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Journal ArticleDOI

2-arachidonoylglycerol reduces chondroitin sulphate proteoglycan production by astrocytes and enhances oligodendrocyte differentiation under inhibitory conditions.

02 Jan 2020-Glia (Glia)-Vol. 68, Iss: 6, pp 1255-1273

TL;DR: Overall, the data obtained favor targeting the endocannabinoid system to neutralize CSPG accumulation and to enhance oligodendrocyte differentiation.

AbstractThe failure to remyelinate and regenerate is a critical impediment to recovery in multiple sclerosis (MS), resulting in severe dysfunction and disability. The chondroitin sulfate proteoglycans (CSPGs) that accumulate in MS lesions are thought to be linked to the failure to regenerate, impeding oligodendrocyte precursor cell (OPC) differentiation and neuronal growth. The potential of endocannabinoids to influence MS progression may reflect their capacity to enhance repair processes. Here, we investigated how 2-arachidonoylglycerol (2-AG) may affect the production of the CSPGs neurocan and brevican by astrocytes in culture. In addition, we studied whether 2-AG promotes oligodendrocyte differentiation under inhibitory conditions in vitro. Following treatment with 2-AG or by enhancing its endogenous tone through the use of inhibitors of its hydrolytic enzymes, CSPG production by rat and human TGF-β1 stimulated astrocytes was reduced. These effects of 2-AG might reflect its influence on TGF-β1/SMAD pathway, signaling that is involved in CSPG upregulation. The matrix generated from 2-AG-treated astrocytes is less inhibitory to oligodendrocyte differentiation and significantly, 2-AG administration directly promotes the differentiation of rat and human oligodendrocytes cultured under inhibitory conditions. Overall, the data obtained favor targeting the endocannabinoid system to neutralize CSPG accumulation and to enhance oligodendrocyte differentiation.

Topics: Oligodendrocyte differentiation (70%), Neurocan (56%), Brevican (52%)

Summary (6 min read)

2.1 | Rat astrocyte cell cultures

  • Rat astrocytes were prepared from postnatal Wistar pups (0–2 days old) as described previously (Mecha et al., 2011).
  • This medium was replaced 3 hr later, supplemented with cytosine-d-arabinofuranoside (#C1768, AraC 10 μM: Sigma-Aldrich Madrid, Spain).
  • The cells harvested at 6 hr were processed for RT-PCR, the ones harvested at 24 hr were processed also for RT-PCR, immunocytochemistry, and CSPGs were analyzed in western blots, whereas the cells harvested at 1 hr were used to study signaling pathways.
  • Supernatants from the cells incubated with the stimuli for 24 hr were analyzed by ELISA.

2.2 | Rat astrocyte-secreted ECM

  • After 3 days in vitro, the cells were incubated for 1 hr in serum-free DMEM prior to treatment for 24 hr with human TGF-β1 (20 ng/ml: Peprotech, London, UK) and the endocannabinoid 2-AG (100 nM: Tocris Bioscience, Bristol, UK).
  • The cell debris was removed by rinsing with PBS and the absence of cells was confirmed by phase-contrast microscopy.
  • The ECM deposited on the 24-well plate was kept hydrated with PBS (Gibco-Invitrogen S.A., Barcelona, Spain) at 4 C until OPCs were seeded.

2.3 | Rat oligodendrocyte cultures

  • Oligodendrocyte cultures derived from P0–P2 Wistar rats were prepared as described previously (Mecha et al., 2011).
  • The cells isolated were plated on coverslips coated with Poly-D-lysine (5 μg/ml) or a CSPG mixture (#CC117, 1 μg/ml containing a mixture of aggrecan, neurocan, phosphacan, and versican: Millipore, Temecula, CA) in 24-well plates at a density of 4 × 105 cells/ml for immunocytochemistry.

2.4 | Human astrocyte cultures

  • Brain tissue from 18- to 20-week-old therapeutically aborted fetuses were used in accordance with the ethics approval from the Research Ethics Board at the University of Calgary.
  • Astrocytes were purified by dissociating the tissue into single cells, as described previously (Giuliani, Goodyer, Antel, & Yong, 2003).
  • The suspension was then washed through a 130 μm pore size filter, and the flowthrough was centrifuged at 1200 rpm for 10 min.
  • The cell pellet was resuspended in PBS, centrifuged again and plated in T-75 flasks coated with 10 μg/ml poly-L-ornithine at a density of 2 × 106 cells/ml in feeding medium (MEM-supplemented with 10% FBS [Gibco-Invitrogen], 0.1% dextrose [Sigma-Aldrich], 1 mM sodium pyruvate [Gibco-Invitrogen], and 1% penicillin/streptomycin, [Gibco-Invitrogen]).
  • The CSPGs in the cells harvested were analyzed in western blots and the supernatants were used for ELISA studies.

2.5 | Isolation of human oligodendrocytes

  • Human adult oligodendrocytes were cultured from resected brain specimens obtained from patients undergoing surgery to treat intractable epilepsy and they were cultured as described previously (Nuttall et al., 2007).
  • Tissue fragments less than 2 mm3 were immersed in saline in the sterile bag at room temperature (23 C), which was transported to the tissue culture laboratory for processing within the hour of completing the surgery at room temperature.
  • The content of the sterile bag was put into 50 ml tubes at room temperature, and the blood, meninges, and clots were removed by washing the tissue several times with PBS, allowing the contents of the brain to settle between washes.
  • The cells were washed twice for 10 min by centrifugation at 1200 rpm in MEM containing 0.2 mM Glutamine, 100 μg/ml penicillin/streptomycin, 1 × nonessential amino acids, 1 × sodium pyruvate, 0.1% dextrose and 10% inactivated FBS (all from Gibco-Invitrogen).
  • Neurons do not survive the isolation procedure while astrocytes are lost in the discarded Percoll fractions.

2.6 | Oligodendrocyte differentiation on a purified CSPG mixture

  • To determine the effects of CSPGs on oligodendrocyte growth in vitro, 96-well plates for human oligodendrocytes and 24-well plates for rat oligodendrocytes were first coated with 10 μg/ml poly-L-lysine overnight and then rinsed with water.
  • The plates were coated for 3–4 hr with purified CSPGs (#CC117) at a concentration of 1 μg/ml diluted in sterile water, then rinsed in water.
  • The cells were fixed with 4% ice-cold paraformaldehyde at 4 C for 20 min, rinsed with PBS, and stored at 4 C until they were processed for immunocytochemistry.
  • In other experiments, rat OPCs were seeded onto rat astrocyte secreted ECM.

