∗p < 0.05 by unpaired Student's t-test. (C,D) Naive CD4+ CD25− CD62L+ CD44− T-cells were isolated from WT/OT-II or DRD5KO/OT-II mice by cell-sorting, stained with 10 µM CFSE and then co-cultured with splenic Dendritic cells (DCs) isolated by positive CD11c+ selection using magnetic beads based kit (from Miltenyi) at a ratio of CD4+ T-cells:DCs = 5:1, in the presence of pOT-II (200 ng/ml) for 48 h or 72 h. (C) Cells were immunostained with fluorochrome-conjugated antibodies for CD4, TCR-Va2 and TCR-Vβ5 and then CFSE-associated fluorescence was analyzed in the CD4+ TCRVa2+ TCRVβ5 + gate. Representative histograms of CFSE-associated fluorescence vs. number of cells (events) are shown. Markers delimiting proliferating cells are shown. Percentages of CD4+ T-cells in the region of proliferating cells are indicated. (D) IL-2 production was determined in the culture supernatant by ELISA. Data from three independent experiments is shown. Values represent mean ± SEM. ∗p < 0.05 by unpaired Student's t-test.

(Contreras et al., 2016). The results show that DRD5-deficiency resulted in attenuated CD4+ T-cell proliferation as indicated by a lower percentage of OT-II cells displaying dilution of CFSE-associated fluorescence (**Figures 2A,B**). To obtain further evidence in the role of DRD5-signaling in CD4+ T-cell activation, we next performed in vitro experiments in which antigen-specific T-cell activation was induced by incubating naive CD4+ T-cells isolated from WT/OT-II or DRD5KO/OT-II in the presence or WT DCs loaded with OVA. To evaluate the potency of T-cell activation, we determined the extent of proliferation as the level of CFSE dilution and the secretion of IL-2 into the culture supernatant, which corresponds to the main T-cell growth factor. The results show that DRD5-deficiency in CD4+ T-cells not only resulted in decreased proliferation (**Figure 2C**) but also in reduced IL-2 production (**Figure 2D**). Thus, these results indicate that DRD5-signaling potentiates CD4+ T-cell activation in vitro and in vivo.

### DRD5-Signaling in CD4+ T-Cells Favors the Acquisition of Th17 Phenotype

To gain deeper insight in the role of DRD5-signaling in the CD4+ T-cell response, we next determined how this receptor affects the differentiation of naive CD4+ T-cells into effector phenotypes. Because the Th1 and Th17 are the main subsets of effector T-cells (Teffs) driving the inflammatory response involved in EAE (Prado et al., 2013) and both of them express DRD5 transcripts (Supplementary Figure S2), we next evaluated the potential role of DRD5-signaling in the acquisition of these functional phenotypes. For this purpose, we performed experiments in which naive CD4+ T-cells were isolated from WT/OT-II or from DRD5KO/OT-II mice and transferred into WT recipients. Afterward, these recipient mice were subcutaneously immunized with pOT-II and then the extent of acquisition of Th1 and Th17 functional phenotypes by the transferred CD4+ T-cells was analyzed in the draining lymph nodes at different time points. The results show that despite there were no significant differences in the absolute number and frequency of Th1 cells (see IFNγ + IL-17− T-cells in **Figure 3A** and in **Figure 3B** middle panels), DRD5-deficiency in CD4+ T-cells resulted in a strong reduction in the absolute number of Th17 cells in the draining lymph nodes 1 week after immunization (see IFN-γ − IL-17+ T-cells in **Figures 3A** and in **Figure 3B** right-bottom panel). DRD5-deficiency in CD4+ T-cells resulted also in a trend of decreased Th17 frequency in the draining lymph nodes 1 week after immunization, although this difference was not statistically significant (**Figure 3B** right-top panel). To evaluate whether difference in the acquisition of functional phenotypes observed between DRD5-deficient and DRD5-suficient CD4+ T-cells was due to altered viability of these cells, we determined the percentage of living cells among transferred CD4+ T-cells. The analyses show that frequencies of viable cells were similar in both genotypes, ruling out this possibility (Supplementary Figure S3A). Interestingly, irrespective of the genotype of CD4+ T-cells, the absolute number of Th1 OT-II CD4+ T-cells in the draining lymph nodes was increased after 2 weeks (**Figure 3B** middle-bottom panel), whilst the number of Th17 OT-II CD4+ T-cells was strongly reduced (**Figure 3B** right-bottom panel), which is probably explained by the faster exit of Th17 cells

FIGURE 3 | DRD5-signaling in CD4+ T-cells favors the acquisition of Th17 phenotype. (A,B) Naive CD4+ CD25− T-cells were isolated from WT/OT-II/cd45.2+/+ or DRD5KO/OT-II/cd45.2+/+ mice by cell-sorting and then i.v. transferred (105 cells/mouse) into WT/cd45.1+/+ recipient mice. One-day later, mice were s.c. immunized with 100 µg of pOT-II in CFA and CD4+ T-cells were analyzed by flow cytometry in the draining lymph nodes (inguinal lymph nodes) 7 and 14 days after immunization. (A) Representative contour-plots analyzing IFN-γ vs. IL-17 in the CD45.2+ CD4+ gated population. Numbers in each quadrant represent the percentage of cells in the corresponding quadrant. (B) Quantification of the percentage (top panels) and absolute number (bottom panels) of total CD4+ T-cells (left panels), Th1 cells (central panels) and Th17 cells (right panels) contained in the CD45.2+ population. Values represent mean ± SEM with n = 4 mice per group. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001 by unpaired Student's t-test.

from the draining lymph nodes after activation (Dardalhon et al., 2008; Reboldi et al., 2009). It is also noteworthy that, irrespective of their phenotype and genotype, the frequency of total OT-II CD4+ T-cells transferred is reduced in the draining lymph nodes 2 weeks after immunization (**Figure 3B** left-top panel) even when the absolute number is increased (**Figure 3B** left-bottom panel). This fact is likely to be due to the activation of endogenous CD4+ T-cells (CD45.1+) with specificity for pOT-II or for different antigens from mycobacterium tuberculosis contained in the CFA used as adjuvant in these experiments. To further explore the role of DRD5-signaling in Teffs, we quantified the transcriptional levels of a panel of different cytokines and transcription factors involved in the effector function of Th1 and Th17 cells. For this purpose, we performed a quantitative RT-PCR array of a set of relevant functional molecules in naive CD4+ T-cells, Th1 and Th17 cells obtained from WT or DRD5KO mice. To gain a deeper insight in the role of DRD5-signaling in Th17 function, we evaluated the transcriptional panel of these molecules in Th17 cells differentiated in pathogenic (Th17p) or non-pathogenic (Th17np) conditions (Lee et al., 2012). The results show that DRD5-deficiency results in decreased IFN-γ transcription by naive CD4+ T-cells or Th17np (**Figure 4A** left and right panels, and **Figure 4B**), increased transcription of IL-10 and IL-22 by Th17p (**Figure 4A** middle-right panel, and **Figure 4B**), exacerbated transcription of IL-17 and IL-22 by Th1 (**Figure 4A** middle-left panel, and **Figure 4B**) and increased transcription of granzyme B and IL-22 by naive CD4+ T-cells (**Figure 4A** left panel, and **Figure 4B**). Of note, we observed similar frequency of viable cells after CD4+ T-cell differentiation (Supplementary Figure S3B), ruling out the possibility that differential expression of cytokines and transcription factors was due to altered cell viability. Taken together, these results suggest that DRD5-defficiency in CD4+ T-cells results in impaired acquisition of the inflammatory Teff phenotypes and reduced expansion of Th17 cells.

# DRD5-Signaling in Tregs Favors Their Suppressive Activity

Since we observed that the transference of DRD5-sufficient total CD4+ T-cells into DRD5KO mice just partially rescues the phenotype on EAE manifestation (**Figure 1C**), whilst the transference of DRD5-sufficient naive CD25− CD4+ CD44−

(A,B) Values are the mean from three independent experiments.

