Matrix metalloproteinases shape the oligodendrocyte (niche) during development and upon demyelination

The oligodendrocyte lineage cell is crucial to proper brain function. During central nervous system development, oligodendrocyte progenitor cells (OPCs) migrate and proliferate to populate the entire brain and spinal cord, and subsequently differentiate into mature oligodendrocytes that wrap neuronal axons in an insulating myelin layer. When damage occurs to the myelin sheath, OPCs are activated and recruited to the demyelinated site, where they differentiate into oligodendrocytes that remyelinate the denuded axons. The process of OPC attraction and differentiation is influenced by a multitude of factors from the cell's niche. Matrix metalloproteinases (MMPs) are powerful and versatile enzymes that do not only degrade extracellular matrix proteins, but also cleave cell surface receptors, growth factors, signaling molecules, proteases and other precursor proteins, leading to their activation or degradation. MMPs are markedly upregulated during brain development and upon demyelinating injury, where their broad functions influence the behavior of neural progenitor cells (NPCs), OPCs and oligodendrocytes. In this review, we focus on the role of MMPs in (re)myelination. We will start out in the developing brain with describing the effects of MMPs on NPCs, OPCs and eventually oligodendrocytes. Then, we will outline their functions in oligodendrocyte process extension and developmental myelination. Finally, we will review their potential role in demyelination, describe their significance in remyelination and discuss the evidence for a role of MMPs in remyelination failure, focusing on multiple sclerosis. In conclusion, MMPs shape the oligodendrocyte (niche) both during development and upon demyelination, and thus are important players in directing the fate and behavior of oligodendrocyte lineage cells throughout their life cycle

Macroglial diversity:white and grey areas and relevance to remyelination

Macroglia, comprising astrocytes and oligodendroglial lineage cells, have long been regarded as uniform cell types of the central nervous system (CNS). Although regional morphological differences between these cell types were initially described after their identification a century ago, these differences were largely ignored. Recently, accumulating evidence suggests that macroglial cells form distinct populations throughout the CNS, based on both functional and morphological features. Moreover, with the use of refined techniques including single-cell and single-nucleus RNA sequencing, additional evidence is emerging for regional macroglial heterogeneity at the transcriptional level. In parallel, several studies revealed the existence of regional differences in remyelination capacity between CNS grey and white matter areas, both in experimental models for successful remyelination as well as in the chronic demyelinating disease multiple sclerosis (MS). In this review, we provide an overview of the diversity in oligodendroglial lineage cells and astrocytes from the grey and white matter, as well as their interplay in health and upon demyelination and successful remyelination. In addition, we discuss the implications of regional macroglial diversity for remyelination in light of its failure in MS. Since the etiology of MS remains unknown and only disease-modifying treatments altering the immune response are available for MS, the elucidation of macroglial diversity in grey and white matter and its putative contribution to the observed difference in remyelination efficiency between these regions may open therapeutic avenues aimed at enhancing endogenous remyelination in either area

Impairing committed cholesterol biosynthesis in white matter astrocytes, but not grey matter astrocytes, enhances in vitro myelination

Remyelination is a regenerative process that is essential to recover saltatory conduction and to prevent neurodegeneration upon demyelination. The formation of new myelin involves the differentiation of oligodendrocyte progenitor cells (OPCs) toward oligodendrocytes and requires high amounts of cholesterol. Astrocytes (ASTRs) modulate remyelination by supplying lipids to oligodendrocytes. Remarkably, remyelination is more efficient in grey matter (GM) than in white matter (WM), which may relate to regional differences in ASTR subtype. Here, we show that a feeding layer of gmASTRs was more supportive to in vitro myelination than a feeding layer of wmASTRs. While conditioned medium from both gmASTRs and wmASTRs accelerated gmOPC differentiation, wmOPC differentiation is enhanced by secreted factors from gmASTRs, but not wmASTRs. In vitro analyses revealed that gmASTRs secreted more cholesterol than wmASTRs. Cholesterol efflux from both ASTR types was reduced upon exposure to pro-inflammatory cytokines, which was mediated via cholesterol transporter ABCA1, but not ABCG1, and correlated with a minor reduction of myelin membrane formation by oligodendrocytes. Surprisingly, a wmASTR knockdown of Fdft1 encoding for squalene synthase (SQS), an enzyme essential for the first committed step in cholesterol biosynthesis, enhanced in vitro myelination. Reduced secretion of interleukin-1β likely by enhanced isoprenylation, and increased unsaturated fatty acid synthesis, both pathways upstream of SQS, likely masked the effect of reduced levels of ASTR-derived cholesterol. Hence, our findings indicate that gmASTRs export more cholesterol and are more supportive to myelination than wmASTRs, but specific inhibition of cholesterol biosynthesis in ASTRs is beneficial for wmASTR-mediated modulation of myelination. (Figure presented.)

