Redox signaling modulates axonal microtubule organization and induces a specific phosphorylation signature of microtubule-regulating proteins

Abstract

Many life processes are regulated by physiological redox signaling, but excessive oxidative stress can damage biomolecules and contribute to disease. Neuronal microtubules are critically involved in axon homeostasis, regulation of axonal transport, and neurodegenerative processes. However, whether and how physiological redox signaling affects axonal microtubules is largely unknown. Using live cell imaging and super-resolution microscopy, we show that subtoxic concentrations of the central redox metabolite hydrogen peroxide increase axonal microtubule dynamics, alter the structure of the axonal microtubule array, and affect the efficiency of axonal transport. We report that the mitochondria-targeting antioxidant SkQ1 and the microtubule stabilizer EpoD abolish the increase in microtubule dynamics. We found that hydrogen peroxide specifically modulates the phosphorylation state of microtubule-regulating proteins, which differs from arsenite as an alternative stress inducer, and induces a largely non-overlapping phosphorylation pattern of MAP1B as a main target. Cell-wide phosphoproteome analysis revealed signaling pathways that are inversely activated by hydrogen peroxide and arsenite. In particular, hydrogen peroxide treatment was associated with kinases that suppress apoptosis and regulate brain metabolism (PRKDC, CK2, PDKs), suggesting that these pathways play a central role in physiological redox signaling and modulation of axonal microtubule organization. The results suggest that the redox metabolite and second messenger hydrogen peroxide induces rapid and local reorganization of the microtubule array in response to mitochondrial activity or as a messenger from neighboring cells by activating specific signaling cascades.

Keywords: Axon; Hydrogen peroxide; Microtubules; Redox signalling; Tau.

Fig. 1

Subtoxic concentrations of hydrogen peroxide decrease microtubule polymer in axon-like processes and increase their dynamics. A. Combined MTT (blue) and LDH (red) assay showing the effect of 3 h H₂O₂ exposure on neuronally differentiated PC12 cells. Mean ± SEM of 4 experiments, each carried out in triplicates, are shown. Statistically significant differences as determined by one-way ANOVA followed by a Dunnett post hoc test showed significant differences from a control at concentrations ≥225 μM. B. Bar graphs showing the increase in intracellular ROS level in response to H₂O₂. Each data point represents an independent experiment normalized to a control. Mean ± SEM is shown. Statistically significant differences between treated and control cells as determined by one-sample t-tests are indicated (∗∗p < 0.01). C. Representative time-lapse images of a fluorescence decay after photoactivation (FDAP) experiment in an axon-like process. A 6 μm long segment (white box) in the middle of a process was photoactivated and the fluorescence decay in this area was monitored over time. A schematic representation of the FDAP approach and the expressed construct is shown on the left. D. FDAP diagrams after photoactivation of PAGFP-α-Tubulin expressing cells show an increased fluorescence decay after treatment with H₂O₂. Mean values ± SEM of 29 (control) and 13 (150 μM H₂O₂) cells are shown. E. Scatterplots of the association (k∗_on) and dissociation rate constants (k_off), determined by modelling the FDAP plots from (D), show that H₂O₂ decreases k∗_on and increases k_off. Statistically significant differences between treated and control cells, determined by unpaired two-tailed Student's t-tests, are indicated. ∗p < 0.05, ∗∗p < 0.01. F. The scatterplot of the amount of polymerized tubulin determined from the association and dissociation constants in (E) shows that H₂O₂ reduces the amount of polymerized tubulin in axon-like processes. Statistically significant differences determined by unpaired two-tailed Student's t-tests are indicated. ∗∗∗p < 0.001. G. A schematic representation of the conversion between the oxidized and reduced forms of SkQ1 as a mitochondria-targeted antioxidant is shown. TPP, which lacks the antioxidant quinone moiety, is indicated. A bar graph is displayed on the right showing that pretreatment with SkQ1 abolished the increase in intracellular ROS levels in response to H₂O₂. H. Schematic representation of the timeline of the experiment to determine the effect of pretreatment with SkQ1 or a control (TPP) on microtubule polymerization. I. Scatterplot of the amount of polymerized tubulin with the control (TPP) and SkQ1, showing that pretreatment with SkQ1 prevents H₂O₂-induced microtubule depolymerization in axon-like processes as determined by FDAP experiments. Shown are mean values ± SEM of 16, 13 (TPP) and 28, 23 (SkQ1) cells for control and H₂O₂-treatment, respectively. Statistically significant differences determined by unpaired two-tailed Student's t-tests are indicated. ∗∗p < 0.01. J. Shown is the time course of the experiment to determine the effect of pretreatment with the microtubule-stabilizer EpoD on H₂O₂-induced microtubule depolymerization. K. Scatterplot of the amount of polymerized tubulin showing that pretreatment with EpoD prevents H₂O₂-induced microtubule depolymerization. Shown are mean values ± SEM of 21, 14 (carrier, 0.01 % DMSO) and 29, 17 (1 nM EpoD) cells for control and H₂O₂-treatment, respectively. Statistically significant differences determined by unpaired two-tailed Student's t-tests are indicated. ∗∗p < 0.01. L. Morphometric analysis of the effect of subtoxic H₂O₂ concentrations on neuronally differentiated PC12 cells, showing that H₂O₂ reduces process length and cell body area. Fluorescence micrographs of representative cells fixed and stained against β-tubulin using a fixation extraction protocol are shown on the left. Violin plots (with mean and standard deviation) and bar graphs (with mean) show total and mean process length, mean process number per cell, and cell body radius. Cells were treated with H₂O₂ (150 μM) for 3 h and compared with untreated controls. Data from 120 cells (150 μM H₂O₂) and 106 cells (control) from 3 independent experiments are shown. Statistically significant differences determined by unpaired two-tailed Student's t-tests are indicated. ∗∗∗p < 0.001. Scale bar, 20 μm.

