Sara Travaglione

Enhancement of learning and memory after activation of cerebral Rho GTPases.

The mechanism whereby the morphology and connectivity of the dendritic tree is regulated depends on an actin dynamics that, in turn, is controlled by Rho GTPases, a family of small GTP-binding proteins encompassing Rho, Rac, and Cdc42 subfamilies. Cytotoxic necrotizing factor 1 (CNF1), a protein toxin from Escherichia coli, constitutively activates Rho GTPases, thus leading to remodeling of the actin cytoskeleton in intact cells. Here, we show that the modulation of cerebral RhoA and Rac1 activity induced by CNF1 in mice leads to (i) rearrangement of cerebral actin cytoskeleton, (ii) enhanced neurotransmission and synaptic plasticity, and (iii) improved learning and memory in various behavioral tasks. The effects persist for weeks and are not observed in mice treated with a recombinant CNF1, in which the enzymatic activity was abolished by substituting serine to cysteine at position 866. The results suggest that learning ability can be improved through pharmacological manipulation of neural connectivity.

Hijacking Rho GTPases by protein toxins and apoptosis: molecular strategies of pathogenic bacteria

AbstractCertain bacterial toxins and type-III-translocated virulence factors have a peculiar property: they exert part of their actions by modulating Rho GTPases. These toxins target the actin cytoskeleton of host cells and reorganize it to their own advantage, either to facilitate macropinocytosis, which is required for invasive bacteria to enter cells, or to block pathogen sequestration by macrophages. In addition, by acting on Rho GTPases, bacteria may also interfere with the fate of host cells, favoring survival or death depending on their needs. Rho GTPases control the activation of NF-κB, which is involved in the expression of antiapoptotic proteins and mediates immunological responses as well. Here, we give a perspective on how NF-κB may participate in linking Rho-acting toxins and apoptosis.

Cytotoxic necrotizing factor 1 hinders skeletal muscle differentiation in vitro by perturbing the activation/deactivation balance of Rho GTPases.

The current knowledge assigns a crucial role to the Rho GTPases family (Rho, Rac, Cdc42) in the complex transductive pathway leading to skeletal muscle cell differentiation. Their exact function in myogenesis, however, remains largely undefined. The protein toxin CNF1 was herein employed as a tool to activate Rho, Rac and Cdc42 in the myogenic cell line C2C12. We demonstrated that CNF1 impaired myogenesis by affecting the muscle regulatory factors MyoD and myogenin and the structural protein MHC expressions. This was principally driven by Rac/Cdc42 activation whereas Rho apparently controlled only the fusion process. More importantly, we proved that a controlled balance between Rho and Rac/Cdc42 activation/deactivation state was crucial for the correct execution of the differentiation program, thus providing a novel view for the role of Rho GTPases in muscle cell differentiation. Also, the use of Rho hijacking toxins can represent a new strategy to pharmacologically influence the differentiative process.

Epithelial cells and expression of the phagocytic marker CD68: scavenging of apoptotic bodies following Rho activation

Macropinocytosis is a ruffling-driven process which drives the ingestion of large particles by both macrophages and epithelial cells. In this context, we have previously described a Rho-activating bacterial toxin from E. coli, the cytotoxic necrotizing factor 1 (CNF1), which allows epithelial cells to macropinocytose not only latex beads and bacteria, but also apoptotic cells in a fashion similar to that of professional phagocytes. We herein report that (i) epithelial cells express the typical phagocytic marker CD68, (ii) Rho activation by CNF1 varies the intracellular localization of CD68, which appears to be co-distributed, as in macrophages, with the homologous lysosomal protein Lamp-1. Together with the capability of digesting apoptotic cells following their internalization, our findings indicate that Rho-activated epithelial cells behave in most respects as professional phagocytes.

Cytotoxic necrotizing factor 1 enhances reactive oxygen species-dependent transcription and secretion of proinflammatory cytokines in human uroepithelial cells

ABSTRACT Uropathogenic Escherichia coli strains frequently produce a Rho-activating protein toxin named cytotoxic necrotizing factor type 1 (CNF1). We herein report that CNF1 promotes transcription and release of tumor necrosis factor alpha, gamma interferon, interleukin-6 (IL-6), and IL-8 proinflammatory cytokines and increases the production of reactive oxygen species (ROS) in uroepithelial T24 cells. The antioxidant N-acetyl-l-cysteine counteracts these phenomena, a fact which suggests a role for ROS-mediated signaling in CNF1-induced proinflammatory cytokine production.

CNF1 increases brain energy level, counteracts neuroinflammatory markers and rescues cognitive deficits in a murine model of Alzheimer's disease.

Overexpression of pro-inflammatory cytokines and cellular energy failure are associated with neuroinflammatory disorders, such as Alzheimers disease. Transgenic mice homozygous for human ApoE4 gene, a well known AD and atherosclerosis animal model, show decreased levels of ATP, increased inflammatory cytokines level and accumulation of beta amyloid in the brain. All these findings are considered responsible for triggering cognitive decline. We have demonstrated that a single administration of the bacterial E. coli protein toxin CNF1 to aged apoE4 mice, beside inducing a strong amelioration of both spatial and emotional memory deficits, favored the cell energy restore through an increment of ATP content. This was accompanied by a modulation of cerebral Rho and Rac1 activity. Furthermore, CNF1 decreased the levels of beta amyloid accumulation and interleukin-1β expression in the hippocampus. Altogether, these data suggest that the pharmacological modulation of Rho GTPases by CNF1 can improve memory performances in an animal model of Alzheimers disease via a control of neuroinflammation and a rescue of systemic energy homeostasis.