times the levels of mRNA obtained in WT duplicates those obtained in DRD5KO. (B) Data represented as Ln of mRNA levels/house keeping (WT vs. DRD5KO). Central diagonal dotted line indicates same mRNA levels. External dotted lines indicate 2-fold mRNA levels and external solid lines indicate 4-fold mRNA levels. CD62L+ T-cells into DRD5KO mice completely rescues the phenotype on EAE manifestation (**Figure 1D**), we next wondered whether DRD5-signaling plays a relevant role in the Tregs (CD25high CD4+) response. To address this question, we first determined the effect of DRD5-signaling in the suppressive activity of Tregs. For this purpose, we performed in vitro assays in which WT naive OT-II CD4+ T-cells (Teff) were loaded with CFSE, activated with OVA-loaded DCs and co-incubated with increasing concentrations of DRD5-defficient or DRD5-sufficient OT-II Tregs and the extent of inhibition of Teff proliferation was quantified. The results show that DRD5-sufficient Tregs exert a stronger inhibition of Teff proliferation than that exerted by DRD5-defficient Tregs (**Figures 5A,B**), thus indicating that DRD5-signaling favors a stronger suppressive activity by Tregs. To evaluate whether DRD5-signaling in Tregs plays a relevant role in the suppressive activity in vivo, we next performed experiments in which DRD5-sufficient or DRD5-defficient Tregs were transferred into WT recipient mice just before EAE induction. In the same direction of conclusions obtained from in vitro experiments (**Figures 5A,B**), these results show that EAE severity was

reduced by DRD5-sufficient Tregs in a higher extent than that exerted by DRD5-defficient Tregs (**Figure 5C**), thus indicating that DRD5-signaling in Tregs favors a more potent suppressive activity in vivo. It is noteworthy that there was not differences in the extent of attenuation of EAE manifestation exerted by DRD5-sufficient and DRD5-defficient Tregs until late stages (after day 15 post-induction) in the time-course of EAE development, indicating that DRD5-signaling in Tregs was relevant only in late stages of EAE manifestation. To gain insight in the mechanism of how DRD5-signaling favors the suppressive activity of Tregs, we next evaluated the transcriptional level of the main cytokines associated to Tregs function, IL-10 and TGF-β, however we did not find any difference between DRD5-sufficient and DRD5-defficient Tregs (**Figure 5D**). To confirm whether IL-10 production was not affected by DRD5-signaling in Tregs we also analyzed the extent of IL-10 production at the level of protein and we found no differences between WT and knockout cells (Supplementary Figure S4). To further explore how DRD5-signaling potentiates the suppressive activity of Tregs, we next evaluated the level of protein expression of a panel of surface molecules and

FIGURE 5 | DRD5-signaling in Tregs favors their suppressive activity in vitro and in vivo. (A,B) Splenic CD4+ CD25− T-cells (Teff) were isolated from WT/OT-II mice by cell-sorting, stained with 5 µM CFSE and then co-cultured with splenic DCs isolated from WT mice by positive CD11c+ selection using magnetic beads based kit (MACS) at a ratio of CD4+ T-cells:DCs = 5:1, in the presence of OVA (5 µM) for 5 days. Splenic CD4+ CD25high T-cells (nTregs) were isolated from WT/OT-II or DRD5KO/OT-II mice by cell-sorting and added to the co-cultures at indicated ratios. (A) Representative histograms of CFSE-associated fluorescence vs. number of cells (events) in the CD4+ TCR-Va2+ TCR-Vβ5 + gate are shown. Markers delimiting proliferating cells are indicated. Percentage of CD4+ T-cells in the region of proliferating cells are indicated. (B) Percentages of suppression of Teff proliferation from three independent experiments are represented in the bars graphs. One-hundred percent proliferation corresponds to the extent of proliferation of Teffs in the absence of nTregs (positive control). % Suppression is calculated as 100% proliferation (from positive control) minus % proliferation observed for each sample. Values represent mean ± SEM. ∗p < 0.05 by unpaired Student's t-test. (C) Splenic CD4+ CD25high T-cells (nTregs) were isolated from WT (gray symbols) or DRD5KO (red symbols) mice by cell-sorting and i.v. transferred into WT recipient mice (7 × 105 cells per mouse). One-day later, EAE was induced by immunization with pMOG in CFA followed by Pertussis Toxin injection and disease severity was daily determined throughout the time-course of disease development. EAE was also induced in untransferred WT (black symbols) as a control group. Values represent mean ± SEM with n = 3–12 mice per group. ##p < 0.01 untransferred WT vs. DRD5KO nTregs transferred into WT mice; ∗∗p < 0.01 and ∗∗∗p < 0.001 untransferred WT vs. WT nTregs transferred into WT mice; &p < 0.05 WT nTregs transferred into WT mice vs. DRD5KO nTregs transferred into WT mice by Mann-Whitney U-test. (D) Splenic CD4+ CD25high T-cells (nTregs) were isolated from WT or DRD5KO mice by cell-sorting and immediately analyzed (resting) or activated with anti-CD3 and anti-CD28 antibodies in the presence of IL-2 for 36 h (activated). Afterward, total RNA was extracted and the levels of transcripts for TGF-β and IL-10 were quantified by real time RT-PCR. Data is represented as the levels of transcripts relative to the house-keeping gapdh. Values represent mean ± SD from at least three independent experiments. No significant differences were found between different genotypes.

transcription factor associated with the suppressive activity of Tregs. The results show that the only difference detected between DRD5-sufficient and DRD5-defficient Tregs was that DRD5-deficiency results in a significant reduction in the expression of GITR (**Figures 6A,B**), a molecule that has been related with the expansion of Tregs (Ronchetti et al., 2015). Of note, this difference in GITR expression was observed only in activated Tregs (**Figure 6B**), but not in resting conditions (**Figure 6C**). Thus, these results together indicate that DRD5-signaling in Tregs plays a relevant role favoring Tregs-mediated suppressive response, an effect that would be exerted at least in part by increasing the expression of GITR.

# DISCUSSION

Our findings here described represent the first genetic evidence indicating a relevant role of DRD5-signaling in the CD4+ T-cell response involved in EAE. Interestingly these findings suggest a dual role of DRD5-signaling, which are manifested at different stages during the time-course of EAE development and trigger opposite outcomes. Whereas the stimulation of DRD5 in naive CD4+ T-cells potentiates the inflammatory Th17-driven response early after the onset of the disease, DRD5-signaling in Tregs favors the suppressive activity of these cells dampening inflammation just once the peak of disease manifestation has been reached. Thus, these findings contribute to a better understanding of the complex regulation of the CD4+ T-cell driven autoimmune response involved in EAE and also illustrate how depending on the timing and on the precise T-cell subset, neuroimmune communications may exert different outcomes.

In a previous study, we showed genetic and pharmacologic evidence indicating that DRD5-signaling in CD4+ T-cells favored T-cell activation in vitro, a process that was mediated by ERK1/2 phosphorylation (Franz et al., 2015). In the same study we appreciated that DRD5-signaling in CD4+ T-cells had no effect in the process of Th1 differentiation in vitro. According with this previous study, our present results indicate that DRD5-signaling is relevant in vivo favoring T-cell activation (**Figure 2**) and without effect in the acquisition of the Th1 phenotype (**Figure 3**). Moreover, in agreement with our results about the role of DRD5-signaling in the effector CD4+ T-cell response in vivo, previous studies have shown pharmacologic evidence suggesting that the systemic antagonism of type I DRs attenuates the potency of Th17-driven responses (Nakano et al., 2008, 2011; Hashimoto et al., 2009; Okada et al., 2009; Nakagome et al., 2011; Nakashioya et al., 2011). In this regard, the systemic administration of the type I DRs antagonist SCH23390 reduces the clinical manifestation of EAE in mice immunized with a peptide derived from myelin proteolipid protein (Nakano et al., 2008). Similarly, subsequent studies showed that the same drug attenuates the inflammatory response in collagen-induced arthritis (Nakano et al., 2011; Nakashioya et al., 2011), allergic asthma (Nakagome et al., 2011; Gong et al., 2013), diabetes (Hashimoto et al., 2009) and nephrotoxic serum nephritis (Okada et al., 2009). Mechanistic analyses suggested that the therapeutic effect exerted by SCH23390 in these inflammatory disorders is due to the type I DRs antagonism in both CD4+ T-cells and DCs. Addressing the molecular mechanism involved in type I DRs signaling in CD4+ T-cells, the evidence suggests that type I DRs stimulation promotes B-cell activating transcription factor activity, favoring RORγt up-regulation and consequently Th17-mediated responses, which is inhibited by SCH23390 (Gong et al., 2013). Nevertheless, the pharmacologic approach used in all those studies does not allow the discrimination between the effects mediated by DRD5 or DRD1 since the drug inhibits both type I DRs with similar affinities (Bourne, 2001). Here we present genetic evidence indicating an important role of DRD5-signaling in promoting the acquisition of Th17 inflammatory phenotype in vivo (**Figure 3**).

Two previous studies have addressed the role of dopaminergic regulation of Tregs function (Kipnis et al., 2004; Cosentino et al., 2007). In apparent controversy with the present work, the studies carried out by Kipnis et al. (2004) and Cosentino et al. (2007) indicated that type I DRs stimulation attenuates Tregs suppressive activity by using a pharmacologic approach. Nonetheless, these studies were performed with drugs that cannot discriminate between their effects mediated by DRD1 or DRD5, as those drugs display very similar affinities for both receptors (Kipnis et al., 2004; Cosentino et al., 2007). Thus, it is likely that the inhibitory effect of dopamine or dopaminergic analogs on Tregs activity appreciated in those studies was mediated by DRD1 rather than DRD5. Here, we show genetic evidence indicating unequivocally that DRD5-deficiency in Tregs results in impaired suppressive activity of these cells in vitro and in vivo (**Figure 5**).

