LAR is a functional receptor for CSPG axon growth inhibitors Shuxin Li Department of Neurology and Neuroscience Program UT Southwestern Medical Center at Dallas
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
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)
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.
Neuronal expression of LAR in the brain and spinal cord
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.
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
LAR deletion overcomes neurite growth inhibition of CSPGs 1 day DRG neuronal cultures from LAR KO mice
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.
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
ELP and ILP stimulate 5-HT axonal growth in SCI mice
ELP and ILP stimulate 5-HT axonal growth in SCI mice: Camera Lucida drawing
ELP and ILP stimulate 5-HT axonal growth in SCI mice 5 weeks post-SCI
ELP and ILP promote behavioral recovery in SCI mice
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.
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.