Targeting Fibronectin to Overcome Remyelination Failure in Multiple Sclerosis:The Need for Brain- and Lesion-Targeted Drug Delivery

Multiple sclerosis (MS) is a neuroinflammatory and neurodegenerative disease with unknown etiology that can be characterized by the presence of demyelinated lesions. Prevailing treatment protocols in MS rely on the modulation of the inflammatory process but do not impact disease progression. Remyelination is an essential factor for both axonal survival and functional neurological recovery but is often insufficient. The extracellular matrix protein fibronectin contributes to the inhibitory environment created in MS lesions and likely plays a causative role in remyelination failure. The presence of the blood-brain barrier (BBB) hinders the delivery of remyelination therapeutics to lesions. Therefore, therapeutic interventions to normalize the pathogenic MS lesion environment need to be able to cross the BBB. In this review, we outline the multifaceted roles of fibronectin in MS pathogenesis and discuss promising therapeutic targets and agents to overcome fibronectin-mediated inhibition of remyelination. In addition, to pave the way for clinical use, we reflect on opportunities to deliver MS therapeutics to lesions through the utilization of nanomedicine and discuss strategies to deliver fibronectin-directed therapeutics across the BBB. The use of well-designed nanocarriers with appropriate surface functionalization to cross the BBB and target the lesion sites is recommended

Investigating demyelination, efficient remyelination and remyelination failure in organotypic cerebellar slice cultures:Workflow and practical tips

Healthy myelin is essential for proper brain function. When the myelin sheath is damaged, fast saltatory impulse conduction is lost and neuronal axons become vulnerable to degeneration. Thus, regeneration of the myelin sheath by encouraging oligodendrocyte lineage cells to remyelinate the denuded axons is a promising therapeutic target for demyelinating diseases such as multiple sclerosis. Ex vivo organotypic cerebellar slice cultures are a useful model to study developmental myelination, demyelination, remyelination and remyelination failure. In these cultures, the cerebellum's three-dimensional architecture and various cell populations remain largely intact, providing a realistic and relatively cost-efficient model that can be easily manipulated by the addition of viral vectors, pharmaceuticals or (transgenic) cells to augment or replace resident cell populations. Moreover, slice cultures can be treated with lysolecithin or polyinosinic:polycytidylic acid to induce demyelination and mimic efficient as well as inefficient remyelination. It can be challenging to set up slice cultures for the first time, as in our experience, seemingly minor differences in technique and materials can make a great difference to the quality of the cultures. Therefore, this report provides an in-depth description for the generation and maintenance of ex vivo organotypic cerebellar cultures for demyelination-remyelination studies with a focus on practical tips for scientists that are new to this technique

TLR3 agonists induce fibronectin aggregation by activated astrocytes:a role of pro-inflammatory cytokines and fibronectin splice variants

Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system which eventually results in axonal loss mainly due to failure of remyelination. Previously we have shown that the persistent presence of stable astrocyte-derived fibronectin aggregates in MS lesions impairs OPC differentiation, and thereby remyelination. Here we set out to discern whether and, if so, how inflammatory mediators as present in MS lesions trigger astrocytes to form fibronectin aggregates. Our findings revealed that in slice cultures only upon demyelination, the TLR3 agonist Poly(I:C) evoked astrocytes to form fibronectin aggregates. Consistently, pro-inflammatory cytokine-pretreated astrocytes were more susceptible to Poly(I:C)-induced fibronectin aggregation, indicating that astrocytes form fibronectin aggregates upon a double hit by inflammatory mediators. The underlying mechanism involves disrupted fibronectin fibrillogenesis at the cell surface as a result of a cytokineinduced increase in relative mRNA levels of EIIIA(pos)-Fn over EIIIBpos-Fn and a Poly(I:C)-mediated decrease in integrin affinity. Remarkably, fibronectin aggregation is exacerbated by white matter astrocytes compared to grey matter astrocytes, which may be a reflection of higher expression levels of EIIIA(pos)-fibronectin in white matter astrocytes. Hence, interfering with alternative fibronectin splicing and/or TLR3-mediated signaling may prevent fibronectin aggregation and overcome remyelination failure in MS lesions

Grey matter OPCs are less mature and less sensitive to IFN gamma than white matter OPCs:consequences for remyelination

