DOCSLIB.ORG

Mechanistic Target of Rapamycin Regulates the Oligodendrocyte Cytoskeleton During Myelination

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text

Load more

Research ReportDevelopment/Plasticity/Repair

Mechanistic Target of Rapamycin Regulates the Oligodendrocyte Cytoskeleton during Myelination

https://doi.org/10.1523/JNEUROSCI.1434-18.2020

Cite as: J. Neurosci 2020; 10.1523/JNEUROSCI.1434-18.2020 Received: 31 May 2018 Revised: 23 February 2020 Accepted: 26 February 2020

This Early Release article has been peer-reviewed and accepted, but has not been through the composition and copyediting processes. The final version may differ slightly in style or formatting and will contain links to any extended data.

Alerts: Sign up at www.jneurosci.org/alerts to receive customized email alerts when the fully formatted version of this article is published.

Copyright © 2020 Musah et al.

12

Mechanistic Target of Rapamycin Regulates the Oligodendrocyte Cytoskeleton during
Myelination

3456789
1Aminat S. Musah, 2Tanya L. Brown, 1Marisa A. Jeffries, 1Quan Shang, 2Hirokazu Hashimoto, 1Angelina V. Evangelou, 3Alison Kowalski, 3Mona Batish, 2Wendy B. Macklin and 1Teresa L.
Wood
1Department of Pharmacology, Physiology & Neuroscience, New Jersey Medical School,
Rutgers University, Newark, NJ U.S.A. 07101, 2Department of Cell and Developmental Biology, University of Colorado School of Medicine, Aurora, CO, U.S.A. 80045, 3Department of Medical and Molecular Sciences, University of Delaware, Newark, DE 19716

10

11 Abbreviated Title: mTOR Regulates the Oligodendrocyte Cytoskeleton

12 13 14 15

Corresponding Author: Teresa L. Wood, PhD, Department Pharmacology, Physiology & Neuroscience, New Jersey Medical School Cancer Center H1200, Rutgers University, 205 S. Orange Ave, Newark, NJ 07101-1709 [email protected]

16 17 18 19 20 21 22

Number of pages: 39 Number of Figures: 10 Number of Tables: 0 Number of Words:
Abstract: 242 Introduction: 687 Discussion: 1075

23 24 25
Conflict of Interest: The authors declare no competing financial interests. Acknowledgements: This work was supported by National Institute of Neurological Disorders & Stroke NS082203 to WM and TLW, National Multiple Sclerosis Society RG5371-A-4 to TLW
26 and NIH Director’s Early Independence Award DP5 OD012160 to Mona Batish. The authors 27 28 29 wish to thank Luke Fritzky for technical assistance and Luipa Khandker for critical reading of the manuscript.

ꢀ

2

30 31 32 33 34 35 36

Abstract

During differentiation, oligodendrocyte precursor cells (OPCs) extend a network of processes that make contact with axons and initiate myelination. Recent studies revealed that actin polymerization is required for initiation of myelination whereas actin depolymerization promotes myelin wrapping. Here, we used primary OPCs in culture isolated from neonatal rat cortices of both sexes and young male and female mice with oligodendrocyte-specific deletion of mechanistic target of rapamycin (mTOR) to demonstrate that mTOR regulates expression of
37 specific cytoskeletal targets and actin reorganization in oligodendrocytes during developmental 38 39 40 41 myelination. Loss or inhibition of mTOR reduced expression of profilin2 and ARPC3, actin polymerizing factors, and elevated levels of active cofilin, which mediates actin depolymerization. The deficits in actin polymerization were revealed in reduced phalloidin and deficits in oligodendrocyte cellular branching complexity at the peak of morphological
42 differentiation and a delay in initiation of myelination. We further show a critical role for mTOR 43 in expression and localization of myelin basic protein (Mbp) mRNA and MBP protein to the 44 cellular processes where it is necessary at the myelin membrane for axon wrapping. Mbp mRNA

  • 45
  • transport deficits were confirmed by single molecule RNA FISH. Moreover, expression of the

46 kinesin family member 1B, an Mbp mRNA transport protein, was reduced in CC1+ cells in the 47 48 49 mTOR cKO and in mTOR inhibited oligodendrocytes undergoing differentiation in vitro. These data support the conclusion that mTOR regulates both initiation of myelination and axon wrapping by targeting cytoskeletal reorganization and MBP localization to oligodendrocyte
50 processes. 51 52 53 54 55 56 57 58 59 60

Significance Statement

Myelination is essential for normal central nervous system (CNS) development and adult axon preservation and function. The mechanistic target of rapamycin (mTOR) signaling pathway has been implicated in promoting CNS myelination; however, there is a gap in our understanding of the mechanisms by which mTOR promotes developmental myelination through regulating specific downstream targets. Here, we present evidence that mTOR promotes the initiation of myelination through regulating specific cytoskeletal targets and cellular process expansion by oligodendrocyte progenitor cells as well as expression and cellular localization of myelin basic protein.

