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Glial ankyrins facilitate paranodal axoglial junction assembly

Abstract

Neuron-glia interactions establish functional membrane domains along myelinated axons. These include nodes of Ranvier, paranodal axoglial junctions and juxtaparanodes. Paranodal junctions are the largest vertebrate junctional adhesion complex, and they are essential for rapid saltatory conduction and contribute to assembly and maintenance of nodes. However, the molecular mechanisms underlying paranodal junction assembly are poorly understood. Ankyrins are cytoskeletal scaffolds traditionally associated with Na+ channel clustering in neurons and are important for membrane domain establishment and maintenance in many cell types. Here we show that ankyrin-B, expressed by Schwann cells, and ankyrin-G, expressed by oligodendrocytes, are highly enriched at the glial side of paranodal junctions where they interact with the essential glial junctional component neurofascin 155. Conditional knockout of ankyrins in oligodendrocytes disrupts paranodal junction assembly and delays nerve conduction during early development in mice. Thus, glial ankyrins function as major scaffolds that facilitate early and efficient paranodal junction assembly in the developing CNS.

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Figure 1: Paranodal AnkB is derived from Schwann cells in the PNS.
Figure 2: No paranodal or axonal abnormalities were observed in sciatic nerves of AnkB-cKO mice.
Figure 3: Paranodal junction assembly is disrupted in AnkG-cKO mice.
Figure 4: Paranodal junctions recover over time in the CNS of the AnkG-cKO and AnkB/G-cKO mice.
Figure 5: Paranodal junction assembly and myelination proceed in the absence of AnkB and AnkG in oligodendrocytes.
Figure 6: AnkB and AnkG interact with NF155 in vivo and can be targeted to paranodes independently of paranodal junctions and NF155.
Figure 7: Schwann cells and oligodendrocytes express 220-kDa isoform of AnkB from multiple promoters.
Figure 8: Oligodendrocytes express the 270-kDa and 190-kDa isoforms of AnkG from the promoter upstream to exon 1b.

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Acknowledgements

E. Peles (Weizmann Institute of Science) provided NfascF/F mice and 4.1G antibodies. K.-A. Nave (Max Planck Institute of Experimental Medicine) provided Cnp-Cre mice. P.M. Jenkins (Duke University) provided AnkG 480/270 antibodies. This work was supported by grants from the US National Institutes of Health (NS069688 and NS044916 to M.N.R.; HL084583, HL083422 and HL114383 to P.J.M.), the National Multiple Sclerosis Society (M.N.R.) and the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation (M.N.R.). V.B. is an investigator of the Howard Hughes Medical Institute.

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Contributions

M.N.R. and K.-J.C. conceived the project, designed the experiments and wrote the manuscript. D.R.Z. and K.S. did electrophysiology experiments and analyzed the data. D.R.Z. conducted the electron microscopy experiments. K.-J.C. did all other experiments and analyzed the data. P.J.M. and M.A.M. designed and constructed the Ank2F/F allele. D.L.S., P.J.B., E.C.C. and V.B. provided reagents and mice.

Corresponding author

Correspondence to Matthew N Rasband.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Paranodal junction assembly is disrupted in the CNS of AnkB/G-cKO mice.

P8 spinal cord (a,b) and P17 brain (corpus callosum shown in c,d) were immunostained with antibodies as indicated. Scale bars = 10 μm (a,b) and 5 μm (c,d).

Supplementary Figure 2 The summary and implications of this study.

(a) The molecular architecture of nodes of Ranvier and paranodes including membrane proteins and cytoskeletal scaffolds. Paranodal ankyrins are located in myelinating glia and interact with NF155. Schwann cells in the PNS express AnkB (B) whereas oligodendrocytes in the CNS mainly express AnkG (G). The shorter tail of paranodal AnkG symbolizes 190-kDa and 270-kDa isoforms and the longer tail of nodal AnkG designates 480-kDa and 270-kDa isoforms. Cntn, contactin. αII/βII, αII/βII spectrin tetramer. βIV, βIV spectrin. This figure was reprinted from Chang & Rasband, Excitable domains of myelinated nerves: axon initial segments and nodes of Ranvier. Curr. Top. Membr. 72, 159-192 (2013) with permission from Elsevier. (b) The cartoon illustrates the differential timing of formation of nodes (red) and paranodal junctions (green) in the PNS, CNS and AnkB/G-cKO CNS.

Supplementary Figure 3 The full immunoblots of Figs. 1e, 6e and 6f.

The same membranes were used for stripping and reprobing in a top-to-bottom order except that the lanes of 19 μg input in Fig. 6e and 30 μg input in Fig. 6f were cut and not probed for NFasc. Dotted lines indicate regions of the blots used in each figure.

Supplementary Figure 4 The full immunoblots of Figs. 3i, 7b, 7c and 8b.

The same membranes were used for stripping and reprobing in a top-to-bottom order except that Fig. 3i Contactin blot was from another immunoblotted membrane. Dotted lines indicate regions of the blots used in each figure.

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Chang, KJ., Zollinger, D., Susuki, K. et al. Glial ankyrins facilitate paranodal axoglial junction assembly. Nat Neurosci 17, 1673–1681 (2014). https://doi.org/10.1038/nn.3858

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