Fig 2: Peripheral Nerve Injury Regulates Raet1 Expression and Injured Sensory Neurons Show Increased Neurite Fragmentation by Stimulated NK Cells(A) Schematic diagram of spinal nerve transection injury site in relation to lumbar DRGs (L3, L4, and L5)(B) Raet1 mRNA expression in ipsilateral L5 DRG after spinal nerve transection relative to contralateral DRG by qPCR. Two-way ANOVA: effect of injury, F(1,12) = 121.25, p = 0001; effect of time, F(1,12) = 5.08, p = 0.0438. Bonferroni post-test, ipsi versus contra: 4 days, t = 6.193, ***p < 0.001; 7 days, t = 9.380, ***p < 0.001. n = 4 samples per time point, n = 2 mice DRG pooled per sample.(C) qPCR shows injury-related increase in Raet1 mRNA expression in adult DRG (1 day in vitro). Student’s unpaired t test, t = 4.994, p = 0.0075. n = 3 mice, 3 replicate cultures.(D) Immunolabeling (ß-tubulin III) of cultured L5 DRG (1 day in vitro) isolated 7 d after sham surgery co-cultured (4 h) with either freshly isolated (control) or IL-2-stimulated NK cells.(E) Quantification of sham DRG neurite fragmentation. One-way ANOVA: F(2,61) = 7.171 p = 0.0016. Bonferroni post-test: DRG only versus control NK, t = 0.4349, nonsignificant (ns) p > 0.05; control NK versus IL-2-stimulated NK, t = 3.221 **p < 0.01; DRG only versus IL-2-stimulated NK, t = 3.180, **p < 0.01.(F) Immunolabeling (ß-tubulin III) of cultured L5 DRG (1 day in vitro) isolated 7 days after L5 spinal nerve transection co-cultured (4 h) with either freshly isolated (control) or IL-2-stimulated NK cells.(G) Quantification of L5x DRG neurite fragmentation. One-way ANOVA: F(2,56) = 95.92, p < 0.0001. Bonferroni post-test: DRG only versus control NK, t = 0.5204, ns p > 0.05; control NK versus IL-2-stimulated NK, t = 11.45, ***p < 0.001; DRG only versus IL-2-stimulated NK, ***t = 11.86, p < 0.001. n = 6–8 regions per coverslip, n = 6 coverslips per group, two independent experimental repeats.(H) Sciatic nerve tissue sections from adult male NKp46-YFP mice 7 days after L5 spinal nerve transection injury immunolabeled with anti-GFP (NKp46, green). Arrows indicate NK cells in sciatic nerve.(I) Quantification of YFP+ events within lymphocyte gate from whole sciatic nerve homogenates. One-way ANOVA: F(3,16) = 10.61, p = 0.0004. Bonferroni post-test: L5x ipsi versus contra, t = 4.710, **p < 0.01; sham ipsi versus contra, t = 0.053, ns p > 0.05; L5x ipsi versus sham ipsi, t = 4.526, **p < 0.01 (n = 5 mice for each group).(J) ELISA quantification of granzyme B content in wild-type whole sciatic nerve after L5x injury. Student’s paired two-tailed t test: 7 days ipsi versus contra L5x, t = 8.088, p = 0.0002 (n = 7 mice).(K) ELISA quantification of granzyme B content in NKp46-DTR mice whole sciatic nerve after L5x injury. Student’s unpaired one-tailed t test: 7 days PBS versus DTx L5x ipsi, t = 11.03, p < 0.0001 (n = 5 mice per treatment).See also Figures S2 and S3.

