Pu et al. Journal of Neuroinflammation(2020) 17:220
https://doi.org/10.1186/s12974-020-01895-1

RESEARCH

Open Access

The glycosyltransferase EXTL2 promotes
proteoglycan deposition and injurious
neuroinflammation following
demyelination
Annie Pu1, Manoj K. Mishra1, Yifei Dong1, Samira Ghorbanigazar1, Erin L. Stephenson1, Khalil S. Rawji1,
Claudia Silva1, Hiroshi Kitagawa2, Stephen Sawcer3 and V. Wee Yong1,4*

Abstract
Background: Chondroitin sulfate proteoglycans (CSPGs) are potent inhibitors of axonal regrowth andremyelination. More recently, they have also been highlighted as a modulator of macrophage infiltration into thecentral nervous system in experimental autoimmune encephalomyelitis, an inflammatory model of multiple
sclerosis.
Methods: We interrogated results from single nucleotide polymorphisms (SNPs) lying in or close to genes
regulating CSPG metabolism in the summary results from two publicly available systematic studies of multiple
sclerosis (MS) genetics. A demyelinating injury model in the spinal cord of exostosin-like 2 deficient (EXTL2-/-) mice
was used to investigate the effects of dysregulation of EXTL2 on remyelination. Cell cultures of bone marrowderived macrophages and primary oligodendrocyte precursor cells and neurons were supplemented with purified
CSPGs or conditioned media to assess potential mechanisms of action.
Results: The strongest evidence for genetic association was seen for SNPs mapping to the region containing theglycosyltransferase exostosin-like 2 (EXTL2), an enzyme that normally suppresses CSPG biosynthesis. Six of these
SNPs showed genome-wide significant evidence for association in one of the studies with concordant andnominally significant effects in the second study. We then went on to show that a demyelinating injury to thespinal cord of EXTL2−/− mice resulted in excessive deposition of CSPGs in the lesion area. EXTL2−/− mice had
exacerbated axonal damage and myelin disruption relative to wild-type mice, and increased representation of
microglia/macrophages within lesions. In tissue culture, activated bone marrow-derived macrophages from
EXTL2−/− mice overproduce tumor necrosis factor α (TNFα) and matrix metalloproteinases (MMPs).
Conclusions: These results emphasize CSPGs as a prominent modulator of neuroinflammation and they highlight
CSPGs accumulating in lesions in promoting axonal injury.
Keywords: CSPG, Multiple sclerosis, Inflammation, Demyelination, Oligodendrocyte, EXTL2* Correspondence: vyong@ucalgary.ca
1
Department of Clinical Neurosciences and the Hotchkiss Brain Institute,
University of Calgary, T2N 4 N1, Calgary, Canada
4
University of Calgary, 3330 Hospital Drive, Calgary, Alberta T3A 4X9, Canada
Full list of author information is available at the end of the article© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate ifchanges were made. The images or other third party material in this article are included in the article's Creative Commonslicence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain
permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to thedata made available in this article, unless otherwise stated in a credit line to the data.

Pu et al. Journal of Neuroinflammation(2020) 17:220

Background
Multiple sclerosis (MS) is an inflammatory disease of thecentral nervous system (CNS) with focal areas of demyelination induced by aberrant immune activation. Associated with demyelination and inflammation are a
multitude of changes in the composition of the CNS
extracellular matrix (ECM) (reviewed in [1, 2]); however,changes in ECM are understudied in the context of MS,
where lesional alterations are largely undefined [3]. Previous reports have highlighted the deposition of chondroitin sulfate proteoglycans (CSPGs) at the borders ofchronic active MS lesions [4]. CSPGs are a class of large,
glycosylated proteins present both on cell surfaces andin the extracellular matrix. Lecticans, a subset of ECM
CSPGs, homeostatically comprise a significant portion ofperineuronal nets as well as the neural interstitial matrix,
serving roles in development, plasticity, and neuronal
function [5, 6]. Lectican CSPG members are versican,
aggrecan, neurocan, and brevican. In demyelinating
injury, they are locally upregulated by several cell types
and deposited near the injury site [7–13]. CSPGs arepotent inhibitors for regenerative processes such asaxonal regeneration and remyelination [14–17].
CSPGs were recently identified by our group as a novel
regulator of neuroinflammation in a mouse model of MS.
In particular, the V1 isoform of versican (in which the coreprotein consists only of the GAGβ segment) is particularly
abundant in inflammatory CNS lesions, and a mixture oflecticans stimulates bone marrow-derived macrophages to
produce inflammatory cytokines and proteases [18]. Others
have reported that the genetic loss of versican in lungmacrophages attenuates their pro-inflammatory response
to polyinosine-polycytidylic acid [19].
As CSPGs potently influence reparative processes in
the CNS, it is important to explore approaches to overcome this impediment to regeneration. The biosynthetic
process of CSPGs has largely been elucidated [20], and anumber of pharmacological inhibitors of CSPG production have been examined for therapeutic effects, such as
xylosides and fluorinated analogues that inhibit activityof CSPG biosynthesis enzymes [21, 22]. Following CNS
insult, chondroitinase ABC (ChABC)-mediated CSPG digestion improved axonal sprouting, remyelination, andcognitive recovery [14–16, 23–27]. Perturbing CSPG signaling through antagonism of receptors (e.g., leukocytecommon antigen related and protein tyrosine phosphatase σ) also increased the population of alternatively
activated macrophages following SCI [25]. However,finding an appropriate approach to removing deposited
CSPGs in diffuse or heterogeneous pathology is difficult,and the regulators of CSPG production are understudied. Understanding the regulatory mechanisms of CSPG
biosynthesis may provide more insight into limiting
CSPG deposition after injury.

Page 2 of 21

We interrogated the summary results from the 2011
MS genome-wide association screen (GWAS) and the2013 MS Immunochip study (Immunochip 2013) focusing on regions containing genes involved in the metabolism of CSPGs, which led to the identification of theglycosyltransferase exostosin-like 2 (EXTL2) gene.
EXTL2 is a glycosyltransferase that limits the productionof CSPGs by terminating its synthesis [28]. Thus, we
asked whether investigating the biology of EXTL2 andits effects on CSPG deposition and repair following a demyelinating injury to the spinal cord of mice could provide insights to endogenous targets for limiting CSPG
production. Our results in mice with constitutive knockout of EXTL2 (EXTL2−/−) in the lysolecithin model of
focal demyelination [29] highlight elevated CSPG deposition in lesions that contribute to the increased recruitment and activation of microglia/macrophages leading
to the exacerbation of axonal loss.

Methods
Analyses of MS genetic databases

The MS GWAS performed jointly by the International
Multiple Sclerosis Genetics Consortium (IMSGC) andthe Wellcome Trust Case Control Consortium (WTCC
C2) involved 9772 cases and 17,376 controls collectedfrom across 15 different countries (all individuals wereof European descent) [30]. The study generated datafrom 465,434 autosomal SNPs and identified 29 novel
genome-wide significant associations. The MS Immunochip study [31] was a follow-up effort focused on genomic regions implicated in autoimmune disease GWAS.
This study was performed by the IMSGC and involved
14,802 cases and 26,703 controls; there was no overlapof samples between these two studies. The Immunochip
study was a focused follow-up effort which did not consider all parts of the genome, so some regions were more
densely covered than others compared to the 2011
GWAS study. The Immunochip study generated datafrom 161,311 autosomal markers and identified 48 novelassociations. Neither of the MS genetics studies considered the sex chromosomes and we were therefore unable
to include sex-linked CSPG genes in this analysis.
Using our knowledge of CSPG biology, we identified alist of 154 autosomal genes (Table 1) involved intimatelyor remotely in the production and degradation of
CSPGs. This includes proteoglycans (Vcan, Acan, Ncan,
Bcan, Bgn, Bsg, Gpc1-6), degradation enzymes (Adam,
Adamts, Mmp), and synthesis-related genes (B3galt6,
B3gat1-3, B4galt7, Chpf, Chpf2, Chst1-15, Chsy1, Chsy3,
Csgalnact1-2, Ext1-2, Extl1-3, Fam20b), among others.
Animals

All experiments were performed in accordance with the
ethical guidelines of the Animal Care Committee at thePu et al. Journal of Neuroinflammation(2020) 17:220

Page 3 of 21

Table 1 Names and description of genes associated with CSPG synthesis and turnover
Gene

Description

Acan

AggrecanAco1

Aconitase 1, soluble

Adam2

ADAM metallopeptidase domain 2

Adam7

ADAM metallopeptidase domain 7

Adam8

ADAM metallopeptidase domain 8

Adam9

ADAM metallopeptidase domain 9

Adam10ADAM metallopeptidase domain 10

Adam11ADAM metallopeptidase domain 11

Adam12

ADAM metallopeptidase domain 12

Adam15ADAM metallopeptidase domain 15

Adam17ADAM metallopeptidase domain 17

Adam18ADAM metallopeptidase domain 18

Adam19ADAM metallopeptidase domain 19

Adam20ADAM metallopeptidase domain 20

Adam21ADAM metallopeptidase domain 21

Adam22

ADAM metallopeptidase domain 22

Adam23

ADAM metallopeptidase domain 23

Adam28ADAM metallopeptidase domain 28

Adam29ADAM metallopeptidase domain 29

Adam30ADAM metallopeptidase domain 30

Adam32

ADAM metallopeptidase domain 32

Adam33

ADAM metallopeptidase domain 33

Adamts1

ADAM metallopeptidase with thrombospondin type 1 motif, 1

Adamts2

ADAM metallopeptidase with thrombospondin type 1 motif, 2Adamts3

ADAM metallopeptidase with thrombospondin type 1 motif, 3Adamts4

ADAM metallopeptidase with thrombospondin type 1 motif, 4Adamts5

ADAM metallopeptidase with thrombospondin type 1 motif, 5Adamts6

ADAM metallopeptidase with thrombospondin type 1 motif, 6

Adamts7

ADAM metallopeptidase with thrombospondin type 1 motif, 7

Adamts8

ADAM metallopeptidase with thrombospondin type 1 motif, 8Adamts9

ADAM metallopeptidase with thrombospondin type 1 motif, 9Adamts10

ADAM metallopeptidase with thrombospondin type 1 motif, 10

Adamts12

ADAM metallopeptidase with thrombospondin type 1 motif, 12

Adamts14

ADAM metallopeptidase with thrombospondin type 1 motif, 14

Adamts15

ADAM metallopeptidase with thrombospondin type 1 motif, 15

Adamts16

ADAM metallopeptidase with thrombospondin type 1 motif, 16

Adamts17

ADAM metallopeptidase with thrombospondin type 1 motif, 17

Adamts18

ADAM metallopeptidase with thrombospondin type 1 motif, 18

Adamts19

ADAM metallopeptidase with thrombospondin type 1 motif, 19

Adamts20

ADAM metallopeptidase with thrombospondin type 1 motif, 20

AppAmyloid beta (A4) precursor protein

B3galt6

UDP-Gal:betaGal beta 1,3-galactosyltransferase polypeptide 6

B3gat1

Beta-1,3-glucuronyltransferase 1 (glucuronosyltransferase P)