With these results in mind arise the question of where and when dopamine is relevant as a source available for CD4+ T-cells. In this regard, relevant sources of dopamine have been described in secondary lymphoid organs, the organs where T-cell responses are initiated, as well as in some target organs, where effector CD4+ T-cells promote inflammation. For instance, secondary lymphoid organs such as lymph nodes and the spleen are highly innervated by sympathetic fibers immunoreactive for dopamine (Weihe et al., 1991; Haskó and Szabó, 1998). In this way, the peripheral nervous system might exert regulation of the initiation of T-cell responses (Ben-Shaanan et al., 2016). According to this possibility, the present results (**Figures 2**, **3**) and previous studies (Pacheco et al., 2014) suggest that dopamine produced in draining lymph nodes shapes the expansion and differentiation of naive CD4+ T-cells toward the different functional T-cell phenotypes. In addition to the sympathetic fibers, DCs and follicular helper T-cells (TFH) may also represent important sources of dopamine in secondary lymphoid organs. Indeed, we have recently described how dopamine contained in DCs plays an important role potentiating Th1 and Th17 responses involved in EAE (Prado et al., 2012, 2018). Furthermore, Papa et al. (2017) recently described how TFH produce high amounts of dopamine and release it upon cognate interaction with B-cells in the germinal center, thus accelerating the production of high-affinity antibodies by plasma-cells. Thereby, dopamine produced in different specific locations inside secondary lymphoid organs might strengthen the initiation and development of adaptive immunity. With regard to the organs target of inflammation as potential relevant sources of dopamine for regulation of effector CD4+ T-cell response, altered levels of dopamine in different structures of the brain have been associated with neuroinflammation involved in some neurodegenerative disorders. For instance, dopamine levels have been found to be dramatically reduced in the nigrostriatal pathway of Parkinson's disease patients as well as in animal models of this disorder (Ehringer and Hornykiewicz, 1960; Brochard et al., 2009). We have previously suggested that this pathologic reduction in dopamine levels results in the selective stimulation of the DR displaying the highest affinity for dopamine, the DRD3 (Pacheco, 2017). In this regard, we have previously found that DRD3-signaling triggers a strong inflammatory behavior of CD4+ T-cells that infiltrate the brain in an animal model of Parkinson's diseases favoring the neurodegenerative process in the nigrostriatal pathway (González et al., 2013). On the other hand, EAE, the animal model used here to study MS and the autoimmune response associated, involves an increase of dopamine levels in the striatum during the peak of the disease (Balkowiec-Iskra et al., 2007). Since we observed a role of DRD5 in Tregs only once the peak of EAE manifestation has been reached, our results here suggest that the increase of striatal dopamine would be relevant stimulating DRD5 in Tregs, thus favoring their suppressive activity and dampening disease manifestation (**Figure 5**). Another potential source of dopamine present in the tissues target of inflammation may be Tregs. According to this idea, Tregs represent an important CD4+ T-cell subset infiltrating the brain and spinal cord during late stages of EAE manifestation, which is associated with the recovery phase of the disease (Petermann et al., 2010). Moreover, these cells have been described to synthesize and store important amounts of dopamine, which can be released upon treatment with reserpine (Cosentino et al., 2007). Despite the endogenous stimulus triggering dopamine release from Tregs has not been found yet, IFN-β has been proposed as a plausible candidate (Cosentino et al., 2012). Taken together these studies suggest that dopamine produced in the secondary lymphoid organs may strengthen the initial development of adaptive immune responses, whilst dopamine present in the inflamed target tissue may shape the effector and suppressive T-cell response depending in the timing and in the precise levels of available dopamine.

Interestingly, not only the CNS may represent a target tissue of inflammation with relevant sources of dopamine, but also the gut mucosa. In this regard, the gut mucosa has been found to be a major source of dopamine in healthy conditions, a source that is strongly reduced upon inflammation (Magro et al., 2002, 2004; Asano et al., 2012). Previous evidence has suggested that in healthy conditions high dopamine levels in the gut promote the stimulation of the low affinity DRs, specially the DRD2, whilst under inflammatory conditions low dopamine levels favors the selective stimulation of DRD3 (Pacheco, 2017). Furthermore, whereas DRD3-stimulation has been shown to promote pro-inflammatory CD4+ T-cell responses in the gut mucosa, including Th1 and Th17-driven immunity (Contreras et al., 2016), DRD2-signaling has been associated with anti-inflammatory effects (Pacheco et al., 2014). Indeed, a genetic polymorphism of DRD2 gene, which results in decreased receptor expression, has been reported as a risk factor for inflammatory bowel diseases (Magro et al., 2006). Accordingly, although the frequency of Tregs was not changed in the gut, suppressive activity of intestinal Tregs was compromised in inflammatory colitis (Wu et al., 2014), a condition associated to decreased dopamine levels (Magro et al., 2004). Interestingly, the impairment of suppressive Tregs function was abolished by the administration of cabergoline, a DRD2 agonist (Wu et al., 2014). Thus, these results together suggest that, whereas DRD2-signaling in Tregs promotes suppressive function in a healthy gut mucosa containing high dopamine levels, the selective DRD3-signaling in the inflamed gut mucosa containing low dopamine levels favors the inflammatory potential of effector CD4+ T-cells promoting further inflammation.

It is noteworthy that one of the main sources of dopamine present in the gut mucosa is given by the commensal gut microbiota. In this regard, it has been described that most dopamine arrives to the gut mucosa as glucuronide conjugated, which is biologically inactive. Nevertheless, Clostridium species present in the gut microbiota express β-glucuronidase activity, which catalyzes the production of free dopamine in the gut mucosa (Asano et al., 2012). In addition, recent studies have shown in vitro evidence indicating that some components of gut microbiota, including Bacillus cereaus, Bacillus mycoides, Bacillus subtilis, Proteus vulgaris, Serratia marcescens, S. aureus, E. coli K-12, Morganella morganii, Klebisella pneumoniae and

#### REFERENCES

Hafnia alvei, can also produce dopamine (Clark and Mach, 2016). Of note, a recent study performed with 34 monozygotic twin pairs discordant for MS has shown that when microbiota from the MS twin is transplanted into a transgenic mouse model of spontaneous brain autoimmunity, mice developed autoimmunity with higher incidence than when transplanted with microbiota coming from the healthy twin (Berer et al., 2017). Importantly, when mice were transplanted with microbiota obtained from MS twins, immune cells present in the gut mucosa produced significantly lower levels of the anti-inflammatory cytokine IL-10 than those immune cells of animals receiving microbiota coming from healthy twins. In addition, the analysis in the composition of gut microbiota shows clear differences between healthy and MS twins (Berer et al., 2017). Taken together, these findings indicate that gut microbiota, which can strongly regulate dopamine levels present in the gut mucosa, constitutes a pivotal factor in the control of gut inflammation and in the triggering of MS.

# AUTHOR CONTRIBUTIONS

RP designed the study and wrote the manuscript. FO-B, CP and FC conducted experiments and acquired data. FO-B, CP, FC and RP analyzed data.

# FUNDING

This work was supported by grants FONDECYT-1170093 (to RP) and FONDECYT-3160383 (to CP) from ''Fondo Nacional de Desarrollo Científico y Tecnológico de Chile'', AFB170004 (to RP) from ''Comisión Nacional de Investigación Científica y Tecnológica de Chile (CONICYT)'' and DI-1224-16/R (to RP) from Universidad Andres Bello.

# ACKNOWLEDGMENTS

We thank Dr. David Sibley for donation of DRD5KO mice and Dr. María Rosa Bono for providing OT-II and B6.SJL-Ptprca (cd45.1+/+) mice. We also thank Dr. Sebastián Valenzuela for his valuable veterinary assistance in our animal facility and María José Fuenzalida for her technical assistance in cell-sorting.

# SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fncel. 2018.00192/full#supplementary-material

noradrenaline changes in the striatum of C57BL mice following myelin oligodendrocyte glycoprotein (MOG) 35–55 and complete Freund adjuvant (CFA) administration. Acta Neurobiol. Exp. 67, 379–388.

encephalomyelitis in mice. Proc. Natl. Acad. Sci. U S A 114, 10719–10724. doi: 10.1073/pnas.1711233114

**Conflict of Interest Statement**: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Osorio-Barrios, Prado, Contreras and Pacheco. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# The Intracellular Cleavage Product of the NG2 Proteoglycan Modulates Translation and Cell-Cycle Kinetics via Effects on mTORC1/FMRP Signaling

#### Tanmoyita Nayak 1 , Jacqueline Trotter 1 and Dominik Sakry 1,2 \*

1 Department of Biology, Molecular Cell Biology, Institute of Developmental Biology and Neurobiology, Johannes Gutenberg University Mainz, Mainz, Germany, 2 Department of Molecular Neurobiology, Max Planck Institute for Experimental Medicine, Göttingen, Germany

#### Edited by:

Marion Baraban, University of Edinburgh, United Kingdom

#### Reviewed by:

Akiko Nishiyama, University of Connecticut, United States Marta Fumagalli, Università degli Studi di Milano, Italy Arthur Butt, University of Portsmouth, United Kingdom

> \*Correspondence: Dominik Sakry<EMAIL_ADDRESS>
Received: 15 March 2018 Accepted: 16 July 2018 Published: 07 August 2018

