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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Research Article Neuroscience

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 7

Cholesterol is required for AGAb uptake in vitro and ex vivo.

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Cholesterol is required for AGAb uptake in vitro and ex vivo. (A) MβCD-t...
(A) MβCD-treated and untreated PC12 cells were labeled with AGAbs at 4°C and incubated for 30 minutes at 37°C to allow endocytosis. AGAbs were detected using secondary Ab and analyzed by fluorescence microscopy. Scale bar: 20 μm. (B) Levels of surface and internalized AGAb were estimated using the quantification module of AxioVision software, and the percentage of internalized AGAb was calculated. Data are mean ± SEM (n = 3; **P < 0.001). (C) MβCD-treated and untreated TS preparations were labeled with anti-GD1b at 4°C and incubated at 37°C for 30 minutes to allow endocytosis. No Ab binding or uptake was observed in parallel preparations receiving anti-TNP IgG3 control Ab. Anti-GD1b was detected using FITC-labeled secondary Ab, and NMJs were detected using Alexa Fluor 555 α-BTx. Scale bar: 20 μm. (D) Surface anti-GD1b Ab overlying the AChR was estimated using ImageJ software. While incubation at 37°C for 30 minutes resulted in significant Ab endocytosis compared with 0 minutes (**P < 0.01), this was completely attenuated in the presence of MβCD, with no significant difference observed compared with TS maintained at 4°C. At least 150 NMJs were analyzed per condition in 2 independent experiments.