61

ꢀ

4

62 63 64 65 66

Introduction

Progression through specific stages of oligodendrocyte differentiation has been well characterized and involves extensive morphological changes in preparation for axon contact and initiation of myelination (for reviews, see (Michalski and Kothary, 2015; Snaidero and Simons, 2017; Brown and Macklin, 2019)). Oligodendrocyte precursor cells (OPCs) transition into
67 immature oligodendrocytes characterized by increased number and length of cellular processes 68 to contact axons and initiate myelination. Final maturation of oligodendrocytes requires the cell 69 membrane to flatten out to wrap axons in the process of myelination (Nawaz et al., 2015; 70 Zuchero et al., 2015). The inability of OPCs to differentiate contributes to impaired 71 developmental myelination as well as to reduced myelin repair after demyelination in diseases 72 73 74 75 76 77 78 such as multiple sclerosis (for reviews, see (Franklin and Ffrench-Constant, 2017; Snaidero and Simons, 2017; Abu-Rub and Miller, 2018)).
The evolving morphology of oligodendrocytes during cellular differentiation and myelination requires a dynamic cytoskeleton (for reviews, see (Michalski and Kothary, 2015; Snaidero and Simons, 2017; Brown and Macklin, 2019; Thomason et al., 2019)). The actin cytoskeleton exists in monomer and filament form; the balance between polymerization and depolymerization is tightly regulated and controls cytoskeletal reorganizations. Numerous
79 laboratories have contributed to our current understanding of cytoskeletal dynamics in 80 81 developing oligodendroglia. Prior reports support two phases of developmental myelination involving opposing cytoskeletal processes in differentiating oligodendrocytes; phase I requires
82 actin polymerization for growth cone formation mediated process extension (Fox et al., 2006; 83 Zuchero et al., 2015), whereas phase II requires actin depolymerization for proper axon wrapping 84 and myelination (Nawaz et al., 2015; Zuchero et al., 2015). Actin binding proteins that regulate

5

85 86 87 88 89 actin cytoskeletal reorganization include the actin polymerizing protein profilin, the ARP2/3 branched actin nucleator complex, and the depolymerizing protein family actin depolymerizing factor (ADF)/cofilin1. The ARP 2/3 complex in oligodendroglia is known to regulate axon ensheathment (Zuchero et al., 2015). In contrast, ADF/cofilin1 regulates filamentous actin (F- actin) turnover for proper myelin wrapping; however, it is hypothesized that F-actin at the
90 membrane leading edge provides the driving force needed for myelin growth (Nawaz et al., 91 2015). Moreover, the involvement of F-actin at the inner tongue of myelinating cells helps create 92 the balance between myelin compaction and cytoplasmic channels (Snaidero and Simons, 2017). 93 The presence of myelin basic protein (MBP) in the oligodendrocyte processes also has been 94 95 proposed to be important in the wrapping phase of myelination to enable MBP to displace cofilin at the membrane (Zuchero et al., 2015). The membrane displacement of cofilin then may
96 facilitate actin depolymerization necessary for wrapping (Zuchero et al., 2015). 97 98
The function of specific intracellular signaling pathways in the developmental progression of oligodendroglia has been the focus of numerous publications (Sperber et al.,
99 2001; Zou et al., 2011; Ishii et al., 2012; Bercury et al., 2014; Dai et al., 2014; Wahl et al., 2014;

  • 100
  • Furusho et al., 2017). However, it is unknown how these pathways orchestrate the morphological

changes observed during differentiation and myelination. Moreover, the studies on actin dynamics in developing oligodendrocytes and myelination did not identify upstream regulators (Nawaz et al., 2015; Zuchero et al., 2015). Studies in a variety of other cell types including neutrophils, fibroblasts and tumor cells demonstrate a role for the mechanistic target of rapamycin (mTOR) pathway in regulating the cytoskeleton (Jacinto et al., 2004; Sarbassov et al., 2004; Liu et al., 2008; He et al., 2013), however, these studies have provided little information on specific cytoskeletal targets downstream of mTOR. Our previous studies revealed that mTOR
101 102 103 104 105 106 107

6

108 109 regulates oligodendrocyte differentiation, initiation of myelination and myelin thickness in the spinal cord (Tyler et al., 2009; Bercury et al., 2014; Wahl et al., 2014). Collectively, these
110 findings led us to hypothesize that mTOR regulates cytoskeletal dynamics in oligodendrocytes 111 112 113 necessary for initiation of myelination and for proper myelin wrapping.
The goal of the studies presented here was to determine the function of mTOR signaling in regulating cytoskeletal reorganization during oligodendrocyte development. We show that loss
114 or inhibition of mTOR alters expression of cytoskeletal proteins involved in actin polymerization 115 in differentiating oligodendrocytes in vitro and in vivo. Moreover, we demonstrate that mTOR in

  • 116
  • oligodendroglia regulates the localization of MBP to oligodendrocyte processes during

117 myelination, through promoting transport of Mbp mRNAs. 118 119 120 121 122 123 124 125 126 127 128

Materials and Methods

Experimental Animals. All mouse protocols were conducted in accordance with Rutgers University Institutional Animal Care and Use Committee and the National Institute of Health guidelines for care and use of laboratory animals. Mice were housed in a barrier facility with a 12/12 light/dark cycle. The CNP-Cre/mTOR conditional knockout (mTOR cKO) mouse line carrying CNP-Cre and floxed alleles for Mtor was described previously (Wahl et al., 2014). Mice homozygous for Mtor floxed and heterozygous for CNP-Cre were used for breeding to generate Cre+ or Cre- littermates for experiments. The PLP-Cre/mTOR inducible cKO (icKO) line was established by crossing Mtor fl/fl mice (Lang et al., 2010a; Lang et al., 2010b; Carr et al., 2012; Wahl et al., 2014) with PLP-CreERT mice (Jackson Laboratory 005975,
129 RRID:IMSR_JAX:005975), henceforth referred to as PLP-mTOR icKO mice. Mice homozygous

  • 130
  • for Mtor floxed and heterozygous for PLP- CreERT were used for breeding to generate Cre+ or