#### Citation:

Nayak T, Trotter J and Sakry D (2018) The Intracellular Cleavage Product of the NG2 Proteoglycan Modulates Translation and Cell-Cycle Kinetics via Effects on mTORC1/FMRP Signaling. Front. Cell. Neurosci. 12:231. doi: 10.3389/fncel.2018.00231 The NG2 proteoglycan is expressed by oligodendrocyte precursor cells (OPCs) and is abundantly expressed by tumors such as melanoma and glioblastoma. Functions of NG2 include an influence on proliferation, migration and neuromodulation. Similar to other type-1 membrane proteins, NG2 undergoes proteolysis, generating a large ectodomain, a C-terminal fragment (CTF) and an intracellular domain (ICD) via sequential action of α- and γ-secretases which is enhanced by neuronal activity. Functional roles of NG2 have so far been shown for the full-length protein, the released ectodomain and CTF, but not for the ICD. In this study, we characterized the role of the NG2 ICD in OPC and Human Embryonic Kidney (HEK) cells. Overexpressed ICD is predominantly localized in the cell cytosol, including the distal processes of OPCs. Nuclear localisation of a fraction of the ICD is dependent on Nuclear Localisation Signals. Immunoprecipitation and Mass Spectrometry followed by functional analysis indicated that the NG2 ICD modulates mRNA translation and cell-cycle kinetics. In OPCs and HEK cells, ICD overexpression results in an mTORC1-dependent upregulation of translation, as well as a shift of the cell population toward S-phase. NG2 ICD increases the active (phosphorylated) form of mTOR and modulates downstream signaling cascades, including increased phosphorylation of p70S6K1 and increased expression of eEF2. Strikingly, levels of FMRP, an RNA-binding protein that is regulated by mTOR/p70S6K1/eEF2 were decreased. In neurons, FMRP acts as a translational repressor under activity-dependent control and is mutated in Fragile X Syndrome (FXS). Knock-down of endogenous NG2 in primary OPC reduced translation and mTOR/p70S6K1 phosphorylation in Oli-neu. Here, we identify the NG2 ICD as a regulator of translation in OPCs via modulation of the well-established mTORC1 pathway. We show that FXS-related FMRP signaling is not exclusive to neurons but plays a role in OPCs. This provides a signal cascade in OPC which can be influenced by the neuronal network, since the NG2 ICD has been shown to be generated by constitutive as well as activity-dependent cleavage. Our results also elucidate a possible role of NG2 in tumors exhibiting enhanced rates of translation and rapid cell cycle kinetics.

Keywords: NG2, ICD, γ-secretase, OPC, FMRP, mTOR, S6K1, eEF2

# INTRODUCTION

NG2 (CSPG4) is a type-1 membrane protein (300 kD MW), belonging to the chondroitin sulfate proteoglycan protein family with a large extracellular region (290 kD) and a very short intracellular domain (8.5 kD). It was first discovered in the rat nervous system (Stallcup, 1977) and has reported homologs in mouse (Niehaus et al., 1999; Schneider et al., 2001; Stegmüller et al., 2002), human (Pluschke et al., 1996), and Drosophila (Estrada et al., 2007; Schnorrer et al., 2007).

In the nervous system, oligodendrocyte precursor cells (OPC) express the NG2 protein, while neurons (Karram et al., 2008; Clarke et al., 2012), astrocytes (Zhu et al., 2008; Huang et al., 2014) and resident microglia (Moransard et al., 2011) do not.  NG2 is expressed by many tumor types, including glioblastoma multiform (GBM) and melanoma (Chekenya et al., 2008; Prestegarden et al., 2010; Al-Mayhani et al., 2011), and is thus considered a potential therapeutic target in cancer.

OPCs are a self-renewing, and proliferating cell population found at all stages of development in gray and white matter. In the adult mammalian brain, around 5% of total neural cells comprise the OPC cell population (Dawson et al., 2003). During differentiation of OPCs into myelinating mature oligodendrocytes, NG2 expression is down-regulated (Nishiyama et al., 2009; De Biase et al., 2010; Kukley et al., 2010). NG2+ OPCs are unique as they are the only glial cell population receiving direct synaptic input from neurons (Bergles et al., 2000; Jabs et al., 2005; Kukley et al., 2008; Mangin and Gallo, 2011; Sakry et al., 2011).

Regulated intramembrane proteolysis (RIP) of type-1 membrane proteins involves sequential cleavage by α- and γ-secretases. The action of endogenous α-secretase on the full-length (FL) protein leads to release of the ectodomain into the extracellular matrix, termed as shedding. Subsequent cleavage of the remaining C- terminal fragment (CTF) by the γ-secretase releases the intracellular region into the cytoplasm, the released peptide is a functional unit and therefore termed intracellular domain (ICD), also referred to as the released ICD in the following text (Brown et al., 2000; Lal and Caplan, 2011; Saftig and Lichtenthaler, 2015). Notch was the first-reported, well-characterized membrane-protein undergoing RIP (Levitan and Greenwald, 1995; Blaumueller et al., 1997; De Strooper et al., 1999) and the released Notch ICD exhibits defined cellular functions, acting as a transcription factor (Bailey and Posakony, 1995; Baker and Zitron, 1995). Although release of the NG2 ectodomain had been previously documented (Nishiyama et al., 1995; Deepa et al., 2006), our published work demonstrated that NG2 undergoes RIP, generating a CTF and ICD in OPCs (Sakry et al., 2014, 2015; Sakry and Trotter, 2016). Recently, RIP has been shown for the neuronal CNS proteins neuroligin-1 and N-cadherin (Malinverno et al., 2010; Suzuki et al., 2012). Interestingly, this process is enhanced by neuronal activity, and similarly, we demonstrated that RIP of NG2 was also enhanced by activity (Sakry et al., 2014; **Figure 1A**). Thus, the cleavage and signaling pathways of these membrane proteins can be potentially modulated by the neuronal network.

The intracellular region of NG2 is known to bind to scaffold proteins such as Mupp1, Syntenin and GRIP1(Barritt et al., 2000; Stegmuller et al., 2003; Chatterjee et al., 2008), and protects cell from oxidative stress by binding to OMI/HtrA2 (Maus et al., 2015). It has also been associated with directed migration of OPCs (Binamé et al., 2013) and has a regulatory influence on the expression of the neuromodulator enzyme PTGDS in OPCs (Sakry et al., 2015).

In this study, we focused on basic cellular functions and signaling pathways which are specifically altered by the released NG2 ICD in OPCs. This is especially important because it would directly demonstrate the NG2 ICD is a functional domain. Additionally, NG2 cleavage-dependent pathways in OPCs are potentially regulated by the neuronal network. The NG2 ICD signaling cascade that we describe may be pertinent for other NG2 expressing cell-types, especially tumors.

#### MATERIALS AND METHODS

#### Cell Culture

The OPC cell-line, Oli-neu, was used as described in Jung et al. (1995). Cells were cultured at 37◦C, with 5% CO2 and expanded in Sato medium containing 1% horse serum [DMEM, with 0.2% (w/v) sodium bicarbonate, 0.01 mg/ml insulin, 0.01 mg/ml transferrin, 220 nm sodium selenite, 100µm putrescine, 500 nm triiodothyronine, 520 nm thyroxine, and 200 nm progesterone]. HEK 293 (HEK, Invitrogen) cells were cultivated at 37◦C in DMEM (Sigma) with 1% pyruvate and 10% FCS with 8% CO2. For primary OPC (pOPC), single cell suspensions were obtained from total brains of the Postnatal day (P) P6-9 of C57Bl/6N mice using the NTDK-P Kit (Miltenyi Biotec). Magnetic isolation (MACS) was performed as described previously (Diers-Fenger et al., 2001). 300,000 cells were plated in each well of a PLL-coated dish (6-well format) and cultured in OPC proliferation medium. OPC proliferation medium consists of NeuroMACS medium (Miltenyi) supplemented with 1:50 of NeuroBrew Miltenyi, 1:100 of L-Glutamine, 1:100 Pen-strep and 1:1,000 of stock conc.

of each of Forskolin (4.2 mg/ml), CNTF (10µg/ml), PDGF (10µg/ml) and NT-3 (1µg/ml). pOPCs were maintained at 37◦C with 8%CO2. All animal experiments were carried out in strict accordance with protocols approved by local Animal Care and Use Committee of Johannes Gutenberg University of Mainz. Mice were sacrificed by decapitation to remove the brain.

#### Expression Vectors and Transfection

NG2 expression vectors were derived from pRK5 constructs used in Sakry et al. (2015) by PCR amplification. NG2 DEL expresses a short recombinant version of full-length NG2 (consisting the signal sequence, one-fourth of the extracellular portion including two laminin G-type motifs, a transmembrane domain, and intracellular domain). Two other constructs are expressing only the intracellular domain (NG2 ICD, AA 2251-2327, UniProt: Q8VHY0) and without the predicted Nuclear Localization Signal (NLS) of ICD (NG2 ICD1NLS, AA 2259–2327). All cDNAs were cloned into the p3X-Flag CMV14 expression vector (c-terminal 3X flag-tag) and used for transfection. Additionally, the H2B-GFP plasmid (gift of Dr. Vijay Tiwari) was co-transfected along with NG2 ICD-Flag expression vector for immunocytochemistry study.