B3gat2

Beta-1,3-glucuronyltransferase 2 (glucuronosyltransferase S)

Pu et al. Journal of Neuroinflammation(2020) 17:220

Page 4 of 21

Table 1 Names and description of genes associated with CSPG synthesis and turnover (Continued)
Gene

Description

B3gat3

Beta-1,3-glucuronyltransferase 3 (glucuronosyltransferase I)

B4galt7

Xylosylprotein beta 1,4-galactosyltransferase, polypeptide 7

Bcan

Brevican

Bgn

BiglycanBsg

Basigin

Chpf

Chondroitin polymerizing factor

Chpf2

Chondroitin polymerizing factor 2

Chst1

Carbohydrate (keratan sulfate Gal-6) sulfotransferase 1

Chst2

Carbohydrate (N-acetylglucosamine-6-O) sulfotransferase 2

Chst3

Carbohydrate (chondroitin 6) sulfotransferase 3

Chst4

Carbohydrate (N-acetylglucosamine 6-O) sulfotransferase 4

Chst5

Carbohydrate (N-acetylglucosamine 6-O) sulfotransferase 5

Chst6

Carbohydrate (N-acetylglucosamine 6-O) sulfotransferase 6

Chst7

Carbohydrate (N-acetylglucosamine 6-O) sulfotransferase 7

Chst8

Carbohydrate (N-acetylgalactosamine 4-0) sulfotransferase 8

Chst9

Carbohydrate (N-acetylgalactosamine 4-0) sulfotransferase 9

Chst10Carbohydrate sulfotransferase 10

Chst11

Carbohydrate (chondroitin 4) sulfotransferase 11

Chst12

Carbohydrate (chondroitin 4) sulfotransferase 12

Chst13

Carbohydrate (chondroitin 4) sulfotransferase 13

Chst14

Carbohydrate (N-acetylgalactosamine 4-0) sulfotransferase 14

Chst15

Carbohydrate (N-acetylgalactosamine 4-sulfate 6-O) sulfotransferase 15

Chsy1

Chondroitin sulfate synthase 1

Chsy3

Chondroitin sulfate synthase 3

Cp

Ceruloplasmin (ferroxidase)

Csgalnact1

Chondroitin sulfate N-acetylgalactosaminyltransferase 1

Csgalnact2

Chondroitin sulfate N-acetylgalactosaminyltransferase 2

Dse

Dermatan sulfate epimerase

Ext1

Exostosin glycosyltransferase 1

Ext2

Exostosin glycosyltransferase 2

Extl1

Exostosin-like glycosyltransferase 1

Extl2

Exostosin-like glycosyltransferase 2

Extl3

Exostosin-like glycosyltransferase 3

Fam20bFamily with sequence similarity 20, member B

Fdx1

Ferredoxin 1

Fth1

Ferritin, heavy polypeptide 1

Ftl

Ferritin, light polypeptide

Ftmt

Ferritin mitochondrial

Galns

Galactosamine (N-acetyl)-6-sulfate sulfatase

Gpc1

Glypican 1

Gpc2

Glypican 2

Gpc3

Glypican 3

Gpc4

Glypican 4

Gpc5

Glypican 5

Pu et al. Journal of Neuroinflammation(2020) 17:220

Page 5 of 21

Table 1 Names and description of genes associated with CSPG synthesis and turnover (Continued)
Gene

Description

Gpc6

Glypican 6

Gsta1

Glutathione S-transferase alpha 1

Gsta2

Glutathione S-transferase alpha 2

Gsta3

Glutathione S-transferase alpha 3

Gsta4

Glutathione S-transferase alpha 4

Gsta5

Glutathione S-transferase alpha 5

Gstk1

Glutathione S-transferase kappa 1

Gstm1

Glutathione S-transferase mu 1

Gstm2

Glutathione S-transferase mu 2 (muscle)

Gstm3

Glutathione S-transferase mu 3 (brain)

Gstm4

Glutathione S-transferase mu 4

Gstm5

Glutathione S-transferase mu 5

Gstp1

Glutathione S-transferase pi 1

Gstt1

Glutathione S-transferase theta 1

Gstt2

Glutathione S-transferase theta 2

Hamp

Hepcidin antimicrobial peptide

Hapln1

Hyaluronan and proteoglycan link protein 1

Hapln2

Hyaluronan and proteoglycan link protein 2

Has1

Hyaluronan synthase 1

Has2

Hyaluronan synthase 2

Has3

Hyaluronan synthase 3

Heph

Hephaestin

Hmox1

Heme oxygenase (decycling) 1

Hmox2

Heme oxygenase (decycling) 2

Hp

Haptoglobin

Hpr

Haptoglobin-related protein

Ilf3

Interleukin enhancer binding factor 3, 90 kDa

Ireb2

Iron-responsive element binding protein 2

Ltf

Lactotransferrin

Mfi2

Antigen p97 (melanoma associated) identified by monoclonal antibodies 133.2 and 96.5

Mgst1

Microsomal glutathione S-transferase 1

Mgst2

Microsomal glutathione S-transferase 2

Mgst3

Microsomal glutathione S-transferase 3

Mmp1

Matrix metallopeptidase 1 (interstitial collagenase)

Mmp2

Matrix metallopeptidase 2 (gelatinase A, 72 kDa gelatinase, 72 kDa type IV collagenase)

Mmp3

Matrix metallopeptidase 3 (stromelysin 1, progelatinase)

Mmp7

Matrix metallopeptidase 7 (matrilysin, uterine)

Mmp8

Matrix metallopeptidase 8 (neutrophil collagenase)

Mmp9

Matrix metallopeptidase 9 (gelatinase B, 92 kDa gelatinase, 92 kDa type IV collagenase)

Mmp10

Matrix metallopeptidase 10 (stromelysin 2)

Mmp11

Matrix metallopeptidase 11 (stromelysin 3)

Mmp12

Matrix metallopeptidase 12 (macrophage elastase)

Mmp13

Matrix metallopeptidase 13 (collagenase 3)

Mmp14

Matrix metallopeptidase 14 (membrane-inserted)Pu et al. Journal of Neuroinflammation(2020) 17:220

Page 6 of 21

Table 1 Names and description of genes associated with CSPG synthesis and turnover (Continued)
Gene

Description

Mmp15

Matrix metallopeptidase 15 (membrane-inserted)

Mmp16

Matrix metallopeptidase 16 (membrane-inserted)

Mmp17

Matrix metallopeptidase 17 (membrane-inserted)

Mmp19

Matrix metallopeptidase 19

Mmp20

Matrix metallopeptidase 20

Mmp21

Matrix metallopeptidase 21

Mmp23bMatrix metallopeptidase 23B

Mmp24

Matrix metallopeptidase 24 (membrane-inserted)

Mmp25

Matrix metallopeptidase 25

Mmp26

Matrix metallopeptidase 26

Mmp27

Matrix metallopeptidase 27

Mmp28

Matrix metallopeptidase 28

Ncan

NeurocanNdst1

N-deacetylase/N-sulfotransferase (heparan glucosaminyl) 1

Ndst2

N-deacetylase/N-sulfotransferase (heparan glucosaminyl) 2

Slc11a1

Solute carrier family 11 (proton-coupled divalent metal ion transporter), member 1

Slc11a2

Solute carrier family 11 (proton-coupled divalent metal ion transporter), member 2

Slc39a14

Solute carrier family 39 (zinc transporter), member 14

Slc40a1

Solute carrier family 40 (iron-regulated transporter), member 1

TfTransferrin

Tfr2

Transferrin receptor 2

Tfrc

Transferrin receptor

Timp3

TIMP metallopeptidase inhibitor 3

Ust

Uronyl-2-sulfotransferase

Vcan

Versican

Xylt1

Xylotransferase I

Xylt2

Xylotransferase II

University of Calgary. Female wild-type C57Bl/6 mice
aged 6 to 8 weeks were purchased from Charles River(Montreal) and Extl2−/− mice were previously described[28, 32] and bred at the University of Calgary Animal
Health Unit. Experiments conducted with C57Bl/6 wildtype mice from our in-house bred colony or purchasedfrom Charles River did not differ in responses when directly compared. The Extl2-null mice were generated bytruncating exon 3 and removal of exon 4 of the Extl2
gene [32]. No defects in breeding or physical impairments were published for the Extl2−/− mice, and we did
not observe any obvious abnormalities including weightchanges in our colony transferred from Japan throughco-author Hiroshi Kitagawa. All experiments werecompleted on female mice aged 8 to 12 weeks.
Lysolecithin-induced demyelination

Experimental demyelination was produced in the ventral
spinal cord by stereotactic injection of the toxin

lysolecithin [29]. A 10 μL glass Hamilton syringe with a
34-gauge needle was preloaded with a 1% solution oflysolecithin. Animals were anesthetized with a mixtureof ketamine/xylazine (100 mg/kg and 10 mg/kg, respectively) administered through an intraperitoneal injection.
The analgesic buprenorphine (0.05 mg/kg) was injectedsubcutaneously prior to surgery. Ophthalmic lubricant
was applied to the eyes to prevent them from drying
during surgery. A small area on the back was shaved
with a razor and the surgical field was disinfected with
70% ethanol and iodine solution. A midline incision was
made, and underlying muscle and adipose tissue separated using retractors. Connective tissue between the T3
and T4 vertebra was cut to reveal the spinal cord. The
dura mater was opened using a 30-gauge needle. The tipof the Hamilton 34-gauge needle was placed at thesurface of the spinal cord, lateral to midline. A baseline
measurement was made, and 1.3 mm was subtracted
from the baseline measurement to obtain the final

