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Transcript

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

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