7

131 132 133 134 135

Cre- littermates for experiments. Tamoxifen was injected intraperitoneally (60 mg/kg) for 4 consecutive days to induce recombination at PND7. Tamoxifen was dissolved in a 9:1 ratio of

sesame oil:100% ethanol. Both males and females were used in all analyses. All strains were on a C57Bl/6 background.
All zebrafish experiments were approved by the Institutional Animal Care and Use
136 Committee at the University of Colorado School of Medicine. Embryos were raised at 28.5°C in 137 embryo media (EM) and staged according to hours post-fertilization (hpf), days post-fertilization 138 139 140
(dpf), and morphological criteria (Kimmel et al., 1995). Rapamycin (Tocris Bioscience) was dissolved in 100% DMSO at a concentration of 20 mM. Drugs were diluted in EM to make a working concentration of 5 mM with a final concentration of 1% DMSO. Control solutions
141 contained 1% DMSO in EM. Tg(nkx2.2a:mEGFP) zebrafish embryos were collected following 142 143 144 145 146 147 148 149 150 151 152 timed matings. Embryos were sorted for GFP, dechorionated and treated with rapamycin or DMSO control. Zebrafish drug treatments were initiated at 48 hpf until 56 hpf, when zebrafish were anesthetized using tricaine (MS-222). Embryos were mounted laterally in 1% low-melt agarose and tricaine and imaged directed above the yolk sac extension on a Leica DM-6000 confocal. Individual oligodendrocytes were analyzed using IMARIS image analysis software (Bitplane).

Preparation and isolation of primary oligodendrocytes

OPCs were purified from cortical mixed glial cultures isolated from postnatal days 0-2
Sprague-Dawley rat pups by established methods and cultured as described previously (McCarthy and Vellis, 1980; Tyler et al., 2009). To initiate OPC differentiation, we followed an
153 established mitogen withdrawal protocol in the presence of 30 ng/ml triiodothyronine (T3) and

8

154 155 156 157 158 159 160 161 162 163 plus or minus the addition of rapamycin (15 nM) as for prior studies (Tyler et al., 2009). In some experiments, we initiated differentiation for 48 hours prior to adding rapamycin. For all experiments, differentiation medium plus/minus rapamycin was replenished every 48 hrs except as noted for Figure 1.

Protein isolation and western immunoblotting

Plated cells from OPC isolation were harvested in TRIS-triton buffer (10mm Tris,
100mM NaCl, 1mM EDTA, 1mM EGTA, 1% Triton X-100, 10% glycerol, 0.1% SDS, 0.5% deoxycholate) supplemented with phosphatase and protease inhibitors (5mM NaF, 1mM PMSF, 1% Protease inhibitor cocktail, 1mM Na3V04), then sonicated. Protein concentrations were
164 determined using the Biorad protein assay and separated by SDS-PAGE. Primary antibodies 165 166 167 168 were as follows: Rb-profilin2 (1:250; Novus Biologicals #NBP1-87426, RRID:AB_11007824), Rb-p-cofilin (1:1000; Cell Signaling #3313, RRID:AB_2080597), Rb-total cofilin (1:1000; Cell Signaling #5175, RRID:AB_10622000), Rb-Kif1b (1:500; Abcam #ab69614, RRID:AB_1860749), Rb-ARPC3 (1:500; Novus Biologicals #NBP1-89016,
169 RRID:AB_11003588), Rb-MBP (1:1000; Abcam #ab40390, RRID:AB_1141521), and Ms-beta170 171 172 173 174 175 176 actin (1:10,000; Sigma #A5441, RRID:AB_476744). Secondary antibodies were as follows: (Gt anti-Rb 1:2000 for all Rb antibodies, Jackson Immunoresearch #111-035-144, RRID:AB 2307391) or (Gt anti-Ms 1:5000 for beta-actin, Jackson Immunoresearch #115-035-003, RRID:AB_10015289).
For F/G actin analysis, proteins were harvested from control or rapamycin treated cells following differentiation in T3 supplemented differentiation media as above. F/G actin ratios were performed according to manufacturer instructions (Cytoskeleton Inc. #BK 037).

9

177 178 179 180

Tissue preparation and immunofluorescent staining

Animals at postnatal day (PND) 7 and 14 were rapidly decapitated and spinal cords were dissected and drop fixed in 3% PFA at 4qC, overnight. Tissues were then dehydrated in 30%
181 sucrose and frozen in OCT for cryosectioning. Mounted cryosections (25 μm) were rinsed in 182 0.1% PBST and incubated in 50 mM ammonium chloride for 1 hour at room temperature. Slides 183 were washed in 0.1% PBST 3 X 10 mins. Sections were then permeabilized and blocked in 10% 184 185
BSA/5% NGS in 0.1% PBST for 1 hour at room temperature. Primary and secondary antibodies were added to 1% BSA/5% NGS in 0.1% PBST. Primary antibody was incubated in a humid
186 chamber, overnight at 4qC. Primary antibodies were obtained as described above and used at the 187 following dilutions: profilin2 (1:50), ARPC3 (1:100), Kif1b (1:200), MBP (1:200), CC1 (1:100; 188 189 190 191 192 193 194 195
Millipore # OP80, RRID:AB_2057371), anti-α-Tubulin (1:500; Sigma #T6199, RRID:AB_477583), Acti-Stain 488 Fluorescent Phalloidin (100 nM; Cytoskeleton #PHDG1), and mouse-neurofilament (NF) 200 (1:20; Sigma #N5389, RRID:AB_260781). Secondary antibodies purchased from Jackson Immunoresearch were: goat anti-rabbit 647 (1:500; #111- 605-144, RRID:AB_2338078), mouse biotin (1:100; #200-002-211, RRID:AB_2339006) and streptavidin 405 (1:300; #016-470-084, RRID:AB_2337248).
Following primary antibody incubation, sections were washed in 0.1% PBST and incubated in the appropriate secondary antibody (described above) for 2 hours at room
196 temperature at the following dilutions: goat anti-rabbit 647 (1:500) or mouse biotin (1:100). 197 Sections were washed in 0.1% PBST and incubated with streptavidin 405 (1:300) for 2 hours at 198 199 room temperature followed by washes in 0.1% PBST and PBS and coverslipped with either Fluorogel or Prolong Gold.