Pu et al. Journal of Neuroinflammation(2020) 17:220

desired measurement for targeting the ventral whitematter. The needle was depressed and injection of lysolecithin was completed with one rotation of the micromanipulator every 5 s for 2 min, for a final volume of
0.5 μL. Animals were sutured and placed in a recovery
chamber and monitored until awake. Postoperative
buprenorphine was administered subcutaneously at 0.05
mg/kg 16 h following surgery.
Tissue harvesting and processing

Animals were sacrificed at 7, 14, and 21 days post-injectionof lysolecithin. Mice were given a lethal dose of ketamine/
xylazine anesthetic. Animals were perfused through the left
ventricle of the heart with 15 mL of phosphate-buffered saline (PBS) and 15 mL of 4% paraformaldehyde (PFA). The
cervical and upper thoracic section of the spinal cord was
harvested. Spinal cords were laid on strips of thick card
stock to maintain structural integrity. For western blot,
spinal cords were immediately flash frozen in liquid nitrogen and stored at – 80 °C until use. For immunohistochemistry, spinal cords were post-fixed overnight in 4% PFA at 4
°C, then transferred into 30% sucrose solution for 72 h.
Spinal cords were then frozen into O.C.T. blocks using dry
ice and blocks were stored at – 20 °C until sectioning on
the cryostat.
Immunohistochemistry

Spinal cords frozen in OCT blocks were cut into 20 μmthick sections and mounted on glass slides. Staining oftissue sections was done in a humidity chamber to prevent evaporation. One section per spinal cord waschosen for staining and analysis. Sections were selectedbased on proximity to the lesion epicenter; the latter was
elucidated as the section in a given mouse containing
the largest lesion as determined using eriochromecyanine staining. The adjacent sections were taken for
immunofluorescent staining. This selection of tissue
sections is similar to our analyses of other featuresdescribed in previous publications (e.g., [21]).
All washes were completed using PBS containing 0.25%
Tween-20. Blocking of tissue sections was done using
horse serum blocking solution (0.01 M PBS, 10% horse
serum, 1% bovine serum albumin (BSA), 0.1% cold fishskin gelatin, 0.1% Triton-X100, and 0.05% Tween-20) for
2 h at room temperature. Blocking solution was removedprior to application of primary antibodies. Primary antibodies were diluted in 0.01 M PBS containing 1% BSA,
0.1% cold fish skin gelatin, and 0.5% Triton-X100. Primary
antibodies were obtained from various sources and used
at dilutions detailed as follows: versican V0/V1 (Millipore,ab1033, 1:200), myelin basic protein (MBP, Abcam,
ab7349, 1:1000), Olig2 (Millipore, AB9610, 1:200),
platelet-derived growth factor receptor α (PDGFRα, R&D
Systems, AF1062, 1:100), adenomatous polyposis coli

Page 7 of 21(APC, Millipore, clone CC-1, OP80, 1:100), neurofilament
heavy chain 200 kD (NF-200, EnCor, RPCA-NF-H, 1:
1000), ionized calcium-binding adaptor molecule 1 (Iba1,
Wako, 019-19741, 1:1000), CD16/32 (Bio-Rad, MCA1075,
1:200), and CD206 (R&D Systems, AF2535, 1:200). Primary antibodies were left on sections overnight at room
temperature. Secondary antibodies were diluted in thesame dilution buffer as primary antibodies and left for 2 h
at room temperature. Slides were mounted with Fluoromount G® (SouthernBiotech). Fluorescence imaging wasdone using the Leica TCS SP8 confocal laser scanningmicroscope.
Versican exists as 4 isoforms: V0, V1, V2, and V3. The
antibody to versican V1 (rabbit anti-mouse versican V0/
V1, Millipore, AB1033) recognizes the GAG beta domain, and the versican V2 antibody (rabbit anti-mouseversican V0/V2, Millipore, AB1032) recognizes the GAG
alpha domain, both of which are present in the versican
V0 splice isoform. However, due to the lack of colocalization of versican V1/V0 and versican V2/V0 in similar
regions (data not shown), we attributed the staining ofversican V1 to primarily V1 isoform, although this is displayed in figures as V0/V1.
Chondroitinase ABC digestion

Chondroitinase ABC (ChABC) digestion was done for
immunohistochemical staining of V0/V1, as the removalof the GAG chains facilitates the binding of antibody tothe core protein. Slides were thawed to room temperature
and washed once with PBS. ChABC was diluted in PBS
and applied to slides at a working concentration of 0.2
units/mL. Slides were incubated in a heated chamber at
37 °C for 30 min. Slides were washed with PBS before
proceeding with staining.
Bone marrow-derived macrophages (BMDM) isolation andcultureC57Bl/6 wild-type or EXTL2−/− animals were euthanizedby overdose of ketamine/xylazine. Femurs and tibia weredissected from the body and soaked briefly in ethanol.
Bone marrow was harvested from bones and centrifuged
at 1200 rpm for 10 min. Cells were resuspended in PBS
and counted. Cells were centrifuged again at 1200 rpm
for 10 min. BMDMs were cultured at 37 °C, 8.5% CO2 in
100 mm plastic, uncoated, tissue culture-treated dishes
in Dulbecco’s modified eagle’s medium (DMEM) containing 10% FBS, 1% penicillin-streptomycin (Gibco), 1%
sodium pyruvate (Gibco), 1% GlutaMAX™ (Gibco), and
10% L929 conditioned media. Cells were cultured for 8–
10 days before harvesting. A half-volume media change
was done on day 5 and full media change was done on
day 7. To harvest BMDMs, media was removed fromplates and cold PBS was applied to the dishes. Cell
scrapers were used to dissociate cells from dishes. Cells

Pu et al. Journal of Neuroinflammation(2020) 17:220

were collected in 50 mL tubes and centrifuged at 1200
rpm for 10 min, then resuspended in same media, except
lacking L929 conditioned media. Cells were replated at adensity of 50,000 cells/100 μL/well in a 96-well plate.
Plates were incubated at 37 °C in 5% CO2. A media
switch to serum-free DMEM with supplements was
performed at 24 h post-plating, and treatments wereadded to wells in 1% FBS in DMEM.
Enzyme-linked immunosorbent assay

Supernatant from BMDM cultures (as described above)
was harvested at 24 h following the initiation of stimulation.
Supernatant was frozen at − 80 °C until use. Samples were
thawed to room temperature and ELISA was performed
according to manufacturer instructions (Invitrogen).
Mouse MMP 5-plex Luminex assay

BMDM supernatant was harvested at 24 h following theinitiation of stimulation. Supernatant was frozen at − 80
°C until the assay was performed. Tubes were stored on
ice until used. Samples were processed by Eve Technologies (Calgary) for a panel of 5 MMP members.
Oligodendrocyte precursor cell isolation and harvest

P0-2 CD-1 mouse pups were sacrificed, and brains wereisolated. Under a dissection microscope, meninges andchoroid plexus tissue were removed from the corticaland ventricular surfaces, respectively. Cortices were dissected away from the hippocampus and cerebellum andcut into 2–3 mm2 pieces. A cocktail of papain, DNase,and L-cysteine was used for tissue digestion. The digestion cocktail was added to tissue in a 50-mL tube andthe tube was placed in a 37 °C water bath for 30 min,with occasional inversion. More DNase was added if tissue became gel-like. Cells were centrifuged at 1200 rpm
for 10 min and resuspended in growth medium. Dissociated cells were plated in T-75 culture flasks pre-coatedwith poly-L-lysine (10 μg/mL, Sigma). The mixed glial
culture was maintained in DMEM containing 10% FBS,
1% GlutaMAX™, 1% sodium pyruvate, and 1% penicillinstreptomycin. Flasks were kept at 37 °C, 8.5% CO2 for 9days. Half media changes were performed every 3 days.
On day 9, flasks were shaken overnight in a 37 °C, 5%
CO2 incubator at 220 rpm to dislodge OPCs and microglia from astrocytes. The supernatant containing OPCs
and microglia was plated on plastic, uncoated 100 mm
dishes for 30 min to allow microglia to adhere. Media
containing enriched OPCs was harvested and plated inoligodendrocyte differentiating media (described below)
at 5000–10,000 cells/100 μL/well in a 96-well plate precoated with poly-L-lysine and other extracellular matrix
molecules when appropriate.
Oligodendrocyte differentiating medium was DMEM
containing 2% (v/v) B27 supplement (Gibco), 1% (v/v)

Page 8 of 21

oligodendrocyte supplement cocktail (see below), 1% (v/v)
GlutaMAX™ (Gibco), 100 μm sodium pyruvate (Gibco),
1% (v/v) penicillin-streptomycin (Gibco), 50 μg/mL holotransferrin (Sigma), 5 μg/mL N-acetyl-L-cysteine (Sigma),
50 ng/mL ciliary neurotrophic factor (PeProTech), and
0.005% (v/v) Trace Elements B (Corning).
Oligodendrocyte supplement cocktail was made from
100 mL DMEM with 1% BSA, 0.06% progesterone(Sigma), 16.1% putrescine (Sigma), 0.005% sodium selenite (Sigma), 0.4% 3,3′,5-triiodo-L-thyronine (Sigma).
Neuron culture and harvest

E16-18 C57Bl/6 pups were obtained from pregnant
C57Bl/6 mice purchased from Charles River. The pregnant mouse was anesthetized with a mixture of ketamineand xylazine and the uterine horns were dissected. Pups
were sacrificed by decapitation and brains were isolated.
Meninges were microdissected from the brain and cortices were cut into small pieces and collected. For tissue
digestion, harvested brain tissue was suspended in HBSS
(Gibco) containing 0.025% trypsin. The suspension was
immersed in a 37 °C water bath with a stir bar tohomogenize tissue. Following digestion, heat inactivated
FBS was added to inactivate trypsin and cells were filtered into a 50 mL tube and centrifuged at 1200 rpm for
10 min. The supernatant was discarded, and cells werethen washed one time with HBSS and centrifuged as before. The supernatant was discarded, and cells were resuspended in growth medium comprised of Neurobasal
Plus Medium (Gibco) containing 2% B27 Plus supplement (Gibco). Cells were seeded at 1 to 2 × 107 cells per
T-25 flasks coated with poly-L-ornithine. Cells weregrown in a 37 °C incubator in 5% CO2 for at least 24 hin the T-25 flask before they were harvested using
0.025% trypsin. Cells were then plated at a density of
100,000 cells/well in a 96-well plate coated with poly-Lornithine in the previously described growth medium.
Neurons were allowed to grow for 24 h prior to treatment with BMDM conditioned media.
Cell staining and imaging

All washing was done using PBS. Plates were placed onan orbital shaker during all incubations. For OPCs,
blocking was done using 10% normal goat serum (NGS)in PBS or Odyssey® Blocking Buffer (Licor) for 1 h atroom temperature. Primary antibody against O4 (Millipore) was diluted 1:50 in blocking buffer and incubatedfor 2 h at room temperature or overnight at 4 °C. An
anti-mouse IgM antibody conjugated to Alexa Fluor®
488 (Jackson ImmunoResearch) as well as nuclear yellow
was added in blocking buffer for 1 h at room
temperature. Plates were washed with PBS and imaged
using the ImageXpress Micro XLS Widefield HighContent Analysis System (Molecular Devices).