2.7 | Reagents and treatments

  • In another subset of experiments, astrocytes were treated for 24 hr with inhibitors of the 2-AG hydrolytic enzymes, the MAGL inhibitors UCM03025 (1 μM: Department of Organic Chemistry, Universidad Complutense de Madrid) and JZL184 (#3836/10, 1 μM: Tocris, Bioscience, Bristol, UK), and an inhibitor of the ABHD6 hydrolase enzyme, WWL70 (#3252/10, 10 μM: Tocris, Bioscience, Bristol, UK).
  • To study cell signaling, TGF-β1, 2-AG, and cannabinoid receptor antagonists were administered for 1 hr, as well as the inhibitor of the TGFβ-SMAD pathway, SB431542 (# S4317, 20 μM, Sigma-Aldrich, Madrid, Spain).

2.8 | Immunocytochemistry

  • Treated and fixed astrocytes and oligodendrocytes that had been maintained in culture for 24 hr were washed in 0.1 M phosphate buffer (PBS), permeabilized in PBS + 0.2% Triton X-100 (Sigma Aldrich, Madrid, Spain) (PBT), and then blocked for 1 hr at room temperature in blocking buffer (PBT plus 5% normal goat serum NGS: Vector Laboratories, Inc., Burlingame, CA).
  • The following day, the cells were rinsed and then incubated for 1 hr with the appropriate fluorescent secondary antibody (Molecular Probes Inc. Eugene, OR): Alexa 594.
  • Fluor-conjugated goat anti-mouse antibody (for neurocan); Alexa 488 Fluor-conjugated goat anti-rabbit antibody (for GFAP); Alexa 488 Fluor-conjugated goat anti-mouse antibody (for MBP); Texas Red 568 Fluor-conjugated donkey anti-mouse IgM (for O4).
  • Finally, the cells were counterstained with DAPI (Sigma Aldrich, Madrid, Spain).

2.9 | Western blotting

  • After treatment, the cultured astrocyte were washed and lysed in RIPA buffer containing a complete protease inhibitor cocktail (Roche Diagnostics; Mannheim, Germany).
  • The reaction was stopped by adding 5 × Laemmli sample buffer and boiling for 5 min.
  • The membranes were washed with TBS and TBS with 0.1% Tween 20 (TBST), and then blocked for 1 hr in LI-COR blocking solution.
  • The signals were normalized to those obtained for tubulin (#T5168, mouse diluted 1:10,000, Sigma-Aldrich, Madrid, Spain) or pan-actin (#D18C11, rabbit diluted 1:1,000, Cell Signaling, MA), and NewBlot IR Stripping (LI-COR) reagent was used for the stripping procedure performed for 30 min at room temperature.

2.10 | ImageXpress acquisition

  • Images of the 96-well plates of primary human oligodendrocytes processed for immunocytochemistry were obtained on a Molecular Device ImageXpress high-content imaging system.
  • The images were collected at 10 × from six fields per well, with appropriate absorption and emission wavelengths for each secondary antibody.

2.11 | Image processing and analysis

  • The images of rat O4 and MBP immunostained oligodendrocytes were acquired on a Leica TCS SP5 confocal microscope.
  • Briefly, 2 μm step size confocal stacks were captured in the z-direction from all cells in 4–5 fields per well and experimental condition, using 20 × objectives, with 2–3 replicates per condition and 3–5 independent experiments were performed.
  • The same threshold was set for all the cells and all the experimental conditions.
  • Oligodendrocyte morphology was studied using the Analyze Skeleton plugin of the ImageJ software (NIH, Bethesda, MD) according to the guidelines of Morrison and Filosa (2013).
  • The number of cells per field corresponds to the average of the images per condition.

2.12 | Reverse transcription (RT) and realtime PCR

  • Total RNA was extracted from rat astrocyte cultures using RNeasy mini columns (Qiagen, Manchester, UK), avoiding genomic DNA contamination by DNase I degradation (DNase I, Sigma-Aldrich).
  • The RNA yield was determined using a Nanodrop spectrophotometer (Thermo Scientific; Wilmington, DE) and the total RNA (1 μg in 20 μl) was reverse transcribed into cDNA using poly-dT primers and a reverse transcription kit (Promega Biotech Ibérica, S.L., Madrid, Spain).
  • The samples were assayed in triplicate on an Applied Biosystems PRISM 7500 Sequence detection system.
  • Gene expression was calculated using the 2−ΔΔCt method and the relative expression was quantified by calculating the ratio between the values obtained for each gene of interest and those of the 18S gene.
  • The results are expressed as a percentage with respect to the control group.

2.13 | ELISA

  • The neurocan and brevican levels in supernatants from primary rat astrocyte cell cultures were measured using specific solid-phase sandwich ELISA kits with antibodies against neurocan and brevican respectively, following the manufacturer's recommendations: for neurocan rat astrocytes - #MBS 450382, My Biosource, San Diego, CA; and for brevican rat astrocytes #MBS732282, My Biosource, San Diego, CA.
  • The neurocan levels in human astrocyte cell cultures were measured by the ELISA kit CSB-EL015513HU Cusabio, Houston, TX, following the manufacturer's recommendations.
  • The minimum detectable concentration of rat neurocan was 0.055 ng/ml, the detection range was 0.156–10 ng/ml, and the intra- and inter-coefficient of variation was 10 and 12%, respectively.
  • The sensitivity of the brevican assay is 1.0 pg/ml.
  • The detectable limit of human neurocan was 0.078 ng/ml, the detection range was 0.312–20 ng/ml, and the intraand inter-coefficient of variation was 8 and 10%, respectively.

2.14 | Statistical analysis

  • All the data are expressed as the mean ± SEM, using the IBM SPSS 24 statistical software (Inc- IBM, Chicago, IL) for the statistical analysis.
  • One-way ANOVA followed by the Bonferroni and Tukey's post hoc tests, or a nonparametric Kruskal-Wallis test was employed for multiple comparisons.

3 | RESULTS

  • 1 | 2-AG treatment diminishes the expression of enzymes involved in the synthesis of CSPGs and the levels of neurocan and brevican in rat astrocytes cultures CSPGs consist of a core protein with attached sulfate GAG side chains (Susarla et al., 2011).
  • The synthesis of the GAG chain is carried out by different xylol transferases (XT-1 and XT-2) that constitute the rate-limiting step of GAG synthesis and by the chondroitin 4 sulfotransferase (C4ST).
  • A dose–response curve of neurocan and brevican protein production reveals that 2-AG 10 nM did not avoid the release of neurocan and brevican at the extracellular F IGURE 1 2-AG treatment reduces the expression of enzymes involved in CSPGs synthesis and the levels of neurocan and brevican in cultured rat astrocytes.
  • Nonparametric Kruskal-Wallis test was applied to analyze RT-PCRs (4–5 independent experiments, 3 replicates per experiment).
  • The maximal effect of 2-AG for the two CSPGs is achieved by the dose of 100 nM (p < .001 vs. TGF-β1).