10

200 201 202 203 204 205
For cell staining in vitro, media was removed from cultured cells in well-slides. Slides were rinsed with PBS, fixed in 3% PFA for 15 mins, washed with PBS and permeabilized with 0.3% triton X-100 for 15 mins. Cells were blocked in 4% BSA/3% goat serum in PBS for 1 hour and incubated overnight with primary antibody in blocking solution. Following washes in PBS, cells were incubated in secondary antibody for 1 hour at room temperature. Cells were then washed in PBS and incubated in DAPI for 5 mins, washed again in PBS and coverslipped with
206 Fluorogel. For F-actin visualization, cells were incubated in 100 nM Acti-Stain 488 Fluorescent 207 208
Phalloidin at room temperature in the dark for 30 min. prior to DAPI incubation.
209 210

In situ hybridization

Mice were intracardially perfused with 4% PFA in PBS; spinal cords were dissected and

  • 211
  • postfixed with 4% PFA overnight, cryoprotected with 30% sucrose-PBS buffer overnight and

212 frozen. Mounted cryosections were prepared at 20 μm thickness with a cryostat. In situ 213 214 hybridization was performed as described previously (Hashimoto et al., 2016) with slight modifications. The following plasmids containing mouse cDNA were used to generate cRNA
215 probes: PLP (full coding region, (Harlow et al., 2014)) and MBP (nucleotides 683-1286 216 217 218 corresponding to NM_010777.3). Briefly, the sections were treated with proteinase K (2 μg/ml for 15 min at room temperature) and hybridized overnight at 63°C with DIG-labeled antisense riboprobes in a hybridization solution consisting of 50% formamide, 20 mM Tris-HCl (pH 7.5),
219 600 mM NaCl, 1 mM EDTA, 10% dextran sulfate, 200 μg/ml yeast tRNA and 1x Denhardt’s 220 solution. After the sections were washed in buffers with decreasing stringency, they were 221 incubated with an alkaline phosphatase‐conjugated anti‐DIG antibody (1:5000; Roche 222 223
Diagnostics). The color was developed in the presence of 4-nitroblue tetrazolium chloride and 5- bromo-4-chloro-3-indolylphosphate (Roche diagnostics) in the dark at room temperature.

11

  • 224
  • Quantification of PLP+ numbers were performed using ImageJ (U.S. National Institutes

225 of Health, Bethesda, Maryland) on 63X control (n=3) and mTOR cKO (n=4) ventral white 226 matter fields. Cells were counted on at least three sections per animal. Quantification of MBP 227 mRNA intensity in the ventral white matter was performed using ImageJ on 63X control (n=3) 228 229 and mTOR cKO (n=4). The average intensity was measured in at least three sections per animal, and the results were expressed as arbitrary units.
230 231 232

FISH

A set of 50 (20 nt long) probes was designed to hybridize to the coding sequence of the

  • 233
  • Mbp mRNA using freely available software at LGC biosearch technologies LLC as described

234 previously (Batish et al., 2011). The oligos were ordered with the 3’ terminal nucleotide having 235 236 237 238 an amino modification. All the modified oligos were pooled in equimolar concentration and coupled en mass with Alexa 594 dye. The labeled probes were purified from unlabeled oligos and unbound dye using a C14 reverse phase hydrophobic HPLC column as described previously (Batish et al., 2011). Primary rat OPC cells were grown on glass coverslips under differentiation
239 conditions as described above plus or minus 15 nM of rapamycin for 7 days with media change 240 every 48 hrs. The cells were fixed using 4% paraformaldehyde for 10 minutes, permeabilized 241 242 243 244 245 246 with 70% ethanol at 4 °C and hybridized overnight at 37 °C with the labeled probes. The coverslips were washed multiple times to remove any unbound probes, stained with DAPI and mounted using deoxygenated media (Batish et al., 2011; Markey et al., 2014). The coverslips were imaged using 100 X oil objective in a Nikon TiE inverted automated fluorescence microscope equipped with cooled CCD Pixis 1024b camera. The z stacks of 3 μm with 0.2 μm apart were acquired using Metamorph software and the images were analyzed using custom

12

247 248 249 250 251 252 253 254 255 written programs in MATLAB (Mathworks Inc) (Batish et al., 2012; Markey et al., 2014). At least 100 cells for each treatment were quantified.

Isolation of O4 positive cells

Spinal cord tissue dissociation was performed according to manufacturer’s instructions using a neural dissociation kit (Miltenyi Biotec #130-092-628). The O4+ oligodendrocytes were isolated from spinal cords using O4 microbeads (Miltenyi Biotec #130-094- 543) and MS columns (Miltenyi Biotec #130-042-201) and used for protein or RNA isolation.

256 RNA isolation and RT-PCR analysis

257 258
RNA isolation from O4 positive cells was completed using RNeasy Plus Micro Kit
(Qiagen, #74034) and the Quick-StartProtocol. RNA concentration was measured using a
259 NanoDrop spectrophotometer (Thermo Scientific); 50 - 80 ng of RNA was used to reverse 260 261 transcribe cDNA using Superscript II (Invitrogen). For RT-PCR, 1.5-2 μl of cDNA per well was used in reactions containing 1 x SYBR Green detection master mix and 1 x Quantitect primer
262 mix to detect mRNA levels. Amplification was normalized to expression levels of β -actin (IDT) 263 264 265 266 267 268 for each sample.