Pu et al. Journal of Neuroinflammation(2020) 17:220

For BMDMs, for detection of intracellular molecules,
cells were permeabilized using 0.025% Triton-X100 for
5 min at room temperature. No permeabilization was required for surface molecules. Blocking was done using
Odyssey® Blocking Buffer (Licor) for 30 min at room
temperature. Primary antibodies were diluted in thesame blocking buffer incubated overnight at 4 °C.
Secondary antibodies were diluted in the same blocking buffer and incubated for 1 h at room temperature.
Nuclear yellow was added in PBS and incubated for
10 min. Plates were washed with PBS and imaged
using the ImageXpress Micro XLS Widefield HighContent Analysis System.

Image analysis

For immunohistochemistry, images were processed andanalyzed using ImageJ. All images were blinded prior to
analysis. For percent of lesion area analyses, lesion area
was demarcated using MBP staining. A disruption in
MBP density was determined to be lesional. The lesionarea was isolated and area of signal of interest was determined using differential thresholding. The area occupiedby the signal was normalized to total area of the lesion
or represented as an absolute area. For axonal counts inthe lesion, the lesion area was demarcated using MBP.
Differential thresholding was used to isolate individualaxons present inside the lesional area and was automatically counted. The number of axons within the lesion
was normalized to total lesion area to obtain density. For
oligodendrocyte-lineage cell counting and scoring, thesame approach to demarcating the lesion area was used.
Images containing Olig2+ nuclei were overlaid with either PDGFRα staining or CC1 staining. Colocalization ofeither PDGFRα or CC1 with Olig2 was determined to be
an OPC or mature oligodendrocyte, respectively. The
density of oligodendrocytes in the lesions was calculated
by isolating the lesion area and counting the number of
Olig2+ nuclei and normalizing counts to the respective
lesion area. Percentage of OPCs or mature oligodendrocytes was calculated using the total number of Olig2+
nuclei. We assessed the mean fluorescence intensity ofthe macrophage activation markers CD16/32 and CD206
within the lesion, internally normalized to the background fluorescence. Mean fluorescence intensity wasassessed within the area of positive immunoreactivity.
For immunocytochemistry, images were analyzed on
the MetaXpress High-Content Image Acquisition andAnalysis Software (Molecular Devices, San Jose, CA).
Appropriate thresholds were determined for true signals.
OPC cultures were analyzed using the Neurite Outgrowth program, and BMDM cultures were analyzed
using the Cell Scoring program. Both processing protocols are from Molecular Devices.

Page 9 of 21

Statistical analysis

All statistical analysis was performed with GraphPad
Prism 8.0. Student’s t test, ordinary one-way ANOVA,
and two-way ANOVA statistical tests were used with analpha of 0.05. Individual tests and post hoc tests are described in figure legends. For assessment of lesions withrespect to versican V1 deposition, oligodendrocytes, lesion area, macrophage/microglia, and axons, between 4
and 12 individual animals were used, and one section
per lesion was quantified. The individual performing thesurgery was not blinded to the genotype of animals;
however, images were blinded prior to analysis on the
ImageJ software. Each animal/lesion was considered an
n of 1. In some cases, the quality of the tissue section or
the staining was not deemed adequate for blinded analysis, and these were omitted. For lesion area analysis
using MBP staining, animals from two sets of surgeries
were combined. Other immunohistological analyses were
performed on one of the two sets of surgeries.
BMDMs were derived from 1 to 2 wild-type or
EXTL2−/− mice. OPCs were derived from between 8 to
12 CD-1 pups. Neurons were derived from 8 to 12
C57Bl/6 prenatal pups aged E13-15. For all cell culture
experiments, each data point was derived from one well.
Subsequent replicates of experiments were performed
and reported in figure captions.

Results
SNP data mining from MS databases implicates EXTL2

Ignoring pseudogenes, we identified 154 autosomal
genes involved in the metabolism of CSPGs (Table 1).
Several of these genes map close to each other so after
allowing a 100-kb extension from both ends, the 154
genes were found to map to 125 distinct genomic regions containing 7382 SNPs in the GWAS (median 42
SNPs per region, range 7–291) and 3139 SNPs (from118 of the 125 regions) in the Immunochip (median 5
SNPs per region, range 1–494).
Both the GWAS and the Immunochip studies included
secondary analyses of disease severity, as measured by
the Multiple Sclerosis Severity Score and the clinicalcourse (relapse onset vs progressive onset). None ofthese secondary analyses identified significant associations so it was unsurprising that no enrichment for association was seen among the SNPs mapping close to
CSPG-related genes.
Six of the SNPs from the GWAS showed genomewide significant evidence for association with all of these
SNPs mapping to the region containing EXTL2. These
SNPs all showed concordant effects on risk in the
Immunochip study with significant but less extreme pvalues (ranging from 1.4e− 4 to 3.7e− 6). All six of the
SNPs mapping to the region containing the EXTL2 gene
were confirmed as associated with genome-widePu et al. Journal of Neuroinflammation(2020) 17:220

Page 10 of 21

significance in the larger MS GWAS meta-analysis(IMSGC 2017 BioRxiv 143933) that involved a total of
14,802 cases and 26,703 controls, including those considered in the 2011 GWAS. Combining all available genetic data showed that there are likely at least two
independent MS susceptibility variants in the region of
the EXTL2 gene (IMSGC 2017 BioRxiv 143933); 5(rs12746227, rs11809572, rs11581062, rs11588568, andrs7541397) of the 6 SNPs were identified tag one ofthese effects and the remaining SNP (rs7552544) tags
the second. In the final combined analysis, there was
highly significant evidence for association with MS in
this region (p = 1.1e− 22). These data thus confirm
beyond any reasonable doubt that genetic variation inthis region influences susceptibility to MS; however, it isimportant to note that EXTL2 is not the only gene in
the region of these associated SNPs. It is therefore notpossible to conclude with certainty that these variants
exert their effects on risk via EXTL2 rather some othergene within the region. Nonetheless, the genetic analyses
provided a solid rationale to evaluate the biology of
EXTL2 in models of MS.

expected a deficiency or delay in remyelination in the
EXTL2−/− mice. In the spinal cord lysolecithin model,
OPCs loss occurs in the acute phase of injury and they
then repopulate the lesion after 3–4 days, with subsequent accumulation of OPCs and eventual differentiation into mature oligodendrocytes and remyelination([29]; Jensen et al., 2018). We analyzed spinal cord sections stained for the transcription factor Olig2 that defines cells of the oligodendrocyte lineage, the surface
receptor PDGFRα that is selective to OPCs, and APC(adenomatous polyposis coli, also called CC1, labelingmature oligodendrocytes) to determine the number ofoligodendrocyte-lineage cells present within the lesion atdays 14 and 21 post-injury (Fig. 2a, e). Unexpectedly, we
found no significant differences in the density of Olig2+
oligodendrocyte-lineage cells within the lesion (Fig. 2b,f), or in proportions of Olig2+PDGFRα+ OPCs (Fig. 2c,g) and Olig2+APC+ oligodendrocytes (Fig. 2d, h) at anyof the assessed time points. No deficiencies in OPC
recruitment to the lesion, nor maturation into oligodendrocytes, was apparent by day 14 and day 21 after
lysolecithin injury.

Increased deposition and persistence of versican V1 andCS-A glycosaminoglycan following injury in the EXTL2−/−
spinal cord

Exacerbated lesion area and decreased axonal density in
EXTL2−/− lesions

Based on previous literature suggesting that EXTL2 is anegative regulator of CSPG synthesis [32], we first asked
whether loss of EXTL2 results in increased CSPG deposition into an injured area during the reparative andremyelinating phases. We induced lysolecithin lesions in
the ventral columns of spinal cords of wild-type and
EXTL2−/− animals and evaluated total CS-A (not shown)and versican V1 deposition into the lesion at 14 and 21days post-lesion (dpl). Versican V1 was focused uponsince this was the predominant lectican CSPG member
produced after lysolecithin injury [21].
Eriochrome cyanine staining shows a demarcated areain the ventral column of the spinal cord with distinctive
loss of myelin in the lesion, corresponding to an increasein V1 immunoreactivity (Fig. 1a, b). In both wild-type
and EXTL2−/− spinal cords, upregulation of V1 waslimited to the lesion site (Fig. 1c). By 14 dpl, there was agreater accumulation of V1 in EXTL2−/− lesions compared to wild-type (Fig. 1d, e). At day 21, V1 was largely
cleared from the wild-type lesion, whereas it remainedin the lesion of EXTL2−/− animals (Fig. 1d, f).

