14-3-3 physically interacts with spastin in vivo and vitro.

(A) Mass spectrum of peptides which was pull-downed by GST-spastin from proteins of the spinal cord tissue (near 1 cm around the T10 level), the representative peptides of 14-3-3 were shown. (B) Immunoprecipitation assay using spastin antibody was performed and Pan 14-3-3 antibody was used for western blotting. (C) Hippocampal neurons were stained with 14-3-3s (green) and spastin (red) antibodies, the growth cone (C1) and axon shaft (C2) profile were shown. (D) Localization of spastin (red line) and 14-3-3 (green line) protein in the hippocampal neurons were analyzed and the intensity of the white dotted line across the neurite compartment were measured. (E) Purified GST and GST-14-3-3s fusion proteins were subjected to sodium dodecyl-sulfate polyacrylamide gel electrophoresis and coomassie blue staining was confirmed the successful purification of relative proteins. (F) GST fusion proteins were used to pull down the lysates of the spinal cord and spastin antibody was used for western blotting. (G) HEK 293T cells were transfected with Flag tagged 14-3-3s and GFP tagged spastin, the cell lysates were subjected to GFP-Trap assay, Flag antibody was used to detect the presence of 14-3-3s.

Ser233 in spastin is essential for the interaction with 14-3-3 and important for neurite outgrowth.

(A) Schematic illustration of the gene truncation strategy for the modular domain of spastin. (B) Truncation mutants of spastin together with Flag tagged 14-3-3 were transfected into HEK 293T cells, and cell lysates were immunoprecipitated by GFP-Trap assay. Flag antibody were used for detecting the flag tagged 14-3-3 fusion protein. (C) The microtubule binding domain (MTBD) truncated GFP tagged spastin were transfected together with Flag tagged 14-3-3 into HEK 293T cells, and subjected to GFP trap assay. (D) HEK 293T cells were transfected with GFP-spastin and then treated with or without staurosporine for 1 hour. Cell lysates were pull down by GST 14-3-3 fusion protein and GFP antibody were used for western blotting. (E) The binding sites between 14-3-3 and spastin were predicted using the scansite 2.0 software and whether these sites could be phosphorylated were confirmed by phosphosite plus website. (F) Serine 233 and Serine 562 of spastin were mutated to alanine, these GFP fusion mutations were transfected together with Flag tagged 14-3-3, then cell lysates were subjected to GFP-trap assay. Flag antibody was used to detect the binding ability between spastin mutations and 14-3-3. (G) Hippocampal neurons were grown on DIV 2 and transfected with spastin Ser233 mutations (Spastin S233A, Spastin S233D), then fixed on DIV 3. Representative images of transfected neurons as indicated were shown. Quantitative analysis of the length of neurites (H) and the number of neurites (I) were shown (n≥25 per group). All error bars are SEM. *P <0.05. Scale bar: 100 μm.

14-3-3 protein protects spastin by Ser233 phosphorylation from degradation through ubiquitin pathway.

(A) GFP tagged spastin together with Flag tagged 14-3-3 (B) or without was transfected into HEK 293T cells. Following the addition of protein synthesis inhibitor (cycloheximide, CHX), cell lysates were subjected to western blot to detect the GFP tagged spastin protein levels. (C, D) Western blot analysis of HEK 293T cell lysates transfected with spastin mutations (S233A or S233D) after treating with cycloheximide at different indicated times. (E) Quantitative analysis of spastin protein levels after treating by cycloheximide with or without 14-3-3 protein. (F) Quantitative analysis of GFP tagged spastin protein levels upon transfected with different spastin mutations. (G) HEK 293T cells were transfected with GFP tagged spastin and Flag tagged ubiquitin, together with R18 or not, cells were harvested and subjected to immunoprecipitation using GFP antibody. Flag antibody was used to detect the ubiquitin band. (H) COS7 cells were transfected with indicated GFP tagged spastin with or without Flag tagged ubiquitin for 24 hours. Then, cells were fixed and stained with tubulin antibody to visualize microtubule arrangements. (I) The normalized quantitative analysis of microtubule fluorescence intensity among indicated groups (n≥20 pictures per group). *P < 0.05, **P < 0.01, ***P < 0.001. Scale bar: 50 μm, 10 μm.

14-3-3 agonist Fusicoccin (FC-A) promotes the repair of neurite outgrowth and regeneration after injury via spastin.

(A) The surface structure and (B) secondary structure of molecular docking model between 14-3-3 protein with FC-A (PDB:2O98) or R18 (PDB:1A38). (C) HEK 293T cells were transfected with GFP tagged spastin and Flag tagged 14-3-3, cell lysates were incubated with FC-A or R18 and then subjected to GFP-trap assay. Flag antibody were used to detect the 14-3-3 binding. (C) Hippocampal neurons (DIV2) were transfected with GFP to visualize its morphology. Neurons were then incubated with glutamate (120 μM) to induce injury, the neurite breakages and swellings were found in C1 and C2. (D) The injured neurons were transfected with spastin and incubated with FC-A, R18 or spastazoline for 24 hours, then cells were fixed and images were taken by confocal microscopy. Neurons were traced using Image J Pro Plus and quantitative analysis of the length of primary neurites (E), secondary neurites (F) and total neurites (G) were performed. Quantitative analysis of the number of primary neurites (H), secondary neurites (I) and total neurites (J). *P < 0.05, **P < 0.01, ***P < 0.001. Scale bar: 50 μm. (K) Primary cortical neurons were grown and scratch assays were performed at DIV7. FC-A or spastazoline were applied and incubated for 36 hours. Then, cells were stained with βIII-tubulin to visualize the morphology of neurites. (L) Quantitative analysis of the normalized axon regeneration rate. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bar: 100 μm.

