Article Figures & Data
Figures
- Figure 1 Autoantibodies of Patients Bind to CNTN1 in Transfected HEK293 Cells and DRG Neurons
(A–D) Immunofluorescence of CNTN1-transfected HEK293 cells shows binding of serum of patient (pat) 1 (B, red), PE material of pat2 (C, red), and serum of pat3 (D, red), whereas no binding was detectable for serum of a HC (A, red). Scale bars: 20 μm. (E–H) Control stainings of HEK293 cells showed no staining of untransfected or GFP-transfected cells (E–F) because of a lack of endogenous CNTN1 expression. GFP (green) demonstrates transfection efficiency. (G) No primary antibody present = negative control 1. (H) No secondary antibody present = negative control 2. (I–L) Similarly, serum of pat1 (J, red), PE material of pat2 (K, red), and serum of pat3 (L, red) showed clear staining at DRG neurons. By contrast, serum of a HC did not lead to immunolabeling of the neurons (I, red). Scale bars: 50 μm. (A–L) All cells were costained with anti-CNTN1 antibody (cyan). Note the overlay of the targeted CNTN1 by patient autoantibodies and the commercial antibody (white signal, arrowheads in magnifications). DAPI was used to stain cell nuclei (blue). CNTN1 = contactin-1; DAPI = 4',6-diamidino-2-phenylindole; DRG = dorsal root ganglia; GFP = green fluorescent protein; HC = healthy control; HEK293 = human embryonic kidney 293; PE = plasma exchange.
- Figure 2 Immunofluorescence of CGNs Shows Reduced Expression of CNTN1 After Long-term Incubation With Serum of Anti-CNTN1–Positive Patients
(A–C) Immunofluorescence of cerebellar granule neurons shows binding of sera of patient (pat) 1 (B) and pat3 (C). No specific binding is detectable for serum of a HC (A). DAPI was used for staining cell nuclei (blue). Scale bars refer to 20 μm. (D) Scheme of the experimental setup. Two to four days after seeding cerebellar granule neurons, cells were incubated for 2 or 4 days with serum of a HC or sera of patients (pat) 1 and 3. In addition, cells were incubated with serum of pat1 and pat3 for 2 days followed by incubation with serum of a HC for 2 days (recovery period, 2R). (E) Representative images of cerebellar granule neurons treated with serum of a HC or serum of pat1 or pat3 for specific periods of time (2 days, 4 days, and 2R) and stained for CNTN1 (red). Signal of CNTN1 is reduced after 2 and 4 days of incubation with pat sera. The recovery phase of 2 days (2R) leads to increased staining of CNTN1 for both patients. DAPI was used for staining of nuclei (blue). Scale bars: 20 μm. CGN = cerebellar granular neurons; CNTN1 = contactin-1; DAPI = 4',6-diamidino-2-phenylindole; HC = healthy control.
- Figure 3 Immunoblotting of CNTN1 in CGN Lysates After Incubation With Anti-CNTN1 Autoantibodies
(A–B) Representative Western blots of CNTN1 expression after incubation of cerebellar granule neuron lysates with serum of a HC or serum of pat1 (A) or pat3 (B) for 2 days, 4 days, or 2R. The 2 CNTN1 isoforms with appropriate molecular weights of around 140 kDa were stained with anti-CNTN1 (black arrowheads). GAPDH (37 kDa) was used as a control protein (white arrowheads). (C) Quantification of CNTN1 expression. Expression was normalized to GAPDH, and expression of neurons treated with serum of a HC for 2 and 4 days was set to 100%. Note that because of the limited sample size of 2 samples per condition, statistical analysis was not performed. (D) Relative cytotoxicity of serum of pat1, pat3, or HC to cerebellar neurons. Triton X-100 was used as high control and set to 100%. A cytotoxic effect of pat3 serum compared with HC serum was visible at 3 and 4 days of incubation. p Values represent the significance level with ***p < 0.001 (t test); error bars represent SD. 2R = 2-day recovery phase; CGN = cerebellar granular neurons; CNTN1 = contactin-1; GAPDH = Glyceraldehyde 3-phosphate dehydrogenase; HC = healthy control.
- Figure 4 Lack of CNTN1 Cross-linking by Generation of Fab Fragment From the Patient IgG Prevents CNTN1 Internalization
(A, B) DRG neurons treated for 2 days with serum of pat1 and pat3 or a HC. (A) Cells were stained for the binding of the patient IgG (red) to the DRG neurons. (B) After binding of patients' CNTN1 autoantibodies, DRG neurons were controlled for the CNTN1 level. Note the CNTN1 signal reduction after incubation with serum from pat1 (more pronounced) and pat3. (C, D) DRG neurons incubated for 2 days with Fab fragments generated from patient IgGs. (C) The CNTN1 level after Fab fragment (HC, pat1, and pat3) binding is shown. (D) The binding of Fab from pat1 and 3 was proven by binding of anti-Fab Cy3 secondary antibodies. The nuclei are always marked by DAPI binding (blue). Scale bar refers to 20 μm. CNTN1 = contactin-1; DAPI = 4',6-diamidino-2-phenylindole; DRG = dorsal root ganglia; HC = healthy control.
