Journal: bioRxiv

Article Title: Local Synthesis of Reticulon-1C Lessens the Outgrowth of Injured Axons by Controlling Spastin Activity

doi: 10.1101/2024.08.11.607514

Figure Lengend Snippet: Axonal Rtn-1 KD increases outgrowth by promoting Spastin’s microtubule severing. A. Representative fluorescent STED images of distal axons from microfluidic chambers containing control cortical axons or forty-four hours post-axotomy, which were stained for Rtn-1C (green), Spastin (red), and F-actin (phalloidin, blue). Insets show colocalization of Rtn-1C and Spastin. Right: Line scan analysis of Rtn-1C and Spastin highlighted in the white rectangle and their distribution along the axon. Bar graphs show the quantification of colocalization in control and axotomy conditions. Scale bar= 5 µm. Data are displayed as mean ± standard error of 6-7 axons. Unpaired t test; ∗∗p < 0.01 B. Representative images of axonal fields from microfluidic chambers 48 hours post-axotomy under different treatments: control sequence (Scrambled, Scr), Rtn-1 knockdown (Rtn-1 siRNA), and Spastin pharmacological inhibition (SPZT), and labeled with β3-tubulin (β3-tub). Scale bar represents 200 µm. C. Quantification of axon outgrowth under the treatments shown in A. The Area under the curve (AUC) was measured as the sum of the relative number of intersections at different distance ranges from the groove in the microfluidic chambers (0-500 µm; 500-1000 µm and 1000-1500 µm). Scale bar represents 10 µm. Data are displayed as mean ± standard error of 22-24 axonal fields for condition from N=3 biological replicates. One-way ANOVA followed by Tukey’s posthoc test; ∗p < 0.05; ∗∗p < 0.01. D. Fluorescence intensity profiles of β3-tubulin, Rtn-1C, and Spastin in axons and quantifications of intensity changes of indicated proteins as the fluorescence AUC fold change of the last 50 µm compared to Scr. Data are displayed as mean ± standard error of 24-40 axons for condition from N=3 biological replicates. Kruskal-Wallis followed by Dunn’s posthoc test; ∗∗∗p < 0.001 E. Axonal kymographs showing dynamic microtubule behavior in neurons with indicated treatments after axotomy, dashes represent the traces of EB3-GFP (end-binding protein 3). Scale bars represent 10 µm and 30 seconds, respectively. F. Analysis of microtubule dynamics parameters in non-injured and injured axons with different treatments: growth rate, track length, and lifetime of microtubule plus-end tracks. Data are displayed as mean ± standard error of 146-377 comets for condition from N=3 biological replicates. Kruskal-Wallis followed by Dunn’s posthoc test; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗∗p < 0.001.

Article Snippet: A 63x objective and GFP single band exciters ET filter set (excitation 470/40, emission 59022m, dichroic 59022BS) were used. mEmerald-EB3-C-20 was a gift from Michael Davidson (Addgene plasmid # 54076; http://n2t.net/addgene:54076 ; RRID:Addgene_54076)

Techniques: Control, Staining, Sequencing, Knockdown, Inhibition, Labeling, Fluorescence, Binding Assay