Recommended publications
  • Sialylated Keratan Sulfate Chains Are Ligands for Siglec-8 in Human Airways

    Sialylated Keratan Sulfate Chains Are Ligands for Siglec-8 in Human Airways

    Sialylated Keratan Sulfate Chains are Ligands for Siglec-8 in Human Airways by Ryan Porell A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland September 2018 © 2018 Ryan Porell All Rights Reserved ABSTRACT Airway inflammatory diseases are characterized by infiltration of immune cells, which are tightly regulated to limit inflammatory damage. Most members of the Siglec family of sialoglycan binding proteins are expressed on the surfaces of immune cells and are immune inhibitory when they bind their sialoglycan ligands. When Siglec-8 on activated eosinophils and mast cells binds to its sialoglycan ligands, apoptosis or inhibition of mediator release is induced. We identified human airway Siglec-8 ligands as sialylated and 6’-sulfated keratan sulfate (KS) chains carried on large proteoglycans. Siglec-8- binding proteoglycans from human airways increase eosinophil apoptosis in vitro. Given the structural complexity of intact proteoglycans, target KS chains were isolated from airway tissue and lavage. Biological samples were extensively proteolyzed, the remaining sulfated glycan chains captured and resolved by anion exchange chromatography, methanol-precipitated then chondroitin and heparan sulfates enzymatically hydrolyzed. The resulting preparation consisted of KS chains attached to a single amino acid or a short peptide. Purified KS chains were hydrolyzed with either hydrochloric acid or trifluoroacetic acid to release acidic and neutral sugars, respectively, followed by DIONEX carbohydrate analysis. To isolate Siglec-8-binding KS chains, purified KS chains from biological samples were biotinylated at the amino acid, resolved by affinity and/or size- exclusion chromatography, the resulting fractions immobilized on streptavidin microwell plates, and probed for binding of Siglec-8-Fc.
  • Immunity to the G1 Globular Domain of the Cartilage Proteoglycan

    Immunity to the G1 Globular Domain of the Cartilage Proteoglycan

    Immunity to the G1 globular domain of the cartilage proteoglycan aggrecan can induce inflammatory erosive polyarthritis and spondylitis in BALB/c mice but immunity to G1 is inhibited by covalently bound keratan sulfate in vitro and in vivo. J Y Leroux, … , S Banerjee, A R Poole J Clin Invest. 1996;97(3):621-632. https://doi.org/10.1172/JCI118458. Research Article Earlier work from this laboratory showed that the human proteoglycan aggrecan from fetal cartilages can induce a CD4+ T cell-dependent inflammatory polyarthritis in BALB/c mice when injected after removal of chondroitin sulfate chains. Adult keratan sulfate (KS)-rich aggrecan does not possess this property. We found that two CD4+ T cell hybridomas (TH5 and TH14) isolated from arthritic mice recognize bovine calf aggrecan and the purified G1 domain of this molecule, which also contains a portion of the interglobular domain to which KS is bound. These hybridoma responses to G1 are enhanced by partial removal of KS by the endoglycosidase keratanase or by cyanogen bromide cleavage of core protein. KS removal results in increased cellular uptake by antigen-present cells in vitro. After removal of KS by keratanase, G1 alone can induce a severe erosive polyarthritis and spondylitis in BALB/c mice identifying it as an arthritogenic domain of aggrecan. The presence of KS prevents induction of arthritis presumably as a result of an impaired immune response as observed in vitro. These observations not only identify the arthritogenic properties of G1 but they also point to the importance of glycosylation and proteolysis in determining the arthritogenicity of aggrecan and fragments thereof.
  • Modulation of Heparin Cofactor II Activity by Histidine-Rich Glycoprotein and Platelet Factor 4

    Modulation of Heparin Cofactor II Activity by Histidine-Rich Glycoprotein and Platelet Factor 4

    Modulation of heparin cofactor II activity by histidine-rich glycoprotein and platelet factor 4. D M Tollefsen, C A Pestka J Clin Invest. 1985;75(2):496-501. https://doi.org/10.1172/JCI111725. Research Article Heparin cofactor II is a plasma protein that inhibits thrombin rapidly in the presence of either heparin or dermatan sulfate. We have determined the effects of two glycosaminoglycan-binding proteins, i.e., histidine-rich glycoprotein and platelet factor 4, on these reactions. Inhibition of thrombin by heparin cofactor II and heparin was completely prevented by purified histidine-rich glycoprotein at the ratio of 13 micrograms histidine-rich glycoprotein/microgram heparin. In contrast, histidine-rich glycoprotein had no effect on inhibition of thrombin by heparin cofactor II and dermatan sulfate at ratios of less than or equal to 128 micrograms histidine-rich glycoprotein/microgram dermatan sulfate. Removal of 85-90% of the histidine-rich glycoprotein from plasma resulted in a fourfold reduction in the amount of heparin required to prolong the thrombin clotting time from 14 s to greater than 180 s but had no effect on the amount of dermatan sulfate required for similar anti-coagulant activity. In contrast to histidine-rich glycoprotein, purified platelet factor 4 prevented inhibition of thrombin by heparin cofactor II in the presence of either heparin or dermatan sulfate at the ratio of 2 micrograms platelet factor 4/micrograms glycosaminoglycan. Furthermore, the supernatant medium from platelets treated with arachidonic acid to cause secretion of platelet factor 4 prevented inhibition of thrombin by heparin cofactor II in the presence of heparin or dermatan sulfate.
  • In Vivo Detection of Intervertebral Disk Injury Using a Radiolabeled Monoclonal Antibody Against Keratan Sulfate

    In Vivo Detection of Intervertebral Disk Injury Using a Radiolabeled Monoclonal Antibody Against Keratan Sulfate

