Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: Neuropathic Pain and Distinct CASPR2 Autoantibody IgG Subclasses Drive Neuronal Hyperexcitability

doi: 10.1212/NXI.0000000000200423

Figure Lengend Snippet: (A) Exemplary immunocytochemical stainings of CASPR2 (cyan) and patient serum (red) binding to membrane of CASPR2-transfected HEK-293 cells and adult DRG neurons. (B) Stainings for different IgG subclasses (cyan) in 2 exemplary total IgG positive (red) patient sera (P31, P39). (C) Distribution of IgG subclasses (IgG4 only or IgG4 plus additional IgG = IgGX) and pain phenotype with pain—red and orange circles, no pain—light and dark blue circles with circle size related to the number of positive patient sera. (D) Distribution of IgG subclass combinations in patients with pain (red) and without pain (blue). CASPR2 = Contactin-associated protein 2; DRG = dorsal root ganglia; IgG = immunoglobulin G.

Article Snippet: For live staining, HEK-293 cells 72 hours posttransfection (eMethods) or adult DRG neurons at DIV3 were incubated with anti-CASPR2 antibodies (AF5145, 1:250, R&D Systems, Minneapolis, MN, US) and/or human patient serum (1:50–1:250) in medium for 1 hour at 4°C.

Techniques: Binding Assay, Membrane, Transfection

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: Neuropathic Pain and Distinct CASPR2 Autoantibody IgG Subclasses Drive Neuronal Hyperexcitability

doi: 10.1212/NXI.0000000000200423

Figure Lengend Snippet: (A) Microfluidic chamber (MFC) with embryonic DRGs to separate somatic and axonal compartments and investigate VGKC complex organization after CASPR2 aAb incubation in separated compartments. (B) Exemplary pictures using lattice SIM 2 microscopy of soma and axons from DRGs in MFCs treated with healthy control serum. Arrows mark the distance between CASPR2 (magenta) and Kv1.2 (green). (C) Quantification of distance between CASPR2 and Kv1.2 for somata (left) and axons (right) after CASPR2 aAb incubation for 2 hours. Data are shown as mean ± SEM. Somatic and axonal complexes: n = 325–1,465 and n = 1872–3,603, respectively. (D) CASPR2 and Kv1.2 density per 100 µm axon (CASPR2 n = 34–37; Kv1.2 n = 33–37). Data are shown as violin plots with individual values, median = bold line, quartiles = dotted lines. Levels of significance: ns: not significant, * p < 0.05, ** p < 0.01, **** p < 0.0001. CASPR2 = Contactin-associated protein 2; DRG = dorsal root ganglia; VGKC complex = voltage-gated potassium channel complex.

Article Snippet: For live staining, HEK-293 cells 72 hours posttransfection (eMethods) or adult DRG neurons at DIV3 were incubated with anti-CASPR2 antibodies (AF5145, 1:250, R&D Systems, Minneapolis, MN, US) and/or human patient serum (1:50–1:250) in medium for 1 hour at 4°C.

Techniques: Incubation, Microscopy, Control

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: Neuropathic Pain and Distinct CASPR2 Autoantibody IgG Subclasses Drive Neuronal Hyperexcitability

doi: 10.1212/NXI.0000000000200423

Figure Lengend Snippet: (A) Exemplary pictures and activity graphs of ROIs after short-term incubation with control serum or serum group with pain and IgGX. Neuronal activity from ∼40 cells is shown in a heatmap. (B) Frequency (left), amplitude (middle), and area under the curve (AUC; right) of spontaneous calcium activity events after short-term incubation with CASPR2 aAbs of different serum subclassifications. Data shown as mean ± SEM, n = 718 (control), n = 682 (no pain/IgG4), n = 476 (no pain/IgGX), n = 566 (pain/IgG4), and n = 727 (pain/IgGX). Levels of significance: * p < 0.05, ** p < 0.01, **** p < 0.0001. aAbs = autoantibodies; CASPR2 = Contactin-associated protein 2; DRG = dorsal root ganglia.

