Fig. 4: Overexpression of DOCK4 decreases heat nociception and alleviates pathological heat hyperalgesia.

a Diagram illustrating the intrathecal injection of CAS9-Easy-SAM lentivirus (LV) vector into mice, followed by behavioral and molecular biology tests conducted 14 days post-injection (Created in BioRender. https://BioRender.com/xs51blc). b–d The impact of DOCK4 overexpression on behavioral responses to Hargreaves, tail-flick, and hot plate experiments. n = 10 mice in control, n = 8 mice in DOCK4-over in b and d; n = 9 mice in control, n = 12 mice in DOCK4-over in c. t₁₆ = 3.662, P = 0.0021 in (b). t₁₉ = 2.819, P = 0.011 in 46 °C; t₁₉ = 2.406, P = 0.0256 in 48 °C; t₁₉ = 5.578, P = 0.000022 in 50 °C; t₁₉ = 3.683, P = 0.0016 in 52°C in (c). t₁₆ = 2.612, P = 0.0189 in 50 °C; t₁₆ = 3.913, P = 0.0012 in 52 °C; t₁₆ = 2.49, P = 0.0242 in 55°C in (d). e Left: the absolute time spent in the dark A chamber before (pre) versus after (post) Hargreaves pairing in control and DOCK4-overexpressed mice. Right: The CPA scores induced by Hargreaves training was assessed in control and DOCK4-overexpressed mice. n = 6 mice per group. t₁₀ = 4.026, P = 0.0024 in left; t₁₀ = 2.434, P = 0.0352 in right. f-i The impact of DOCK4 overexpression on the pathological thermal hyperalgesia induced by CFA and SNL (Created in BioRender. https://BioRender.com/xs51blc). n = 6 mice/group. F_{(1, 40)} = 63.57, P = 0.049 in 0 d, P = 0.0103 in 1 d, P = 0.000019 in 3 d, P = 0.000082 in 5 d in g; F_{(1, 50)} = 170.8, P = 0.0015 in 0 d, P = 0.000007 in 4 d, P = 0.000003 in 7 d, P = 0.00000007 in 10 d, P = 0.000000007 in 14 d in i. j, k The impact of DOCK4 overexpression on the thermal hyperalgesia induced by CFA and SNL (normalized to 0 day). n = 6 mice/group. F_{(1, 40)} = 22.99, P = 0.0016 in 3 d, P = 0.0051 in 5 d in j; F_{(1, 50)} = 39.9, P = 0.0219 in 4 d, P = 0.028 in 7 d, P = 0.0015 in 10 d; P = 0.0003 in 14 d in k. l-q The impact of DOCK4 overexpression on the pathological thermal hyperalgesia induced by PTX and LLC (Created in BioRender. https://BioRender.com/xs51blc). n = 8 mice in control, n = 9 mice in DOCK4-over in m and n; n = 9 mice in control, n = 10 mice in DOCK4-over in p and q. F_{(1, 60)} = 183.5, P = 0.000041 in 0 d, P = 0.000000002 in 5 d, P = 0.0000000004 in 7 d, P = 0.00000001 in 14 d in m; F_{(1, 60)} = 27.76, P = 0.0029 in 5 d, P = 0.0005 in 7 d, P = 0.0231 in 14 d in n; F_{(1, 68)} = 94.76, P = 0.0048 in 0 d, P = 0.0001 in 7 d, P = 0.00001 in 10 d, P = 0.00000005 in 14 d in p; F_{(1, 68)} = 18.08, P = 0.0325 in 10 d, P = 0.0005 in 14 d in q. r-t Schematic diagram for the in vivo calcium imaging of sensory neurons and the representative images of GCaMP6 fluorescence as a measure of intracellular calcium following stimulation to the hindpaw (Created in BioRender. https://BioRender.com/xs51blc). Scale bar, 100 μm. u In comparison to Dock4^WT mice, small, medium, and large-sized DRG neurons in AvCreERT2; Dock4^CKO mice exhibited no significant changes in their responses to brush stimulation ( < 500 μm² Dock4^WT, n = 33 cells; AvCreERT2; Dock4^CKO, n = 35 cells; 500-1000 μm² Dock4^WT, n = 28 cells; AvCreERT2; Dock4^CKO, n = 25 cells; >1000 μm² Dock4^WT, n = 11 cells; AvCreERT2; Dock4^CKO, n = 12 cells). v Compared to Dock4^WT mice, all sized DRG neurons in AvCreERT2; Dock4^CKO mice showed no significant changes in response to pinch stimulation ( < 500 μm² Dock4^WT, n = 105 cells; AvCreERT²; Dock4^CKO, n = 116 cells; 500-1,000 μm² Dock4^WT, n = 52 cells; AvCreERT²; Dock4^CKO, n = 61 cells; >1,000 μm² Dock4^WT, n = 13 cells; AvCreERT2; Dock4^CKO, n = 15 cells). w In comparison to Dock4^WT, small and medium-sized DRG neurons in AvCreERT2; Dock4^CKO mice showed significantly increased responses to heat stimulation ( < 500 μm² Dock4^WT, n = 61 cells; AvCreERT2; Dock4^CKO, n = 132 cells; 500-1,000 μm² Dock4^WT, n = 9 cells; AvCreERT2; Dock4^CKO, n = 25 cells; >1,000 μm² Dock4^WT, n = 3 cells; AvCreERT2; Dock4^CKO, n = 5 cells). t₁₉₁ = 3.535, P = 0.0005 in <500 μm²; t₃₂ = 3.415, P = 0.0018 in 500-1,000 μm². x Compared to Dock4^WT mice, all sized DRG neurons in AvCreERT2; Dock4^CKO mice showed no significant alters in response to capsaicin stimulation ( < 500 μm² Dock4^WT, n = 43 cells; AvCreERT2; Dock4^CKO, n = 51 cells; 500-1,000 μm² Dock4^WT, n = ² cells; AvCreERT2; Dock4^CKO, n = 5 cells; >1,000 μm² AvCreERT2; Dock4^CKO, n = 2 cells). b-e, u-x, Two-tailed Independent Student’s t test; g, i–k, m, n, p, q, Two-way ANOVA followed by Bonferroni’s multiple comparisons test. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, n.s. means not significant. Data are presented as mean ± SEM. Complete sample size and sex is provided in the Supplementary Data. Source data are provided as a Source Data file.