3.2 | 2-AG-induced reduction of neurocan is dependent on the CB2 signaling system in astrocyte cultures

  • Furthermore, exposure of 2-AG alone, without TGF-β1 stimulation did not produce any increase in CSPGs but rather, there was a reduction in neurocan relative to the control condition.
  • Similarly, no effect was observed when cannabinoid receptor antagonists were administered alone.
  • As such, TGF-β1 stimulated astrocytes were treated for 24 hr with two MAGL inhibitors (the reversible inhibitor UCM03025, 1 μM, and an irreversible inhibitor JZL184, 1 μM), and one inhibitor of the ABHD6 enzyme (WWL70, 10 μM).
  • The generation of neurocan and brevican was reduced significantly by inhibitors of 2-AG hydrolysis when measured by western blots.

3.4 | 2-AG treatment reduces the phosphorylation of SMAD2 in TGF-β1 treated astrocytes, an effect possibly mediated through CB2 receptor signaling

  • The TGF-β1-SMAD pathway is a canonical signaling pathway involved in CSPG synthesis by cultured astrocytes (Schachtrup et al., 2010; Susarla et al., 2011).
  • To assess how 2-AG provokes a reduction in CSPGs, the authors examined the phosphorylation of SMAD2 and SMAD3 in TGF-β1 stimulated astrocytes treated with 2-AG (100 nM) for 1 hr, assessing the potential involvement of the CB1R and CB2R through the addition of specific antagonists (SR141716A, 1 μM and AM630, 1 μM) 30 min prior to 2-AG treatment.
  • The authors results confirmed that the TGF-β1–SMAD signaling pathway is activated in the stimulated astrocytes as the phosphorylation of both SMAD2 and SMAD3 proteins was enhanced in these conditions .
  • SMAD2 phosphorylation was significantly dampened by 2-AG treatment, yet it reverted to TGF-β1 levels in cells also exposed to the CB2R antagonist .
  • No significant effect was observed in the case of SMAD3 phosphorylation .

3.5 | 2-AG treatment reduces CSPG expression and extracellular neurocan levels in human astrocytes in culture

  • Human astrocytes are known to synthesize CSPGs in culture when stimulated (Keough et al., 2016), so the authors examined the CSPGs produced by primary human astrocytes isolated from therapeutically aborted human fetuses that were treated for 24 hr with TGF-β1 (10 ng/ml) and 2-AG at concentrations of 10, 50, and 100 nM.
  • In western blots probed with the CS-56 antibody, the expression of CSPGs was induced in TGF-β1 stimulated astrocytes, although this effect was significantly weakened by the administration of 2-AG at 50 and 100 nM F IGURE 2 2-AG treatment reduces the expression of CSPGs in cultured rat astrocytes and the reduction in neurocan is dependent on the CB2 receptor signaling.
  • 2-AG, 2-arachidonoylglycerol; CSPGs, chondroitin sulfate proteoglycans .
  • Both neurocan and brevican expression and its extracellular levels were analyzed by western blots (a and b, respectively) and ELISA assays (c and d, respectively).

3.6 | The matrix generated by astrocytes exposed to 2-AG is less inhibitory to oligodendrocyte differentiation

  • The authors next tested the effect of restricting rat astrocyte CSPG synthesis on rat OPC differentiation in culture.
  • Rat OPCs were then plated on the ECM F IGURE 6 The 2-AG treated astrocyte matrix is less inhibitory for oligodendrocyte differentiation.
  • 2-AG, 2-arachidonoylglycerol generated and the outgrowth of processes was analyzed after 24 hr by immunocytochemistry for O4 staining , while the morphological complexity of the OPCs was assessed by measuring their branches, junctions and end-points .
  • No effect was observed on the initial cell adhesion under any experimental condition .

3.7 | 2-AG treatment promotes oligodendrocyte differentiation under inhibitory conditions

  • To study whether 2-AG directly promotes the differentiation of OPCs plated onto an inhibitory ECM, rat OPCs were enriched from mixed glial cultures and they were seeded onto 24-well plates coated with a commercial mixture of CSPGs (#CC117; 1 μg/ml, mostly containing neurocan, phosphacan, versican, and aggrecan).
  • The number of O4 positive cells/field (c) Branches, junctions, and end-points obtained from the skeleton data.
  • Nonparametric Kruskal-Wallis test (five independent experiments, two replicates per experiment).
  • By contrast, human OPCs plated on CSPGs and treated with 2-AG (2 μM) displayed significantly more elaborated arborizations , but again, there was no effect on their adhesion .

4 | DISCUSSION

  • In recent years, the interest in astrocytes as potential targets for neurotherapies has grown, particularly given their role as primary mediators of reactive gliosis and in maintaining immune homeostasis in the brain, along with microglia.
  • In order to promote reparative mechanisms, combined therapies that neutralize the inhibitory environment around the demyelinated lesions and that enhance oligodendrocyte differentiation would favor the remyelination processes.
  • Regarding the endocannabinoid system, the authors convincingly show that 2-AG diminished the production of CSPGs by rat and human astrocytes.
  • Moreover, activation of CB2Rs provokes anti-fibrotic responses through the inhibition of the TGF-β1/SMAD pathway in a Nrf2 dependent manner (Li et al., 2016).
  • Here, treating cultured astrocytes with 2-AG reduces the inhibitory ECM generated by astrocytes, enhancing oligodendrocyte outgrowth.

ACKNOWLEDGMENTS

  • None of the funding bodies played any role in the study design, data collection, and analysis, the decision to publish, or the preparation of the manuscript.
  • The authors have no competing financial interests to declare.