Escherichia coli Cytotoxic Necrotizing Factor 1 Blocks Cell Cycle G2/M Transition in Uroepithelial Cells

ABSTRACT Evidence is accumulating that a growing number of bacterial toxins act by modulating the eukaryotic cell cycle machinery. In this context, we provide evidence that a protein toxin named cytotoxic necrotizing factor 1 (CNF1) from uropathogenic Escherichia coli is able to block cell cycle G2/M transition in the uroepithelial cell line T24. CNF1 permanently activates the small GTP-binding proteins of the Rho family that, beside controlling the actin cytoskeleton organization, also play a pivotal role in a large number of other cellular processes, including cell cycle regulation. The results reported here show that CNF1 is able to induce the accumulation of cells in the G2/M phase by sequestering cyclin B1 in the cytoplasm and down-regulating its expression. The possible role played by the Rho GTPases in the toxin-induced cell cycle deregulation has been investigated and discussed. The activity of CNF1 on cell cycle progression can offer a novel view of E. coli pathogenicity.

Free radical release in C6 glial cells enriched in hexacosanoic acid: implication for X-linked adrenoleukodystrophy pathogenesis.

Free radicals have been implicated in the etiopathology of some neurological and demyelinating diseases. To evaluate their involvement in the cerebral form of X-linked adrenoleukodystrophy (cerALD) disorder, characterised by very long chain fatty acid (VLCFA) accumulation, we utilised an in vitro model using rat C6 glial cells, enriched in hexacosenoic acid (C26:0, HA). Modified cells were incubated in presence of oxidative stressors, such as bacterial endotoxin lipopolisaccharides (LPS) and human oxidised low-density lipoprotein (ox-LDL), and the production of proinflammatory cytokines, nitrite, nitrate and superoxide was determined in the supernatants. The results show that modified cells produce higher amounts of nitric oxide (NO) products and superoxide compared to native C6 cells, supporting the role of free radicals as important pathophysiological modulator of the neuroinflammatory response in ALD. This hypothesis suggests that the cerebral damage in ALD could be due to intracellular signalling activated by interaction of exogenous factors with the particular membrane fatty acid composition.

CNF1 Improves Astrocytic Ability to Support Neuronal Growth and Differentiation In vitro

Modulation of cerebral Rho GTPases activity in mice brain by intracerebral administration of Cytotoxic Necrotizing Factor 1 (CNF1) leads to enhanced neurotransmission and synaptic plasticity and improves learning and memory. To gain more insight into the interactions between CNF1 and neuronal cells, we used primary neuronal and astrocytic cultures from rat embryonic brain to study CNF1 effects on neuronal differentiation, focusing on dendritic tree growth and synapse formation, which are strictly modulated by Rho GTPases. CNF1 profoundly remodeled the cytoskeleton of hippocampal and cortical neurons, which showed philopodia-like, actin-positive projections, thickened and poorly branched dendrites, and a decrease in synapse number. CNF1 removal, however, restored dendritic tree development and synapse formation, suggesting that the toxin can reversibly block neuronal differentiation. On differentiated neurons, CNF1 had a similar effacing effect on synapses. Therefore, a direct interaction with CNF1 is apparently deleterious for neurons. Since astrocytes play a pivotal role in neuronal differentiation and synaptic regulation, we wondered if the beneficial in vivo effect could be mediated by astrocytes. Primary astrocytes from embryonic cortex were treated with CNF1 for 48 hours and used as a substrate for growing hippocampal neurons. Such neurons showed an increased development of neurites, in respect to age-matched controls, with a wider dendritic tree and a richer content in synapses. In CNF1-exposed astrocytes, the production of interleukin 1β, known to reduce dendrite development and complexity in neuronal cultures, was decreased. These results demonstrate that astrocytes, under the influence of CNF1, increase their supporting activity on neuronal growth and differentiation, possibly related to the diminished levels of interleukin 1β. These observations suggest that the enhanced synaptic plasticity and improved learning and memory described in CNF1-injected mice are probably mediated by astrocytes.

Rho-activating Escherichia coli cytotoxic necrotizing factor 1: macropinocytosis of apoptotic bodies in human epithelial cells

Some pathogenic Escherichia coli strains produce a protein toxin, named cytotoxic necrotizing factor 1 (CNF1), which permanently activates proteins belonging to the Rho family. In epithelial cells, the consequence of this activation is the rearrangement of the actin cytoskeleton and the promotion of an intense and generalized ruffling activity. This leads, in turn, to the induction of a phagocytic-like behavior called macropinocytosis that, in the case of CNF1, depends on the coordinate activation of Rho, Rac and Cdc42. Following internalization, the ingested material is discharged into Rab-7 and Lamp-1-positive acidic vesicles where it probably undergoes degradation. By exerting this activity, CNF1-activated epithelial cells might support the scavenging activity of macrophages during bacterial overgrowth.