Mechanism, and treatment of anti-CV2/CRMP5 autoimmune pain

Abstract

Paraneoplastic neurological syndromes arise from autoimmune reactions against nervous system antigens due to a maladaptive immune response to a peripheral cancer. Patients with small cell lung carcinoma or malignant thymoma can develop an autoimmune response against the CV2/collapsin response mediator protein 5 (CRMP5) antigen. For reasons that are not understood, approximately 80% of patients experience painful neuropathies. Here, we investigated the mechanisms underlying anti-CV2/CRMP5 autoantibodies (CV2/CRMP5-Abs)-related pain. We found that patient-derived CV2/CRMP5-Abs can bind to their target in rodent dorsal root ganglia (DRG) and superficial laminae of the spinal cord. CV2/CRMP5-Abs induced DRG neuron hyperexcitability and mechanical hypersensitivity in rats that were abolished by preventing binding to their cognate autoantigen CRMP5. The effect of CV2/CRMP5-Abs on sensory neuron hyperexcitability and mechanical hypersensitivity observed in patients was recapitulated in rats using genetic immunization providing an approach to rapidly identify possible therapeutic choices for treating autoantibody-induced pain including the repurposing of a monoclonal anti-CD20 antibody that selectively deplete B-lymphocytes. These data reveal a previously unknown neuronal mechanism of neuropathic pain in patients with paraneoplastic neurological syndromes resulting directly from CV2/CRMP5-Abs-induced nociceptor excitability. CV2/CRMP5-Abs directly sensitize pain responses by increasing sensory neuron excitability and strategies aiming at either blocking or reducing CV2/CRMP5-Abs can treat pain as a comorbidity in patients with paraneoplastic neurological syndromes.

Figure 1:. CV2/CRMP5-Abs label nociceptive structures and induce mechanical hypersensitivity and DRG neuron hyperexcitability.

(A) Micrographs of rat dorsal root ganglia (DRG) and spinal cord immunolabelled with anti-CV2 sera from three patients. CV2/CRMP5-Abs positively labelled neuronal soma in DRG and superficial laminae in the dorsal horn of the lumbar spinal cord. The right panels show a magnification of boxed regions of spinal cord sections. (B) Representative recordings of evoked action potentials recorded from small-diameter DRG neurons in response to depolarizing current injection of 30, 60, and 90 picoamperes (pA). Female rat DRG neurons were treated overnight with serum from patient #4 or #6 (1/100 dilution). (C) Quantification of the number of evoked action potentials in response to 0–100 pA of injected current. *p<0.05, multiple Mann-Whitney tests. Representative traces (D) and (E) bar graph with scatter plot showing a decreased rheobase in cells treated with the serum from patient #4 or #6. n=11 cells per condition; error bars indicate mean ± SEM, *p<0.05, Mann-Whitney test. (F) Graph showing the paw withdrawal threshold of male rats injected intrathecally (i.th.) with 10 μL of the indicated positive CV2/CRMP5-Abs or (G) depleted (cross-adsorbed with purified CRMP5) sera. CV2/CRMP5-Abs containing sera consistently elicited mechanical hypersensitivity while depleted sera had no significant effect. n = 12; 3 rats per CV2/CRMP5-Abs serum; 4 different CV2/CRMP5-Abs sera. The black line shows the average of all patients tested. Error bars indicate mean ± SEM, *p<0.05, two-way ANOVA. (H) Bar graph with scatter plot showing the area under the curve for the data in F and G, *p<0.05, Mann-Whitney test.

Figure 2:. Blocking CV2/CRMP5-Abs with epitope peptides prevents the sensitization of sensory neurons and mechanical hypersensitivity in rats.

(A) Heatmap of the immunoreactivity of CV2/CRMP5-Abs sera hybridized on a CRMP5 peptide array mapping the entire sequence of the protein in 15-mer peptides with 3 amino acid increments. Four main epitopes on CRMP5 are targeted by the CV2/CRMP5-Abs. (B) Bar graph with scatter plot showing that peptides 53, 142 and 146 can block the binding of CV2/CRMP5-Abs from patients #8 and #4 to purified CRMP5. 0.03% DMSO is the vehicle, purified CRMP5 was used as a positive control to achieve maximal displacement of the CV2/CRMP5-Abs. n=3 independent measures from an average of 3 repeats. (C) Micrograph of a rat DRG immunolabelled with CV2/CRMP5-Abs positive serum (1/100) from patient #1 and then with blocking peptides 53, 142, 146. Blocking peptides abolished the immunoreactivity of CV2/CRMP5-Abs for their protein target CRMP5. (D) Representative recordings of evoked action potentials recorded from small-diameter DRG neurons treated with serum from patient #1 (1/100 dilution) in combination with 100 ng/ml of peptides 53, 142 and 146 as indicated, overnight in response to depolarizing current injection of 30, 60, and 90 pA. (E) Quantification of the number evoked action potentials in response to 0–100 pA of injected current. n=9–10 cells per condition, *p<0.05, multiple Mann-Whitney tests. (F) traces and (G) bar graph with scatter plot showing unchanged rheobase in cells treated with the serum from patient #1 with blocking peptides as indicated. Error bars indicate mean ± SEM, *p<0.05, Mann-Whitney test. (H) Graph showing the paw withdrawal threshold of rats injected with 15 μl of the indicated CV2/CRMP5-Abs positive sera (1/10 dilution) or with (I) blocking peptides 53, 142 and 146 (300 ng/ml) in the paw. Anti-CV2 containing sera elicited mechanical hypersensitivity which was prevented by blocking the CV2/CRMP5-Abs with their epitope peptides. Error bars indicate mean ± SEM, *p<0.05, two-way ANOVA. (J) bar graph with scatter plot showing the area under the curve of the data in G and H and color coded per treatment groups (n=3 each), error bars indicate mean ± SEM, *p<0.05, Mann-Whitney. Experimenters were blind to the treatment groups.

Figure 3:. CRMP5 auto-immunity induces mechanical allodynia and hyperexcitability in both male and female rats.

Rats were immunized by 3 injections (indicated by arrows) of 50 μg of pCMV2 plasmid allowing for the expression of CRMP5 or control (empty) in the spinodeltoidus muscle. Rats received an intramuscular injection of a plasmid (pCMV2-CRMP5) carrying the coding sequence for CRMP5 on day 0 and then 2 booster shots at weeks 2 and 4 after the first injection. Graph showing the paw withdrawal threshold of (A) male and (E) female rats injected with the CRMP5 coding plasmid compared to the control empty plasmid (n=8 animals per group; *p<0.05, two-way ANOVA). In both sexes, rats injected with the CRMP5 coding plasmid developed mechanical hypersensitivity. Bar graph with scatter plot showing the area under the curve for the mechanical thresholds in (B) male and (F) female rats injected as described above (n=8 animals per group; *p<0.05, Mann-Whitney test). Rats were tested for their thermal thresholds using the Hargreave’s test and no difference was found in (C) male and (G) female rats injected with the CRMP5 coding plasmid compared to the control empty plasmid (n=8 animals per group; *p<0.05, two-way ANOVA). Bar graph with scatter plot showing the area under the curve for the thermal thresholds in (D) male and (H) female rats injected as described above (n=8 animals per group; *p<0.05, Mann-Whitney test. Error bars represent mean ± SEM. (I) Representative recordings in response to a depolarizing current step to evoke action potentials (APs) in sensory neurons from male and female rats injected with plasmid expressing CRMP5 or a control plasmid. (J) Summary of the number of APs in the indicated conditions (n=10 cells per condition). (K) Representative recordings in response to various steps of depolarizing current to measure rheobase in sensory neurons prepared from rats with CRMP5 autoimmunity or control. (L) Summary of the measured rheobase in indicated conditions (n=10 each). Asterisks indicate significance compared with control *p<0.05, Mann-Whitney test. Error bars represent mean ± SEM.

Figure 4:. The anti-CD20 monoclonal antibody reverses CRMP5 autoimmunity induced mechanical hypersensitivity and hyperexcitability.

Rats were immunized against CRMP5 and developed stable mechanical hypersensitivity up to day 86 after the first intramuscular injection. (A) Graph showing the paw withdrawal threshold of rats over time. Black arrows show the 3 plasmid injections necessary for the induction of the model. Anti-CD20 injection (4 mg/kg, i.p.) is indicated at days 56 and 63 by an antibody symbol. Data are mean ± SEM, N=6–8 rats per group, *p<0.05 two-way ANOVA (B) Bar graph with scatter plot showing the area under the curve for each individual (n=8) from the indicated treatment groups. (C) Representative recordings of evoked action potentials recorded from rat small-diameter DRG neurons cultured from the indicated treatment groups in response to depolarizing current injection of 30, 60, and 90 pA. (D) Quantification of the number of current-evoked action potentials in response to 0–100 pA injected current. CRMP5 autoimmunity increased action potential firing compared to control DRG neurons. Anti-CD20 reversed this phenotype back to the level of control sensory neurons. *p<0.05, multiple Mann-Whitney tests. N=8–9 cells each, mean ± SEM.