Regulation of oligodendrocyte precursor maintenance by chondroitin sulphate glycosaminoglycans.

Chondroitin sulfate proteoglycans (CSPGs) have been proven to inhibit morphological maturation of oligodendrocytes as well as their myelination capabilities. Yet, it remained unclear, whether CSPGs and/or their respective chondroitin sulfate glycosaminoglycan (CS-GAG) side chains also regulate the oligodendrocyte lineage progression. Here, we initially show that CS-GAGs detected by the monoclonal antibody 473HD are expressed by primary rat NG2-positive oligodendrocyte precursor cells (OPCs) and O4-positive immature oligodendrocytes. CS-GAGs become down-regulated with ongoing oligodendrocyte differentiation. Enzymatic removal of the CS-GAG chains by the bacterial enzyme Chondroitinase ABC (ChABC) promoted spontaneous differentiation of proliferating rat OPCs toward O4-positive immature oligodendrocytes. Upon forced differentiation, the enzymatic removal of the CS-GAGs accelerated oligodendrocyte differentiation toward both MBP-positive and membrane forming oligodendrocytes. These processes were attenuated on enriched CSPG fractions, mainly consisting of Phosphacan/RPTPß/ζ and to less extent of Brevican and NG2. To qualify CS-GAGs as universal regulators of oligodendrocyte biology, we finally tested the effect of CS-GAG removal on OPCs from different sources such as mouse cortical oligospheres, mouse spinal cord neurospheres, and most importantly human-induced pluripotent stem cell-derived radial glia-like neural precursor cells. For all culture systems used, we observed a similar inhibitory effect of CS-GAGs on oligodendrocyte differentiation. In conclusion, this study clearly suggests an important fundamental principle for complex CS-GAGs to regulate the oligodendrocyte lineage progression. Moreover, the use of ChABC in order to promote oligodendrocyte differentiation toward myelin gene expressing cells might be an applicable therapeutic option to enhance white matter repair.

A LewisX glycoprotein screen identifies the low density lipoprotein receptor-related protein 1 (LRP1) as a modulator of oligodendrogenesis in mice.

In the developing and adult CNS multipotent neural stem cells reside in distinct niches. Specific carbohydrates and glycoproteins are expressed in these niche microenvironments which are important regulators of stem cell maintenance and differentiation fate. LewisX (LeX), also known as stage-specific embryonic antigen-1 or CD15, is a defined carbohydrate moiety expressed in niche microenvironments of the developing and adult CNS. LeX-glycans are involved in stem cell proliferation, migration, and stemness. A few LeX carrier proteins are known, but a systematic analysis of the targets of LeX glycosylation in vivo has not been performed so far. Using LeX glycosylation as a biomarker we aimed to discover new glycoproteins with a potential functional relevance for CNS development. By immunoaffinity chromatography we enriched LeX glycoproteins from embryonic and postnatal mouse brains and used one-dimensional nLC-ESI-MS/MS for their identification. We could validate phosphacan, tenascin-C, and L1-CAM as major LeX carrier proteins present in vivo. Furthermore, we identified LRP1, a member of the LDL receptor family, as a new LeX carrier protein expressed by mouse neural stem cells. Surprisingly, little is known about LRP1 function for neural stem cells. Thus, we generated Lrp1 knock-out neural stem cells by Cre-mediated recombination and investigated their properties. Here, we provide first evidence that LRP1 is necessary for the differentiation of neural stem cells toward oligodendrocytes. However, this function is independent of LeX glycosylation.

Differential expression of micro-heterogeneous LewisX-type glycans in the stem cell compartment of the developing mouse spinal cord.