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RESEARCH ARTICLE
2-arachidonoylglycerol reduces chondroitin sulphate
proteoglycan production by astrocytes and enhances
oligodendrocyte differentiation under inhibitory conditions
Ana Feliu
1
| Leyre Mestre
1
| Francisco J. Carrillo-Salinas
1
| V. Wee Yong
2
|
Miriam Mecha
1
| Carmen Guaza
1
1
Functional and Systems Neurobiology
Department, Neuroimmunology Group,
Instituto Cajal, CSIC, Madrid, Spain
2
Hotchkiss Brain Institute and the Department
of Clinical Neurosciences and Oncology,
University of Calgary, Calgary, Alberta, Canada
Correspondence
Miriam Mecha Rodríguez and Carmen Guaza,
Instituto Cajal, CSIC, Av. Doctor Arce
37, 28002 Madrid, Spain.
Email: miriammecha@cajal.csic.es (M. M. R.)
and
Email: cgjb@cajal.csic.es (C. G)
Present address
Francisco J. Carrillo-Salinas, Department of
Immunology, Tufts University School of
Medicine, Boston, MA.
Funding information
Ministry of the Economy and Competition,
Grant/Award Numbers: SAF2013-42784-R,
SAF2016-76449-R; Red Española de Esclerosis
Múltiple, Grant/Award Number:
RD16/0015/0021; Ministerio de Economía y
Competitividad, Grant/Award Number: BES-
2014-068459
Abstract
The failure to remyelinate and regenerate is a critical impediment to recovery in mul-
tiple sclerosis (MS), resulting in severe dysfunction and disability. The chondroitin sul-
fate proteoglycans (CSPGs) that accumulate in MS lesions are thought to be linked to
the failure to regenerate, impeding oligodendrocyte precursor cell (OPC) differentia-
tion and neuronal growth. The potential of endocannabinoids to influence MS pro-
gression may reflect their capacity to enhance repair processes. Here, we
investigated how 2-arachidonoylglycerol (2-AG) may affect the production of the
CSPGs neurocan and brevican by astrocytes in culture. In addition, we studied
whether 2-AG promotes oligodendrocyte differentiation under inhibitory conditions
in vitro. Following treatment with 2-AG or by enhancing its endogenous tone
through the use of inhibitors of its hydrolytic enzymes, CSPG production by rat and
human TGF-β1 stimulated astrocytes was reduced. These effects of 2-AG might
reflect its influence on TGF-β1/SMAD pathway, signaling that is involved in CSPG
upregulation. The matrix generated from 2-AG-treated astrocytes is less inhibitory to
oligodendrocyte differentiation and significantly, 2-AG administration directly pro-
motes the differentiation of rat and human oligodendrocytes cultured under inhibi-
tory conditions. Overall, the data obtained favor targeting the endocannabinoid
system to neutralize CSPG accumulation and to enhance oligodendrocyte
differentiation.
KEYWORDS
2-AG, multiple sclerosis, astrocytes, CSPGs, oligodendrocytes
1 | INTRODUCTION
Multiple sclerosis (MS) is a neurodegenerative disease in which
inflammation, demyelination, axonal damage, and gliosis affect the
central nervous system (CNS), leading to progressive neurological
decline and permanent disability (Compston & Coles, 2008; Lassmann,
2017; Lassmann, Bruck, & Lucchinetti, 2001). Repair in MS takes place
spontaneously in the form of remyelination, yet as the disease pro-
gresses remyelination fails (Chang et al., 2012; Chang, Tourtellotte,
Rudick, & Trapp, 2002; Franklin, 2002; Goldschmidt, Antel, Konig,
Bruck, & Kuhlmann, 2009). It appears that this failure of remyelination
is not generally due to a loss of oligodendrocytes at the demyelinated
MS lesions, the CNS cells that produce myelin (Chang et al., 2002,
2012), but rather, to an impediment in the successful remyelination of
damaged axons (Franklin, 2002). Hence, remyelination may be
inhibited by factors in the lesion's extracellular milieu and astrogliosis.
Astrocytes become reactive in MS lesions, facilitating bloodbrain
barrier (BBB) repair, secreting immunosuppressive molecules and
Received: 19 June 2019 Revised: 22 November 2019 Accepted: 17 December 2019
DOI: 10.1002/gli a.23775
Glia. 2019;119. wileyonlinelibrary.com/journal/glia © 2020 Wiley Periodicals, Inc. 1

exerting neuroprotective properties. However, astrocytes can also
exacerbate the insult by secreting pro-inflammatory molecules and
they are essential for glial scar formation at demyelinated lesions
(Frischer et al., 2015), sites where there is an increase in the synthesis
and accumulation of chondroitin sulfate proteoglycans (CSPGs) (Back
et al., 2005; Chang et al., 2012; Sobel & Ahmed, 2001).
A basal level of CSPG production is necessary to maintain the
extracellular matrix (ECM) in a state that favors synaptic stabilization
and plasticity (Carulli et al., 2006; Galtrey & Fawcett, 2007). Following
injury, CSPGs are immediately upregulated to limit tissue damage
(Silver & Miller, 2004), yet when CSPG accumulation persists, sponta-
neous repair mechanisms are inhibited due to the formation of a non-
permissive environment that restricts oligodendrocyte precursor cell
(OPC) differentiation (Chang et al., 2012; Karus et al., 2016; Lau et al.,
2012; Pendleton et al., 2013; Siebert & Osterhout, 2011; Sobel &
Ahmed, 2001). Importantly, CSPG clearance through the digestion of
their glycosaminoglycan (GAG) chains, or the blockage of CSPG syn-
thesis and the disruption of CSPG receptors, all provoke remyelination
and axon regeneration in culture, as well as in different experimental
conditions of demyelination and spinal cord injury (SCI) (Bartus et al.,
2014; Dyck et al., 2015; Dyck, Kataria, Akbari-Kelachayeh, Silver, &
Karimi-Abdolrezaee, 2019; Keough et al., 2016; Lang et al., 2015; Lau
et al., 2012; Rolls et al., 2008; Siebert & Osterhout, 2011).
Cannabinoid based therapies are of particular interest in MS, but
also in other pathological scenarios like adenoleukodystrophies, SCI
and a variety of neurodegenerative diseases that require reparative/
remyelination therapeutic approaches. In animal models of MS,
cannabinoids have been found to display the potential to relieve
symptoms and to control inflammation, neurodegeneration, and
demyelination. In general, exogenous and endogenous cannabinoids
can behave as agents with multiple targets acting as an antioxidant
(phytocannabinoids), anti-excitotoxic and neuroprotective lipids
(Fernández-Ruiz, García, Sagredo, Gómez-Ruiz, & de Lago, 2010; Loría
et al., 2010; Mecha et al., 2015; Pryce et al., 2003). Indeed, cannabi-
noid signaling may enhance the activity of reparative mechanisms, as
they can protect OPCs from inflammatory and excitotoxic damage
(Bernal-Chico et al., 2015; Gómez et al., 2010; Mecha et al., 2012;
Molina-Holgado et al., 2002). In particular, the endocannabinoid
2-arachidonoylglycerol (2-AG) promotes the proliferation and differ-
entiation of OPCs (Gómez et al., 2010, 2011, 2015) and it regulates
their migration in culture (Sánchez-Rodríguez, Gómez, Esteban, Gar-
cía-Ovejero, & Molina-Holgado, 2018). Actually, in vivo studies also
demonstrated that inhibiting 2-AG catabolism through the inhibition
of monoacylglycerol lipase (MAGL) or the direct administration of
2-AG attenuates symptomatology and promotes remyelination in the
autoimmune EAE model (Bernal-Chico et al., 2015; Hernández-Torres
et al., 2014; Lourbopoulos et al., 2011), as well as in the Theiler virus
model of MS (Feliu et al., 2017; Mecha et al., 2019). The effects of
2-AG on disease activity and remyelination in the latter progressive
model of MS are dependent on the CB1 and CB2 cannabinoid recep-
tors (CB1R and CB2R), and they involve immune modulation in con-
junction with a reduction in astrogliosis and CSPG deposition (Feliu
et al., 2017).
In the present study, we reveal an effect of 2-AG and the inhibi-
tion of its hydrolytic enzymes on the production of the CSPGs
neurocan and brevican by astrocytes in culture. In addition, we
assessed whether 2-AG promotes oligodendrocyte differentiation
under inhibitory conditions in order to evaluate the potential of the
endocannabinoid system to enhance the endogenous repair mecha-
nisms that are dampened in conditions of chronic demyelination.
2 | MATERIALS AND METHODS
2.1 | Rat astrocyte cell cultures
Rat astrocytes were prepared from postnatal Wistar pups (02 days
old) as described previously (Mecha et al., 2011). After isolation, the
astrocytes were plated in Poly-D-lysine (#P7280;5μg/ml, Sigma-
Aldrich, Madrid, Spain) coated 6-well plates at a density of 1 × 10
6
cells/ml for western blot and RT-PCR analysis, or on coverslips in
24-well plates at a density of 3 × 10
5
cells/ml for immunocytochemis-
try and they were grown at 37
C in an atmosphere of 5% CO
2
and in
DMEM medium (Lonza Ibérica S.A., Barcelona, Spain) supplemented
with 5% horse serum, 5% fetal bovine serum (FBS; Vector Laborato-
ries Inc, Burlingame, CA), 100 U/ml penicillin and 100 mg/ml strepto-
mycin (Gibco-Invitrogen S.A., Barcelona, Spain). This medium was
replaced 3 hr later, supplemented with cytosine-d-arabinofuranoside
(#C1768, AraC 10 μM: Sigma-Aldrich Madrid, Spain). After 3 days
in vitro, the cells were incubated for 1 hr in serum-free DMEM prior
to the 1, 6, and 24 hr pharmacological treatments. The cells harvested
at 6 hr were processed for RT-PCR, the ones harvested at 24 hr were
processed also for RT-PCR, immunocytochemistry, and CSPGs
were analyzed in western blots, whereas the cells harvested at 1 hr
were used to study signaling pathways. Supernatants from the cells
incubated with the stimuli for 24 hr were analyzed by ELISA.
2.2 | Rat astrocyte-secreted ECM
To generate an anchored astrocyte ECM, astrocytes were seeded on
coverslips in 24-well plates at a density of 3 × 10
5
cells/ml in DMEM
medium supplemented with 5% horse serum, 5% FBS, 100 U/ml peni-
cillin and 100 mg/ml streptomycin. The medium was replaced 3 hr
later, adding AraC (10 μM). After 3 days in vitro, the cells were incu-
bated for 1 hr in serum-free DMEM prior to treatment for 24 hr with
human TGF-β1 (20 ng/ml: Peprotech, London, UK) and the endo-
cannabinoid 2-AG (100 nM: Tocris Bioscience, Bristol, UK). The cells
were then rinsed with PBS, and treated with Versene (#15040066;
0.2 g/ml EDTA, Gibco-Invitrogen S.A., Barcelona, Spain) for 30 min at
37
C and in 5% CO
2
, and dislodging the cells with a 200 ml micropi-
pette. The cell debris was removed by rinsing with PBS and the
absence of cells was confirmed by phase-contrast microscopy. The
ECM deposited on the 24-well plate was kept hydrated with PBS
(Gibco-Invitrogen S.A., Barcelona, Spain) at 4
C until OPCs were
seeded.
2 FELIU ET AL.