HEK and Oli-neu cells were plated 1 day before transfection and transfected using either PEI or Fugene HD reagent (Promega) at a ratio of 1:3 (2 µg DNA: 6 µl Fugene for the 3 cm dish). 48 h after transfection, cells were harvested and processed for analysis.

Primary OPCs were transfected after 1 day in vitro (DIV 1) using Lipofectamine RNAiMAX reagent (Thermo Fisher) according to the protocol. 120 pmol siRNA (final concentration) was used per well (6-well format), and the medium was changed 5–6 h after transfection. Cells were processed for analysis at DIV 2.

#### Cell Lysates, SDS PAGE, and Western Blotting

Cells were washed with PBS and scraped with a rubber policeman into lysis buffer (PBS, 1% TX-100, 1X protease inhibitor (PI) cocktail from Roche) from the culture plate on ice. After incubation for 20 min on the rotor at 4◦C, cells were spun down by centrifugation at 1,000 g, 10 min, 4◦C. Supernatants were defined as postnuclear (PN) cell-lysates (lysates). The same volume of lysis buffer was used per sample, and all samples were diluted with 4x SDS or LDS (Invitrogen) sample buffer, heated to 80◦C for 10 min and resolved on 4–12% NuPage Bis-Tris gradient gel in combination with MES or MOPS running buffer (Invitrogen). Western blotting (WB) was done with NuPage Blot system utilizing a PVDF membrane (Millipore). The latter was blocked for 30 min in PBS containing 0.1% Tween 20 (PBST) and 4% nonfat milk or 4% BSA.

Blocked membranes were incubated with primary antibodies (AB) overnight at 4◦C in blocking solution, followed by three washes (PBST). Subsequently, they were incubated with 1:10,000 HRP-conjugated secondary AB (Dianova) in blocking solution for 1 h and washed for three times again. Signal detection was carried out using enhanced chemiluminescence (ECL) assay solution (Millipore) and hyperfilms (GE). ImageJ 1.46 (NIH) was used for signal quantification, and all protein levels were normalized against GAPDH from the same sample. In some experiments, for checking total loaded protein level, membranes were stained with Ponceau S solution for 5 min on a shaker and later rinsed with deionized water three times for 5 min each.

#### Sub-cellular Fractionation Assay

For subcellular fractionation, cells were plated 1 day before transfection and transfected with NG2 ICD or ICD1NLS-Flag plasmids. After 48 h, cells were lysed with cytosolic lysis buffer (1X PBS+ 1% NP-40, 1X PI) on ice for 30 min and centrifuged at 2,000 g for 10 min. Supernatants which were enriched with cytosolic fraction were collected. The pelleted nuclei were further digested with nuclear lysis buffer [20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 10 mM MgCl2, 1% TX-100, 2.5 mM Beta-glycerophosphate, 1 mM NaF, 1 mM DTT, 2 mM EDTA, 10% glycerol 10U of Benzonase, 1X Protease inhibitor cocktail]. for 1 h on a rotor at 4◦C. Samples were centrifuged at 7,000 g for 10 min, and the supernatant was collected which comprises nuclear proteins.

#### Immunoprecipitation (IP) and Mass Spectrometry (MS)

Cells were plated in 100 mm dishes, and at ∼80% confluency were transiently transfected with 8 µg plasmid DNA of ICD and BAP-flag tagged constructs. After 48 h, cells were washed, lysed (1x PBS+ 0.5% TX-100+ 1x protease inhibitor), scraped off and centrifuged (3000 g). Prior to IP, the supernatant was precleared at 12,000 g for 10 min and incubated with anti-flag M2 magnetic beads (Sigma) for 2 h on a rotor at 4◦C. The beads were collected and washed three times (with PBS+0.3% TX-100) and heated at 85◦C for 10 min with 2X LDL sample buffer. Later, the IPed samples were used for various functional assays (coomassie staining, Mass Spec, CoIP). Mass-spectrometry based analysis of the IPed samples was done by chemical labeling method (DML labeling) in both forward and reverse way (IMB, Mainz). Data was analyzed by Maxquant software where the potential contaminants were removed, and the threshold was set to a minimum of 2X enrichment.

#### Primary Antibodies

The following antibodies are used for WB, ICC or FC: 1:200 (WB) of rabbit anti-NG2 cyto (Stegmüller et al., 2002), 1:1,000 (WB) of mouse anti-Flag (Sigma, F1804); 1: 200 (FC) of anti-Flag-FITC (Sigma, F4049); 1:1,000 (WB) of rabbit anti-FMRP (Sigma, F4055); 1:1,000 (WB) of rabbit anti-eEEF2 antibody (Abcam, ab40812); 1:1,000 (WB) of rabbit anti-eIF4B (CST, 3592T); 1:1,000 (WB) of rabbit anti-phospho (Ser422) eIF4B (CST, 3591S); 1:200 (ICC) of rabbit anti-PCNA (NEB, 13110); 1:1,000 (WB) of mouse anti-Puromycin (Millipore, MABBE343); 1:5,000 (WB) of rabbit anti-GAPDH (Biomol, A-300-641A); 1:400 (WB) of mouse anti-Cyclin E (SCBT, E-4), 1:1,000 (WB) of rabbit anti-p70S6K1 (CST, 9202). For studying mTOR and p70S6K1 phosphorylation, substrate sampler kit (CST, 9862T) was used and the antibody dilutions were made for WB according to kit suggestions.

#### Immunocytochemistry

Oli-neu cells were cultured on poly-L-Lysine coated coverslips, washed with PBS and fixed with 4% PFA for 15 min at RT, washed and permeabilized for 5 min with PBS containing 0.3% TX-100. Blocking was with PBS+10%HS for 30 min at RT. Primary AB incubation was done overnight at 4◦C, and secondary antibodies were applied for 30 min at RT. Coverslips were mounted in Moviol. For PCNA staining, cells were washed (1X PBS) and incubated with PBS+ 0.3% Triton-X on ice for 5 min and immediately fixed with acetone: methanol (1:1) solution for 10 min at −20◦C. Later, cells were blocked with PBS containing 3% goat serum for 1 h at RT. Primary and secondary AB incubation was done as described above. DAPI (0.1µg/ml) was added along with secondary antibodies. The following secondary AB were used: goat or donkey anti mouse and anti-rabbit coupled with Alexa488, 546 or 647 (Invitrogen).

Images were taken with a Leica DM6000 fluorescent microscope or an SP5 laser scanning confocal microscope (IMB, Mainz). Image processing was done with Leica LAS AF and ImageJ (NIH), specifically the ImageJ3DViewer. Nuclear morphology of immunostained cells were also analyzed for two indices; area and roundness with ImageJ.

#### SUnSET Assay

HEK293 and Oli-neu cells were transiently transfected with either NG2 ICD or empty flag control vector. After 48 h, cells were incubated with puromycin (10µg/mL) for 15 min in the culture medium and lysed afterwards for WB as described above. Anisomycin, a translation blocker, was used as a negative control (40µM final concentration) to confer puromycin incorporation only in active ongoing nascent polypeptide chains. The same protocol was repeated with siCNT, and siNG2 transfected primary OPCs and puromycin incubation was carried out 24 h post-transfection.

In an additional and independent experiment, Oli-neu cells were treated with a mTORC1 inhibitor, Temsirolimus (ab141999). Cells, overexpressing control or ICD, were treated with Temsirolimus (TM) at a final concentration of 12µM for 24 h and then treated with Puromycin as previously described. Cells were subsequently lysed (PBS + 0.5% TX-100 + 1X PI), and lysates were used for WB and signals developed after incubation with the anti-puromycin antibody. Before Western-Blotting, total loading of puromycin-treated protein samples was checked by Ponceau S staining.

# FACS

HEK293 cells were transfected with NG2 ICD or BAP-flag (as a positive control) and pelleted in a falcon after 48 h by centrifugation (800 rpm, 10 min, 4◦C). Cells were fixed with pre-chilled 80% ethanol in a dropwise manner and incubated for 10 min on ice. After permeabilization (PBS, 0.3% TX-100, 1X PI) and blocking with PBS+10% HS for 1 h, cells were incubated with the primary anti-Flag-FITC conjugated antibody (in permeabilization buffer) for 2 h at 4◦C. For DNA content analysis, cells were treated with RNase (10µg/mL) and stained with propidium iodide (50µg/mL) for 30 min at dark. Cell cycle distribution was checked only for Flag+ cell population, and data were analyzed by FlowJo software (IMB, Mainz).

#### Statistics

Each experiment was repeated independently at least three or more times. The numerical data of multiple experiments is expressed as mean ± standard error of the mean (SEM). Statistical analysis was done using Excel and Graph Pad Prism. For significance analysis, normal distribution was tested by the Shapiro-Wilk Normality test. For parametric distributed datasets two tailed t-test was applied: In the case of non-parametric distribution, Mann-Whitney or Wilcoxon Signed rank test was used. Significance was classified as follows: ∗p ≤ 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; n.s. p > 0.05.