14-3-3/spastin pathway is involved in nerve regeneration after spinal cord injury by targeting MTs.

(A) After spinal cord contusion, the spinal cord tissues of the lesion site (near 1 cm) at indicated times (1, 3, 7 14, and 30 DPI) were ground and lysed, then subjected to western blot. (B) Quantitative analysis of the 14-3-3 protein expression (n=3 per group). (C) The lesion site of spinal tissue in the sham group and the SCI group were stained with Pan 14-3-3 (red) and βIII tubulin (green). The arrows mean the elevated 14-3-3 protein in the neuronal compartment. (D) 14-3-3 agnoist FC-A and spastin inhibitor spastazoline were admisnistrated after spinal cord contusion. At 45 DPI (Days post-injury), the tissues were fixed and embedded, then cut horizontally and longitudinally. H&E and LFB (Luxol fast blue stain) were stained. The spinal cord demyelination was shown inside the area of the dotted line. Scale bar, 200 μ m. (E) The slices were then subjected to immunofluorescence staining, and GFAP (green) was stained to label astrocytes, NF (red) was stained to label neurons and 5-HT (magenta) was stained to label monoaminergic axons. The lesion site in different groups was boxed and enlarged in (E1)(E2)(E3)(E4). Scale bar, 400 μm, and 100 μm. (F) The spinal cord slices were stained with acetylated tubulin (Stable MTs which lack of dynamics) and β-tubulin (total tubulin). Scale bar, 50 μm. (G) The normalized intensity of NF was quantified (n=5 animals per group). Mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. (H) The normalized ratio of acetylation tubulin to total tubulin was calculated by the intensity value of acetylated tubulin divided by total tubulin (n=5 pictures per group). Mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

14-3-3/spastin pathway coordinates locomotor recovery after spinal cord contusion.

(A) Schematic illustration of relative treatments and examinations at different indicated times. (B) The max contact areas of the right hindlimb were recorded and analyzed by the Catwalk software. (C) The footfall patterns were visualized by the Catwalk software. The blue dot represents the start of a pattern. The red dot represents the gait was not part of a pattern. The yellow dot represents that the gait was not taken into account. The green one means the gait belongs to a normal part of a pattern. (D) BMS score at different indicated days after injury and data were represented as mean±SEM. (n=5 group, data were analyzed by one way ANOVA and Bonferroni’s multiple-comparison test were used for post hoc comparisons. (E) The quantitative analysis of the hindlimb (right) max contact area. (n=5 animals per group). (F) Footprint analysis of the mice at 6 weeks after spinal cord contusion. The forelimbs were marked with blue and the hindlimbs were marked with red. Scale bar: 2.5 cm. (G) The normalized regularity index of the footfall pattern calculated by Catwalk software (∼100 % in the sham group, n=5 animals per group). (H) Quantatitive analysis of the stride length in the footprint assay. The mean stride length of each walk was counted, n=5 animals per group). (I) Motor evoked potentials (MEPs) were recorded at 7 weeks after spinal cord contusion. The craniotomy was performed and the M1 region of the sensorimotor cortex were stimulated by single square-wave stimulus of 0.5 mA, 0.5 ms duration, 2 ms time delay, and 1 Hz. The MEP were recorded with the signals detected by the electrode on the gastrocnemius muscle. The representative histograms of the amplitude-times are shown. The arrows indicated the stimulus. (J) The current amplitudes of the MEP were quantified, n=5 animals per group. Mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

FC-A promotes axon regeneration in a T10 lateral hemisection spinal cord injury model.

(A) The spinal cords of adult mice were subjected to lateral hemisection and administrated by intraperitoneal injection with FC-A or spastazoline. Sagittal view of 5-HT-immunoreactive raphespinal fibers in the spinal cord on 17 DPI. Scale bar, 500 μm and 100 μm. (B) Sagittal view of neurofilaments which was MBP positive in the white manner of the lesion site of the spinal cord. Scale bar, 50 μm. (C) The normalized quantification of 5-HT immunoreactive fluorescence intensity (0.5 mm caudal to the lesion site, n=5 animals per group). (D) The normalized quantification of NF (Neurofilament) immunoreactive fluorescence intensity in the lesion site (n=5 animals per group). (E) The locomotor function of mice after SCI were scored at the indicated time points according to the standard of Basso Mouse Scale (BMS). (F) Foot fault test was performed and the total steps and steps drop out of the right hindlimb were recorded. The rate of the foot errors was analyzed, n=5 animals per group. *P < 0.05, **P < 0.01, ***P < 0.001. (G) (H)The footprint assay was performed and stride length (G) and stride width (H) quantitative analyzed. Data were presented as mean ± SEM, n=5 animals per group, the mean value of each animal’ s walk were calculated.