Fig 3: Injury-Dependent Regulation of Raet1 mRNA and RAE1 Protein Expression in Sensory Neurons In Vivo, Related to Figure 4(A) The size of all NeuN+ neuronal profiles in each DRG are shown as a histograms in blue (50 µm2 bins). The number of in situ ‘spots’ per NeuN+ neuronal profile are overlayed as a scatterplot. The majority of de novo Raet1-expressing NeuN+ profiles in ipsilateral DRG appear to be in the small-medium size range (200-500 µm2). A large number of Raet1 in situ spots are also seen NeuN+ profiles of 500 µm2 and greater in ipsilateral DRG. The overall size distribution of NeuN+ profiles was not different between DRG (Kolmogorov-Smirnov test: p = 0.0513, KS D-value = 0.04502).(B) Maximum projection images of RAE1 (green) co-localized with neuronal injury marker STMN2 (magenta) in sciatic nerve 3 days after tight ligation. Note lack of STMN2 immunolabeling in uninjured contralateral nerves. Scale bars, 50 µm.(C) western blots of ligated and proximal regions of sciatic nerve tissue 7 days after tight ligation (35 µg protein loading) with pan-RAE1 antibody and neuronal cadherin as loading controls. Blots from two independent experiments.

Fig 4: Raet1 Expression Is Upregulated in Dissociated Adult DRG In Vitro and Confers NK Cell-Mediated Neurite Fragmentation(A) Microfluidic culture of adult DRG (5 days in vitro) exposed to freshly isolated (control) or IL-2-stimulated NK cells (4 h) in the neurite compartment immunolabeled with ß-tubulin III.(B) Quantification of DRG neurite density. n = 6–7 regions per microfluidic device, n = 3 devices per group, two independent experimental repeats. Student’s unpaired t test; t = 5.448, p < 0.0001.(C) Raet1 mRNA expression in adult DRG cultures by qPCR. One-way ANOVA; F (3,15) = 25.94, ***p < 0.0001 with Bonferroni post-test: #p < 0.05, **p < 0.001, ***p < 0.0001; n = 3–6 mice, or replicate cultures per time point.(D) RAE1 protein expression in adult DRG cultures 1 and 2 days in vitro (representative of three independent experiments). 25 µg protein loading.(E) Adult DRG culture (2 days in vitro) alone or co-cultured with IL-2-stimulated NK cells immunolabeled with ß-tubulin III.(F) Quantification of DRG neurite fragmentation. Student’s unpaired t test; t = 12.74, p < 0.0001. n = 6–8 regions per coverslip, n = 3 coverslips per group, two independent experimental repeats.(G) siRNA knockdown of Raet1 mRNA expression in adult DRG in vitro. Two-way ANOVA: effect of siRNA, F(1,16) = 54.02, ***p < 0.0001 with Bonferroni post-test: **p < 0.001, ***p < 0.0001. n = 3–5 cultures per group.(H) siRNA-transfected adult DRG culture (2 days in vitro) alone or co-cultured with IL-2-stimulated NK cells (4 h) immunolabeled with ß-tubulin III.(I) Quantification of DRG neurite fragmentation. One-way ANOVA; F (3,201) = 51.16, p < 0.0001 with Bonferroni post-test: ***p < 0.0001; n = 6–8 regions per coverslip, n = 6 coverslips per group, two independent experimental repeats.See also Figure S2.

Fig 5: Specific Overexpression of Raet1 in Nociceptive (TrpV1-Expressing) Neurons Reduces Sensitivity to Noxious Heat but Not Touch, Related to Figure 7(A) qPCR shows increase in Raet1 mRNA expression in DRG from TrpV1-Rae1 mice. One-way ANOVA; F (2,18) = 9.346; p = 0.0016 with Bonferroni post-test, t = 3.885, **p < 0.01. n = 5 (C57BL/6), 8 (littermates and TrpV1-Rae1 mice).(B) Latency to withdrawal from a 49°C hotplate. Cut-off time was set at 60 s. TrpV1-Rae1 mice showed reduction on heat pain perception compare to littermate controls. One-way ANOVA; F (2,21) = 22.31; p < 0.0001 with Bonferroni post-test, t = 4.831, ***p < 0.001). n = 8 mice per genotype.(C) TrpV1-Rae1 mice did not differ from littermate or wild-type controls in their mechanical sensitivity, as measured by the 50% threshold with von Frey filaments of different forces applied to the plantar surface of the hind paw. One-way ANOVA; n = 8 mice per genotype (ns, not significant).