Effects of the Rho GTPase-activating toxin CNF1 on fibroblasts derived from Rett syndrome patients: A pilot study

Abstract

The bacterial product CNF1, through its action on the Rho GTPases, is emerging as a modulator of crucial signalling pathways involved in selected neurological diseases characterized by mitochondrial dysfunctions. Mitochondrial impairment has been hypothesized to have a key role in paramount mechanisms underlying Rett syndrome (RTT), a severe neurologic rare disorder. CNF1 has been already reported to have beneficial effects in mouse models of RTT. Using human RTT fibroblasts from four patients carrying different mutations, as a reliable disease-in-a-dish model, we explored the cellular and molecular mechanisms, which can underlie the CNF1-induced amelioration of RTT deficits. We found that CNF1 treatment modulates the Rho GTPases activity of RTT fibroblasts and induces a considerable re-organization of the actin cytoskeleton, mainly in stress fibres. Mitochondria of RTT fibroblasts show a hyperfused morphology and CNF1 decreases the mitochondrial mass leaving substantially unaltered the mitochondrial dynamic. From a functional perspective, CNF1 induces mitochondrial membrane potential depolarization and activation of AKT in RTT fibroblasts. Given that mitochondrial quality control is altered in RTT, our results are suggestive of a reactivation of the damaged mitochondria removal via mitophagy restoration. These effects can be at the basis of the beneficial effects of CNF1 in RTT.

Keywords: CNF1; Rett syndrome; Rho GTPases; actin; fibroblasts; mitochondria.

FIGURE 1

Effects of CNF1 on RTT fibroblasts cytoskeleton. (A) Fluorescence micrographs of WT and RTT1 fibroblasts treated with CNF1 for 4 and 24 h and stained with FITC‐phalloidin to visualize the actin cytoskeleton. (B) Graph showing the actin fluorescence intensity (Fold activation). (C) Fluorescence micrographs of CNF1‐ and mCNF1‐treated RTT1 cells stained with TRITC‐phalloidin. (D and E) Immunoblot showing the amount of activated Rho (D) and Rac (E) after different times of CNF1 exposure, obtained by pull‐down assays of fibroblasts from patient RTT1. Immunoblots were analysed by densitometry, as described in Materials and Methods. The graphs (right panels) report the relative activity of Rho/Rac normalized as a function of the total amount of Rho/Rac protein loaded. Data are expressed relative to the values of untreated cells (control = 1, dashed line). Data are median (interquartile range) ±1.5*interquartile range. Statistical significance was assessed using One‐way anova + Dunnett's post hoc test or Kruskal–Wallis + Dunn's post hoc tests. (*) p < 0.1; *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 2

CNF1 reduces mitochondrial mass in RTT fibroblasts. (A) Fluorescence micrographs of WT and RTT fibroblasts treated with CNF1 and stained with Mitotracker Red to visualize mitochondria. (B and C) Flow cytometry analysis of Mitotracker Green: (B) representative image and (C) graph showing the intensity of fluorescence. (D) Digital droplet PCR and (E) real‐time PCR showing the ratio of the mtDNA/nDNA in RTT1 and RTT2 fibroblasts treated with CNF1. Data in (C) are expressed relative to the values of untreated cells (control = 1, dashed line). Data are mean ± SEM or median (interquartile range) ±1.5*interquartile range. Statistical significance was assessed using One‐way anova + Dunnett's post hoc test or Kruskal–Wallis + Dunn's post hoc tests. *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 3

CNF1 activity on mitochondrial functionality. (A) Flow Cytometry analysis of JC‐1 stained cells. (B) Fluorescence micrographs of RTT1 fibroblasts stained with Hoechst 33258 to visualize nuclei. (C and D) Graph showing the percentage of (C) depolarized and (D) hyperpolarized WT and RTT fibroblasts following CNF1 exposure, as obtained by flow cytometry analysis. (E and F) Western blot analysis of phosphor‐AKT (pAKT) in whole‐cell lysates of RTT1 fibroblasts after treatment with CNF1. Data are expressed relative to the values of untreated cells (control = 1, dashed line). Data are median (interquartile range) ±1.5*interquartile range. Statistical significance was assessed using One‐way anova + Dunnett's post hoc test or Kruskal–Wallis + Dunn's post hoc tests. Data are mean ± SEM. Statistical significance was assessed using the Kruskal–Wallis test. (*) p < 0.1; ***p < 0.001.

FIGURE 4

CNF1 induces autophagic markers in RTT fibroblasts. Western blot analysis of the autophagic markers (A) PINK1 and (C) LC3. In the boxplots (B and D), PINK1 and LC3 were normalized as a function of the GAPDH protein. Data are expressed relative to the values of untreated cells (control = 1, dashed line). Data are median (interquartile range) ±1.5*interquartile range. Statistical significance was assessed using One‐way anova + Dunnett's post hoc test or Kruskal–Wallis + Dunn's post hoc tests. Data are mean ± SEM. Statistical significance was assessed using the Kruskal–Wallis test. *p < 0.05; **p < 0.01.

FIGURE 5

p62 co‐localizes with small mitochondria in CNF1‐treated RTT fibroblasts. Western blot analysis of the autophagic markers p62 (A) with the respective boxplots (B) normalized as a function of the GAPDH protein. Fluorescence micrographs of control and CNF1 treated RTT1 fibroblasts showing colocalization of the autophagic marker p62 and mitochondria (C). Insets show a magnification of small mitochondria colocalized with p62. Data are expressed relative to the values of untreated cells (control = 1, dashed line). Data are median (interquartile range) ±1.5*interquartile range. Statistical significance was assessed using One‐way anova + Dunnett's post hoc test or Kruskal–Wallis + Dunn's post hoc tests. ***p < 0.001.