# RESULTS

# NG2 Expression Constructs

As a tool for analyzing the cellular localization and functions of NG2 ICD, the following expression constructs were used (for details see the Methods section). NG2 DEL (65 kD) mimics a shortened version of the full-length NG2 protein, NG2 ICD (12 kD) encodes the intracellular domain of NG2 and ICD1NLS (10 kD) encodes the ICD lacking the first nine amino acids which consist the predicted Nuclear Localization Signal(s). All these constructs were c-terminally tagged with three flag epitopes (p3X-Flag-CMV-14, Sigma) and are shown in detail in **Figures 1B,C**.

#### NG2 ICD Localizes Predominantly to the Cytosol and Distal Processes With Limited Nuclear Expression

First, we checked the expression of the different NG2 constructs and their size-difference pattern in a gradient gel by western blot analysis (WB). The HEK 293 cell-line (HEK), as well as the OPC cell line, Oli-neu and primary OPCs (pOPC) from total brain (mouse), were routinely used for transfection. The small endogenous NG2 ICD has an MW of 8.5 KD and thus, like other gamma-secretase products, was unstable and hard to detect in WB with a specific antibody recognizing the intracellular part of NG2 (NG2-cyto). To initially validate the expression of ICD constructs, we used sodium butyrate to enhance the expression levels. However, sodium butyrate was not used in any other experiments. Expression of ICD1NLS only lead to sufficient protein levels in WB if sodium butyrate was added, while NG2 ICD levels were high even in the absence of butyrate. NG2 CTF (15 kD) derived from NG2 Del expression construct was the predominant, membrane-bound cleavage product recognized by the NG2 cyto antibody (**Figure 2A**). The localization of NG2 ICD and function of the predicted NLS was analyzed by performing subcellular fractionation of ICD over-expressing Oli-neu cells. NG2 ICD was detected in the nuclear fraction (Nuclear) as well as in the cytosolic fraction (Cyto) while expression of ICD1NLS was restricted to the cytosolic fraction (**Figure 2B**). These observations were confirmed when Oli-neu cells transiently expressing the NG2 ICD and ICD1NLS were immunostained and analyzed by confocal microscopy (**Figures 2C,D**). However, the cytosolic levels of ICD were always higher than the levels in the nucleus.

# Identification of the NG2 ICD Interactome

Since the major fraction of the ICD was present in the cell cytosol and the NG2 ICD has several predicted protein binding motifs, we characterized the interactome of the NG2 ICD. HEK cells were transiently transfected with NG2 ICD or BAP (Flag-tagged control protein) and immunoprecipitation was carried out using magnetic anti-Flag antibody conjugated beads (Scheme shown in **Figure 3A**). Proteins that co-precipitated with NG2 ICD were identified by mass spectrometry (MS) from total IP samples run on gradient SDS-PAGE. Red arrows in **Figure 3B** indicate heavy (55 kD) and light chain (25 kD) of the Flag IgG used for IP. The NG2 ICD (around 12 kD) is also shown on the gel image, while BAP (control) protein (around 51 kD) overlaps with the IgG heavy chain and is indistinguishable from the latter on the Coomassie gel. Additionally, in an independent experiment, observed unique bands in ICD IP lane from the Coomassie-stained gradient gel were excised between 70 and 100 kD and sent for mass spectrometry-based band identification analysis (indicated by black arrows in **Figure 3B**). Band identification revealed that peptides corresponding to two translation factors eEF2 (100 kD) and eIF4B (70 kD) were highly represented in these excised bands (**Table 1**). Strikingly, KEGG biological pathway analysis based on Gene ontology from whole NG2 ICD-IPs also suggested an enrichment of DNA replication, translation regulators, PI3K-Akt-mTOR signaling pathways and cell-cycle stages (**Figure 3C**). Additionally, a functional annotation clustering analysis of the putative interaction partners of NG2 ICD was performed with FUNRICH proteomic analysis tool. This analysis also confirmed that protein clusters involved in translation regulation, cell-cell adhesion RNA metabolism, and DNA replication were highly represented in the identified annotated peptide list (**Table 2**).

### NG2 ICD Increases Translation Rate in Cultured OPCs in an mTORC1-Dependent Manner

The GO-based analysis strongly suggested an influence of the NG2 ICD on translation. To test this directly, we pulselabeled Oli-neu and HEK cells with puromycin followed by immunoblotting with an anti-puromycin antibody, a technique known as SUnSET assay (Schmidt et al., 2009). Low concentrations of puromycin are incorporated into nascent

expression-constructs have been transiently transfected for 48 h before cell-lysis. Flag-tagged constructs show their size-based differences, NG2 ICD runs at 11 kD, and NG2 ICD1NLS (NLS was deleted) runs just below ICD at 10 kD. NG2 DEL (65 kD) strongly shows processed membrane-bound CTF (15 kD) at the shown lower molecular masses. Na-butyrate was used only in this experiment to enhance the expression of ICD constructs. (B) Sub-cellular fractionation assay was done from Oli-neu after overexpressing ICD or ICD1NLS for 48 h. Lamin B and α-tubulin were used as nuclear and cytosolic markers respectively. (C) Confocal laser scanning microscope images (z-stack, max. intensity projection) of Oli-neu expressing NG2 ICD or ICD1NLS (flag; red) fixed after 48 h of transfection. Cells were immunostained with anti-flag antibody. NG2 ICD shows a distinguishable homogeneous staining pattern including nucleus where in case of ICD1NLS, expression remained cytosolic. H2B (green) and DAPI (blue) were used as nuclear markers. (D) Three-dimensional orthogonal view of a z-stack of an Oli-neu showed the presence of NG2 ICD (red) within the nucleus, whereas ICD1NLS expression was limited to the cytosol and peri-nuclear area. Lamin B (green) was used as a nuclear envelope marker. (Scale bar = 10µm).

polypeptide chains labeling the newly synthesized proteome. Increased puromycin incorporation reflects a higher translation rate (**Figure 4A**). The translation blocker Anisomycin was used as negative control. In cells overexpressing ICD, puromycin incorporation was increased by ∼90% in HEK and ∼70% in Oli-neu cells compared to controls, indicating an increased translation rate (**Figure 4B**). To validate that the observed increased translation rate is specific for cleaved NG2 ICD, NG2 DEL was used. NG2 DEL mimics membrane-bound NG2 and is cleaved in the cell yielding small amounts of ICD. Expression of NG2 DEL in Oli-neu yielded a smaller increase in the puromycin signal compared to control (∼22%). Specific inhibition of mTORC1 by temsirolimus (TM) completely blocked the NG2 ICD dependent increase of translation in Oli-neu (ICD + TM) compared to the control (TM) (**Figure 4B**) and reduced the translation rate to slightly below control levels. Knock-down of endogenous NG2 (siNG2) in primary OPC by siRNA reduced NG2 levels by 40% and total translation was also reduced by around 30% in pOPC transfected with siNG2 compared to control (**Figures 4C,D**).

#### NG2 ICD Increases Cell Population in S-Phase

The proteomics data showed that DNA replication was one of the top altered processes in cells overexpressing ICD. Furthermore, exaggerated protein synthesis often leads to cell proliferation (Bilanges and Stokoe, 2007). We thus investigated whether cleaved NG2 ICD plays a role in cell-cycle regulation.

TABLE 1 | Unique band identification of IPed samples by MS revealed enrichment of two translation factors in ICD IP lane.

TABLE 2 | Functional annotation clusters of putative interactome of cleaved NG2 ICD by FUNRICH analysis software from whole IP/MS data.

HEK cells were transfected with NG2 ICD or BAP-Flag (Flag positive control). After 24 h, the cells were fixed and stained with anti-Flag-FITC conjugated antibody after permeabilization and analyzed by FACS after staining with PI.

Only FITC+ cells were gated for PI (DNA-binding dye) signal measurement. From the cell cycle distribution, in the ICD transfected population, 45.95 ± 6.53% of the cells were in S-phase compared to 35.41 ± 6.7% in S phase in the control population (**Figures 5A,B**). Due to low transfection efficiency and technical difficulties, FACS experiments could not be performed on Oli-neu. However, to address this issue in Oli-neu, cells were immunostained for PCNA (Proliferating cell nuclear antigen) which is an S-phase marker (**Figure 5C**). PCNA-staining revealed that 21.04 ± 1.26% Oli-neu cells were PCNA+ (out of total DAPI+ cells) in the ICD-transfected population. In the control population, 9.33 ± 2.01% Olineu were PCNA+ (**Figure 5D**), resulting in a 2-fold increase of Oli-neu cells in S-phase after ICD overexpression. 10% more Oli-neu were in S-phase after ICD expression, similar to increase in HEK cells. Levels of the G1 to S-phase regulator Cyclin E, increased around 2-fold on the protein level in Oli-neu total cell lysates, after NG2 ICD overexpression (**Figures 5E,F**).

