Regulation of myelin structure and conduction velocity by perinodal astrocytes

Abstract

The speed of impulse transmission is critical for optimal neural circuit function, but it is unclear how the appropriate conduction velocity is established in individual axons. The velocity of impulse transmission is influenced by the thickness of the myelin sheath and the morphology of electrogenic nodes of Ranvier along axons. Here we show that myelin thickness and nodal gap length are reversibly altered by astrocytes, glial cells that contact nodes of Ranvier. Thrombin-dependent proteolysis of a cell adhesion molecule that attaches myelin to the axon (neurofascin 155) is inhibited by vesicular release of thrombin protease inhibitors from perinodal astrocytes. Transgenic mice expressing a dominant-negative fragment of VAMP2 in astrocytes, to reduce exocytosis by 50%, exhibited detachment of adjacent paranodal loops of myelin from the axon, increased nodal gap length, and thinning of the myelin sheath in the optic nerve. These morphological changes alter the passive cable properties of axons to reduce conduction velocity and spike-time arrival in the CNS in parallel with a decrease in visual acuity. All effects were reversed by the thrombin inhibitor Fondaparinux. Similar results were obtained by viral transfection of tetanus toxin into astrocytes of rat corpus callosum. Previously, it was unknown how the myelin sheath could be thinned and the functions of perinodal astrocytes were not well understood. These findings describe a form of nervous system plasticity in which myelin structure and conduction velocity are adjusted by astrocytes. The thrombin-dependent cleavage of neurofascin 155 may also have relevance to myelin disruption and repair.

Keywords: myelin plasticity; neurofascin 155; node of Ranvier; spike-time–dependent plasticity; thrombin.

Fig. 1.

Secretion of PN1 from astrocytes inhibits thrombin-mediated cleavage of NF155 to regulate attachment of myelin to the axon. (A and B) The myelin sheath is attached to the axon by NF155 in septate junctions (B arrowheads). B is a TEM of adult optic nerve. Astrocytes (*) contact the node. (C) Thrombin cleavage of NF155, inhibited by PN1 and secreted from astrocytes, would sever NF155 attachment of paranodal loops to the axon. Thrombin proteolytic site AA924 (red), short fragment (NF30) (blue), and long fragment (NF125) (green). (D) Reducing exocytosis from astrocytes in culture, by expressing a dominant-negative vesicle-associated membrane protein 2 [(gfap)dnVAMP2)], reduces Mastoparan-induced PN1 secretion (ANOVA, F_{2, 19} = 8.15, P = 0.003). (E–H) Increased cleavage of NF155 in subcortical white matter of mice expressing (gfap)dnVAMP2 (t test, t₁₁ = 3.315, E and F) is reversed by the thrombin inhibitor Fondaparinux (t test, t₄ = 2.9, G and H). Neurofascin 186, lacking a thrombin cleavage site, and neuron-specific enolase, are controls. EGFP is a reporter of dnVAMP2 expression. Predicted molecular structures of paranodal septate junction proteins in C were determined by I-TASSER (Materials and Methods; Movie S1; SI Appendix, Figs. S1 and S2). A single asterisk (*) indicates a significant difference.

Fig. 2.

Detachment of outermost paranodal loops of myelin is regulated by vesicular release of PN1 from astrocytes. (A–C) Detachment of paranodal loop (A), expanded cytoplasm (B), and displacement from the node (C) are shown by EM in mice with exocytosis from astrocytes reduced by (gfap)dnVAMP2 expression (yellow arrows). Septate junctions distal to the perinodal astrocytes remain intact (A–C, green arrows). (D–F) A 3D reconstruction from serial block face scanning EM showing detachment of outermost paranodal loops and retraction of the outer spirals of myelin from the axon (yellow arrow) in the (gfap)dnVAMP2 condition. Compact myelin, magenta; axon, gray; cytoplasmic pocket of paranodal loop, tan; perinodal astrocyte, aqua. (G) Transverse section through an axon in the (gfap)dnVAMP2 condition where the outermost layers of myelin are filled with cytoplasm for one and one-half wraps around the axon (arrows) in withdrawing over compacted myelin. (H and I) Detachment of paranodal loops was reduced in WT and after terminating (gfap)dnVAMP2 expression (recovery). (J) TEM analysis of 617 adult optic nerve axons showing percentage of nodes with detached paranodal loops under various conditions [P < 0.0001, χ²_{(7, n = 617)} = 31.34]. Detachment increased significantly in the (gfap)dnVAMP2 condition compared with −dnVAMP2 [χ²_{(2, N = 350)} = 15.882, P = 0.0001]. The increase could be induced in the adult [adult onset vs. (gfap)dnVAMP2, χ²_{(2, N = 246)} = 0.764, P = 0.382, n.s.]. Increased paranodal loop detachment was reversed after terminating expression of the transgene (recovery, 30 d) or by injecting Fondaparinux for 21 d [(gfap)dnVAMP2 +Fond]. The percentage of nodes with paranodal loops detached in WT mice was not significantly different from mice with (gfap)dnVAMP2 expression blocked (−dnVAMP2) (Movies S2 and S3). A single asterisk (*) indicates a significant difference in J and a perinodal astrocyte in H and I.