Multiple sclerosis (MS) is a chronic inflammatory disease characterized by the formation of demyelinated lesions in the central nervous system. At later stages of the disease repair in the form of remyelination often fails, which leads to axonal degeneration and neurological disability. For the regeneration of myelin, oligodendrocyte progenitor cells (OPCs) have to migrate, proliferate and differentiate into remyelinating oligodendrocytes. Remyelination occurs faster and is more extensive in grey matter (GM) lesions than in white matter (WM) lesions. Here, we examined differences in neonatal OPCs from GM (gmOPCs) and WM (wmOPCs), both intrinsically and in response to environmental (injury) signals. We show that gmOPCs are less mature than wmOPCs, both on morphological and on gene-expression level. Additionally, gmOPCs proliferate more and differentiate slower than wmOPCs. When exposed to astrocyte-secreted signals wmOPC, but not gmOPC, migration decreases. In addition, wmOPCs are more sensitive to the detrimental effects of IFNγ treatment on proliferation, differentiation, and process arborisation, which is potentiated by TNFα. Our results demonstrate that OPCs from GM and WM differ both intrinsically and in response to their environment, which may contribute to the difference in remyelination efficiency between GM and WM MS lesions

Regulation of cell proliferation by nucleocytoplasmic dynamics of postnatal and embryonic exon-II-containing MBP isoforms

AbstractThe only known structural protein required for formation of myelin, produced by oligodendrocytes in the central nervous system, is myelin basic protein (MBP). This peripheral membrane protein has different developmentally-regulated isoforms, generated by alternative splicing. The isoforms are targeted to distinct subcellular locations, which is governed by the presence or absence of exon-II, although their functional expression is often less clear. Here, we investigated the role of exon-II-containing MBP isoforms and their link with cell proliferation. Live-cell imaging and FRAP analysis revealed a dynamic nucleocytoplasmic translocation of the exon-II-containing postnatal 21.5-kDa MBP isoform upon mitogenic modulation. Its nuclear export was blocked upon treatment with leptomycin B, an inhibitor of nuclear protein export. Next to the postnatal MBP isoforms, embryonic exon-II-containing MBP (e-MBP) is expressed in primary (immature) oligodendrocytes. The e-MBP isoform is exclusively present in OLN-93 cells, a rat-derived oligodendrocyte progenitor cell line, and interestingly, also in several non-CNS cell lines. As seen for postnatal MBPs, a similar nucleocytoplasmic translocation upon mitogenic modulation was observed for e-MBP. Thus, upon serum deprivation, e-MBP was excluded from the nucleus, whereas re-addition of serum re-established its nuclear localization, with a concomitant increase in proliferation. Knockdown of MBP by shRNA confirmed a role for e-MBP in OLN-93 proliferation, whereas the absence of e-MBP similarly reduced the proliferative capacity of non-CNS cell lines. Thus, exon-II-containing MBP isoforms may regulate cell proliferation via a mechanism that relies on their dynamic nuclear import and export, which is not restricted to the oligodendrocyte lineage

Classically and alternatively activated bone marrow derived macrophages differ in cytoskeletal functions and migration towards specific CNS cell types

<p>Abstract</p> <p>Background</p> <p>Macrophages play an important role in neuroinflammatory diseases such as multiple sclerosis (MS) and spinal cord injury (SCI), being involved in both damage and repair. The divergent effects of macrophages might be explained by their different activation status: classically activated (CA/M1), pro-inflammatory, macrophages and alternatively activated (AA/M2), growth promoting, macrophages. Little is known about the effect of macrophages with these phenotypes in the central nervous system (CNS) and how they influence pathogenesis. The aim of this study was therefore to determine the characteristics of these phenotypically different macrophages in the context of the CNS in an <it>in vitro </it>setting.</p> <p>Results</p> <p>Here we show that bone marrow derived CA and AA macrophages have a distinct migratory capacity towards medium conditioned by various cell types of the CNS. AA macrophages were preferentially attracted by the low weight (< 10 kD) fraction of neuronal conditioned medium, while CA macrophages were attracted in higher numbers by astrocyte- and oligodendrocyte conditioned medium. Intrinsic motility was twice as high in AA macrophages compared to CA macrophages. The adhesion to extracellular matrix molecules (ECM) was significantly enhanced in CA macrophages compared to control and AA macrophages. The actin cytoskeleton was differentially organized between CA and AA macrophages, possibly due to greater activity of the GTPases RhoA and Rac in CA macrophages. Phagocytosis of myelin and neuronal fragments was increased in CA macrophages compared to AA macrophages. The increase in myelin phagocytosis was associated with higher expression of CR3/MAC-1 in CA macrophages.</p> <p>Conclusion</p> <p>In conclusion, since AA macrophages are more motile and are attracted by NCM, they are prone to migrate towards neurons in the CNS. CA macrophages have a lower motility and a stronger adhesion to ECM. In neuroinflammatory diseases the restricted migration and motility of CA macrophages might limit lesion size due to bystander damage.</p