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.

OBJECTIVE: This study evaluated the involvement of Rho GTPases proteins in the regulation of cytodifferentiation of the SCC-4 human oral squamous cell carcinoma cell line.
METHODS: Cytokeratin and vimentin immunofluorescence and F-actin staining, assays were performed with control cells and Clostridium difficile 1, 2 and 4 μg/mL Toxin A (Rho GTPases inhibitor) treated SCC-4 cells on three-dimensional MatrigelTM for 24 h. Samples were analyzed by using confocal laser microscopy. Significances were p.

Most neurons elaborate a characteristic dendritic arbor which is physiologically important for receiving and processing of synaptic inputs. Pathologically, disturbances in the regulation of dendritic tree complexity are often associated with mental retardation and other neurological deficits. Rho GTPases are major players in the regulation of dendritic tree complexity. They are involved in many signal transduction cascades, activated at the neuronal plasma membrane, and relayed to intracellular proteins that directly rearrange the cytoskeleton. The use of siRNA technology combined with morphometric and imaging techniques allows the roles of individual Rho GTPases, such as Rac1, in dendritic branching to be examined. In this chapter we describe the establishment, transfection, and processing of a primary hippocampal cell culture. Methods to assess the complexity of dendritic arbors like the Sholl analysis, and techniques to investigate Rac1 activity in hippocampal cells, and specifically in neuronal dendrites, such as fluorescence resonance energy transfer (FRET) imaging are presented.

Bacterial pathogens developed several strategies to overcome defense systems of eukaryotic hosts. Within the infection process they need to attach to and cross through epithelial layers, escape from the innate and adaptive immune response, and find a physiological niche to survive. For this purpose bacteria developed toxins that specifically target central eukaryotic proteins, for example actin or Rho GTPases as regulators of the actin cytoskeleton. Some bacterial toxins catalyze a covalent modification of Rho GTPases to keep these molecular switches in a constitutive active or inactive state. This leads to rearrangement of the actin cytoskeleton. Toxin-treated cells show typical morphological changes depending on substrate specificity and action of the toxins. In this chapter I describe methods to illustrate how bacterial toxins may help to study the involvement of Rho GTPases in physiological and pathophysiological processes.