Recent literature implies that nuclear morphology is correlated with distinct phases of cell-cycle and it is reported that most G1 phase cell nuclei appear round. As cells progress from late S-phase to mitosis, the nuclear shape also changes to a more elongated or dumbbell-shaped appearance (Wang et al., 2016). We examined the nuclear morphology by nuclear area and roundness of cells expressing the ICD. We found a significant difference between mean nuclear area and roundness of ICD transfected cell nuclei compared to controls (**Figures 6A,B**). While roundness was slightly reduced in ICD overexpressing Oli-neu cells, the nuclear area was increased around 1.6 fold in ICD overexpressing cells.

#### NG2 ICD Increases Translation by Modulating mTOR Signaling Components

We further investigated molecular mechanisms behind the translational and cell-cycle effects mediated by the NG2 ICD. To address this, we studied the expression of the global translation and proliferation regulator mTOR and components of the related signal-cascade. As described before, HEK and Oli-neu cells were transiently transfected with ICD or mock vector, and after 48 h, cell lysates were prepared and run on an SDS-PAGE gradient gel followed by WB analysis. In ICD-overexpressing cells, levels of phospho-mTOR (Ser2481) were increased and levels of the phosphorylated form (Thr 389) of the downstream target of mTOR, p70S6K1 were also increased, suggesting that this signaling pathway is a target of NG2 ICD (**Figures 7A,B**). Active (phosphorylated) S6K1 phosphorylates and inactivates eEF2K resulting in an upregulation of eEF2 protein, and it also phosphorylates and activates eIF4B. In support of this, we found an increase of phospho-eIF4B (Ser422) in ICD-transfected cells (**Figures 7A,B**). Knock-down of total NG2 protein in Oli-neu by siRNA (siNG2) resulted in a decrease of mTOR and p70S6K1 phosphorylation of around 20–25% compared to control (siCNT) (**Figures 7C,D**).

In the nervous system, S6K1 has been reported to phosphorylate FMRP, a protein acting as a general repressor of translation (Narayanan et al., 2008). Mutation of FMRP is the causal link to the intellectual disorder; Fragile-X Mental Syndrome (Santos et al., 2014). FMRP is an RNA binding protein with a wide range of substrates including dendritic mRNAs (Nalavadi et al., 2012) thus FMRP plays a key role in maintaining local translation and neuronal development (Zalfa et al., 2003; Nalavadi et al., 2012; Pasciuto and Bagni, 2014b). In recent studies, Eef2 mRNA has been recognized as an additional substrate of FMRP (Pasciuto and Bagni, 2014a; Richter et al., 2015) and FMRP/Eef2 signaling has been suggested to regulate translation of mRNAs involved in LTD in neurons (Park et al., 2008). Furthermore, FMRP-KO mice show exaggerated protein synthesis (Darnell et al., 2011) as a result of hyperactive S6K1 signaling (Bhattacharya et al., 2012). Intrigued by this reported molecular interplay, we checked the level of FMRP and found significant downregulation (∼70%) in ICD-overexpressing Oli-neu cells (**Figures 8A,B**). Downregulation of FMRP was much lower in HEK (∼30%) compared to Oli-neu. Moreover, the total cellular levels of eEF2 were increased in both NG2 ICD-transfected (**Figures 8A,B**) cell-types.

NG2 knockdown (siRNA transfection) was performed in cultured primary OPCs (pOPCs) to check the effect on translation. (D) NG2 knockdown (siNG2) in pOPCs revealed a knockdown efficiency of around 40% compared to control (siCNT), translation (puromycin level) was reduced by ∼30% by NG2 knock-down. [Data represents mean ±SEM. Statistical analysis was done by two-tailed paired t-test after checking the data is normally distributed by Shapiro-Wilk normality test by PRISM (GraphPad)]. Significance was classified as defined in the Materials and Methods part.

#### DISCUSSION

#### NLS-Dependent Localization of the NG2 ICD

An initial study by our group investigated the localization of the NG2 ICD. After transfection, most of the protein was localized in the cytoplasm, but lower nuclear levels were also reported (Sakry et al., 2015); in the present study, we confirm this localization pattern. However, immunofluorescent staining of cells expressing an NLS-truncated ICD construct showed only cytoplasmic and no nuclear localization, suggesting an NLS-dependent transport of the NG2 ICD into the cell nucleus. In contrast to the NOTCH ICD which contains a DNA binding domain, the much smaller NG2 ICD does not contain such conserved DNA/TF binding domains implying that a direct function of the NG2 ICD as a transcription factor is unlikely. According to ELM motif database, the NG2 intracellular sequence harbors a predicted site for MAPK interaction, suggesting additional involvement in cell signaling pathways. So far, no nuclear function or binding partner has been identified for the released NG2 ICD, but the predicted intracellular WW4 binding sequence of NG2 (**Figure 1C**) could be of interest, since WW4 domain-containing proteins have distinct roles, including nuclear functions (Sudol et al., 2001). In this study, we did not use the NLS deletion

construct for further experiments because of its low detectable protein levels, reflecting a much lower expression or a much higher degradation rate compared to the NG2 ICD expression construct.

# NG2 ICD Increases Translation and DNA Replication

Based on the results from the ontology analysis of our proteomics data, we investigated the effect of the NG2 ICD on mRNA translation and cell-cycle kinetics.

Strikingly, total translation rate was increased by at least ∼70% after NG2 ICD overexpression in Oli-neu and HEK cells, but not increased in the presence of an inhibitor of mTORC1. These results demonstrate an essential role of mTORC1 signaling in the increase in translation due to overexpression of the NG2 ICD. Additionally, NG2 DEL expression only slightly increased translation (∼22%) in Oli-neu. In our present and previous studies (Sakry et al., 2014, 2015) we show that the NG2 CTF but not the ICD, is the major fragment generated after NG2 DEL expression, resulting in much lower levels of

overexpressed NG2 ICD protein after NG2 DEL transfection compared to expression of the NG2 ICD construct. The much lower translational increase observed with NG2 DEL compared to NG2 ICD (22 vs. 70%), suggests a proteinlevel dependent effect of the released NG2 ICD on mTORC1 dependent translational regulation. In support of this, we found that the reduction of total endogenous NG2 protein levels in primary OPCs by siRNA knock-down decreased total translation rate.

Cell cycle analysis demonstrated a shift toward S-phase of cells expressing the NG2 ICD, where the percentage of the cellpopulation in S-phase increased by about 10% both for Olineu and HEK cells, reflecting increased DNA replication after NG2 ICD overexpression. We also detected changes in nuclear morphology of ICD-transfected Oli-neu concomitant with the observed alterations in nuclear shape reported in S-phase cells (Wang et al., 2016). In the case of Oli-neu, the cell number in S-phase increased by 2-fold, interestingly we found that Cyclin E, the major Cyclin upregulated during G1 to S-phase shift (Sgambato et al., 2003; Loeb et al., 2005), is increased around 2 fold on the protein level in Oli-neu under the same experimental conditions.

To date, NG2 protein or cleavage fragments have not been reported to modulate translation. However, NG2 expression is characteristic for proliferating cells, both in normal tissue and tumors. OPCs are the only proliferating cell population in the adult unlesioned mammalian CNS, apart from stem cells (Simon et al., 2011; Dimou and Götz, 2014). The ICD-mediated drive toward S-phase progression in OPCs that we report is striking as it has been recently published that stimulation of neuronal circuits causes an increase in adjacent neuronal progenitor proliferation, including NG2+ OPCs (Gibson et al., 2014). Since neuronal activity stimulates NG2 cleavage leading to increased release of the NG2 ICD in the cell cytoplasm (Sakry et al., 2014), our observation of ICD stimulation of cell-cycle progression is of interest in this context.

### NG2 ICD Regulates mTOR and FMRP Signaling

Since mRNA translation and cell-cycle kinetics were the major biological processes altered upon ICD overexpression, we investigated the potential underlying molecular mechanism behind these regulated biological processes. We started by analyzing the mTOR signaling cascade, as the mTOR pathway is a central regulator of cell growth, proliferation, and survival and growing evidence also supports its role in cell-cycle progression and tumorigenesis (Fingar et al., 2003; Ohanna et al., 2005; Laplante and Sabatini, 2012).

