Anti-ganglioside antibody internalization attenuates motor nerve terminal injury in a mouse model of acute motor axonal neuropathy
Simon N. Fewou, … , Jaap J. Plomp, Hugh J. Willison
Simon N. Fewou, … , Jaap J. Plomp, Hugh J. Willison
Published February 6, 2012
Citation Information: J Clin Invest. 2012;122(3):1037-1051. https://doi.org/10.1172/JCI59110.
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Anti-ganglioside antibody internalization attenuates motor nerve terminal injury in a mouse model of acute motor axonal neuropathy

Abstract

In the Guillain-Barré syndrome subform acute motor axonal neuropathy (AMAN), Campylobacter jejuni enteritis triggers the production of anti-ganglioside Abs (AGAbs), leading to immune-mediated injury of distal motor nerves. An important question has been whether injury to the presynaptic neuron at the neuromuscular junction is a major factor in AMAN. Although disease modeling in mice exposed to AGAbs indicates that complement-mediated necrosis occurs extensively in the presynaptic axons, evidence in humans is more limited, in comparison to the extensive injury seen at nodes of Ranvier. We considered that rapid AGAb uptake at the motor nerve terminal membrane might attenuate complement-mediated injury. We found that PC12 rat neuronal cells rapidly internalized AGAb, which were trafficked to recycling endosomes and lysosomes. Consequently, complement-mediated cytotoxicity was attenuated. Importantly, we observed the same AGAb endocytosis and protection from cytotoxicity in live mouse nerve terminals. AGAb uptake was attenuated following membrane cholesterol depletion in vitro and ex vivo, indicating that this process may be dependent upon cholesterol-enriched microdomains. In contrast, we observed minimal AGAb uptake at nodes of Ranvier, and this structure thus remained vulnerable to complement-mediated injury. These results indicate that differential endocytic processing of AGAbs by different neuronal and glial membranes might be an important modulator of site-specific injury in acute AGAb-mediated Guillain-Barré syndrome subforms and their chronic counterparts.

Authors

Simon N. Fewou, Angie Rupp, Lauren E. Nickolay, Kathryn Carrick, Kay N. Greenshields, John Pediani, Jaap J. Plomp, Hugh J. Willison

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Figure 2

Internalization of AGAb from presynaptic membranes at NMJs occurs rapidly at physiological temperature (37°C).

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Internalization of AGAb from presynaptic membranes at NMJs occurs rapidl...
(A) TS muscle was labeled with anti-GD1b Ab on ice and subsequently incubated at 37°C for 30 and 60 minutes to allow endocytosis. Surface anti-GD1b Ab was detected with secondary Ab and analyzed by fluorescence microscopy. Arrowheads indicate the expected site of surface anti-GD1b Ab signal overlying the acetylcholine receptor (AChR) signal at the NMJ upon incubation at 37°C. (B) Following microscopic analysis, the same TS preparations were permeabilized (with Triton X-100), and the total internalized and surface anti-GD1b Ab was imaged using secondary Ab. (C and D) Fluorescence images for anti-GD1b Ab were quantitated using ImageJ software. (C) Anti-GD1b Ab was rapidly depleted from the cell surface over time. (D) Following permeabilization, no significant difference in total anti-GD1b Ab signal overlying the NMJ was observed, compared with surface Ab levels at t = 0. (E and F) For anti-GD1a Ab (images not shown) rapid clearance by internalization from the cell surface was also observed (E), and by 60 minutes, there was also a mild but significant overall reduction in total anti-GD1a Ab over the NMJ (F, 60-minute time point), suggesting clearance away from this site. At least 150 NMJs were analyzed per time point, and results are from 3 independent experiments per Ab. n = 3; **P < 0.005. Scale bars: 20 μM. Control tissues exposed to secondary Ab alone showed no significant binding or uptake (for anti-TNP IgG3 control data, see Figures 7 and 8).