In our characterization of the EXTL2−/− phenotype following injury, we found an exacerbated injury area demarcated by loss of MBP immunoreactivity (Fig. 3a).
Analysis of the total area of MBP disruption in bothwild-type and EXTL2−/− lesioned spinal cords showed alarger area of demyelination in EXTL2−/− spinal cords at
day 14 (Fig. 3b) and a similar but non-statistically significant trend at day 21 (Fig. 3c) following injection of lysolecithin compared to those of wild-type animals. We
then hypothesized that there could be a corresponding
loss of axons in the demyelinated area. To identify
axons, we used an antibody against the 200 kD neurofilament (NF200), which reliably labels axons in the ventral
column (Fig. 3d). Qualitatively, there was close correlation between the area of axonal loss to area of demyelination (Fig. 3d). At day 14, we observed a decreased
axonal density in the EXTL2−/− lesions, corroborating
our finding of a larger area of demyelination (Fig. 3e, f).
Lesions from day 21 animals had too much backgroundto be evaluated for axons. Overall, we observed a larger
lesion and a greater loss of axons in the EXTL2−/− spinal
cord with injection of lysolecithin.

EXTL2−/− mice exhibit no overt differences in OPC
repopulation and maturation following injury

Increased accumulation of microglia and macrophages
within lesions of EXTL2−/− animals

We next asked whether the extensive deposition andpersistence of CSPGs in the EXTL2−/− animals impacted
the remyelination process. As there is extensive literature on the inhibitory nature of CSPGs on OPCs, weGiven our previous report of CSPGs being a stimulatorof macrophages in EAE [18] and our previous finding ofexacerbated injury in the EXTL2−/− spinal cord, weinvestigated whether there were corresponding pro-Pu et al. Journal of Neuroinflammation(2020) 17:220

Page 11 of 21

Fig. 1 Immunolabeling of versican V0/V1 in wild-type and EXTL2−/− lesions. a Representative images of tissue sections stained with the anti-versican V0/V1
antibody (left) and the secondary antibody control. Scale bar = 200 μm. b Representative lesion from wild-type day 14 animal stained with eriochromecyanine R (scale bar = 100 μm) and c immunofluorescence staining for versican V0/V1, MBP, and merged image. Scale bar on immunofluorescent image =200 μm. Inset shows high magnification image of versican and MBP. d Representative images of versican V0/V1 in wild-type and EXTL2−/− lesions at 14
and 21 dpl. White outline indicates approximate lesion area. Scale bar = 200 μm. Quantification of versican area within the lesions, in terms of percent areaof the whole lesion determined separately by MBP staining, and in terms of absolute area within the isolated lesion are shown in e for day 14 and f forday 21. Graphs show mean with standard deviation. Two-tailed, unpaired Student’s t test. *p < 0.05, **p < 0.01

inflammatory phenotypes in lesional microglia/macrophages in EXTL2−/− animals. Macrophages and microglia
were labeled by Iba1 (ionized calcium-binding adaptor
molecule 1) (Fig. 4a). We determined that there was agreater area of lesion covered by Iba1 immunoreactivity at
day 14 (Fig. 4b), indicating an increase in macrophages/
microglia density within the lesion, in the EXTL2−/−
animals. Greater Iba1 immunoreactivity was still observed
at day 21 (Fig. 4c), suggesting macrophages/microglia
persisted for longer in the EXTL2−/− lesions.

for M1-like pro-inflammatory and M2-like antiinflammatory surface markers, CD16/32 and CD206 [33],respectively (Fig. 5a, d); the pro-inflammatory enzyme inducible nitric oxide synthase (iNOS) was attempted but did not
result in reliable staining. For both CD16/32 and CD206,
intensity was not significantly different between wild-type
and EXTL2−/− lesions (Fig. 5b, c, e, f), indicating that expression levels of these receptors are similar and that there was
no apparent alterations in microglia/macrophage phenotype
in EXTL2−/− animals.

No difference in the surface marker expression of CD16/
32 and CD206 in lesional Iba1+ cells in wild-type and
EXTL2−/− mice

Cultured EXTL2−/− BMDMs upregulate iNOS, secrete more
TNFα, and differentially regulate MMPs in response to
CSPGs

To further resolve whether differences in polarization exist in
the Iba1+ cells in the wild-type and EXTL2−/− lesions, weprobed tissue sections using immunofluorescence staining

Due to limitations of assessing the activity of Iba1+ cells in
the lesion using fixed tissue sections, we cultured murinederived BMDMs to examine functional or phenotypicPu et al. Journal of Neuroinflammation(2020) 17:220

Page 12 of 21

Fig. 2 Immunostaining and quantification of oligodendrocyte lineage cells following injury. Representative images of lesions labeled with Olig2,
PDGFRα, and APC (CC1) are shown in a and e, for days 14 and 21, respectively. Scale bar = 200 μm. Insets are high magnification images ofindividual Olig2+ nuclei co-labeled with PDGFRα or APC. Density of all Olig2+ nuclei are quantified for day 14 in b and day 21 in f. Proportion of
Olig2+PDGFRα+ OPCs at both day 14 and 21, calculated as a fraction of all Olig2+ cells, are shown in c and g; c shows day 14 and g shows day
21 analyses. Graphs show mean with standard deviation. Two-tailed, unpaired Student’s t test

differences between wild-type and EXTL2−/− macrophages
upon stimulation. As monocyte-derived macrophages constitute a significant portion of immune cells present in thelysolecithin-induced lesion (unpublished observations), we

reasoned that BMDMs are an appropriate population ofcells to study as a model for macrophages in the lesion. We
hypothesized that excessive deposition and persistence of
CSPGs in EXTL2−/− animals underlies the prolonged

Pu et al. Journal of Neuroinflammation(2020) 17:220

Page 13 of 21

Fig. 3 Assessment of area of lesion by loss of MBP immunoreactivity and changes in axonal density in wild-type vs EXTL2−/− lesions at day 14. a
Representative images of MBP immunofluorescent staining in wild-type and EXTL2−/− mice. Scale bar = 200 μm. Lesion area determined by lossof MBP immunoreactivity was quantified for both day 14 (b) and day 21 (c). Two-tailed, unpaired Student’s t test. d Representative images fromday 14 lesions labeled with MBP and NF200 to denote demyelinated area and axons, respectively. Scale bar = 200 μm. e Total number of axons
per lesion. f Density of axons calculated by dividing number of axons over lesion area. Graphs show mean with standard deviation. Two-tailed,unpaired Student’s t test. *p < 0.05

myelin disruption and macrophage/microglia accumulationor activation, which consequently contributes to exacerbated injury. BMDMs were harvested from both wild-type
C56Bl/6 and EXTL2−/− mice and stimulated with a commercial CSPG mixture containing versican for 24 h. Basally,

wild-type and EXTL2−/− BMDMs were comparable withregard to surface expression of CD45, F4/80, CD11b, andCD68. Both groups of naïve BMDMs also exhibited
similarly low expression of CD206, MHC II, and CD11c(data not shown).

Pu et al. Journal of Neuroinflammation(2020) 17:220

Page 14 of 21

Fig. 4 Staining and quantification of Iba1 immunoreactivity in the lesion. Iba1 staining is shown in representative images for day 14 and day 21
in a. Scale bar = 200 μm. Comparison of total Iba1+ area within the lesion in wild-type and EXTL2−/− mice for day 14 (b) and day 21 (c) are
shown. Graphs show mean with standard deviation. Two-tailed, unpaired Student’s t test. **p < 0.01

Cultured BMDMs were stained for the proinflammatory marker iNOS (Fig. 6a), and number ofcells with enhanced iNOS expression was quantified.
While similar numbers of unstimulated wild-type and
EXTL2−/− BMDMs expressed iNOS, CSPG stimulation
resulted in a marked increase in iNOS+ EXTL2−/−
BMDMs (Fig. 6b).
Multiplex analysis to detect the presence of secreted
MMP members was performed on supernatant harvested from stimulated wild-type and EXTL2−/− BMDMs(Fig. 6c–g). We found that EXTL2−/− BMDMs secreted
more MMP8 relative to wild-type BMDMs (Fig. 6e). We
observed a similar trend in MMP2 secretion; however,this did not reach statistical significance (Fig. 6c). No
significant differences in MMP3 secretion were detectedin any group (Fig. 6d). Upon stimulation with CSPGs,
pro-MMP9 was the only MMP to be significantly
increased in the wild-type BMDM conditioned medium(Fig. 6f). Unexpectedly, wild-type BMDMs decreased
secretion of MMP12 in response to CSPG stimulation,
and EXTL2−/− BMDMs exhibited a baseline decrease in
MMP12 production and did not alter MMP12 production with stimulation (Fig. 6g).
Assessment of the BMDM conditioned media using
enzyme-linked immunosorbent assay (ELISA) analysis
revealed significantly increased amounts of TNFα in the
supernatant of EXTL2−/− BMDMs compared to wildtype BMDM supernatant in response to CSPG stimulation (Fig. 6h). To corroborate this in vitro finding, we
stained tissue from wild-type and EXTL2−/− animals 14
days post-injury for TNFα (Fig. 6i). We found greater
areas of immunoreactivity of TNFα in EXTL2−/− spinal

cords than wild-type (Fig. 6j), and pattern of staining
appears to correlate closely with CD68, a surface marker
highly expressed by microglia/macrophages [34].
Conditioned media from EXTL2−/− BMDMs do not killcultured neurons or OPCs

Given that in the presence of CSPGs, EXTL2−/−
BMDMS exhibited a more pro-inflammatory phenotype
in culture, we next asked whether the pro-inflammatory
nature of these macrophages may underlie the exacerbated lesion size and axonal loss observed in EXTL2−/−
animals following lysolecithin injection (Fig. 3). To exclude the possible contribution of contact-mediated cell
death, we harvested conditioned media from cultured
wild-type and EXTL2−/− BMDMs stimulated with
CSPGs. These conditioned media were applied to neurons in a 1:1 ratio with neuron culture media for 24 h.
Although some cultures showed attrition of neuronalnumbers qualitatively when exposed to the conditioned
media of EXTL2−/− BMDM, analyses of pooled results
across 3 experiments (quadruplicate cultures each) did
not show a quantitative loss of neurons when comparedto cultures exposed to conditioned media from wild-type
BMDMs (Fig. 7a, b).
Next, conditioned media were applied to primary OPC
cultures. Cells were cultured for 24 h before fixing andassessment of process outgrowth using the oligodendrocyte lineage marker O4 (Fig. 7c). EXTL2−/− and wildtype BMDM conditioned media had the same effect on
OPC growth (Fig. 7c, d). The conditioned media of bothwild-type and EXTL2−/− BMDMs stimulated with
CSPGs impaired outgrowth, likely due to the CSPGs

