Journal: Acta Neuropathologica Communications
Article Title: Small molecule modulation of the p75 neurotrophin receptor promotes dendritic spine resilience to pathogenic tau species and reduces their accumulation
doi: 10.1186/s40478-026-02263-5
Figure Lengend Snippet: p75 NTR modulation mitigates oTau-induced alterations in PKC, RhoA, LIMK1, and cofilin signaling. A – I Hippocampal neurons at 21 days in vitro were collected one hour after the indicated treatments. A – E , G Neurons were treated with culture medium (CM) or recombinant oTau ± concomitant LM11A-31 (C31, 100 nM). F , H , I Neurons were treated with CM or hippocampal S1p fractions (designed to capture oTau) from Wt or PS19 mice ± LM11A-31 (100 nM) concomitantly added to the cultured neurons. A Representative Western blot images are shown ( B – D , G – I ). Western blots were quantitated as ratios of phospho (p)-protein to total protein or actin; or for calpain activity, as a ratio of cleaved (~ 145 kDa) to uncleaved (~ 250 kDa) α-fodrin. All values were subsequently normalized to the CM condition. E , F Activation of RhoA was measured using a G-LISA assay kit. Statistical significance was assessed in E , F using Kruskal–Wallis testing with Dunn’s multiple comparisons test, for E, H = 17.36, p = 0.0002; for F, H = 21.18, p = 0.0003, n = 8 protein preparations from 8 independent experiments; in B – D and G using ordinary one-way ANOVA with Dunnett’s multiple comparisons testing (JNK, F (2, 33) = 0.08885, p = 0.9152) and Kruskal–Wallis testing with Dunn’s multiple comparisons [(calpain (H = 0.5687, p = 0.7458), PKC (H = 19.76, p < 0.0001), and cofilin (H = 14.49, p = 0.0007)] , n = 13 individual protein preparations for each condition from 13 independent experiments; and in H , using Kruskal–Wallis with Dunn’s multiple comparisons test (H = 14.99, p = 0.0018), n = 8 individual protein preparations for each condition from 8 independent experiments; in I using Kruskal–Wallis testing with Dunn’s multiple comparisons test (H = 19.52, p = 0.0006), n = 10 individual protein preparations for each condition from 10 independent experiments. J , K Hippocampal neurons at 20 days in vitro were treated with CM or the slingshot inhibitor (SSHi) D3 (5 µM), or the LIMK inhibitor (LIMKi) BMS-3 (5 nM) ± oTau and/or ± C31 (100 nM). Neurons were fixed 24 h following treatment. Quantitation of dendritic spine density (number of spines per length of dendrite segment) displayed as a J batch-corrected spines/µm and K cumulative frequency distribution. Bars in B – I represent mean ± SE. Bars in J show estimated marginal mean ± SE. Statistical significance was determined using a linear model (for J , INT Spine density ~ group + batch, F(13, 166) = 14.279, p < 2e−16) followed by pairwise comparison of estimated marginal means (Bonferroni p-adj shown in figure), or in K using Kolmogorov–Smirnov testing of indicated comparisons. J , K n = 15 neurons per condition from 3 independent experiments (n = 3–6 neurons per condition per experiment). INT–inverse normal transformation
Article Snippet: For analysis of RhoA activation, a G-LISA RhoA Activation Assay Biochem kit and a total RhoA ELISA kit (Cytoskeleton, Inc. Denver, CO) were used according to the manufacturer’s instructions.
Techniques: In Vitro, Recombinant, Cell Culture, Western Blot, Activity Assay, Activation Assay, Quantitation Assay, Comparison, Transformation Assay
Cytoskeleton Inc is a verified supplier 
Cytoskeleton Inc manufactures this product 
![p75 NTR modulation mitigates oTau-induced alterations in PKC, <t>RhoA,</t> LIMK1, and cofilin signaling. A – I Hippocampal neurons at 21 days in vitro were collected one hour after the indicated treatments. A – E , G Neurons were treated with culture medium (CM) or recombinant oTau ± concomitant LM11A-31 (C31, 100 nM). F , H , I Neurons were treated with CM or hippocampal S1p fractions (designed to capture oTau) from Wt or PS19 mice ± LM11A-31 (100 nM) concomitantly added to the cultured neurons. A Representative Western blot images are shown ( B – D , G – I ). Western blots were quantitated as ratios of phospho (p)-protein to total protein or actin; or for calpain activity, as a ratio of cleaved (~ 145 kDa) to uncleaved (~ 250 kDa) α-fodrin. All values were subsequently normalized to the CM condition. E , F Activation of RhoA was measured using a G-LISA assay kit. Statistical significance was assessed in E , F using Kruskal–Wallis testing with Dunn’s multiple comparisons test, for E, H = 17.36, p = 0.0002; for F, H = 21.18, p = 0.0003, n = 8 protein preparations from 8 independent experiments; in B – D and G using ordinary one-way ANOVA with Dunnett’s multiple comparisons testing (JNK, F (2, 33) = 0.08885, p = 0.9152) and Kruskal–Wallis testing with Dunn’s multiple comparisons [(calpain (H = 0.5687, p = 0.7458), PKC (H = 19.76, p < 0.0001), and cofilin (H = 14.49, p = 0.0007)] , n = 13 individual protein preparations for each condition from 13 independent experiments; and in H , using Kruskal–Wallis with Dunn’s multiple comparisons test (H = 14.99, p = 0.0018), n = 8 individual protein preparations for each condition from 8 independent experiments; in I using Kruskal–Wallis testing with Dunn’s multiple comparisons test (H = 19.52, p = 0.0006), n = 10 individual protein preparations for each condition from 10 independent experiments. J , K Hippocampal neurons at 20 days in vitro were treated with CM or the slingshot inhibitor (SSHi) D3 (5 µM), or the LIMK inhibitor (LIMKi) BMS-3 (5 nM) ± oTau and/or ± C31 (100 nM). Neurons were fixed 24 h following treatment. Quantitation of dendritic spine density (number of spines per length of dendrite segment) displayed as a J batch-corrected spines/µm and K cumulative frequency distribution. Bars in B – I represent mean ± SE. Bars in J show estimated marginal mean ± SE. Statistical significance was determined using a linear model (for J , INT Spine density ~ group + batch, F(13, 166) = 14.279, p < 2e−16) followed by pairwise comparison of estimated marginal means (Bonferroni p-adj shown in figure), or in K using Kolmogorov–Smirnov testing of indicated comparisons. J , K n = 15 neurons per condition from 3 independent experiments (n = 3–6 neurons per condition per experiment). INT–inverse normal transformation](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_7901/pmc13077901/pmc13077901__40478_2026_2263_Fig6_HTML.jpg)