2.3 | Rat oligodendrocyte cultures
Oligodendrocyte cultures derived from P0P2 Wistar rats were pre-
pared as described previously (Mecha et al., 2011). The cells isolated
were plated on coverslips coated with Poly-D-lysine (5 μg/ml) or a
CSPG mixture (#CC117, 1 μg/ml containing a mixture of aggrecan,
neurocan, phosphacan, and versican: Millipore, Temecula, CA) in
24-well plates at a density of 4 × 10
5
cells/ml for immunocytochemis-
try. The cells were maintained for 24 hr at 37
C and 5% CO
2
in Oligo-
dendrocyte medium: serum-free DMEM containing B27, BSA
(0.1 mg/ml: Gibco-Invitrogen, Barcelona, Spain), progesterone (#P6149;
6 ng/ml: Sigma-Aldrich, Madrid, Spain), putrescine (#P7505;1μg/ml:
Sigma-Aldrich, Madrid, Spain), sodium selenite (#S9133; 5 ng/ml:
Sigma-Aldrich, Madrid, Spain), T3 (#T5516; 40 ng/ml: Sigma-Aldrich,
Madrid, Spain), Glutamax (#35050061; 2 mM: Gibco-Invitrogen, Barce-
lona, Spain), insulin (#I1882;5μg/ml: Sigma-Aldrich, Madrid, Spain),
apotransferrin (#T1147;25μg/ml: Sigma-Aldrich, Madrid, Spain), biotin
(#B4639;2.44μg/ml: Sigma-Aldrich, Madrid, Spain), hydrocortisone
(#H2270; 10 ng/ml: Sigma-Aldrich, Madrid, Spain), penicillin/strepto-
mycin (#15070063; 1%: Gibco-Invitrogen, Barcelona, Spain), and
sodium pyruvate (#11360070; 1 mM: Gibco-Invitrogen, Barcelona,
Spain). In another set of experiments, isolated oligodendrocytes were
plated for 24 hr in the same media on a rat astrocyte-derived matrix.
2.4 | Human astrocyte cultures
Brain tissue from 18- to 20-week-old therapeutically aborted fetuses
were used in accordance with the ethics approval from the Research
Ethics Board at the University of Calgary. Astrocytes were purified by
dissociating the tissue into single cells, as described previously
(Giuliani, Goodyer, Antel, & Yong, 2003). Briefly, 515 g of brain tis-
sue was cut into 1 mm fragments with a pair of scalpels and incubated
for 15 min at 37
C in 40 ml PBS containing 0.25% trypsin (Gibco-Invi-
trogen) and 200 g/ml DNase I (Roche Boehringer). The suspension
was then washed through a 130 μm pore size filter, and the flow-
through was centrifuged at 1200 rpm for 10 min. The cell pellet was
resuspended in PBS, centrifuged again and plated in T-75 flasks
coated with 10 μg/ml poly-L-ornithine at a density of 2 × 10
6
cells/ml
in feeding medium (MEM-supplemented with 10% FBS [Gibco-Invi-
trogen], 0.1% dextrose [Sigma-Aldrich], 1 mM sodium pyruvate
[Gibco-Invitrogen], and 1% penicillin/streptomycin, [Gibco-Invi-
trogen]). The cultures were passaged seven to eight times over
45 weeks, depleting the neurons and achieving 95% astrocyte purity.
The flasks were washed with PBS and trypsinized to obtain the astro-
cytes, which were then plated in poly-L-ornithine (10 μg/ml) (Sigma-
Aldrich) coated 6-well plates at a density of 1 × 10
6
cells/ml, and they
were grown in feeding medium at 37
C and in an atmosphere of 5%
CO
2
. The medium was replaced 3 hr later, adding AraC (10 μM:
Sigma-Aldrich), and after 3 days in vitro, the cells were incubated for
1 hr in serum-free DMEM prior to their pharmacological treatment for
24 hr. The CSPGs in the cells harvested were analyzed in western
blots and the supernatants were used for ELISA studies.
2.5 | Isolation of human oligodendrocytes
Human adult oligodendrocytes were cultured from resected brain
specimens obtained from patients u nder goi ng su rgery to treat
intractable epi lepsy and they were cultured as described previously
(Nuttall et al., 200 7). The tissues used were adjacent t o the epileptic
foci in cortical areas and this mate rial was used in accordance wit h
the ethics approval obtained from the Research Ethics Board at the
Uni vers ity of Calgary. In brief, the pieces of the brain targeted for
excision were suctioned into a sterile bag using a Cavitron Ultrasonic
Aspirator. Tissue fragments less than 2 mm
3
were i mmersed in saline
in the st eri le bag at room temperatur e (23
C), which was trans-
ported to the tissue culture laboratory for processing within the
hour of c ompleting the surgery at room temperature. The content of
the sterile bag was put into 50 ml tubes at room temperature, and
the blood, meninges, and clots were removed by washing the tissue
several times with PBS, allowing the contents of the brain t o settle
between washes. The tissue was t ransfer red to a ster ile 100 ml glass
bottle for digestion with DNase I (0.1 mg/ml, Roche Boehringer) and
0.25% trypsin in PBS, and it was incubated at 37
C for 25 min. FBS
was added to inactivate the trypsin and the sample was filtered
through sterile 130 μm mesh. After centrifugation a t 1200 rpm for
10 min, the sample was diluted in 21 ml of PB S and laid over 9 ml of
Percoll (GE Healthca re Bio-Sciences AB) in ster ile 68 polycarbon-
ate ultra-centrifuge tubes. The sample was homogenized using a
pipette and the cells recovered by centrifugation in a Beckman
J221 M centrifuge at 15,000 rpm for 30 min at 4
Cwithnobrakes.
After Percoll gradient centrifugation, the myelin was removed by
aspiration from the uppe r layer, and the viable cell layer was pip-
etted and diluted 1:1 in PBS. The cells were washed twice for
10 min by centri fugat ion at 1200 rpm in MEM containing 0.2 mM
Glutamine, 100 μg/ml p enicillin/streptomycin, 1 × nonessential
amino acids, 1 × sodium pyruvate, 0.1% dextrose and 10%
inactivated FBS (all from Gibco-Invitrogen). Finally, the cells were
suspended in medium, plated at 2 x 10
6
cells/ml in 5 mL uncoated
T25 Flask and incubated for 48 hr at 37
Cinanatmosphereof5%
CO
2
. Microglia strongly adhere to the flask while oligodendrocytes
remained in suspension or were loosely attached. Neurons do not
survive the isolation procedure while astrocytes are lost in the dis-
carded Percoll fr actions . The floating oligodendrocytes w ere col-
lected, centrifuged and plated onto pol y-L-ornithine (10 μg/ml) or on
aCSPGmixture(#CC1 17, 1 μg/ml, Sigma-Aldrich) in 96-well coated
plates at a d ensity of 10
5
cells/well in human oligode ndrocyte
medium: DMEM/ F12 (Gibco-Invitrogen) medium conta ining N2 sup-
plement, Glutamax (2 mM, Gibco-I nvitrogen), sodium pyruvate
(1 mM, Gibco- Invitrogen), p enicillin/streptomycin (1%, Gibco-Invi-
trogen) , insulin (5 μg/ml, Gibco-Invitrogen), holo-transferrin
(50 μg/ml: Sigma-Aldrich), N- acetylcyst eine (5 μg/ml: Sigma-Ald rich),
biotin (0.01 μg/ml: Sigma-Ald rich), Trace Elements B, dextrose
(1 mg/ml : Sigma-Aldrich), BSA (0.1 mg/ml: Sigma-Aldri ch), proges-
terone (60 ng /ml, Sigma-Aldrich), putrescine (16 μg/ml, Sigma-
Aldrich), Sodium Selenite (30 nM: Gibco-Invitrog en), and T3 (30 nM:
Sigma-Aldrich).
FELIU ET AL. 3