- Figure 5 Reduced Immunofluorescence of CNTN1 in Transfected HEK293 Cells After Treatment With Anti-CNTN1 Autoantibodies
Representative images of the appropriate conditions (2 days, 4 days, and 2R) shown for cells incubated with HC serum (1:500), pat1 (1:500, red) or pat3 (1:500, red). Note the reduced signal of CNTN1 in cells incubated with serum of pat1 for 2 and 4 days. DAPI was used to mark the nucleus (blue). Scale bar refers to 20 μm. 2R = 2-day recovery phase; CNTN1 = contactin-1; DAPI = 4',6-diamidino-2-phenylindole; HC = healthy control; HEK293 = human embryonic kidney 293.
- Figure 6 Quantitative Analysis of CNTN1 Expression Levels Shows Reduced CNTN1 Surface Expression After Long-term Exposure to Mainly IgG3 Anti-CNTN1 Autoantibodies in Transfected HEK293 Cells
(A) Representative Western blots of the whole-cell CNTN1 expression after 2 days of the incubation period with pat sera or a HC. Staining was performed with CNTN1 antibody (140 kD, black arrowheads) and β-actin as control protein (46 kD marked by white arrowheads). (B) Whole-cell fractions of condition 4 days of incubation and 2R. Same HC, as well as pat1 and pat3 serum were used. Dotted lines in (A, B) indicate that the lane was cut but from the same Western blot. (C) Quantification of the whole-cell CNTN1 expression. CNTN1 expression was normalized to β-actin, and the expression with HC serum at 2 days, 4 days, and 2R was always set to 100%. (D, E) Same as in (A, B), but surface fractions were stained. ATPase was used as control protein for the cellular membrane (100 kD, white arrowheads). Note the reduction of CNTN1 protein in fractions incubated with pat1 serum at 2- and 4-day presence of the autoantibodies. Dotted lines in (D, E) label cut lanes from the same Western blot. (F) Quantification of the surface CNTN1 protein normalized to ATPase levels. Again, the HC incubations at 2 days, 4 days, and 2R were set to 100% and compared with patient serum presence. Value of significance, *p < 0.05. 2R = 2-day recovery phase; CNTN1 = contactin-1; HC = healthy control; HEK293 = human embryonic kidney 293.
- Figure 7 Analysis of Signal Intensities and Colocalization of CNTN1 and Pan-Nav Using SIM
(A) SIM recordings on adult DRG neurons immunostained for CNTN1 and pan-NaV and incubated with single pat sera or HC sera (controls = pooled) for 72 hours. Single stretch along the axon taken from a large field of view recording. Scale bar: 2 μm. (B) Exemplary profile plots along 6 μm of axon (taken from [A]) for showing the level of coincidence of CNTN1 and pan-NaV signals in representative SIM recordings after incubation with serum of HC (B.a) or pat1 (B.b). Note the different y-axis scale. (C) Colocalization analysis of CNTN1 and pan-NaV depicting Pearson correlation coefficients. (D) Analysis of signal density (sum intensity/summed area in μm2) of CNTN1 (D.a) and pan-NaV (D.b) over CNTN1 ROIs. Values of significance, *p < 0.05, **p < 0.01, and ***p < 0.001. CNTN1 = contactin-1; DRG = dorsal root ganglia; HC = healthy control; ROI = region of interest; SIM = structured illumination microscopy.
- Figure 8 Reduced Sodium Currents for DRG Neurons After Long-term Incubation With Serum of Anti-CNTN1–Positive Patients
(A) Scheme of short-term and long-term incubation experiments. DRG neurons were incubated with serum of pat1, PE material of pat2, or serum of pat3 for specific periods of time (1, 2, 24, 48, and 72 hours). (B) Representative sodium current traces at –40 mV for DRG neurons treated with patients' serum or PE sample or serum of a HC for 1 (B.a) or 72 hours (B.b). The voltage step protocol consisted of a prepulse potential of –120 mV and of 200 ms steps ranging from –80 to +60 mV in 10 mV increments at a holding potential of –70 mV. (C) Average peak current densities for sodium channels at –40 mV after incubation with serum of a HC, serum of pat1, PE material of pat2, or serum of pat3 for specific periods of time. Short-term incubation (1 hour, C.a; 2 hours, C.b; 24 hours, C.c; 48 hours, C.d) showed no effects on sodium currents, whereas incubation with serum of pat1 and pat3 for 72 hours showed a significant reduction of sodium current density (C.e). Error bars represent standard errors of the mean (SEM). (D) Current-voltage relationships of sodium current densities from DRG neurons treated with serum of a HC, serum of pat1, PE material of pat2, or serum of pat3 for 1 hour (D.a) or 72 hours (D.b). p Value represents the significance level with *p < 0.05 and **p < 0.01 (Mann-Whitney U test); error bars represent SD. CNTN1 = contactin-1; DRG = dorsal root ganglia; HC = healthy control; PE = plasma exchange.
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