Fig. 3.

Nodal morphology, nodal Na⁺ channel distribution, and myelin sheath thickness regulated by vesicular release of PN1 from astrocytes. (A and B) Nodal gap length increased when astrocyte exocytosis was reduced by (gfap)dnVAMP2 expression throughout development or in adult mice (adult onset), but not when thrombin proteolysis was inhibited by Fondaparinux (ANOVA F_{6, 310} = 31.83, P < 0.0001). Nodal gap length recovered 30 d after terminating (gfap)dnVAMP2 expression, but not by 5 d. Caspr1 marking paranode, red; Na⁺ channels, blue. (C) Over all experimental conditions, nodal gap length was linearly related to frequency of nodal gap detachment (r² = 0.978, P < 0.0001, y = 0.0107× + 0.431). (D) Astrocyte-specific expression of EGFP-tagged TeLC virus in corpus callosum of WT rats (green), colocalized with astrocyte marker GFAP (red). (E) Nodal gap length in regions encompassed by TeLC-transfected astrocytes was longer than in nodes outside regions of transfected astrocytes and in contralateral brain regions injected with control virus expressing EGFP (ANOVA F_{2, 87} = 50.18, P < 0.0001). (F) Na⁺ channel distribution was more dispersed in nodes following (gfap)dnVAMP2 expression (red) and was returned to normal after treatment with Fondaparinux (blue). Half maximal width of distributions: 0.48 ± 0.0445, 0.80 ± 0.0411, and 0.54 ± 0.0239 nm for black, red, and blue traces, respectively [ANOVA, F_{2, 78} = 20.50, P < 0.0001, n.s., −dnVAMP2 vs. (gfap)dnVAMP2, t₃₁ = 1.32, P = 0.58, Bonferroni posttest]. (G) Thinning of myelin when astrocyte exocytosis was reduced in adult mice [(gfap)dnVAMP2]. (Inset) Normal compaction of myelin sheath. (H) Thinner myelin sheath (g-ratio) in mice expressing (gfap)dnVAMP2 (t test, t₃₆₂ = 11.69, P < 0.001). (I) Myelin sheath was thinner when astrocyte exocytosis was reduced throughout development [−dnVAMP2 vs. (gfap)dnVAMP2]; recovered 30 d after transgene expression was terminated and was thinned when astrocyte exocytosis was reduced in adults (adult onset). Myelin sheath thickness recovered after injection of Fondaparinux for 21 d (ANOVA, F_4,190 = 33.4, P < 0.001). n = sample size, N = number of animals. A single asterisk indicates a significant difference.

Fig. 4.

Myelin plasticity changes conduction velocity, spike-time arrival in visual cortex, and visual acuity. (A–E) Conduction times were slower for all components of the compound action potential [G2 (t test, t₁₁ = 2.21), G3 (t test, t₁₁ = 2.96), and G4 (t test, t₁₁ = 3.05)] that could be measured in excised optic nerves in the (gfap)dnVAMP2, compared with the −dnVAMP2 condition. (F) VEPs in visual cortex. (G–I) Latency to peak VEP was 6.74 ms longer in the (gfap)dnVAMP2 condition (G and H, t test, t₅₀ = 4.52) and restored by Fondaparinux treatment (I, t test, t₃₈ = 3.17). G shows summed waveforms for all data in H. (J–L) Visual acuity measured by computerized striped-drum assay was reduced in the (gfap)dnVAMP2 condition (K, t test, t₅₀ = 2.23), and the effect was reversed by Fondaparinux (L, t test, t₃₈ = 2.49). A single asterisk (*) indicates a significant difference.