    In Vivo Detection of Intervertebral Disk Injury Using a Radiolabeled Monoclonal Antibody Against Keratan Sulfate Kalevi J.A. Kairemo, Anu K. Lappalainen, Eeva Ka¨a¨pa¨, Outi M. Laitinen, Timo Hyytinen, Sirkka-Liisa Karonen, and Mats Gro¨nblad Departments of Clinical Chemistry, Physical Medicine and Rehabilitation, and Thoracic and Cardiovascular Surgery, Helsinki University Central Hospital, Helsinki; and Faculty of Veterinary Medicine, Department of Clinical Veterinary Sciences, University of Helsinki, Helsinki, Finland been suggested to be related to back pain (1,2). None of In the intervertebral disk, proteoglycans form the major part of the present in vivo methods, however, show more de- the extracellular matrix, surrounding chondrocytelike disk cells. tailed pathology coupled with intervertebral disk degenera- Keratan sulfate is a major constituent of proteoglycans. Meth- tion or disk injury. In particular, at present no reliable in ods: We have radioiodinated a monoclonal antibody raised vivo methods are available for revealing annulus fibrosus against keratan sulfate. This antibody was injected into rats (n ϭ 6), and the biodistribution was studied. A model of intervertebral pathology. disk injury was developed, and two tail disks in each animal with If successful, specific in vivo targeting of the interverte- both acute (2 wk old) and subacute (7 wk old) injuries were bral disk by labeled antibodies, directed to specific molec- studied for in vivo antibody uptake. Results: The biodistribution ular structures, will provide clinicians with a means for at 72 h was as follows: blood, 0.0018 percentage injected dose studying mechanisms of disk pathology. The ability to fol- per gram of tissue (%ID/g); lung, 0.0106 %ID/g; esophagus, low in vivo reparative and degenerative processes prospec- 0.0078 %ID/g; kidney, 0.0063 %ID/g; liver, 0.0047 %ID/g; tively may also become possible.
  • Trabecular Meshwork Glycosaminoglycans in Human and Cynomolgus Monkey Eye Ted S

    Trabecular Meshwork Glycosaminoglycans in Human and Cynomolgus Monkey Eye Ted S

    Trabecular Meshwork Glycosaminoglycans in Human and Cynomolgus Monkey Eye Ted S. Acorr,*t Mary Wesrcorr,* Michael 5. Posso,* and E. Michael Van Duskirk* The glycosaminoglycans (GAGs) extractable from the trabecular meshworks (TM) of human and non- human primate eye have been analyzed by sequential enzymatic degradation and cellulose acetate elec- trophoresis. For comparison, similar extracts of the cornea, sclera, iris, and ciliary body have also been analyzed. The distribution of glycosaminoglycans in human and in cynomolgus monkey TMs are similar, although not identical. The human TM contains hyaluronic acid (HA), chondroitin- 4-sulfate and/or 6- sulfate (CS), dermatan sulfate (DS), keratan sulfate (KS), heparan sulfate (HS), and an unidentified band of Alcian Blue staining material, which is resistant to the enzymes that we used. Based upon quantitation of the Alcian Blue staining intensities of extracted GAGs, which have been corrected by a relative dye-binding factor, the GAGs of the human TM include: 29.0% HA, 14.1% CS, 21.5% DS, 20.3% KS, and 15.0% HS. The cynomolgus monkey trabecular GAGs include: 12.8% HA, 14.3% CS, 15.2% DS, 42.1% KS, and 15.6% HS. Invest Ophthalmol Vis Sci 26:1320-1329, 1985 The preponderance of evidence suggests that the We have examined the extracellular matrix GAGs primary site of aqueous outflow resistance resides of the human and nonhuman primate trabecular within the trabecular meshwork and possibly within meshwork as the initial step in studies intended to clar- the deep portion of the corneoscleral meshwork and/ ify the role that GAGs play in the regulation of aqueous or the amorphous juxtacanalicular basement mem- outflow.
  • Cartilage Proteoglycans

    Cartilage Proteoglycans

    seminars in CELL & DEVELOPMENTAL BIOLOGY, Vol. 12, 2001: pp. 69–78 doi:10.1006/scdb.2000.0243, available online at http://www.idealibrary.com on Cartilage proteoglycans Cheryl B. Knudson∗ and Warren Knudson The predominant proteoglycan present in cartilage is the tural analysis. The predominate glycosaminoglycan large chondroitin sulfate proteoglycan ‘aggrecan’. Following present in cartilage has long been known to be its secretion, aggrecan self-assembles into a supramolecular chondroitin sulfate. 2 However, extraction of the structure with as many as 50 monomers bound to a filament chondroitin sulfate in a more native form, as a of hyaluronan. Aggrecan serves a direct, primary role pro- proteoglycan, proved to be a daunting task. The viding the osmotic resistance necessary for cartilage to resist revolution in the field came about through the compressive loads. Other proteoglycans expressed during work of Hascall and Sajdera. 3 With the use of the chondrogenesis and in cartilage include the cell surface strong chaotropic agent guanidinium hydrochlo- syndecans and glypican, the small leucine-rich proteoglycans ride, the proteoglycans of cartilage could now be decorin, biglycan, fibromodulin, lumican and epiphycan readily extracted and separated into relatively pure and the basement membrane proteoglycan, perlecan. The monomers through the use of CsCl density gradient emerging functions of these proteoglycans in cartilage will centrifugation. This provided the means to identify enhance our understanding of chondrogenesis and cartilage and characterize the major chondroitin sulfate pro- degeneration. teoglycan of cartilage, later to be termed ‘aggrecan’ following the cloning and sequencing of its core Key words: aggrecan / cartilage / CD44 / chondrocytes / protein. 4 From this start, aggrecan has gone on to hyaluronan serve as the paradigm for much of proteoglycan c 2001 Academic Press research.
  • Chondroitin Sulfate Safety and Quality