Pu et al. Journal of Neuroinflammation(2020) 17:220

Page 15 of 21

contained within, but they did so to the same extent(Fig. 7d).
Three days following the application of BMDM conditioned media to OPCs, cells were assessed for MBP expression as a determinant of viability and maturation.
Between wild-type and EXTL2−/− conditions, the total
number of cells was highly comparable, and the majority
were viable and MBP-expressing (Fig. 7e). Measurementof the mean intensity of MBP staining per cell as well as
the mean area of MBP staining per cell did not reveal obvious differences in both intensity (not shown) and area of
MBP staining (Fig. 7f) in cultures exposed to conditioned
media from wild-type or EXTL2−/− BMDMs.
Finally, the presence of several growth factors normally
added to the growth medium of OPC cultures may obscure differences found in the injury milieu in vivo. We
therefore conducted the same 24-h OPC experiment asshown in Fig. 7 c and d, but without fetal bovine serumand growth factors added to the standard OPC growth
medium. In this minimal medium (MM), OPC growth
was poor in comparison to standard OPC medium (Fig.
7g, h). OPCs grown in MM were more sensitive to inhibition of process outgrowth when exposed to CSPGstimulated BMDM conditioned media; however, there
were no differences in OPC growth when exposed towild-type and EXTL2−/− conditioned media (Fig. 7i).
Overall, the OPC results in tissue culture support thein vivo observations that repopulation of lysolecithin lesions by OPCs and oligodendrocytes was not impairedin EXTL2−/− mice. In contrast, using neurons in culture,
we were unable to mimic the conditions in vivo whereby
an exacerbated axonal loss occurred in EXTL2−/−demyelinated lesions.

Fig. 5 Immunolabeling of microglia/macrophages and surface
markers CD16/32 and CD206. Tissue sections were probed for both
CD16/32 and CD206, and representative images are shown in a for
CD16/32 and Iba1, and d for CD206 and Iba1. Scale bar = 200 μm.
Insets in third image are 63X objective lens magnified images oflesional Iba1+ (white) cells merged with either CD16/32 (red) or
CD206 (green) and nuclear yellow (blue). Scale bar in inset = 20 μm.
b Average lesion intensity of CD16/32 in lesions from 14 dpl. c
Average lesion intensity of CD16/32 in lesions from 21 dpl. e,f)Average lesion intensity for CD206 in day 14 and day 21 lesions,
respectively. Graphs show mean with standard deviation. Two-tailed,unpaired Student’s t test

Discussion
In the present study, we show that dysregulation of
CSPG production following CNS injury profoundly impacts the inflammatory state of a demyelinated lesion.
Loss of the enzyme EXTL2, a regulatory mechanism
serving to limit CSPG synthesis, resulted in more extensive versican deposition into a lysolecithin-induced
demyelinated injury, a larger area of myelin disruption
with an associated decrease in axonal density within thelesion, and greater accumulation of macrophages andmicroglia (schematic shown in Fig. 8). The increased
versican load and microglia/macrophage representation
within the demyelinated lesion of EXTL2−/− mice may
contribute to the expansion of lesion and axonal loss,
but we were not able to show in culture that macrophages from EXTL2−/− mice exposed to CSPGs promote
neuronal or OPC death. While our results support theincreasing recognition that an elevated load of CSPGs
can be pro-inflammatory and injurious in vivo, in
addition to their known roles in inhibiting axonalPu et al. Journal of Neuroinflammation(2020) 17:220

Page 16 of 21

Fig. 6 Assessment of iNOS expression in cultured BMDMs following CSPG stimulation; MMP 5-plex assay and ELISA analyses of conditioned
media from cultured BMDMs. Cultured BMDMs from wild-type and EXTL2−/− mice were stained for iNOS and nuclear yellow (NY). Representative
images for unstimulated and CSPG-stimulated cells are shown in a. Scale bar = 100 μm. b Comparison of number of iNOS+ cells between wildtype and EXTL2−/− cells, unstimulated or with CSPG stimulation. n = 4 wells per condition. Two-way ANOVA with Sidak’s multiple comparisonstest. c-g Fold change of MMP2, MMP8, MMP3, pro-MMP9, and MMP12 detected in conditioned media from both wild-type and EXTL2−/− BMDMs,
unstimulated and CSPG stimulated, respectively. Ordinary one-way ANOVA with Tukey’s multiple comparisons test. Raw quantities of MMPs in pg/
mL ranged from 37.1 to 58.1 for MMP2, 0.1 to 10.3 for MMP3, 305.7 to 490.0 for MMP8, 157.6 to 6265.0 for pro-MMP9, and 3936.5 to 11,558.3 for
MMP12. Fold change is calculated over unstimulated cells. h TNFα levels in BMDM conditioned media as determined by ELISA. Two-way ANOVA
with Sidak’s multiple comparisons test. i Representative images of TNFα and CD68 staining in day 14 lysolecithin-injured spinal cord tissue. Scalebar = 200 μm. j Analysis of absolute TNFα immunoreactive area within lesions at day 14 in wild-type and EXTL2−/− spinal cords. All graphs aremean with SEM, except for panel j (mean with standard deviation). Two-tailed, unpaired Student’s t test. *p < 0.05, **p < 0.01

Pu et al. Journal of Neuroinflammation(2020) 17:220

Page 17 of 21

Fig. 7 Effect of BMDM conditioned media on cultured neurons and OPCs. a Fold change in number of MAP2+ cells assessed for cultured
neurons 24 h following application of CSPG-stimulated BMDM conditioned media (CM) (data pooled from 3 independent experiments ofquadruplicate cultures each). Unpaired, two-way Student’s t test. b Images of neurons labeled with MAP2 and nuclear yellow; scale bar = 100 μm.
c Representative images of wild-type (WT) OPCs labeled with O4 and nuclear yellow 24 h following treatment with BMDM conditioned media.
Scale bar = 200 μm. d Comparison of wild-type and EXTL2−/− BMDM conditioned media on OPC outgrowth. Fold change represents data pooled
from 3 independent experiments. Two-way ANOVA with Tukey’s multiple comparisons test. n.s. = not significant. *p < 0.05. e Representative
images of MBP+ oligodendrocytes 72 h following seeding into a 96-well plate with BMDM conditioned media. Scale bar = 100 μm. f Comparisonof wild-type and EXTL2−/− BMDM conditioned media on OPC differentiation 72 h after seeding. Two-way ANOVA with Sidak’s multiple
comparisons test. n.s. = not significant. g Comparison of number of OPCs seeded either in the standard OPC media (as described in the methods
section) or minimal media (MM) of DMEM containing only GlutaMAX™, sodium pyruvate, and penicillin/streptomycin. h Representative images of
O4-stained OPCs 24 h post-seeding in either standard OPC media or MM. Scale bar = 100 μm. i Comparison of OPC process outgrowth 24 h postseeding in MM, treated with conditioned media from unstimulated or CSPG-stimulated BMDMs. All graphs are mean with SEM. Two-way ANOVA
with Sidak’s multiple comparisons test. n.s. = not significant

regeneration and remyelination, the precise mechanisms by which EXTL2 deficiency promotes injury
remain elusive.

Results from genetic studies strongly implicate genes
from the region containing the EXTL2 gene but cannot
define with precision which of the genes from the region

Pu et al. Journal of Neuroinflammation(2020) 17:220

Page 18 of 21

Fig. 8 Schematic showing the phenotypes observed in EXTL2−/− mice with lysolecithin-induced lesions. EXTL2−/− animals have greater versican
deposition into the lesion following demyelination, and this is correlated to a larger area of damage and exacerbated axon dropout within thelesion. In addition, greater amounts of Iba1 immunoreactivity was seen in EXTL2−/− lesions, indicative of a more inflammatory milieu compared tothat of wild-type animals

is relevant. To date, there are no well-powered studies ofMS progression so it is not possible to infer geneticallywhether EXTL2 (or any other gene) might be involvedin the neurodegenerative aspects of the disease. In asmall study of 147 patients, Saigoh et al. (2016) analyzed
SNPs in the region of the related gene chondroitin sulfate β-1,4-N-acetylgalactosaminyltransferase-1 (ChGn-1),
an enzyme also involved in CSPG synthesis, and suggested that the coding SNP (cSNP: rs140161612) might
influence progression, particularly in male patients. In
short, in the absence of well-powered GWAS for MS
progression, it is challenging to assess geneticallywhether genes that regulate CSPG metabolism might beinvolved in MS progression.
To our knowledge, this is the first study to explore therole of EXTL2 within the CNS. Several questions remainunanswered. While we observed greater CSPG production and deposition into the lesion, the cellular sourcesof these CSPGs have yet to be confirmed. Multiple cell
types are known to produce CSPG members, including
astrocytes, OPCs, and microglia/macrophages. However,for versican V1, its major source is cells of myeloid origin [35]. In previous work, we found that CSPGs in lysolecithin lesions were expressed within Iba1+ microglia/
macrophages [22]; versican V1 is the lectican CSPG
member elevated within these lesions [21]. Moreover, byin situ hybridization, versican V1 transcript was detectedin CD45+ cells (mostly microglia/macrophages) within
lesions [18]. Whether expression within the lysolecithin
lesions could be contributed by circulating versican is

unknown. Another unanswered question is whether the
enzymatic activity of EXTL2 is altered in the lysolecithin
lesion, as this should provide further insight into theregulation of CSPG synthesis following injury.
Others have shown that inhibition of CSPG signaling
through perturbation of the CSPG receptor PTPσ skews
the microglia/macrophage response towards an antiinflammatory “M2-like” state with associated increases
in IL-10 [25]. Given that CSPGs, particularly versican,
have a known role in the modulation of inflammation[19, 36], whether CSPGs deposited by microglia/macrophages locally can act in an autocrine or paracrine fashion to further promote inflammation while reducing“M2-like” condition is another unanswered question. In
our study, we found no difference in the expression of
CD16/32 and CD206, presumed “M1-like” and “M2-like,” respectively, in lesional macrophages/microglia (Fig. 5).
However, previous work has suggested that a populationof mononuclear phagocytes co-expresses the “M1-like”
iNOS and the “M2-like” Arginase-1 simultaneously [37].
Thus, the use of only two markers in our study does notadequately reflect differences in macrophage/microglia
phenotype, and cannot exclude the possibility thatfurther analysis using a more extensive panel of markers
or employing techniques such as RNA sequencing may
reveal more subtle changes induced the EXTL2−/− mice.
The Extl2 gene lies close to the Csf-1 gene on chromosome 11q11-12, so there is a possibility of mutating theCsf-1 gene as a result of the Extl2 manipulation. A lossof function mutation in the Csf-1 gene in mice producesPu et al. Journal of Neuroinflammation(2020) 17:220