Fig. 2

Subtoxic hydrogen peroxide modulates the structure of the axonal microtubule array and reduces microtubule mass. A. Shown on the left is a schematic representation of the single molecule localization microscopy (SMLM) approach to quantify MT organization. An indication of the average microtubule spacing in axon-like processes of differentiated PC12 is included. A total internal reflection fluorescence (TIRF) image in the highly inclined and laminated optical sheet (HILO) mode of the 3D-rendered SML data and a 150 nm optical section with fire color code are shown in the middle. Microtubule filaments as extracted from a selected ROI using SIFNE (single-molecule localization microscopy image filament network extractor) are shown in the rainbow color code on the right. Scale bar 10 μm. B. Boxplots showing the mass, density, mean length and straightness of microtubules. Each data point represents the average of a single cell (control, 6 cells with n = 902 individual microtubules; H₂O₂, 7 cells with n = 457 individual microtubules). H₂O₂ treatment (150 μM for 3 h) reduces microtubule mass and increases mean microtubule length. Statistically significant differences determined by unpaired two-tailed Student's t-tests are indicated. ∗p < 0.05, ∗∗p < 0.01. C. Distribution of microtubule lengths in a relative abundance histogram. Dotted lines show mean microtubule length under control conditions and with H₂O₂. The bin width was set to 0.5 μm and the area of the histogram bars is 1.0. H₂O₂ shifts the length distribution by reducing the proportion of short microtubules (0.5–2.5 μm length) and increasing the number of long microtubules (>8 μm). D. Extracted microtubules from an axon section of a SMLM image of H₂O₂-treated dorsal root ganglion (DRG) neurons compared to a control cell are shown on the left. Boxplots on the right show that treatment with 150 μM H₂O₂ for 3 h reduces microtubule mass but does not alter microtubule density, mean length, and straightness compared to an untreated control group. Each data point represents the average of a single cell (control, 13 cells with n = 378 individual microtubules; H₂O₂, 12 cells with n = 427 individual microtubules). Statistically significant differences determined by unpaired two-tailed Student's t-tests are indicated. ∗p < 0.05. Scale bar, 5 μm.

Fig. 3

Hydrogen peroxide-induced changes in the microtubule array reduce the proportion of mobile vesicles but have no effect on the speed and velocity of vesicle transport, or the interaction of the axonal tau protein with microtubules. A. Tracking APP vesicles in a PC12 cell process using eGFP-tagged APP and an autoregressive motion algorithm. The first micrograph shows an overview of a cell expressing APP-eGFP. The images on the right show selected times of the APP vesicle movement from the part of the process marked by the white box in the overview image. Arrows point to a moving (white) and a stationary vesicle (red). Scale bars, 10 μm (overview) and 1 μm (time lapse). B. The bar graph shows proportions of mobile vesicles in the axon-like process under control conditions and in response to H₂O₂. Velocity and speed of mobile vesicles are shown in the scatter plots on the right (mean ± SEM of n = 25 cells with 1595 trajectories (control) and 23 cells with 1209 trajectories (H₂O₂); each point represents an average value for one analyzed cell). Statistically significant differences determined by unpaired two-tailed Student's t-tests are indicated. ∗∗p < 0.01. C. Fraction of mobile vesicles in cells pretreated with 1 nM EpoD according to the timeline shown in Fig. 2D. Comparison with vehicle (0.01 % DMSO) shows no effect of EpoD on the fraction of mobile vesicles. Each data point represents an average value for a corresponding cell (mean ± SEM of 20–25 cells with 1276–1720 trajectories is shown). D. Effect of H₂O₂ on the interaction of tau with microtubules. A schematic representation of the FDAP approach and the expressed tau construct is shown on the left. FDAP plots after photoactivation of PAGFP-Tau expressing cells (middle) show a similar fluorescence decay with and without H₂O₂. Likewise, the scatterplots of the association (k∗_on) and dissociation rate constants (k_off) (right) show no statistically significant differences. Mean values ± SEM of 25 (control) and 14 (H₂O₂) cells are shown.