Article Snippet: For live staining, HEK-293 cells 72 hours posttransfection (eMethods) or adult DRG neurons at DIV3 were incubated with anti-CASPR2 antibodies (AF5145, 1:250, R&D Systems, Minneapolis, MN, US) and/or human patient serum (1:50–1:250) in medium for 1 hour at 4°C.

Techniques: Activity Assay, Incubation, Control

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: Neuropathic Pain and Distinct CASPR2 Autoantibody IgG Subclasses Drive Neuronal Hyperexcitability

doi: 10.1212/NXI.0000000000200423

Figure Lengend Snippet: (A) Bar plot of potassium channel activity at different voltage steps from −80 to +40 mV. Normalized current compared with the healthy control is shown. (B) I–V (current-voltage relation) plot measured after short-term aAb application on DRG neurons under the condition: pain/no IgG4, N = 2, n = 10. (C and D) Replacement of the CASPR2 aAbs by healthy control serum led to a rescue of affected potassium channel current (patients with pain and patients without pain), N = 2, n = 10–11. Levels of significance: * p < 0.05, ** p < 0.01, *** p < 0.001. aAbs = autoantibodies; CASPR2 = Contactin-associated protein 2; DRG = dorsal root ganglia; Kv = voltage-gated potassium.

Article Snippet: For live staining, HEK-293 cells 72 hours posttransfection (eMethods) or adult DRG neurons at DIV3 were incubated with anti-CASPR2 antibodies (AF5145, 1:250, R&D Systems, Minneapolis, MN, US) and/or human patient serum (1:50–1:250) in medium for 1 hour at 4°C.

Techniques: Activity Assay, Control

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: Neuropathic Pain and Distinct CASPR2 Autoantibody IgG Subclasses Drive Neuronal Hyperexcitability

doi: 10.1212/NXI.0000000000200423

Figure Lengend Snippet: (A and B) Representative potassium channel current recording and the effect of specific potassium channel subunit composition blocker α-dendrotoxin Dtx-A and κM-RIIIJ (red traces) shown in the bar plot (at +40 mV). Data shown as mean ± SEM. (C) Domain architecture of CASPR2 including the discoidin domain, fibrinogen-like domain, and laminin domains. Patch clamp recordings using a voltage step protocol from −80 to +40 mV in the presence of CASPR2 monoclonal autoantibodies against the discoidin domain (left) demonstrating significant alterations of the potassium channel activity and against the laminin domain (right) with no significant alterations. N = 3, n = 13–16. Level of significance: * p < 0.05, **** p < 0.0001. CASPR2 = Contactin-associated protein 2; DRG = dorsal root ganglia.

Article Snippet: For live staining, HEK-293 cells 72 hours posttransfection (eMethods) or adult DRG neurons at DIV3 were incubated with anti-CASPR2 antibodies (AF5145, 1:250, R&D Systems, Minneapolis, MN, US) and/or human patient serum (1:50–1:250) in medium for 1 hour at 4°C.

Techniques: Patch Clamp, Activity Assay

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: Neuropathic Pain and Distinct CASPR2 Autoantibody IgG Subclasses Drive Neuronal Hyperexcitability

doi: 10.1212/NXI.0000000000200423

Figure Lengend Snippet: DRG neurons are active mediators in developing neuropathic pain: healthy condition (left) and disease condition in the presence of CASPR2 autoantibodies (aAbs, right). Sensory neuron hyperexcitability is driven by pain-associated CASPR2 aAbs. This causes enhanced neuronal activity and decreased function of the associated Kv channels. Pathologic activity of sensory neurons is mainly promoted by CASPR2 aAbs of the IgG4 subtype. Created in BioRender. Villmann, C. (2025) BioRender.com/ . CASPR2 = Contactin-associated protein 2; DRG = dorsal root ganglia.

Article Snippet: For live staining, HEK-293 cells 72 hours posttransfection (eMethods) or adult DRG neurons at DIV3 were incubated with anti-CASPR2 antibodies (AF5145, 1:250, R&D Systems, Minneapolis, MN, US) and/or human patient serum (1:50–1:250) in medium for 1 hour at 4°C.

Techniques: Activity Assay