the op/op model for osteopetrosis, which is accompanied by a number of overt manifestations such as a lackof teeth, stunted growth, a domed skull, and distinct reduction in the number of macrophages across several
tissues. Our EXTL2−/− animals have similar abundanceand distribution of Iba1+ cells in the spinal cord, and do
not exhibit the distinctive lack of teeth. As we have notobserved these outward presentations, we concluded
that deleterious alterations to the Csf-1 gene are unlikely
in our Extl2-deficient animals. We attempted to measure
CSF-1 production by BMDM from wild-type or
EXTL2−/− animals but were unsuccessful in detecting its
expression using ELISA.
Unexpectedly, we did not find any deficiency in the extent of OPC repopulation of the lesion in the EXTL2−/−
animals compared to wild-type, in contrast to several
lines of evidence supporting the inhibitory nature of
CSPGs to remyelination. A recent study by the Yang
group provides evidence that the CSPG-PTPσ signaling
pathway is a suppressor of OPC migration and maturation. The authors report that perturbation of CSPG signaling (by using the PTPσ inhibitor intracellular sigma
peptide) significantly accelerated recovery from dorsally
injected lysolecithin and decreased severity of experimental autoimmune encephalomyelitis (EAE) [38]. It is
important to note that EXTL2 is not specific to versicansynthesis, and that versican was the focus of our current
work because of its pronounced upregulation following
injury in the lesion after lysolecithin demyelination,
whereas other lectican CSPG members were not [21].
NG2, a non-lectican member of the CSPG family, ishighly expressed by OPCs and upregulated upon injury.
There is evidence to suggest that NG2 plays a key rolein the injury response of OPCs, where loss of NG2 in
OPCs reduces their mitotic index and delays remyelination [39]. Loss of EXTL2 in OPCs may similarly affect
NG2 expression, thus counter-balancing the inhibitory
effects of lesional versican.
Although the formation of the glial scar, largely contributed to by CSPGs, is thought to be protective in limiting the area of injury, we found a significantly greater
lesional area in the EXTL2−/− mice. Previous work fromothers has shown that the disruption of CSPG deposition in the acute phase of injury (1–3 dpl) exacerbates
injury and impedes remyelination and resolution of inflammation [40]. The time points we selected for ourcurrent work do not necessarily reflect the acute injury
phase, and future studies may focus on early time points
to determine whether disruption of glial scar formation
occurs in EXTL2−/− mice. However, a report of CSPG
members in active MS lesions suggests a less clear roleof CSPGs. In chronic active lesions, CSPGs are seen at
the borders of the lesion; however, foamy macrophages
were present in the lesion core with punctate staining ofPage 19 of 21

CSPGs associated with the cells [4]. CSPGs, at an earlier
time, may have been present uniformly throughout thelesion and are being degraded and endocytosed by macrophages from the lesion center. This, supported by ourprevious study in EAE [18], implies that CSPGs may be
exacerbating inflammation rather than serving a neuroprotective role.
This study did not address the closely related group of
ECM to the CSPGs, the heparan sulfate proteoglycans(HSPGs). Due to the N-acetylglucosaminyltransferase
activity of EXTL2, it has been proposed that EXTL2 also
plays a role in HSPG synthesis, where it is thought to bea promoter of HSPG production rather than a regulator.
There is some evidence to suggest that knockdown of
EXTL2 may reduce HSPG synthesis [41, 42]. HSPGs arelargely regarded as a growth-permissive ECM member.
Whether HSPGs are downregulated as a result of EXTL2
knockout was not determined, and consequently, what
impact reduced HSPG production may have on inflammation and repair.
Our work and others have shown that approaches to reducing CSPGs may be key in promoting regeneration in
the CNS. The bacteria-derived enzyme chondroitinase
ABC has previously been used in SCI and white matter ischemia models, where treated animals exhibited histological and functional improvements over untreatedanimals [14, 24, 27]. Pharmacological agents that reduce
biosynthesis of CSPGs have been proposed as an alternative
to CSPG-cleaving enzymes [21, 22]. Protamine is a heparan
neutralizing drug that was also found to block CSPGmediated inhibition on OPC growth in culture [43].
Overall, our data supports previous reports that EXTL2
is a negative regulator of CSPG biosynthesis. We haveshown that the loss of EXTL2 results in the excessive production of versican in response to demyelination, whichconsequently results in protracted response from microglia/macrophages. This study provides the first evidence
for a role of EXTL2 in regulating the responses to CNS
demyelination and identifies a novel target for future studies investigating CSPGs and their role in CNS injury. Our
results also set the stage to evaluate the mechanisms bywhich the CSPG-elevated microenvironment leads toexacerbation of lesion size following demyelination.

Conclusions
This study suggests a potential genetic MS risk association with the Extl2 gene, which encodes for EXTL2, anegative regulator of CSPG biosynthesis. We conclude
from the present study that CSPGs may be a potent
modulator of neuroinflammation in the context of a demyelinating injury. Dysregulation of the EXTL2 enzyme
results in increased accumulation of CSPGs within thewhite matter injury also associated with a decreased
axonal density and exacerbated injury area. We highlightPu et al. Journal of Neuroinflammation(2020) 17:220

the accumulation of CSPGs as a promoter of axonal
injury, through the potential augmentation of theinflammatory macrophage response. In vitro cell culture
results suggest that CSPGs are able to promote an
inflammatory response through induction of TNFα
secretion, as well as some MMPs. In addition to their
better described inhibitory roles for remyelination andaxonal repair in the CNS, these results indicate that
CSPGs are also promoters of detrimental neuroinflammation in white matter demyelination.
Abbreviations
BMDM: Bone marrow-derived macrophages; ChABC: Chondroitinase ABC;
CSPGs: Chondroitin sulfate proteoglycans; ECM: Extracellular matrix;
EXTL2: Exostosin-like 2; HSPGs: Heparan sulfate proteoglycans; MBP: Myelin
basic protein; MMPs: Matrix metalloproteinases; NF200: 200 kDa
neurofilament; OPC: Oligodendrocyte precursor cell; PBS: Phosphate-bufferedsaline; PFA: Paraformaldehyde; SNP: Single nucleotide polymorphism; TNFα: Tumor necrosis factor α
Acknowledgements
VWY acknowledges Canada Research Chair (Tier 1) support.
Authors’ contributions
AP contributed to the design of this work, generated, and analyzed all of thein vivo mouse data, as well as the majority of cell culture data. MKM, YF, andSG contributed to generating cell culture data. KSR contributed to theinterpretation of data. CS contributed to the provision and maintenance ofreagents. HK provided the EXTL2−/− transgenic mouse line. SS provided datafrom the GWAS study by the International Multiple Sclerosis Genetics
Consortium. VWY conceptualized this work and provided funding and allmaterials for this study. All authors read and approved the final manuscript.
Funding
This study was supported by operating grants from the Canadian Institutesof Health Sciences, the AIHS Alberta/Novartis Translational Research Fund,and the Multiple Sclerosis Society of Canada. YD acknowledges postdoctoral
fellowship support from the Alberta MS Collaboration. KSR and ELS are
Canada Vanier Scholars and acknowledge additional doctoral support from
the Multiple Sclerosis Society of Canada and the Hotchkiss Brain Institute.
Availability of data and materials
All data generated or analyzed during this study are included in thispublished article.
Ethics approval and consent to participate
All animal experiments were performed with ethics approval from theAnimal Care Committee at the University of Calgary, following guidelines of
the Canadian Council of Animal Care. There are no human studies in thismanuscript.
Consent for publication
All authors have consented to the publication of this manuscript.
Competing interests
The authors declare that they have no competing interests.
Author details
1
Department of Clinical Neurosciences and the Hotchkiss Brain Institute,
University of Calgary, T2N 4 N1, Calgary, Canada. 2Kobe Pharmaceutical
University, Kobe, Japan. 3University of Cambridge, Cambridge, UK. 4Universityof Calgary, 3330 Hospital Drive, Calgary, Alberta T3A 4X9, Canada.

