LAR is a functional receptor for CSPG axon growth inhibitors<br />Shuxin Li<br />Department of Neurology and Neuroscience ...
Upregulation of  CSPGs after the CNS injury<br /> Core protein<br />GAG <br />chains<br />Exp Neurol. 2003;182(2):399-411<...
CSPGs contribute to axon growth failure in the CNS<br />Nature 416: <br />636-640;  2002<br />Molecular mechanisms for CSP...
LAR and LAR subfamily of proteins<br />• The heparan sulfate proteoglycansyndecan is an in vivo ligand for the Drosophila ...
Neuronal expression of LAR in the brain and spinal cord<br />
CSPGs bind to LAR in COS-7 cells with high affinity<br />•  Incubate cells with CSPGs 7.5 ug/ml.<br />•  Stained CSPG sign...
CSPG & chondroitin sulfate enhance activity of LAR <br />phosphatasein COS-7 cells <br />• COS7 transfection: 2 days<br />...
LAR deletion overcomes neurite growth <br />inhibition of CSPGs<br />1 day  DRG neuronal cultures from LAR KO mice<br />
LAR-targeting peptides overcome CSPG inhibition  <br />• The extracellular LAR peptide (ELP): 37 residues,  corresponding ...
Systemic treatments with ELP/ILP in SCI mice<br />Hemisection at T7<br />Dorsal<br />Lesioned<br />Adult mouse<br />Ventra...
ELP and ILP stimulate 5-HT axonal growth in SCI mice   <br />
ELP and ILP stimulate 5-HT axonal growth in SCI mice:<br />Camera Lucida drawing   <br />
ELP and ILP stimulate 5-HT axonal growth in SCI mice   <br />5 weeks post-SCI<br />
ELP and ILP promote behavioral recovery in SCI mice   <br />
Conclusions<br />CSPGs bind LAR and interact with LAR with high affinity.<br />CSPG stimulates LAR phosphatase activity in...
Acknowledgements <br />Neuronal cultures<br />neurite/axon growth assay and quantification <br />mouse breeding & genotypi...
Li, Shuxin
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Li, Shuxin

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Li, Shuxin

  1. 1. LAR is a functional receptor for CSPG axon growth inhibitors<br />Shuxin Li<br />Department of Neurology and Neuroscience Program<br />UT Southwestern Medical Center at Dallas<br />
  2. 2. Upregulation of CSPGs after the CNS injury<br /> Core protein<br />GAG <br />chains<br />Exp Neurol. 2003;182(2):399-411<br />Both GAG and core <br />protein are inhibitory<br />GAG: glycosaminoglycan <br />KSPG: Keratan<br />
  3. 3. CSPGs contribute to axon growth failure in the CNS<br />Nature 416: <br />636-640; 2002<br />Molecular mechanisms for CSPG inhibitions<br />• Negative charges of sugar chains and interrupting cell adhesion.<br />• Blocking functions of positive molecules: laminin & integrins.<br />• As receptor for Sema 5A. <br />• Binding to specific receptors for CSPGs. RPTPσ as CSPG receptor (Science, 2009, 326:592-596)<br />
  4. 4. LAR and LAR subfamily of proteins<br />• The heparan sulfate proteoglycansyndecan is an in vivo ligand for the Drosophila LAR receptor tyrosine phosphatase. Current Biology, 2005; 15:1701.<br />• The HSPGs Syndecan and Dallylike Bind the Receptor Phosphatase LAR and Exert Distinct Effects on Synaptic Development. Neuron 49, 517–531, 2006.<br />ST3: mutant<br />Transmembrane proteins<br />Extracell: Ig & fibronectin type IIIdomains<br />Intracell: D1 and D2 domains; D1-activity; D2-regulating ligandbinding<br />Major members: LAR, RPTPσ & RPTPδ<br />Physical ligands and functions are unclear.<br />
  5. 5. Neuronal expression of LAR in the brain and spinal cord<br />
  6. 6. CSPGs bind to LAR in COS-7 cells with high affinity<br />• Incubate cells with CSPGs 7.5 ug/ml.<br />• Stained CSPG signal with Ab.<br />• Delta D2: deletion in D2 domain: regulating ligand binding.<br />• C1522S: mutant in D1 domain: regulating activity, not binding.<br />
  7. 7. CSPG & chondroitin sulfate enhance activity of LAR <br />phosphatasein COS-7 cells <br />• COS7 transfection: 2 days<br />• Cell lysates treated with CSPG or CS<br />• LAR activity by measuring phasphatase-driven generation of phosphate using a PTP kit<br />
  8. 8. LAR deletion overcomes neurite growth <br />inhibition of CSPGs<br />1 day DRG neuronal cultures from LAR KO mice<br />
  9. 9. LAR-targeting peptides overcome CSPG inhibition <br />• The extracellular LAR peptide (ELP): 37 residues, corresponding to the fifth LAR fibronectin type III (FN-III) domain. <br />• The intracellular LAR peptide (ILP): 35 amino acids, including 24 residues derived from sequence located between the membrane proximal region and the D1catalytic domain, and membrane-penetrating sequence (TAT) domain. <br />
  10. 10. Systemic treatments with ELP/ILP in SCI mice<br />Hemisection at T7<br />Dorsal<br />Lesioned<br />Adult mouse<br />Ventral<br />Peptide application:<br />• Starting 2 days post-SCI<br />• Dose: 140 (ELP) or 75 (ILP) <br />mg/Kg/day<br />• Infusion time: 10 days<br />Tissue processing<br />Lesion<br />7 mm<br />4mm<br />7 mm<br />4mm<br />R<br />C<br />Cross<br />Parasagittal<br />Cross<br />
  11. 11. ELP and ILP stimulate 5-HT axonal growth in SCI mice <br />
  12. 12. ELP and ILP stimulate 5-HT axonal growth in SCI mice:<br />Camera Lucida drawing <br />
  13. 13. ELP and ILP stimulate 5-HT axonal growth in SCI mice <br />5 weeks post-SCI<br />
  14. 14. ELP and ILP promote behavioral recovery in SCI mice <br />
  15. 15. Conclusions<br />CSPGs bind LAR and interact with LAR with high affinity.<br />CSPG stimulates LAR phosphatase activity in vitro.<br />Transgenic/pharmacological blockade of LAR reverses CSPG-mediated growth inhibition.<br />4. LAR blockade promotes axon growth and functional recovery in adult SCI mice.<br />CSPG<br />LAR<br />Conclusions: <br />LAR is an important receptor for CSPG inhibitors. <br />LAR blockade has a great therapeutic potential for CNS axon injury disorders.<br />LAR peptides may become a feasible treatment for axon injury patients.<br />
  16. 16. Acknowledgements <br />Neuronal cultures<br />neurite/axon growth assay and quantification <br />mouse breeding & genotyping<br />LAR mutation<br />Mikey Hoang<br />Daniel Fisher<br />John Dill<br />Ankur Patel<br />Zhenze Zhao <br />Xiao-Li Yang<br />Hui Li<br />Bin Xing<br />In vivo axon regeneration, behaviors and histology<br />Frank Longo (Stanford U): LAR KO & over-expression mice<br />Morgan Sheng (MIT): LAR plasmids<br />Jerry Silver (Case Western Reserve U): confirm LAR peptides using CSPG spot assay<br />Grant supports: <br />ParalyzedVeterans of America, NIH (1R21NS066114-01A1), Morton Cure Paralysis Fund and Christopher and Dana Reeve Foundation.<br />

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