    Chondroitin Sulfate Safety and Quality

    molecules Review Chondroitin Sulfate Safety and Quality Nicola Volpi Department of Life Sciences, Laboratory of Biochemistry and Glycobiology, University of Modena and Reggio Emilia, 41125 Modena, Italy; [email protected]; Tel.: +39-59-2055543; Fax: +39-59-2055548 Academic Editor: Cédric Delattre Received: 7 March 2019; Accepted: 9 April 2019; Published: 12 April 2019 Abstract: The industrial production of chondroitin sulfate (CS) uses animal tissue sources as raw material derived from different terrestrial or marine species of animals. CS possesses a heterogeneous structure and physical-chemical profile in different species and tissues, responsible for the various and more specialized functions of these macromolecules. Moreover, mixes of different animal tissues and sources are possible, producing a CS final product having varied characteristics and not well identified profile, influencing oral absorption and activity. Finally, different extraction and purification processes may introduce further modifications of the CS structural characteristics and properties and may lead to extracts having a variable grade of purity, limited biological effects, presence of contaminants causing problems of safety and reproducibility along with not surely identified origin. These aspects pose a serious problem for the final consumers of the pharmaceutical or nutraceutical products mainly related to the traceability of CS and to the declaration of the real origin of the active ingredient and its content. In this review, specific, sensitive and validated analytical
  • Differential Relative Sulfation of Keratan Sulfate Glycosaminoglycan in the Chick Cornea During Embryonic Development

    Differential Relative Sulfation of Keratan Sulfate Glycosaminoglycan in the Chick Cornea During Embryonic Development

    Cornea Differential Relative Sulfation of Keratan Sulfate Glycosaminoglycan in the Chick Cornea during Embryonic Development Melody Liles,1 Barbara P. Palka,1 Anthony Harris,2 Briedgeen Kerr,2 Clare Hughes,2 Robert D. Young,1 Keith M. Meek,1 Bruce Caterson,2 and Andrew J. Quantock1 PURPOSE. To investigate structural remodeling of the develop- embryonic day (E)14, the chick cornea transmits only approx- ing corneal stroma concomitant with changing sulfation pat- imately 40% of white light, but then begins to increase in terns of keratan sulfate (KS) glycosaminoglycan (GAG) transparency so that, at E19, transmission is more than 95%, epitopes during embryogenesis and the onset of corneal trans- similar to the adult condition.4 The transparency increase after parency. E14 is accompanied by significant dehydration of the cornea, METHODS. Developing chick corneas were obtained from em- flattening of keratocytes, and compaction of stromal collagen 5,6 bryonic day (E)12 to E18 of incubation. Extracellular matrix fibrils into a spatially ordered array. This restructuring of the 7,8 composition and collagen fibril spacing were evaluated by stroma is key for the acquisition of corneal transparency. synchrotron x-ray diffraction, hydroxyproline assay, ELISA Small leucine-rich proteoglycans (PGs) interact with colla- (with antibodies against lesser and more highly sulfated KS), gen fibrils in the corneal stroma and are thought to help 9 and transmission electron microscopy with specific proteogly- control fibril size and spatial organization. PGs are composed can staining. of a core protein covalently bound to sulfated glycosaminogly- can (GAG) side chains. The major GAG in the cornea is keratan RESULTS.
  • Immunolocalization of Keratan Sulfate in Rat Spinal Tissues Using

    Immunolocalization of Keratan Sulfate in Rat Spinal Tissues Using

    cells Article Immunolocalization of Keratan Sulfate in Rat Spinal Tissues Using the Keratanase Generated BKS-1(+) Neoepitope: Correlation of Expression Patterns with the Class II SLRPs, Lumican and Keratocan Anthony J. Hayes 1 and James Melrose 2,3,4,* 1 Bioimaging Research Hub, Cardiff School of Biosciences, Cardiff University, Cardiff CF10 3AX, Wales, UK; HayesAJ@cardiff.ac.uk 2 Graduate School of Biomedical Engineering, University of New South Wales, Sydney, NSW 2052, Australia 3 Raymond Purves Laboratory, Institute of Bone and Joint Research, Kolling Institute of Medical Research, Northern Sydney Local Health District, Faculty of Medicine and Health, University of Sydney Royal North Shore Hospital, St. Leonards, NSW 2065, Australia 4 Sydney Medical School, Northern, University of Sydney at Royal North Shore Hospital, St. Leonards, NSW 2065, Australia * Correspondence: [email protected] Received: 8 February 2020; Accepted: 28 March 2020; Published: 30 March 2020 Abstract: This study has identified keratan sulfate in fetal and adult rat spinal cord and vertebral connective tissues using the antibody BKS-1(+) which recognizes a reducing terminal N-acetyl glucosamine-6-sulfate neo-epitope exposed by keratanase-I digestion. Labeling patterns were correlated with those of lumican and keratocan using core protein antibodies to these small leucine rich proteoglycan species. BKS-1(+) was not immunolocalized in fetal spinal cord but was apparent in adult cord and was also prominently immunolocalized to the nucleus pulposus and inner annulus fibrosus of the intervertebral disc. Interestingly, BKS-1(+) was also strongly associated with vertebral body ossification centers of the fetal spine. Immunolocalization of lumican and keratocan was faint within the vertebral body rudiments of the fetus and did not correlate with the BKS-1(+) localization indicating that this reactivity was due to another KS-proteoglycan, possibly osteoadherin (osteomodulin) which has known roles in endochondral ossification.