Complex glycan structures and their respective carrier molecules are often expressed in a cell type specific manner. Thus, glycans can be used for the enrichment of specific cell types such as neural precursor cells (NPCs). We have recently shown that the monoclonal antibodies 487(LeX) and 5750(LeX) differentially detect the LewisX (LeX) glycan on NPCs in the developing mouse forebrain. Here, we analysed the staining pattern of both antibodies during late embryonic mouse spinal cord development. At E13.5 both antibodies strongly label the central canal region. Along these lines they detect the LeX glycan primarily on Nestin-positive NPCs at that age. Moreover, we show that spinal cord NPCs cultured as free floating neurospheres display a high immunoreactivity to both antibodies. In that context, we also demonstrate that the 487(LeX) antibody can be used to deplete a subpopulation of neurosphere forming NPCs from a mixed E13.5 spinal cord cell suspension. Towards the end of embryogenesis the overall immunoreactivity to both antibodies increases and the staining appears very diffuse. However, the 5750(LeX) antibody still labels the central canal region. The increase in immunoreactivity correlates with an expression increase of the extracellular matrix molecules Tenascin C and Receptor Protein Tyrosine Phosphatase ß/ζ, two potential LeX carrier proteins. In line with this, immunoprecipitation analyses confirmed Tenascin C as a LeX carrier protein in the embryonic mouse spinal cord. However, the immunoreactivity to both antibodies appears only to be marginally affected in the absence of Tenascin C, arguing against Tenascin C being the major LeX carrier. In conclusion our study gives some novel insights into the complex expression of LeX glycans and potential carrier proteins during the development of the mouse spinal cord.

Structurally distinct LewisX glycans distinguish subpopulations of neural stem/progenitor cells.

There is increasing evidence that the stem and progenitor cell population that builds the central nervous system is very heterogeneous. Stem cell markers with the potential to divide this cell pool into subpopulations with distinct characteristics are sparse. We were looking for new cell type-specific antigens to further subdivide the progenitor pool. Here, we introduce the novel monoclonal antibody clone 5750. We show that it specifically labels cell surfaces of neural stem and progenitor cells. When 5750-expressing cells were isolated by fluorescence-activated cell sorting from embryonic mouse brains, the sorted population showed increased neurosphere forming capacity and multipotency. Neurospheres generated from 5750-positive cells could self-renew and remained multipotent even after prolonged passaging. Carbohydrate binding assays revealed that the 5750 antibody specifically binds to LewisX-related carbohydrates. Interestingly, we found that the LewisX epitope recognized by clone 5750 differs from those detected by other anti-LewisX antibody clones like 487(LeX), SSEA-1(LeX), and MMA(LeX). Our data further reveal that individual anti-LewisX clones can be successfully used to label and deplete different subpopulations of neural cells in vivo and in vitro. In conclusion, we present a new tool for the isolation and characterization of neural subpopulations and provide insights into the complexity of cell surface glycosylation.

Novel conserved oligodendrocyte surface epitope identified by monoclonal antibody 4860.

Oligodendrocytes are the myelinating cells of the central nervous system. They differentiate from oligodendrocyte precursor cells through several intermediate states that can be followed by characteristic morphological changes and the expression of marker molecules. However, most oligodendrocyte lineage markers demarcate either the precursor or the differentiated oligodendrocyte in restricted subcellular compartments. Here, we describe a novel marker of the oligodendrocyte lineage recognised by the monoclonal antibody clone 4860. It selectively labels the surfaces of differentiated oligodendrocytes in culture and clearly differs from other oligodendrocyte markers. Importantly, the 4860 epitope highlights developing white matter tracts in rodent and avian brains and thus represents a useful and conserved feature. The 4860 epitope is not associated with protein backbones as revealed by the related 487/L5 antibody. Furthermore, the epitope disappears upon lipid extraction from cryosections or inhibition of sphingolipid synthesis in cultured oligodendrocytes. Thus, we conclude that mAb 4860 represents a novel lipid-based oligodendrocyte marker.

LewisX: a neural stem cell specific glycan?

LewisX (LeX) detecting antibodies are routinely used for cell sorting of neural stem- and progenitor cells (NSPCs). Applications include the enrichment of NSPCs after neural differentiation of human induced pluripotent- or embryonic stem cells, as well as their direct isolation from mouse neural tissue. Nevertheless, only little is known about the role of LeX in the central nervous system. Here we review the current knowledge on LeX-containing glycans expressed by neural stem cells and their progeny. New LeX-carrier proteins and ligands have recently been identified which reveal further insights into the potential function(s) of LeX-glycans. Moreover, evidence accumulates that individual LeX detecting antibody clones vary in their suitability as neural stem cell specific biomarker. Each antibody clone detects a unique LeX-containing glycan epitope. This allows a versatile utilization of anti-LeX antibodies that goes beyond neural stem cell sorting applications.