2.6 | Oligodendrocyte differentiation on a purified
CSPG mixture
To determine the effects of CSPGs on oligodendrocyte growth
in vitro, 96-well plates for human oligodendrocytes and 24-well plates
for rat oligodendrocytes were first coated with 10 μg/ml poly-L-lysine
overnight and then rinsed with water. The plates were coated for
34 hr with purified CSPGs (#CC117) at a concentration of 1 μg/ml
diluted in sterile water, then rinsed in water. The control wells con-
tained poly-L-lysine alone. Enriched OPCs were seeded as described
previously and the plates were incubated for 24 hr. The cells were
fixed with 4% ice-cold paraformaldehyde at 4
C for 20 min, rinsed
with PBS, and stored at 4
C until they were processed for immunocy-
tochemistry. In other experiments, rat OPCs were seeded onto rat
astrocyte secreted ECM.
2.7 | Reagents and treatments
Rat astrocytes were exposed to the cytokine human TGF-β1
(#10021C, 20 ng/ml: Peprotech, London, UK), bFGF (10 ng/ml:
Peprotech, London, UK), the endocannabinoid 2-AG (#1298/10,at
10, 50, 100 nM and 500 nM: Tocris, Bioscience, Bristol, UK), and the
CB1R (SR141716A, 1 μM: Sanofi Recherche, Montpellier, France) and
CB2R (#1120/10, AM630, 1 μMor#5039/10, SR144528 [SR2],
1 μM, both from Tocris Bioscience, Bristol, UK) antagonists for 24 hr,
administered 30 min before 2-AG treatment. In another subset of
experiments, astrocytes were treated for 24 hr with inhibitors of the
2-AG hydrolytic enzymes, the MAGL inhibitors UCM03025 (1 μM:
Department of Organic Chemistry, Universidad Complutense de
Madrid) and JZL184 (#3836/10,1μM: Tocris, Bioscience, Bristol, UK),
and an inhibitor of the ABHD6 hydrolase enzyme, WWL70
(#3252/10,10μM: Tocris, Bioscience, Bristol, UK). To study cell sig-
naling, TGF-β1, 2-AG, and cannabinoid receptor antagonists were
administered for 1 hr, as well as the inhibitor of the TGFβ-SMAD
pathway, SB431542 (# S4317,20μM, Sigma-Aldrich, Madrid, Spain).
Primary human astrocytes were treated for 24 hr with TGF-β1
(10 ng/ml) and 2-AG at 10, 50, and 100 nM. Primary rat oligodendro-
cytes were exposed to 2-AG at concentrations of 100, 500, 1, and
2 μM at time of plating on the CSPG matrix ( #CC117, 1 μg/ml), and
the cells were then cultured for 24 hr. Primary human oligodendro-
cytes were exposed to 2-AG at 2 μM at the time of plating on the
CSPG matrix (#CC117, 1 μg/ml) and then cultured for 24 hr.
2.8 | Immunocytochemistry
Treated and fixed astrocytes and oligodendrocytes that had been
maintained in culture for 24 hr were washed in 0.1 M phosphate
buffer (PBS), permeabilized in PBS + 0.2% Triton X-100 (Sigma
Aldrich, Madrid, Spain) (PBT), and then blocked for 1 hr at room tem-
perature in blocking buffer (PBT plus 5% normal goat serum NGS:
Vector Laboratories, Inc., Burlingame, CA). Rat astrocytes were then
stained overnight at 4
C with antibodies against neurocan (#1F6 mouse
neurocan diluted 1:1,000, Developmental studies Hybridoma Bank, IA)
and GFAP (#G9269 rabbit GFAP diluted 1:1,000: Sigma-Aldrich,
Madrid, Spain), while rat and human oligodendrocytes were stained
with antibodies against O4 (#MAB345, mouse IgM diluted 1:500:
Millipore, Temecula, CA) and MBP (#MAB382, mouse MBP diluted
1:250: Millipore, Temecula, CA). The following day, the cells were
rinsed and then incubated for 1 hr with the appropriate fluorescent
secondary antibody (Molecular Probes Inc. Eugene, OR): Alexa
594 Fluor-conjugated goat anti-mouse antibody (for neurocan);
Alexa 488 Fluor-conjugated goat anti-rabbit antibody (for GFAP); Alexa
488 Fluor-conjugated goat anti-mouse antibody (for MBP); Texas Red
568 Fluor-conjugated donkey anti-mouse IgM (for O4). Finally, the cells
were counterstained with DAPI (Sigma Aldrich, Madrid, Spain).
2.9 | Western blotting
After treatment, the cultured astrocyte were washed and lysed in
RIPA buffer containing a complete protease inhibitor cocktail
(Roche Diagnostics; Mannheim, Germany). To detect CSPGs, cell
lysates were diluted in chondroitinase ABC buffer (50 mM Tris
[pH 8.0], 60 mM sodium acetat e and 0.02 % BSA) an d then t reated
for 3 hr at 37
Cwith0.1U/mlchondroitinase ABC from Proteus
vulgaris (#C3667, Sigma-Aldrich, Madrid Spain). To detect the CS-
56 signal, incubation with ch ondroiti nase ABC was omi tted. The
reaction was stopped by adding 5 × Laemmli sample buffer and
boiling for 5 min. Equal amounts of pr otein (15 μg) were resolved
on a 6% s odi um dodecy l s ulf ate -po lyacrylamide gel (SDS-PAGE) to
examine CSPGs and on a 1012% SDS-PAGE for signaling proteins,
and then transferred to a nitrocellulose membrane at 4
C
(Amersham Biosciences, Pisca taway, NJ). The membranes were
washedwithTBSandTBSwith0.1%Tween20(TBST),andthen
blocked for 1 hr in LI-COR blocking solution. After blocking, the
membranes were washed in TB ST and probed overnight with anti-
bodies against: neurocan (# 1F6, mouse, Developmental studies
Hybridoma Bank), brevican (#610894, mouse, BD Bioscience, San
Diego, C A), SMAD2 (#MAS-15112, mouse, Thermofisher, MA), p-
SMAD2 (#3108, rabbit , Cell Signaling, MA), SMAD3 (#Ab75512,
mouse, Abcam, Cambri dge, UK ) and p-SMAD3 (#95 20, ra bbit, C ell
Signaling, MA), all diluted 1:1,000; and CS-56 diluted 1:500
(#C8035, mouse IgM, Sig ma-Aldrich, Madrid, Spain). The mem-
branes were then washed in blockin g solution and i ncubated for
1 hr wi th the secondary conjugated f luorophores diluted 1:15, 000
(IRDye 800CW anti-mouse; IRDye680LTantimouseIgM;IRDye
680LT anti-rabb it). The membranes were then washed again with
TBST and T BS, and the signa l was detected on an Odyssey
®
CLx
Imaging System ( LI-COR Biotechnology, Germany). The signa ls
were normalized to those obtained for tubulin (#T516 8, mou se
diluted 1:10,0 00, Sig ma-Aldrich , Madr id, Spa in) or pa n-actin
(#D18C11, rabbit diluted 1:1,000, Cell Sign aling, MA), and NewBlot
IR Stripping (LI-COR) reagent was used for the stripping procedure
performed for 30 min at room temperature.
4 FELIU ET AL.