We found an increase of phosphorylated (p-ser2481, active) mTOR in ICD-overexpressing Oli-neu and HEK cells, indicating activation by PI3K, which is responsible for phosphorylation of this specific residue (Soliman et al., 2010). Active mTOR, part of the mTOR complex1 (mTORC1), is known to phosphorylate

mTORC1 signaling cascades were checked, and p-mTOR and p-S6K1 both were reduced (∼20–25%) in NG2 knockdown (efficacy 80%) samples compared to control. [Data represents mean ± SEM. Statistical analysis was done by two-tailed paired t-test after checking the data is normally distributed by Shapiro-Wilk normality test by PRISM (GraphPad)]. Significance was classified as defined in the Materials and Methods part.

the downstream target S6K1 at the Thr389 residue (Saitoh et al., 2002). Phosphorylated (active) S6K1 phosphorylates several downstream targets including the translation regulators eIF4B and eEF2K (Wang et al., 2001; Holz et al., 2005). Phosphorylated eIf4B promotes initiation of cap-dependent translation (Dennis et al., 2012), while phosphorylated eEF2K (the inactive form) leads to an increased level of the eEF2 protein resulting in elevated translation rates (Wang et al., 2001). Focusing on mTOR downstream targets, we found significantly increased levels of phosphorylated S6K1 in both cell lines after NG2 ICD overexpression. Additionally, total amounts of eEF2 protein and levels of p-eIF4B (the active form) were upregulated in both cell-lines, supporting the activation of the mTOR signaling cascade. Knock-down of endogenous NG2 in Oli-neu lead to decreased levels of phosphorylated mTOR/S6K1.

In addition to acting on the translation factors as described above, active S6K1 has been reported to phosphorylate FMRP, a protein highly expressed in the brain (Narayanan et al., 2008). Furthermore, eEF2 is a reported target of FMRP (Darnell and Klann, 2013). In FMRP knockout mice, total eEF2 protein and levels of p-eIF4B were higher than in wild-type mice (Bhattacharya et al., 2012). In line with this observation, we found significant downregulation of FMRP protein levels of

70% compared to the normal protein levels in NG2 ICDoverexpressing Oli-neu, but in HEK cells overexpressing the NG2 ICD, FMRP protein levels were only 30% lower. These NG2 ICD-mediated effects on FMRP were thus much stronger in Oli-neu, in contrast to the ICD mediated effects on the mTOR and S6K1 pathway which were similar in both cell types.

Fragile X protein (FMRP) is an RNA binding protein with demonstrated roles in mRNA transport, localization, stability, and translation. Mutations in FMRP are a major cause of the human cognitive disorder Fragile-X syndrome. In FMRP knockout mice, increased S6K1 signaling was reported (Bhattacharya et al., 2012) leading to exaggerated protein synthesis, similar to observations in FXS. Reduced expression of functional FMRP in both FXS patients (Qin et al., 2013) and animal models (Weiler et al., 2004; Gross et al., 2010) leads to excessive protein synthesis in neurons, suggesting that FMRP acts as a general repressor of translation. Since we found reduced FMRP levels and increased translation after NG2 ICD overexpression in Oli-neu, it is likely that FMRP also acts as a translational repressor in OPCs, as well as in neurons.

In our study of OPC, we identified two translation-related pathways affected by the NG2 ICD. One is responsible for general translation regulation in cells (mTORC1/S6K1 signaling cascade), and the other is important for regulating local translation and synaptic strength including LTD (FMRP-eEF2) in neurons (Park et al., 2008). Recent studies have shown that FMRP also activates the mTOR-signaling pathway in neurons (Sharma et al., 2010), combining these two signaling cascades. We thus suggest a new signaling paradigm for the NG2 protein in OPCs involving the neuronal network, as summarized in the graphical illustration in **Figure 9**.

#### NG2 ICD Signaling in OPCs

Our findings establish a model for NG2 ICD dependent modulation of FMRP/mTOR signaling cascades in OPCs with associated increased translation rate and an increased fraction of the cell population in the S-phase. OPCs are the only glial celltype that receives direct synaptic input from neurons in all major brain areas (Bergles et al., 2000; Jabs et al., 2005; Mangin and Gallo, 2011; Sakry et al., 2011). Furthermore, cleavage of NG2 leading to the cytoplasmic release of NG2 ICD has been shown to be increased by neuronal activity (Sakry et al., 2014).

The mTOR pathway has been shown to be crucial for OPC differentiation to myelinating oligodendrocytes (Tyler et al., 2009; Zou et al., 2014). However, NG2 is downregulated during early OPC differentiation (Nishiyama et al., 2009; De Biase et al., 2010; Kukley et al., 2010) making it unlikely that regulation of mTOR by NG2 is contributing to OPC differentiation.

#### NG2 ICD Signaling in Tumors

Interestingly, dysregulation of the FMRP/Akt/mTOR pathway has been reported to promote tumorigenesis (Rajasekhar et al., 2003; Lucá et al., 2013) and impaired mTOR cascade has especially been linked to Glioblastoma, the most common form of primary brain tumor (Akhavan et al., 2010; Li et al., 2016). NG2 is abundantly expressed by highly proliferative tumor cells in melanomas and gliomas (Chekenya et al., 2008; Al-Mayhani et al., 2011). The migration-promoting function of NG2 (Binamé et al., 2013) as well as the binding to OMI/HtrA2 (Maus et al., 2015) are features favoring NG2 expression by tumor cells promoting invasion and increasing resistance to oxidative stress. NG2 is included in a pool of several antigens used in a vaccine therapy against glioblastoma multiform, which reduces tumor growth (Poli et al., 2013). Additionally, OPCs are discussed as the major cells of origin for gliomas (Liu and Zong, 2012). NG2 is also involved in regulating symmetric vs. asymmetric cell-division of OPCs, the mode of division influences the likelihood of an OPC to become a tumor cell (Sugiarto et al., 2011).

Our findings of increased translation and DNA-synthesis (Sphase) by NG2 ICD correlates with the high proliferation and expression rates of these NG2-expressing tumors and is therefore likely to be one contributing factor to increased translation and DNA-synthesis within these cancers. One could speculate, that targeting NG2 as an antigen in the vaccine therapy described

FIGURE 9 | Model for NG2 ICD-mediated intracellular signaling in OPCs. (A) We propose the signaling pathway shown for OPCs, based on our previous NG2 cleavage study (Sakry et al., 2014; Sakry and Trotter, 2016) and the present NG2 ICD overexpression and NG2 knock-down study (highlighted in part B). The mTOR pathway shown has been suggested by Bhattacharya et al. (2012) for neurons (without NG2 signaling). In OPCs, NG2 full-length (FL) protein undergoes regulated intramembrane proteolysis (RIP) leading to the release of an ectodomain into the extracellular matrix (ECM) and generation of a c-terminal fragment (CTF) by α-secretase activity. γ-secretase activity on the NG2 CTF releases the NG2 intracellular domain (ICD) into the cytoplasm. This NG2 cleavage occurs constitutively in OPCs and can be increased by neuronal activity (Sakry et al., 2014). Under normal conditions, FMRP, which is a translation repressor and target of the mTOR-S6K1 signaling cascade, binds to eEF2 on ribosomal subunits and stalls translation, as suggested for neurons (Bhattacharya et al., 2012). (B) Increased NG2 cleavage generates an increased amount of NG2 ICD by γ-secretase activity. NG2 ICD overexpression mimics an increase in products of intracellular NG2 cleavage, elevating levels of phosphorylated (active) p-mTOR/p-S6K1/p-eIF4B, all known downstream components of the mTORC1 signal cascade, and thus indicating an increase in p-eIF4B dependent translation initiation. P-S6K1 phosphorylates and inactivates eEF2K leading to increased levels of active eEF2 protein favoring translation elongation. Increasing levels of NG2 ICD resulted in a higher levels of eEF2 and a reduction of FMRP protein, leading to a reduction of stalled FMRP-eEF2 complexes and relieving translation inhibition. These combined effects on the signaling cascade favor the observed mTORC1-dependent increase of overall translation in OPCs (Figures 4A,B) after ICD overexpression. A reduction of NG2 protein level in primary OPCs leads to a reduction of the total translation rate (Figures 4C,D).

above may affect NG2 signaling and modulate the signalingpathway presented here. It could further explain part of the reduction in tumor growth observed during such therapy by reduced translation and proliferation effected by NG2 signaling.

#### SUMMARY

In summary, our results add new insights to the role of NG2 cleavage in OPC and tumor signaling pathways. In addition to effects of the released NG2 ectodomain on the glutamatergic properties of neurons (Sakry et al., 2014), we show here that the released NG2 ICD can signal as a functional domain affecting translation and cell-cycle kinetics. These effects are important both for OPC under normal physiological conditions as well as NG2-expressing tumors. Our results imply translational regulation of localized (synaptic) mRNAs by FMRP in OPCs that can be influenced by the neuronal network.

#### AUTHOR CONTRIBUTIONS

TN and DS conceived and designed the experiments. TN performed the experiments and analyzed the data. DS and JT supervised the experiments. TN, DS, and JT wrote the manuscript.

#### FUNDING

This study was supported by the Deutsche Forschungsgemeinschaft (DFG TR 231/8-1 to JT; CRC TRR 128: TPB07 to JT) and a graduate stipendium to TN in the

#### REFERENCES

International PhD Programme on Gene Regulation, Epigenetics & Genome Stability, Mainz, Germany.

#### ACKNOWLEDGMENTS

We thank Dr. Eva-Maria Albers for her critical and valuable input and Dr. Vijay Tiwari for sharing a plasmid and thoughts on experiment design. We acknowledge the support and guidance provided by the Core facilities of Microscopy, Proteomics and Flow cytometry of the Institute of Molecular Biology, Mainz. We are grateful to Cornelia Braun for technical support.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.