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NgR1 and NgR3 are receptors for chondroitin sulfate proteoglycans

Abstract

In the adult mammalian CNS, chondroitin sulfate proteoglycans (CSPGs) and myelin-associated inhibitors (MAIs) stabilize neuronal structure and restrict compensatory sprouting following injury. The Nogo receptor family members NgR1 and NgR2 bind to MAIs and have been implicated in neuronal inhibition. We found that NgR1 and NgR3 bind with high affinity to the glycosaminoglycan moiety of proteoglycans and participate in CSPG inhibition in cultured neurons. Nogo receptor triple mutants (Ngr1−/−; Ngr2−/−; Ngr3−/−; which are also known as Rtn4r, Rtn4rl2 and Rtn4rl1, respectively), but not single mutants, showed enhanced axonal regeneration following retro-orbital optic nerve crush injury. The combined loss of Ngr1 and Ngr3 (Ngr1−/−; Ngr3−/−), but not Ngr1 and Ngr2 (Ngr1−/−; Ngr2−/−), was sufficient to mimic the triple mutant regeneration phenotype. Regeneration in Ngr1−/−; Ngr3−/− mice was further enhanced by simultaneous ablation of Rptpσ (also known as Ptprs), a known CSPG receptor. Collectively, our results identify NgR1 and NgR3 as CSPG receptors, suggest that there is functional redundancy among CSPG receptors, and provide evidence for shared mechanisms of MAI and CSPG inhibition.

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Figure 1: Loss of all three NgRs results in enhanced growth on CNS myelin.
Figure 2: NgR1 and NgR3, but not NgR2, contain two discontinuous and evolutionarily conserved sequence motifs that are necessary for binding to brain tissue.
Figure 3: NgR1 and NgR3 interact directly with specific GAGs.
Figure 4: Binding sites for MAIs and CSPGs on NgR1 are distinct and dissociable.
Figure 5: Nogo receptors mediate CSPG inhibition.
Figure 6: Binding of soluble NgR1-Fc and NgR3-Fc to optic nerve is enhanced by injury.
Figure 7: Retinal stratification, optic nerve myelination and RGC central projections appear to be normal in Ngr1−/−; Ngr2−/−; Ngr3−/− mice.
Figure 8: Ngr1−/−; Ngr2−/−; Ngr3−/− and Ngr1−/−; Ngr3−/−; Rptpσ−/− compound mutants show enhanced fiber regeneration following crush injury to the optic nerve.

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Acknowledgements

We thank M. Tremblay for Rptpσ−/− mice, M. Greenberg for Ngr3−/− mice, B. Pierchala for p75NTR−/− mice, B. Bates, D. Howland and M.L. Mercado for their assistance in the generation and initial analysis of Ngr1−/−; Ngr2−/−; Ngr3−/− mice, U. Rutishauser for Endo-N, D. Figge and Y. Yasui for assistance in ELISA binding assays, Y. Yin for training in optic nerve surgery, Y. Duan for generation of the RPTPσ(Ig1–3)-Fc construct, J. Barbieri for technical assistance and M.M. Zaleska for project administration. This work was supported by Neuroscience Training Grant T32EY017878 and the University of Michigan Rackham Merit Fellowship (T.L.D.), Cellular and Molecular Biology Training Grant T32GM007315 (K.T.B. and Y.A.M.), National Research Service Award Ruth Kirschstein Fellowship F31NS061589 (S.J.R.), the New York State Spinal Cord Injury Research Program, the Dr. Miriam and Sheldon G. Adelson Medical Foundation on Neural Repair and Rehabilitation, the US Department of Veterans Affairs (1I01RX000229-01), the National Institute of Neurological Disorders and Stroke (R56NS047333, R.J.G.) and the National Eye Institute (L.I.B.).

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R.J.G. conceived the study. T.L.D., L.I.B., H.M.G. and R.J.G. designed the experiments. T.L.D., K.T.B., Y.A.M., Y. Koriyama, S.J.R., C.D.L., Y. Katagiri and R.J.G. performed the experiments. T.L.D., K.T.B. and Y. Koriyama contributed to data analysis and figure preparation. K.L.A., A.W., C.G.G. and B.Z. generated and provided mice or reagents for the study, T.L.D. and R.J.G. wrote the manuscript. All of the authors read and agreed on the final version of the manuscript.

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Correspondence to Roman J Giger.

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Dickendesher, T., Baldwin, K., Mironova, Y. et al. NgR1 and NgR3 are receptors for chondroitin sulfate proteoglycans. Nat Neurosci 15, 703–712 (2012). https://doi.org/10.1038/nn.3070

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