2.10 | ImageXpress acquisition
Images of the 96-well plates of primary human oligodendrocytes
processed for immunocytochemistry were obtained on a Molecular
Device ImageXpress high-content imaging system. The images were
collected at 10 × from six fields per well, with appropriate absorption
and emission wavelengths for each secondary antibody.
2.11 | Image processing and analysis
The images of rat O4 and MBP immunostained oligodendrocytes
were acquired on a Leica TCS SP5 confocal microscope. Briefly, 2 μm
step size confocal stacks were captured in the z-direction from all cells
in 45 fields per well and experimental condition, using 20 × objec-
tives, with 23 replicates per condition and 35 independent experi-
ments were performed. The same threshold was set for all the cells
and all the experimental conditions. Oligodendrocyte morphology was
studied using the Analyze Skeleton plugin of the ImageJ software
(NIH, Bethesda, MD) according to the guidelines of Morrison and
Filosa (2013). Confocal images were acquired and the maximum inten-
sity projection of the O4 or MPB positive channel was used as a noise
filter to eliminate single-pixel background fluorescence, converting
the resulting images to a binary image in order to skeletonize it use
the ImageJ software. All the cells per field and image were analyzed
and averaged to a single data point for each replicate and condition,
collecting the data for the number of branches (total number of ramifi-
cations), joints (bifurcations of ramifications), and end-points (final
points of the ramifications) as a morphological measure of oligoden-
drocyte complexity. The number of cells per field corresponds to the
average of the images per condition.
2.12 | Reverse transcription (RT) and real-
time PCR
Total RNA was extracted from rat astrocyte cultures using RNeasy
mini columns (Qiagen, Manchester, UK), avoiding genomic DNA con-
tamination by DNase I degradation (DNase I, Sigma-Aldrich). The RNA
yield was determined using a Nanodrop spectrophotometer (Thermo
Scientific; Wilmington, DE) and the total RNA (1 μgin20μl) was
reverse transcribed into cDNA using poly-dT primers and a reverse
transcription kit (Promega Biotech Ibérica, S.L., Madrid, Spain). Real-
time PCR was carried out with SYBR
®
using the followed oligonucleo-
tide primer sequences (Applied Biosystems, Warrington, UK): XT-1
forward 5
0
- GGGAATGCAGAAATGGGGGA-3
0
, XT-1 reverse 5´-
GAAGGTCAGAGGTGCGACAA-3
0
; XT-2 forward 5´-GGGTGAGACCC
GCTTCCT-3
0
, XT-2 reverse 5´-CTGAGAGGTAGTTTGCGGTTG-3
0
;
C4ST forward 5´-GGCGCTGCTGGAAGTGAT-3
0
, C4ST reverse
5-AAGATAAAGGATCCGAAGCAA-3
0
; Brevican forward 5´-CCATCC
AGAACCCACGAGA-3
0
, Brevican reverse 5´-ACCCACCACTCC
GTAATTCC-3
0
; 18S forward 5
0
-ATGCTCTTAGCTGAGTGTCCCG-3
0
,
18S reverse 5
0
-ATTCCTAGCTGCGGTATCCAGG-3
0
. After an initial
incubation at 50
C for 2 min and 95
C for 10 min, PCR amplification
was performed over 40 cycles of 95
C for 15 s and 60
C for 1 min.
The samples were assayed in triplicate on an Applied Biosystems
PRISM 7500 Sequence detection system. To rule out genomic DNA
contamination, a control sample using RNA that had not been
reversed transcribed was used as the template for each set of extrac-
tions. Gene expression was calculated using the 2
ΔΔCt
method and
the relative expression was quantified by calculating the ratio
between the values obtained for each gene of interest and those of
the 18S gene. The results are expressed as a percentage with respect
to the control group.
2.13 | ELISA
The neurocan and brevican levels in supernatants from primary rat
astrocyte cell cultures were measured using specific solid-phase sand-
wich ELISA kits with antibodies against neurocan and brevican respec-
tively, following the manufacturer's recommendations: for neurocan
rat astrocytes - #MBS 450382, My Biosource, San Diego, CA; and for
brevican rat astrocytes #MBS732282, My Biosource, San Diego,
CA. The neurocan levels in human astrocyte cell cultures were mea-
sured by the ELISA kit CSB-EL015513HU Cusabio, Houston, TX, fol-
lowing the manufacturer's recommendations. The minimum
detectable concentration of rat neurocan was 0.055 ng/ml, the detec-
tion range was 0.15610 ng/ml, and the intra- and inter-coefficient of
variation was 10 and 12%, respectively. The sensitivity of the brevican
assay is 1.0 pg/ml. The detectable limit of human neurocan was
0.078 ng/ml, the detection range was 0.31220 ng/ml, and the intra-
and inter-coefficient of variation was 8 and 10%, respectively.
2.14 | Statistical analysis
All the data are expressed as the mean ± SEM, using the IBM SPSS
24 statistical software (Inc- IBM, Chicago, IL) for the statistical analy-
sis. One-way ANOVA followed by the Bonferroni and Tukey's post
hoc tests, or a nonparametric Kruskal-Wallis test was employed for
multiple comparisons. The level of significance was set at p .05.
3 | RESULTS
3.1 | 2-AG treatment diminishes the expression of
enzymes involved in the synthesis of CSPGs and the
levels of neurocan and brevican in rat astrocytes
cultures
CSPGs consist of a core pro tein with attached sulfate GAG side
chains (Susarla et al., 2011). The synthesis of the GAG chain is car-
ried out by diffe rent xylol transferases (XT-1 and XT-2) that consti-
tute the rate-limiting step of GAG synthesis and by the chondroitin
4 sulfotransferase (C4ST). Here first, we show that stimulated
FELIU ET AL. 5

