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 8

MβCD treatment of TS preparations increases AGAb-dependent MAC deposition and tissue injury at NMJs by attenuating Ab uptake.

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MβCD treatment of TS preparations increases AGAb-dependent MAC depositio...
MβCD-treated and untreated TS preparations from WT (A–C) and GD3s–/– (D–F) mice were labeled at 4°C with anti-GD1b and anti-GM1 Abs, respectively. Ab-labeled TS was either directly exposed to NHS (0 minutes) at 37°C or further incubated for 30 minutes at 37°C (to allow Ab internalization) and then exposed to NHS (30 minutes). MAC deposits at NMJs were detected with anti–C5b-9 and TRITC-labeled secondary Ab. NMJs were identified using α-BTx-488 that binds AchR. Representative fluorescence images are shown. Scale bar: 20 μm. (B and E) Quantitative analysis of MAC deposits at NMJs of MβCD-treated and untreated TS from confocal images using ImageJ software. Incubation of Ab-labeled TS for 30 minutes at 37°C (t = 30 minutes) in comparison to immediate exposure to NHS (t = 0 minutes) resulted in greatly reduced MAC deposition for both Abs; **P < 0.0001. Pretreatment with MβCD attenuated both anti-GD1b and -GM1 Ab uptake, and as a consequence, MAC deposition was enhanced, even to levels greater than those seen with immediate exposure to NHS (t = 0 minutes); P < 0.001 for both Abs. (C and F) NMJ integrity was assessed by counting α-BTx–positive NMJs that retained cytosolic CFP (healthy NMJs). A decrease in the number of healthy NMJs in tissues pretreated with MβCD was observed. P= 0.01 for anti-GD1b Ab; P= 0.02 for anti-GM1 Ab. At least 150 NMJs were analyzed per condition in 3 independent experiments. Control studies using anti-TNP Ab (IgG3) were conducted, and no effects were induced. Data are expressed as median values and interquartile ranges.