Fig. 4

Subtoxic hydrogen peroxide modulates the phosphorylation state of microtubule-regulating proteins. A. Schematic representation of the approach for proteomics and phosphoproteomics analyses of differentiated model neurons treated with hydrogen peroxide compared to control. B. Bar plot with the top enriched GO-terms for molecular function of all differentially regulated proteins in response to H₂O₂. The length of the bar indicates the p-value, reflecting the enrichment significance of each term. Note an enrichment of proteins mostly involved in RNA processing and redox modulation. C. Volcano plot showing up- and down-regulated proteins in hydrogen peroxide-treated cultures compared to controls. Log₂ fold changes are plotted against -log10 p-values. Significant upregulation upon H₂O₂ treatment is shown in blue, downregulation in dark grey. Members of different groups of microtubule-related proteins are indicated. The axes are cut for representation purposes (x-axis from −3 to 3, y-axis from 0 to 60); all the proteins above the limits are shown as points at each limit border. Members of different groups of microtubule-related proteins are indicated in different colors, with significantly changed ones labelled with their gene names. Only one protein of the more than 30 identified tubulin and microtubule-regulating proteins (MAPRE3) shows a slight upregulation. D. Bar plot with the top enriched GO-terms for cellular components of all proteins with upregulated phosphosites in response to H₂O₂. The length of the bar indicates the p-value, reflecting the enrichment significance of each term. Note enrichment of nuclear components, organelles and proteins of the cytoskeleton. E. Volcano plot showing up- and down-regulated phosphosites in hydrogen peroxide-treated cultures compared to controls. The axes are cut for representation purposes and all phosphosites above the limits are shown as points at each limit border. Coloring and labelling as in C.

Fig. 5

Hydrogen peroxide and arsenite induce a specific phosphorylation signature of MAP1B as a major target of microtubule-regulating proteins. A. Volcano plot showing upregulated phosphosites in arsenite-treated cells compared to control. Members of different groups of microtubule-regulating proteins are indicated by the same color code as in Fig. 4C. Log₂ fold changes are plotted against -log10 p-values. Significant upregulation upon arsenite treatment is shown in orange, downregulation in dark grey. The axes are cut for representation purposes and all phosphosites above the limits are shown as points at each limit border. B. Venn diagram showing low overlap of phosphosites of microtubule-related proteins upregulated in response to hydrogen peroxide or arsenite. C, D. Distribution of upregulated phosphosites on different microtubule-related proteins in response to hydrogen peroxide (C) or arsenite (D). Note that MT-binding proteins and especially MAP1B are the main target. A list of all altered phosphorylation sites in microtubule-regulating proteins can be found in Supplementary Table 1 (hydrogen peroxide) and Supplementary Table 2 (arsenite). E. Graphical representation of the different phosphosites on MAP1B that are upregulated in response to hydrogen peroxide or arsenite. The blue bar shows the microtubule-binding region according to the deletion study by Ref. [80]. Phosphorylated epitopes recognized by the monoclonal antibody SMI-31 [71], which detects disease-associated mode I phosphorylation sites, which cause a loss of the microtubule stabilizing activity, are indicated by the dark green bar.

Fig. 6

Cell-wide phosphoproteome analysis of predicted upstream kinases in neuronally differentiated cells reveals a pattern of inversely regulated kinases by hydrogen peroxide and arsenite. A. Kinase enrichment analysis of the phosphoproteomics data to identify the pattern of kinases responsible for increased phosphorylation of all cellular proteins in response to hydrogen peroxide (left) and arsenite (right). Note that there is no overlap between significantly upregulated upstream kinases with hydrogen peroxide and arsenite. B. Venn diagrams showing upstream kinases leading to a reverse change in phosphorylation, i.e. induction of increased phosphorylation with hydrogen peroxide and reduced phosphorylation with arsenite (top) and increased phosphorylation with arsenite and reduced phosphorylation with hydrogen peroxide (bottom). The respective upstream kinases with reverse change are indicated below the corresponding Venn diagram. C. Phosphatase mapping to identify specific phosphatase activity based on the dephosphorylated residues of all proteins in response to hydrogen peroxide and arsenite. Note that the three highest ranked protein phosphatases identified are the same after treatment with hydrogen peroxide and arsenite.

Fig. 7

Schematic representation of the effect of hydrogen peroxide on axonal microtubule organization and microtubule-dependent transport. Hydrogen peroxide diffuses or distributes through cells and tissues by passive transport and can reach neighboring axons when produced in oligodendrocytes or astrocytes. In addition, it is also produced by mitochondria in the axons. In axons, H₂O₂ causes a reorganization of the microtubule cytoskeleton toward a lower microtubule mass, which reduces the efficiency of vesicle transport. Figure created with BioRender.com.