Passive immunization with anti-ganglioside antibodies directly inhibits axon regeneration in an animal model

Abstract

Recent studies have proposed that neurite outgrowth is influenced by specific nerve cell surface gangliosides, which are sialic acid-containing glycosphingolipids highly enriched in the mammalian nervous system. For example, the endogenous lectin, myelin-associated glycoprotein (MAG), is reported to bind to axonal gangliosides (GD1a and GT1b) to inhibit neurite outgrowth. Clustering of gangliosides in the absence of inhibitors such as MAG is also shown to inhibit neurite outgrowth in culture. In some human autoimmune PNS and CNS disorders, autoantibodies against GD1a or other gangliosides are implicated in pathophysiology. Because of neurobiological and clinical relevance, we asked whether anti-GD1a antibodies inhibit regeneration of injured axons in vivo. Passive transfer of anti-GD1a antibody severely inhibited axon regeneration after PNS injury in mice. In mutant mice with altered ganglioside or complement expression, inhibition by antibodies was mediated directly through GD1a and was independent of complement-induced cytolytic injury. The impaired regenerative responses and ultrastructure of injured peripheral axons mimicked the abortive regeneration typically seen after CNS injury. These data demonstrate that inhibition of axon regeneration is induced directly by engaging cell surface gangliosides in vivo and imply that circulating autoimmune antibodies can inhibit axon regeneration through neuronal gangliosides independent of endogenous regeneration inhibitors such as MAG.

Figure 1.

Biosynthetic pathways for major mammalian nervous system gangliosides. The biosynthetic relationships between major nervous system gangliosides and their precursors is shown schematically [ganglioside nomenclature of Svennerholm (1994)]. The block in ganglioside biosynthesis because of disruption at the Galgt1 or Siat8a loci is indicated by double lines. Galgt1-null mice lack all complex major nervous system gangliosides (within brackets), expressing instead a corresponding increase in GM3 and GD3. Siat8a-null mice do not express b-series gangliosides but instead have a corresponding increase in a-series gangliosides (box), particularly GM1 and GD1a.

Figure 2.

Anti-glycan Ab-mediated inhibition of axon regeneration in peripheral nerves. A, Schematic diagram showing the site of nerve crush and its relationship with different nerve segments (S1-S4) as outlined in the text. B–E, Light (scale bar, 20 μm) (B, C) and electron (scale bar, 2 μm) (D, E) micrographs of sciatic nerve S3 segments. B, Many regenerating myelinated axons in sham-treated animal (arrows). C, Rare regenerating nerve fibers in GD1a/GT1b-2b-treated animal (arrows). D, Both myelinated and unmyelinated fibers (arrow) are present in sham-treated animals. E, GD1a/GT1b-2b-treated nerve with a dystrophic sprout (arrow) and unmyelinated fibers (arrowhead), but no myelinated fibers in this field. F, Significant decrease in numbers of MFs in GD1a/GT1b-2b-treated (gray bars; n = 9 each) sciatic (SN) (283 ± 74) and tibial (TN) (21 ± 3) nerves compared with sham Ab-treated (black bars; n = 7 each) sciatic (2540 ± 52) and tibial (651 ± 92) nerves. G, Significant decrease in numbers of UMFs in GD1a/GT1b-treated nerves at the tibial but not the sciatic level (n = 5 each of sciatic and tibial nerves/group). *p < 0.05. Error bars indicate SEM.

Figure 3.

Anti-glycan Abs induce formation of dystrophic regenerating sprouts. A, Light micrograph showing abortive sprouting characterized by dystrophic sprouts (arrows); these sprouts are not myelinated and are surrounded by Schwann cell nuclei (*) (scale bar, 20 μm). B, EM showing dystrophic growth cone with a core of neurofilaments and a surrounding pellet with membranous organelles (scale bar, 2 μm).

Figure 4.

IgG deposition in nerve ligation model. A, B, Increased IgG deposition in GD1a/GT1b-2b-treated nerves (B) compared with sham Ab-treated nerves (A). Scale bar: (in B) A, B, 0.5 mm. C, Quantification shows a gradient of IgG staining from tip (1 mm) to more proximal segments of the nerve (4 mm) in GD1a/GT1b-2b-treated group (black bars) compared with sham-Ab-treated group (gray bars). Error bars indicate SEM.

Figure 5.

GD1a/GT1b-2b binds to injured axon tips. GD1a/GT1b-2b-treated (A, B) and sham-treated (C, D) nerves. Double labeling studies for β-III tubulin (A, C) and immunoglobulins (B, D) show IgG binding to axons in GD1a/GT1b-2b-treated nerves compared with controls. Scale bar, 20 μm.

Figure 6.

Anti-glycan Abs decrease target reinnervation of muscle. A, CMAP amplitudes recorded in hindpaws of mice at baseline and day 16 after nerve crush. GD1a/GT1b-2b-treated (Ab) animals have inexcitable nerves; sham Ab-treated (Cont) animals have delayed and dispersed CMAP amplitudes consistent with muscle reinnervation. B, 3D reconstructions of sham Ab-treated (Cont; left panels) and GD1a/GT1b-2b-treated (Ab; right panels) calf muscles showing muscle atrophy of GD1a/GT1b-2b-treated animal.

Figure 7.

Effects of anti-glycan Abs on axon regeneration in mice with altered ganglioside or C5 expression. A, Galgt1-null mice. Numbers of regenerating MF in GD1a/GT1b-2b-treated (gray bars; n = 3 each) sciatic (2921 ± 204) and tibial (262 ± 51) nerves were similar to sham-Ab treated (black bars; n = 3 each) sciatic (2963 ± 79) and tibial (250 ± 24) nerves. B, Siat8a-null mice. Numbers of regenerating MF in GD1a/GT1b-2b-treated (gray bars; n = 3 each) sciatic (604 ± 158) and tibial (43 ± 23) nerves were severely reduced compared with sham Ab-treated (black bars; n = 3 each) sciatic (2829 ± 137) and tibial (852 ± 33) nerves. C, C5-deficient mice. Numbers of regenerating MF in GD1a/GT1b-2b-treated (gray bars; n = 4 each) sciatic (1390 ± 185) and tibial (127 ± 31) nerves were significantly reduced compared with sham Ab-treated (black bars) sciatic (3400 ± 321) and tibial (1127 ± 31) nerves. *p < 0.05. Error bars indicate SEM.