spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online September 3, 2008
doi: 10.1242/10.1242/jcs.032649

Journal of Cell Science 121, 3083-3091 (2008)
Published by The Company of Biologists 2008
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Supplementary Material
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Related articles in JCS
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wang, H.
Right arrow Articles by Geller, H. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wang, H.
Right arrow Articles by Geller, H. M.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

Chondroitin-4-sulfation negatively regulates axonal guidance and growth

Hang Wang1,*, Yasuhiro Katagiri1,*,{ddagger}, Thomas E. McCann1, Edward Unsworth2, Paul Goldsmith2, Zu-Xi Yu3, Fei Tan1, Lizzie Santiago1, Edward M. Mills4, Yu Wang5, Aviva J. Symes5 and Herbert M. Geller1

1 Developmental Neurobiology Section, National Institutes of Health, Bethesda, MD 20892, USA
3 Pathology Core, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA
2 Basic Research Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
4 Division of Pharmacology/Toxicology, College of Pharmacy, The University of Texas at Austin, Austin, TX 78712, USA
5 Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, USA


Figure 1
View larger version (39K):
[in this window]
[in a new window]

  		Fig. 1. CS-A, but not CS-C, repels axons in a 4-sulfation-dependent manner. (A-F) Axonal guidance spot assays were performed with (A) chicken CSPG, (B) chicken CSPG treated with cABC, (C) CS-A, (D) CS-A partially digested with cABC, (E) CS-C, (F) CS-A treated with chondro-4-sulfatase. Each test substance was immobilized onto a PLL-coated coverslip and the behavior of axons at the interface between PLL and the sample (red) was quantified as described in the Materials and Methods. (G) Quantitative analysis of axonal behavior at the interface. Data are expressed as the mean ± s.d. Scale bar: 25 µm.

 

Figure 2
View larger version (40K):
[in this window]
[in a new window]

  		Fig. 2. Reactive astrocytes induced by TGFβ1 produce more CSPGs. (A) Monolayers of astrocytes were cultured with (bottom) or without (top) TGFβ1 for 7 days in the absnce of serum. CGNs were plated onto the astrocyte monolayers. After 48 hours, neurons were visualized and axonal length was analyzed. Scale bar: 25 µm. (B) Conditioned medium derived from TGFβ1-treated astrocytes inhibited axonal outgrowth. Conditioned medium was collected from untreated astrocytes (Control) or astrocytes treated with TGFβ1 for 7 days. Dissociated CGNs were then cultured for 48 hours in conditioned medium, alone or with the addition of SB-431542. Relative axonal length was measured and analyzed (*P<0.01; Student's t-test). (C,D) Increased production of CSPG by TGFβ1-treated astrocytes detected by immunoblot (C) and ELISA (D) using CS-56. Open column, control conditioned medium; filled column, TGFβ1-treated conditioned medium. Data are expressed as the mean ± s.d.

 

Figure 3
View larger version (33K):
[in this window]
[in a new window]

  		Fig. 3. Increased production of CSPGs by reactive astrocytes is responsible for reduced neuronal growth. (A,B) Astrocytes were treated with TGFβ1 for 7 days and conditioned medium was collected and concentrated. After incubation at 37°C for 3 hours with or without cABC, the conditioned medium was immobilized on PLL-coated coverslips, and axonal guidance spot assays were performed. (A) Although axons did not cross the interface between PLL and conditioned medium (arrows, left), many axons cross onto conditioned medium after cABC treatment (right). Scale bar: 25 µm. (B) Quantitative analysis of axonal behavior at the interface. The percentage of axons crossing onto the immobilized conditioned medium was calculated (*,#P<0.05; ANOVA). (C) Inhibition of GAG chain biosynthesis restores neuronal growth. Confluent monolayers of astrocytes were pretreated with TGFβ1, with or without the GAG-chain synthesis inhibitors for 3 days, and neurons were co-cultured for 2 days, followed by the analysis of relative axon length. PNP, p-nitro-phenyl-β-D-xylopyranoside (1 mM); Mu, methyl-umbelliferyl β-D-xyloside (1 mM); SC, sodium chlorate (20 mM) (*P<0.01 compared with TGFβ1-treated astrocytes; ANOVA). Data are expressed as the mean ± s.d.