  • Proteoglycans & Glycosaminoglycans

    Proteoglycans & Glycosaminoglycans Glycobiology Research Hyaluronic Acid - Chondroitinase ABC - Assays - Enzymes - Antibodies www.amsbio.com | www.proteoglycan.info Table of Contents Structure of Glycosaminoglycans........................................................................... 03 Systemic Analysis of Glycosaminoglycan (GAG) Structure ............................. 04 Structure of the Unsaturated Disaccharides .................................................. 04 Disaccharide Composition of Heparan Sulfate and Heparin........................... 05 Disaccharide Composition of Chondroitin Sulfate and Dermatan Sulfate ...... 05 Proteoglycan (GAG Side Chain) Degrading Enzymes .............................................. 06 Chondroitinase Enzymes .............................................................................. 06 Perineuronal Net Removal by Chondroitinase ABC ............................... 07 Chondroitinase ABC Citations .............................................................. 07 Immunoprecipitation for Chondroitin Sulfate Proteoglycan ................. 05 Heparinase Enzymes ..................................................................................... 09 K5 Heparan Lyase Enzyme ............................................................................. 10 Hyaluronidase ............................................................................................... 10 Keratanase Enzymes ..................................................................................... 11 Glycobiology Antibodies .......................................................................................
  • Glcnac6st-1 Regulates Sulfation of N-Glycans and Myelination in the Peripheral Nervous System

    Glcnac6st-1 Regulates Sulfation of N-Glycans and Myelination in the Peripheral Nervous System

    www.nature.com/scientificreports OPEN GlcNAc6ST-1 regulates sulfation of N-glycans and myelination in the peripheral nervous system Received: 30 September 2016 Takeshi Yoshimura1,2,†,*, Akiko Hayashi3,*, Mai Handa-Narumi1,2, Hirokazu Yagi4, Accepted: 05 January 2017 Nobuhiko Ohno1,2, Takako Koike1, Yoshihide Yamaguchi3, Kenji Uchimura5, Kenji Kadomatsu5, Published: 10 February 2017 Jan Sedzik1,6,✠, Kunio Kitamura7, Koichi Kato4,8, Bruce D. Trapp9, Hiroko Baba3 & Kazuhiro Ikenaka1,2 Highly specialized glial cells wrap axons with a multilayered myelin membrane in vertebrates. Myelin serves essential roles in the functioning of the nervous system. Axonal degeneration is the major cause of permanent neurological disability in primary myelin diseases. Many glycoproteins have been identified in myelin, and a lack of one myelin glycoprotein results in abnormal myelin structures in many cases. However, the roles of glycans on myelin glycoproteins remain poorly understood. Here, we report that sulfated N-glycans are involved in peripheral nervous system (PNS) myelination. PNS myelin glycoproteins contain highly abundant sulfated N-glycans. Major sulfated N-glycans were identified in both porcine and mouse PNS myelin, demonstrating that the 6-O-sulfation of N-acetylglucosamine (GlcNAc-6-O-sulfation) is highly conserved in PNS myelin between these species. P0 protein, the most abundant glycoprotein in PNS myelin and mutations in which at the glycosylation site cause Charcot-Marie-Tooth neuropathy, has abundant GlcNAc-6-O-sulfated N-glycans. Mice deficient in N-acetylglucosamine-6-O-sulfotransferase-1 (GlcNAc6ST-1) failed to synthesize sulfated N-glycans and exhibited abnormal myelination and axonal degeneration in the PNS. Taken together, this study demonstrates that GlcNAc6ST-1 modulates PNS myelination and myelinated axonal survival through the GlcNAc-6-O-sulfation of N-glycans on glycoproteins.
  • Keratan Sulfate Proteoglycan Phosphacan Regulates Mossy Fiber Outgrowth and Regeneration

    Keratan Sulfate Proteoglycan Phosphacan Regulates Mossy Fiber Outgrowth and Regeneration

    462 • The Journal of Neuroscience, January 14, 2004 • 24(2):462–473 Development/Plasticity/Repair Keratan Sulfate Proteoglycan Phosphacan Regulates Mossy Fiber Outgrowth and Regeneration Christy D. Butler,2 Stephanie A. Schnetz,1 Eric Y. Yu,1 Joseph B. Davis,1 Katherine Temple,1 Jerry Silver,2 and Alfred T. Malouf1,2 Departments of 1Pediatrics and 2Neurosciences, Case Western Reserve University, Cleveland, Ohio 44106 We have examined the role of chondroitin sulfate proteoglycans (CSPGs) and keratan sulfate proteoglycans (KSPGs) in directing mossy fiber (MF) outgrowth and regeneration in rat hippocampal slice cultures. MFs normally exhibit a very specific innervation pattern that is restricted to the stratum lucidum (SL). In addition, MFs in hippocampal slice cultures will regenerate this specific innervation pattern after transection. CSPGs are one of the best characterized inhibitory axon guidance molecules in the CNS and are widely expressed in all areas of the hippocampus except SL. KSPGs are also widely expressed in the hippocampus, but their role in axon outgrowth has not been extensively studied in the CNS where phosphacan is the only protein that appears to contain KS-GAGs. Cultured hippocampal slices were treated with either chondroitin ABC lyase or keratanases to reduce the inhibitory axon guidance properties of CS and KS proteoglycans, respectively. The ability of transected MFs to regenerate their normal innervation pattern after digestion of CS and KS-GAGS sugars with these enzymes was examined. Only keratanase treatment resulted in misrouting of MFs. Identifying the mechanism by which keratanase produced MF misrouting is complicated by the presence of splice variants of the phosphacan gene that include the extracellular form of phosphacan and the transmembrane receptor protein tyrosine phosphatase ␤/␨ (RPTP␤/␨).