Research Article: Defining the role of NG2-expressing cells in experimental models of multiple sclerosis. A biofunctional analysis of the neurovascular unit in wild type and NG2 null mice

Date Published: March 14, 2019

Publisher: Public Library of Science

Author(s): Francesco Girolamo, Mariella Errede, Giovanna Longo, Tiziana Annese, Carlotta Alias, Giovanni Ferrara, Sara Morando, Maria Trojano, Nicole Kerlero de Rosbo, Antonio Uccelli, Daniela Virgintino, Mária A. Deli.

http://doi.org/10.1371/journal.pone.0213508

Abstract

During experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis associated with blood-brain barrier (BBB) disruption, oligodendrocyte precursor cells (OPCs) overexpress proteoglycan nerve/glial antigen 2 (NG2), proliferate, and make contacts with the microvessel wall. To explore whether OPCs may actually be recruited within the neurovascular unit (NVU), de facto intervening in its cellular and molecular composition, we quantified by immunoconfocal morphometry the presence of OPCs in contact with brain microvessels, during postnatal cerebral cortex vascularization at postnatal day 6, in wild-type (WT) and NG2 knock-out (NG2KO) mice, and in the cortex of adult naïve and EAE-affected WT and NG2KO mice. As observed in WT mice during postnatal development, a higher number of juxtavascular and perivascular OPCs was revealed in adult WT mice during EAE compared to adult naïve WT mice. In EAE-affected mice, OPCs were mostly associated with microvessels that showed altered claudin-5 and occludin tight junction (TJ) staining patterns and barrier leakage. In contrast, EAE-affected NG2KO mice, which did not show any significant increase in vessel-associated OPCs, seemed to retain better preserved TJs and BBB integrity. As expected, absence of NG2, in both OPCs and pericytes, led to a reduced content of vessel basal lamina molecules, laminin, collagen VI, and collagen IV. In addition, analysis of the major ligand/receptor systems known to promote OPC proliferation and migration indicated that vascular endothelial growth factor A (VEGF-A), platelet-derived growth factor-AA (PDGF-AA), and the transforming growth factor-β (TGF-β) were the molecules most likely involved in proliferation and recruitment of vascular OPCs during EAE. These results were confirmed by real time-PCR that showed Fgf2, Pdgfa and Tgfb expression on isolated cerebral cortex microvessels and by dual RNAscope-immunohistochemistry/in situ hybridization (IHC/ISH), which detected Vegfa and Vegfr2 transcripts on cerebral cortex sections. Overall, this study suggests that vascular OPCs, in virtue of their developmental arrangement and response to neuroinflammation and growth factors, could be integrated among the classical NVU cell components. Moreover, the synchronized activation of vascular OPCs and pericytes during both BBB development and dysfunction, points to NG2 as a key regulator of vascular interactions.

Partial Text

Oligodendrocyte precursor cells (OPCs) are proliferating elements of the oligodendroglia lineage that remain in the adult brain at the end of oligodendrogenesis as a unique type of potentially self-renewing glia and generate oligodendrocytes throughout life [1, 2]. Similarly to what occurs during central nervous system (CNS) development, in adult CNS, OPCs express high levels of nerve/glial antigen 2 (NG2) chondroitin sulfate proteoglycan 4 constitutively, and are often indicated as NG2-glia, the two terms being interchangeable in the literature [3]. OPCs/NG2-glia are the fourth glial type in adult CNS, making up 5–8% of the total glia in grey and white matter, where these cells develop functional relations at synapses and Ranvier’s nodes [4, 5]. After myelin damage due to white matter traumatic injury or neuroinflammation, OPCs/NG2-glia reload a proliferative phenotype and differentiate, becoming a potential source for replacement of degenerating oligodendrocytes [6–9]. As described previously in cerebral cortex of mice affected with experimental autoimmune encephalomyelitis (EAE), OPCs proliferate during EAE and are present in close vicinity to damaged blood-brain barrier (BBB) microvessels [10]. The same model of EAE repeated in NG2 knock-out mice leads to a milder disease characterized by a less inflammatory profile of pathogenic Th1 lymphocytes, reduced inflammatory infiltrate, and preserved BBB function [11]. Overall, these data suggest that if vascular OPCs are actually members of the neurovascular unit (NVU), they could be involved in the alteration of BBB function during neuroinflammation, and NG2 could interact with endothelial cells engaging the tight junctions (TJs) in the disease response. This idea is supported by recent data on OPC/vessel mutual relationships [12, 13] and by growing evidence of the importance of NG2 proteoglycan molecular interactions and biological functions [14, 15]. During neurohistogenesis, OPCs migrate along brain microvessels according to their tissue fate map [12], and are involved in vessel sprouting, as well as in the establishment of a proper brain vascular network [13]. The structure of NG2, as a type 1 integral membrane glycoprotein, enables it to work in a cis modality as a self-sensor/transducer, which interacts through the cytoplasmic PDZ (PSD-95, DISC-large, ZO-1)-binding domain with scaffolding proteins to control cell migration, but also as a non-self-regulator through trans interactions with the accompanying cells established by its extracellular domain [16, 17]. NG2 trans activity has been extensively studied in the vascular system, where the proteoglycan, expressed by immature pericytes, influences endothelial cell adhesion, spreading and migration [16]. In this study, we used immunoconfocal morphometry and TJ protein immunohistochemistry to quantify vascular OPCs/NG2-glia (hereinafter referred to as OPCs) and investigate whether a vascular contingent of NG2-bearing OPCs may contribute to TJ organization in the cerebral cortex of developing and adult naïve wild-type (WT) and NG2 knock-out (NG2KO) mice, as well as after EAE induction in WT and NG2KO mice. The expression of the growth factors revealed by immunohistochemistry was also analysed by real time-PCR on isolated cortex microvessels and by dual RNAscope immunohistochemistry/in situ hybridization (IHC/ISH) on cortex section from EAE-affected WT mice. The results demonstrate that OPCs are a dynamic, integral cell component of the developing and adult NVU, and support the hypothesis of NG2 as the molecule involved in TJ regulation during BBB development and dysfunction.

As demonstrated by in vivo time-lapse imaging in mouse brain, OPCs are highly motile cells that actively migrate and extend processes to survey the homeostasis of their own territory in both physiological and pathological conditions [29]. The basic idea of this study is that such a dynamic type of glial cells may contribute to nervous tissue development and/or response to injury [30–32] through their recruitment and active participation to the NVU, and that part of the biological functions related to OPC/vessel interactions may be mediated by NG2 proteoglycan. Interestingly, in the CNS, two cell types primarily express NG2: OPCs, which express NG2 during development (proper OPCs) and continue to express the proteoglycan in adulthood (NG2-glia) [33], and pericytes, which upregulate NG2 expression according to their developmental (‘immature’ pericytes) and pathological (re-activated pericytes) status, and downregulate the proteoglycan in adult brain [25, 34].

 

Source:

http://doi.org/10.1371/journal.pone.0213508

 

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