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TL;DR: Astrocyte functions in healthy CNS, mechanisms and functions of reactive astrogliosis and glial scar formation, and ways in which reactive astrocytes may cause or contribute to specific CNS disorders and lesions are reviewed.
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TL;DR: Chondroitin and keratan sulphate proteoglycans are among the main inhibitory extracellular matrix molecules that are produced by reactive astrocytes in the glial scar, and they are believed to play a crucial part in regeneration failure.
Abstract: After injury to the adult central nervous system (CNS), injured axons cannot regenerate past the lesion. In this review, we present evidence that this is due to the formation of a glial scar. Chondroitin and keratan sulphate proteoglycans are among the main inhibitory extracellular matrix molecules that are produced by reactive astrocytes in the glial scar, and they are believed to play a crucial part in regeneration failure. We will focus on this role, as well as considering the behaviour of regenerating neurons in the environment of CNS injury.

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  • ...…et al., 2008), evidence that this cytokine enhances the expression of a whole battery of enzymes responsible for CSPG production by astrocytes (Asher et al., 2000; Gris et al., 2007; Hamel et al., 2005; Smith & Strunz, 2005; Wang et al., 2008) in agreement with the results of the present study....

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TL;DR: The growing understanding of TGFbeta signaling through the Smad pathway provides general principles for how animal cells translate complex inputs into concrete behavior.
Abstract: Smad transcription factors lie at the core of one of the most versatile cytokine signaling pathways in metazoan biology-the transforming growth factor-beta (TGFbeta) pathway. Recent progress has shed light into the processes of Smad activation and deactivation, nucleocytoplasmic dynamics, and assembly of transcriptional complexes. A rich repertoire of regulatory devices exerts control over each step of the Smad pathway. This knowledge is enabling work on more complex questions about the organization, integration, and modulation of Smad-dependent transcriptional programs. We are beginning to uncover self-enabled gene response cascades, graded Smad response mechanisms, and Smad-dependent synexpression groups. Our growing understanding of TGFbeta signaling through the Smad pathway provides general principles for how animal cells translate complex inputs into concrete behavior.

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TL;DR: Deyelinated plaques in multiple sclerosis consists mostly of scar-type astrocytes and naked axons, but astroCytes inhibit the migration of both oligodendrocyte precursors and Schwann cells which must restrict their access to demyelinated axons.
Abstract: Damage to the central nervous system (CNS) results in a glial reaction, leading eventually to the formation of a glial scar. In this environment, axon regeneration fails, and remyelination may also be unsuccessful. The glial reaction to injury recruits microglia, oligodendrocyte precursors, meningeal cells, astrocytes and stem cells. Damaged CNS also contains oligodendrocytes and myelin debris. Most of these cell types produce molecules that have been shown to be inhibitory to axon regeneration. Oligodendrocytes produce NI250, myelin-associated glycoprotein (MAG), and tenascin-R, oligodendrocyte precursors produce NG2 DSD-1/phosphacan and versican, astrocytes produce tenascin, brevican, and neurocan, and can be stimulated to produce NG2, meningeal cells produce NG2 and other proteoglycans, and acitivated microglia produce free radicals, nitric oxide, and arachidonic acid derivatives. Many of these molecules must participate in rendering the damaged CNS inhibitory for axon regeneration. Demyelinated plaques in multiple sclerosis consists mostly of scar-type astrocytes and naked axons. The extent to which the astrocytosis is responsible for blocking remyelination is not established, but astrocytes inhibit the migration of both oligodendrocyte precursors and Schwann cells which must restrict their access to demyelinated axons.

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Frequently Asked Questions (1)
Q1. What contributions have the authors mentioned in the paper "2-arachidonoylglycerol reduces chondroitin sulphate proteoglycan production by astrocytes and enhances oligodendrocyte differentiation under inhibitory conditions" ?

Here, the authors investigated how 2-arachidonoylglycerol ( 2-AG ) may affect the production of the CSPGs neurocan and brevican by astrocytes in culture. In addition, the authors studied whether 2-AG promotes oligodendrocyte differentiation under inhibitory conditions in vitro.