Page 20 of 21

Received: 5 April 2020 Accepted: 13 July 2020

References
1. Burnside ER, Bradbury EJ. Review: manipulating the extracellular matrix andits role in brain and spinal cord plasticity and repair. Neuropathol Appl
Neurobiol. 2014;40:26–59.
2. Pu A, Stephenson EL, Yong VW. The extracellular matrix: focus on
oligodendrocyte biology and targeting CSPGs for remyelination therapies.
Glia. 2018;66:1809–25..
3. Mohan H, Krumbholz M, Sharma R, Eisele S, Junker A, Sixt M, Newcombe J,
Wekerle H, Hohlfeld R, Lassmann H, Meinl E. Extracellular matrix in multiple
sclerosis lesions: fibrillar collagens, biglycan and decorin are upregulated
and associated with infiltrating immune cells. Brain Pathol. 2010;20:966–75.
4. Sobel RA, Ahmed AS. White matter extracellular matrix chondroitin sulfate/dermatan sulfate proteoglycans in multiple sclerosis. J Neuropathol Exp
Neurol. 2001;60:1198–207.
5. Lau LW, Cua R, Keough MB, Haylock-Jacobs S, Yong VW. Pathophysiology ofthe brain extracellular matrix: a new target for remyelination. Nat Rev
Neurosci. 2013;14:722–9.
6. Suttkus A, Rohr SO, Weigel S, Glöckner P, Arendt T, Morawski M. Aggrecan,link protein and tenascin-R are essential components of the perineuronal
net to protect neurons against iron-induced oxidative stress. Cell Death Dis.
2014;5:e1119.
7. Asher RA, Morgenstern DA, Fidler PS, Adcock KH, Oohira A, Braistead JE,
Levine JM, Margolis RU, Rogers JH, Fawcett JW. Neurocan isupregulated in injured brain and in cytokine-treated astrocytes. J
Neurosci. 2000;20:2427–38.
8. Asher RA, Morgenstern DA, Shearer MC, Adcock KH, Pesheva P, Fawcett JW.
Versican is upregulated in CNS injury and is a product of oligodendrocyte
lineage cells. J Neurosci. 2002;22:2225–36.
9. Beggah AT, Dours-Zimmermann MT, Barras FM, Brosius A, Zimmermann DR,
Zurn AAD. Lesion-induced differential expression and cell association ofneurocan, brevican, versican V1 and V2 in the mouse dorsal root entry zone.
Neuroscience. 2005;133:749–62.
10. Hallmann R, Zhang X, Di Russo J, Li L, Song J, Hannocks MJ, Sorokin L. The
regulation of immune cell trafficking by the extracellular matrix. Curr Opin
Cell Biol. 2015;36:54–61.
11. Jones LL, Margolis RU, Tuszynski MH. The chondroitin sulfate proteoglycans
neurocan, brevican, phosphacan, and versican are differentially regulated
following spinal cord injury. Exp Neurol. 2003;182:399–411.
12. Jones LL, Yamaguchi Y, Stallcup WB, Tuszynski MH. NG2 is a majorchondroitin sulfate proteoglycan produced after spinal cord injury and isexpressed by macrophages and oligodendrocyte progenitors. J Neurosci.
2002;22:2792–803.
13. Properzi F, Carulli D, Asher RA, Muir EM, Camargo LM, Van Kuppevelt TH,
Ten Dam GB, Furukawa Y, Mikami T, Sugahara K, Toida T, Geller HM, Fawcett
JW. Chondroitin 6-sulphate synthesis is up-regulated in injured CNS,
induced by injury-related cytokines and enhanced in axon-growth
inhibitory glia. Eur J Neurosci. 2005;21:378–90.
14. Deng Y-P, Sun Y, Hu L, Li Z-H, Xu Q-M, Pei Y-L, Huang Z-H, Yang Z-G, Chen
C. Chondroitin sulfate proteoglycans impede myelination byoligodendrocytes after perinatal white matter injury. Exp Neurol. 2015;269:
213–23.
15. Karimi-Abdolrezaee S, Schut D, Wang J, Fehlings MG, Zheng JC.
Chondroitinase and growth factors enhance activation and oligodendrocyte
differentiation of endogenous neural precursor cells after spinal cord injury.
PLoS One. 2012;7.
16. Siebert JR, Osterhout DJ. The inhibitory effects of chondroitin sulfate
proteoglycans on oligodendrocytes. J Neurochem. 2011;119:176–88.
17. Wang H, Katagiri Y, McCann TE, Unsworth E, Goldsmith P, Yu Z-X, Tan
F, Santiago L, Mills EM, Wang Y, Symes AJ, Geller HM. Chondroitin-4sulfation negatively regulates axonal guidance and growth. J Cell Sci.
2008;121:3083–91.
18. Stephenson EL, Mishra MK, Moussienko D, Laflamme N, Rivest S, Ling C-C,
Yong VW. Chondroitin sulfate proteoglycans as novel drivers of leucocyte
infiltration in multiple sclerosis. Brain. 2018;141:1094–110.
19. Kang I, Harten IA, Chang MY, Braun KR, Sheih A, Nivison MP, Johnson PY,
Workman GA, Kaber G, Evanko SP, Chan CK, Merrilees MJ, Ziegler SF, Kinsella
MG, Frevert CW, Wight TN. Versican deficiency significantly reduces lungPu et al. Journal of Neuroinflammation

20.
21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.
36.

37.

38.

39.

(2020) 17:220

inflammatory response induced by polyinosine-polycytidylic acidstimulation. J Biol Chem. 2017;292:51–63.
Prydz K, Dalen KT. Synthesis and sorting of proteoglycans. J Cell Sci. 2000;
113:193–205.
Keough MB, Rogers JA, Zhang P, Jensen SK, Stephenson EL, Chen T,
Hurlbert MG, Lau LW, Rawji KS, Plemel JR, Koch M, Ling C-C, Yong VW. An
inhibitor of chondroitin sulfate proteoglycan synthesis promotes central
nervous system remyelination. Nat Commun. 2016;7.
Lau LW, Keough MB, Haylock-Jacobs S, Cua R, Döring A, Sloka S, Stirling DP,
Rivest S, Yong VW. Chondroitin sulfate proteoglycans in demyelinatedlesions impair remyelination. Ann Neurol. 2012;72:419–32.
Barritt AW, Davies M, Marchand F, Hartley R, Grist J, Yip P, Mcmahon SB,
Bradbury EJ. Chondroitinase ABC promotes sprouting of intact and injured
spinal systems after spinal cord injury. J Neurosci. 2006;26:10856–67.
Bradbury EJ, Lawrence DFM, Popat RJ, King VR, Bennett GS, Patel PN,
Fawcett JW, McMahon SB. Chondroitinase ABC promotes functional
recovery after spinal cord injury. Nature. 2002;416:636–40.
Dyck S, Kataria H, Alizadeh A, Santhosh KT, Lang B, Silver J, KarimiAbdolrezaee S. Perturbing chondroitin sulfate proteoglycan signaling
through LAR and PTPσ receptors promotes a beneficial inflammatory
response following spinal cord injury. J Neuroinflammation. 2018;15.
Massey JM, Hubscher CH, Wagoner MR, Decker JA, Amps J, Silver J, Onifer
SM. Chondroitinase ABC digestion of the perineuronal net promotes
functional collateral sprouting in the cuneate nucleus after cervical spinal
cord injury. J Neurosci. 2006;26:4406–14.
Siebert JR, Stelzner DJ, Osterhout DJ. Chondroitinase treatment following
spinal contusion injury increases migration of oligodendrocyte progenitor
cells. Exp Neurol. 2011;231:19–29.
Kitagawa H, Shimakawa H, Sugahara K. The tumor suppressor EXT-like gene
EXTL2 encodes an α1,4-N-acetylhexosaminyltransferase that transfers Nacetylgalactosamine and N-acetylglucosamine to the common
glycosaminoglycan-protein linkage region. J Biol Chem. 1999;274:13933–7.
Keough MB, Jensen SK, Yong VW. Experimental demyelination andremyelination of murine spinal cord by focal injection of lysolecithin. J Vis
Exp. 2015:e52679.
International Multiple Sclerosis Genetics Consortium, Wellcome Trust Case
Control Consortium 2. Genetic risk and a primary role for cell-mediatedimmune mechanisms in multiple sclerosis. Nature. 2011;476:214–9.
International Multiple Sclerosis Genetics Consortium. Analysis of immunerelated loci identifies 48 new susceptibility variants for multiple sclerosis.
Nat Genet. 2013;45:1353–60.
Nadanaka S, Zhou S, Kagiyama S, Shoji N, Sugahara K, Sugihara K, Asano M,
Kitagawa H. EXTL2, a member of the EXT family of tumor suppressors,
controls glycosaminoglycan biosynthesis in a xylose kinase-dependent
manner. J Biol Chem. 2013;288:9321–33.
Kigerl KA, Gensel JC, Ankeny DP, Alexander JK, Donnelly DJ, Popovich PG.
Identification of two distinct macrophage subsets with divergent effects
causing either neurotoxicity or regeneration in the injured mouse spinal
cord. J Neurosci. 2009;29:13435–44.
Zhang Y, Chen K, Sloan SA, Bennett ML, Scholze AR, O’Keeffe S, Phatnani
HP, Guarnieri XP, Caneda C, Ruderisch N, Deng S, Liddelow SA, Zhang C,
Daneman R, Maniatis T, Barres BA, Jia X, Wu Q. An RNA-sequencing
transcriptome and splicing database of glia, neurons, and vascular cells of
the cerebral cortex. J Neurosci. 2014;34:11929–47.
Su AI, Wiltshire T, Batalov S, et al. A gene atlas of the mouse and humanprotein-encoding transcriptomes. Proc Natl Acad Sci USA. 2004;101(16):6062.
Kim S, Takahashi H, Lin WW, Descargues P, Grivennikov S, Kim Y, Luo JL,
Karin M. Carcinoma-produced factors activate myeloid cells through TLR2 to
stimulate metastasis. Nature. 2009;457:102–6.
Locatelli G, Theodorou D, Kendirli A, Jordão MJC, Staszewski O, Phulphagar
K, Cantuti-Castelvetri L, Dagkalis A, Bessis A, Simons M, Meissner F, Prinz M,
Kerschensteiner M. Mononuclear phagocytes locally specify and adapt theirphenotype in a multiple sclerosis model. Nat Neurosci. 2018;21:1196–208.
Luo F, Tran AP, Xin L, Sanapala C, Lang BT, Silver J, Yang Y. Modulation ofproteoglycan receptor PTPσ enhances MMP-2 activity to promote recovery
from multiple sclerosis. Nat Commun. 2018;9:4126.
Kucharova K, Stallcup WB. NG2-proteoglycan-dependent contributions ofoligodendrocyte progenitors and myeloid cells to myelin damage andrepair. J Neuroinflammation. 2015;12.

Page 21 of 21

40. Rolls A, Shechter R, London A, Segev Y, Jacob-Hirsch J, Amariglio N, Rechavi G,
Schwartz M. Two faces of chondroitin sulfate proteoglycan in spinal cord
repair: a role in microglia/macrophage activation. PLoS Med. 2008;5:1262–77.
41. Canals I, Benetó N, Cozar M, Vilageliu L, Grinberg D. EXTL2 and EXTL3
inhibition with siRNAs as a promising substrate reduction therapy for
Sanfilippo C syndrome. Sci Rep. 2015;5.
42. Kaidonis X, Liaw WC, Roberts AD, Ly M, Anson D, Byers S. Gene silencing of
EXTL2 and EXTL3 as a substrate deprivation therapy for heparan sulphatestoring mucopolysaccharidoses. Eur J Hum Genet. 2009;18:194–9.
43. Kuboyama K, Tanga N, Suzuki R, Fujikawa A, Noda M. Protamine neutralizes
chondroitin sulfate proteoglycan-mediated inhibition of oligodendrocyte
differentiation. PLoS One. 2017:12.

Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations.