Fig 1: Recombinant Cbln1 administration into the central amygdala rescues behavioral hypersensitivity in an inflammatory pain model in a GluD1-dependent manner. (A) Experimental interventions. Mice underwent surgery for intra-CeA or intraventricular cannula implantation. After recovery, mechanical sensitivity was tested using von Frey test for paw withdrawal threshold under baseline condition followed by intraplantar injection of CFA. Effect of intra-CeA administration of Cbln1 (250 ng in 0.5 µL per side) was assessed. Paw withdrawal threshold following Cbln1 injection was measured at 6, 24, 48, 72, 96 h, and 1-week timepoints. (B) Alleviation of pain sensitivity after bilateral intra-CeA Cbln1 administration. Paw withdrawal threshold increased after a single Cbln1 injection and lasted up to a week; Two-way repeated measures ANOVA, treatment F (4, 22) = 68.54, p < 0.0001, Bonferroni’s post hoc test, WT-vehicle (n = 6 mice) vs. WT-Cbln1 (n = 7 mice); 6 h, * p = 0.012, 24 h–1 week, **** p < 0.0001. This improvement was not observed in GluD1 KO (GluD1 KO-vehicle (n = 4 mice), GluD1 KO-Cbln1 (n = 6 mice)). Cbln2 was not able to reverse the CFA-induced mechanical hypersensitivity (n = 4 mice). (B’) No change in mechanical threshold in von Frey analysis in control (non-CFA injected) paw. (C) Rescue of inflammatory pain by Cbln1 injection is lateralized. Recombinant Cbln1 first injected into the left CeA did not show any rescue in mechanical hypersensitivity. However, injection into the right CeA was able to rescue mechanical hypersensitivity; Two-way repeated measures ANOVA, treatment F (1, 9) = 44.15, p < 0.0001; Bonferroni’s post hoc test WT CFA-vehicle (n = 4 mice) vs. WT CFA-Cbln1 (n = 7 mice); 48 h and 72 h **** p < 0.0001. (C’) No change in mechanical threshold in von Frey analysis in control (non-CFA injected) paw. (D) D-serine (30 µg in 0.5 µL) opposes the antihyperalgesic effect of recombinant Cbln1 (250 ng in 0.5 µL). Two-way repeated measures ANOVA, treatment F (1, 6) = 5.35, p = 0.060, Bonferroni’s post hoc test WT-Cbln1 (n = 4 mice) vs. WT-Cbln1+D-serine (n = 4 mice); 24 h, ** p = 0.008; 48 h, *** p = 0.0001.
Fig 2: Cbln2 and Cbln2 are upregulated in TG neurons from IS model of headache. A-B Phylogenetically conserved expression patterns of Cbln2 predominantly in Aδ-fiber neurons based on human and mouse TG cell atlases. Human NF3_pctExpress: 56.34%, mouse NF3_pctExpress: 66.62%. NF3 = neurofilament + LTMR enriched for Aδ; NF2 = neurofilament + A-LTMR enriched for Aβ; cLTMR = c-fiber LTMR. C-D No significant sex difference was observed in the expression profile of Cbln2 in TG of mice. Male mouse Aδ-LTMRs_pctExpress: 69.47%, female mouse Aδ-LTMRs_pctExpress: 72.92%. Ntrk3low + Ntrk2 = Aδ-LTMRs, Ntrk3high + Ntrk2 = Aβ-LTMRs, Th = cLTMRs. E-J Expression profiling of cerebellins in two established migraine models, inflammatory soup (IS) and cortical spreading depression (CSD). Both Cbln1 and Cbln2 were robustly detected in TG neurons across models. IS model exhibited significant upregulation of Cbln1, Cbln2 and Cbln4 compared to naïve (E-F, H-I). CSD model showed no significant alterations in Cbln1/Cbln2 expression (G, J)
Fig 3: pIONT model of trigeminal neuralgia increases CBLN2 expression in TG. A Expression data of four cerebellin members in a transcriptional cell atlas of mouse TG. Cbln2 was prominent in TG neurons, particularly in NF3 cluster. B RNA sequencing of TG revealed upregulation of Cbln2 at day 3 and day 10 post-pIONT, while Cbln1 remained unchanged. C qRT-PCR analysis of Cbln2 in TG of male and female mice 3 days post-pIONT or sham. Cbln2 was upregulated in both sexes after pIONT with no sex difference. ***P < 0.001, one-way ANOVA with Bonferroni’s test, n = 6 mice/group. D Cbln2 mRNA expression in TG of naïve, sham-, and pIONT-operated mice. ***P < 0.001 vs. corresponding sham group, two-way ANOVA followed by Bonferroni’s test, n = 8 mice/group. E-F Western blot analysis showed increased CBLN2 protein in TG at 10 days post-pIONT. **P < 0.01 vs. sham, Student’s t-test, n = 4 mice/group. G ELISA analysis showed CBLN2 protein upregulation at day 10 post-pIONT. *p < 0.05 vs. sham, Student’s t-test, n = 6 mice/group. H-N Representative immunofluorescence images of CBLN2 in TG. Staining intensity increased significantly at 1, 3, 10, 21 d post-pIONT. **P < 0.01, ***P < 0.001, one-way ANOVA followed by Bonferroni’s test, n = 3 mice/group
Fig 4: Differential expression of CBLN2 is partly mediated by promoter methylation. A Prediction of the CpG island upstream of Cbln2 transcriptional start site. B-C MSP assay revealed significantly reduced methylation in the CBLN2 promoter region of pIONT mice compared to sham-operated controls. *P < 0.05, Student’s t-test, n = 3 mice/group. D-E BSP sequencing targeted a 117-bp fragment containing 11 CpG sites. F pIONT mice exhibited decreased CpG methylation at the CBLN2 promoter compared to sham controls. **P < 0.01, two-way ANOVA followed by Bonferroni’s test, n = 3 mice/group. G Tet3 mRNA expression in TG of naïve, sham-, and pIONT-operated mice. ***P < 0.001 vs. corresponding sham group, two-way ANOVA followed by Bonferroni’s test, n = 8 mice/group. H Screening of Tet3-targeting siRNA in Neuro-2a cells. **p < 0.01, ***p < 0.001, Student’s t-test, n = 6 wells/group. I-J Intra-TG injection of Tet3 siRNA at 10 days post-pIONT relieved mechanical allodynia at 24 h post-injection, evaluated with 0.02 g von Frey filaments (I) and 0.60 g von Frey filaments (J). ***p < 0.001, two-way RM ANOVA followed by Bonferroni’s test, n = 6 mice/group. K-L Tet3 siRNA suppressed Tet3 and Cbln2 mRNA in TG. **P < 0.01, Student’s t-test, n = 6 mice/group
Fig 5: MEK inhibitor reduces CBLN2-induced neuronal hypersensitivity. A-B Intra-TG injection of PD98059 attenuated CBLN2-induced mechanical allodynia measured using 0.02 g von Frey filaments (A) and 0.60 g von Frey filaments (B). **p < 0.01, ***p < 0.001, two-way RM ANOVA followed by Bonferroni’s test, n = 7 mice/group. C The representative traces of APs evoked by a rheobase current from CBLN2-, CBLN2 + PD98059-treated TG neurons. D PD98059 increased the rheobase current required to evoke an AP compared with vehicle group. **p < 0.01 vs. vehicle, Student’s t-test, n = 12 neurons/group from 3–4 mice. E PD98059 reduced AP frequency. ***P < 0.001, two-way RM ANOVA followed by Bonferroni’s test, n = 12 neurons/group from 3–4 mice. F The representative traces of APs evoked by depolarizing current steps recorded from CBLN2-, CBLN2 + PD98059-treated TG neurons. G PD98059 decreased the number of APs in response to 160 pA, 1000 ms current injection. ***p < 0.001, two-way RM ANOVA followed by Bonferroni’s test, n = 12 neurons/group from 3–4 mice
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