 

Figure 4
View larger version (37K):
[in this window]
[in a new window]

  		Fig. 4. Reactive astrocytes produce more 4-sulfated than 6-sulfated chondroitin sulfate GAG chains. (A) Conditioned medium from 3 day culture was concentrated and digested with cABC, followed by immunoblot analysis. (B) Conditioned medium collected at indicated time was treated with cABC and subjected to ELISA without concentration. Monoclonal antibodies 2B6/MAB2030 and 3B3/MAB2035 were used against 4-sulfated and 6-sulfated GAG chains, respectively. (C) C4ST1 mRNA was rapidly increased upon induction of reactive astrocytes by TGFβ1. RNA was prepared from astrocytes at indicated time after TGFβ1 treatment and quantitative RT-PCR was performed. Open column, control; filled column, TGFβ1. (D) Transcript of the gene encoding C4ST1 but not C6ST1 was upregulated 3 days after TGFβ1 treatment of astrocytes. Open column, control; filled column, TGFβ1. (E) Upregulation of C4ST1 protein was detected with chicken anti-C4ST1 peptide antibody 3 days after TGFβ1 treatment. Lysates from HeLa cells transfected with pTracer-C4ST1 were used as a positive control.

 

Figure 5
View larger version (26K):
[in this window]
[in a new window]

  		Fig. 5. Depletion of C4ST1 in TGFβ1-treated astrocytes reduces the production of 4-sulfated GAG chains as well as repellent activity of conditioned medium. (A) Conditioned medium derived from C4ST1 siRNA-transfected astrocytes treated with or without TGFβ1 was subjected to ELISA to measure the amount of 4-sulfated GAG chains with MAB2030. (B) C4ST1 protein level in the cell lysates was checked by immunoblotting with anti-C4ST1 peptide antibody. Actin was used as loading control. (C) Disaccharide composition analyses of conditioned medium (CM). The values obtained from three experiments were used to calculate pmol of {Delta}HexUA-GalNAc(4-O-sulfate) ({Delta}Di-4S) per 1 ml conditioned medium. Data are expressed as the mean ± s.d. (D) Axonal guidance spot assay. More axons that crossed the interface were observed in C4ST1 siRNA conditioned medium compared with TGFβ1 conditioned medium (*P<0.01 compared with scrambled siRNA without TGFβ1 and C4ST1 siRNA with TGFβ1; ANOVA).

 

Figure 6
View larger version (23K):
[in this window]
[in a new window]

  		Fig. 6. Depletion of C6ST1 by siRNA does not alter the production of 4-sulfated chondroitin sulfate GAG chains nor repellent activity in reactive astrocytes. Astrocytes transfected with C6ST1 siRNA were treated with or without TGFβ1 as in Fig. 5, and RNA and conditioned medium were prepared. Quantitative RT-PCR for (A) C4ST1, (B) C6ST1. ELISA to measure the amount of (C) 4-sulfated GAG chains with MAB2030, (D) 6-sulfated GAG chains with MAB2035. (E) Axonal guidance spot assay was performed (*P<0.01 compared with scrambled siRNA without TGFβ1; ANOVA). Although C6ST1 siRNA reduced C6ST1 mRNA and production of 6-sulfated GAG chains, induction of C4ST1 mRNA and 4-sulfated GAG chains by TGFβ1 were unaffected, and repellent activity of TGFβ1-treated conditioned medium was observed.

 

Figure 7
View larger version (27K):
[in this window]
[in a new window]

  		Fig. 7. C4ST1 overexpression renders astrocytes far less permissive to neuronal growth. Wild-type or inactive form of C4ST1 was transfected into astrocytes and neurons were co-cultured on monolayers. Expression levels of recombinant C4ST1 (A) and 4-sulfated GAG chains (B) were examined in immunoblot with anti-DsRed antibody and LY111 (anti-4-sulfated GAG), respectively. Internal cleavage of DsRed fusion protein was detected. (C) Disaccharide composition analyses of conditioned medium (CM) were performed as in Fig. 5. (D) Relative axonal length was measured. (*P<0.01 compared with vector; ANOVA.) Axonal growth was reduced by increased production of 4-sulfated GAG chains in astrocytes.

 

Figure 8
View larger version (106K):
[in this window]
[in a new window]

  		Fig. 8. 4-sulfated GAG chains are acutely upregulated in spinal cord lesion. One day after spinal cord injury, cryosections were prepared and stained with LY111 (green, anti 4-sulfated GAG in A-C), MC21C (green, anti 6-sulfated GAG in D) and anti-GFAP antibody (red). Arrows indicate the injury sites. The area far from the injury site (asterisk) showed lower levels of LY111 immunoreactivity. Boxed regions in A are shown at higher magnification (B,C), revealing colocalization of 4-sulfated GAG with GFAP, an astrocyte marker (arrowheads). Scale bars: 200 µm (A), 40 µm (B). (E) Coronal cryosections were prepared from uninjured and injured spinal cords and treated with cABC. Disaccharide composition analysis was performed. Upregulation of {Delta}Di-4S was observed upon spinal cord injury.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2008