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

et al. (2012) CNF1 Improves Astrocytic Ability to Support Neuronal Growth and Differentiation In vitro. PLoS ONE 7(4): e34115. doi:10.1371/journal.pone.0034115 CNF1 Improves Astrocytic Ability to Support Neuronal Growth and Differentiation In vitro Fiorella Malchiodi-Albedi 0 Silvia Paradisi 0 Michela Di Nottia 0 Daiana Simone 0 Sara Travaglione 0 Loredana Falzano 0 Marco Guidotti 0 Claudio Frank 0 Alessandro Cutarelli 0 Alessia Fabbri 0 Carla Fiorentini 0 Nicoletta Landsberger, University of Insubria, Italy 0 1 Department of Cell Biology and Neuroscience, Istituto Superiore di Sanita` , Rome , Italy , 2 Department of Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanita` , Rome , Italy , 3 Departmrent of Veterinary Public Health and Food Safety, Istituto Superiore di Sanita` , Rome , Italy , 4 National Centre for Rare Diseases, Istituto Superiore di Sanita` , Rome , Italy 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 1b, 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 1b. These observations suggest that the enhanced synaptic plasticity and improved learning and memory described in CNF1-injected mice are probably mediated by astrocytes. - Funding: This work was partially supported by Italia-USA Collaboration Program, Fascicolo 11US/1 (to Dr. Fiorentini) and by the Cure_FXS project (www.curefxs.eu) under the E-Rare program (to Dr. Fiorentini and Dr. Malchiodi-Albedi). No additional external funding received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Proteins belonging to the Rho GTPases family, including Rho, Rac and Cdc42 subfamilies, act as molecular switches that cycle between a GDP-bound inactive and a GTP-bound active state to transduce extracellular signals to the actin cytoskeleton. Their ability to modulate the organization of the actin network [1] plays important roles in the morphogenesis of the dendritic spines of neurons in the brain [2,3,4] and synaptic plasticity [5,6,7,8,9,10]. Although the picture is not fully resolved yet, it appears that Rac and Cdc42, which induce actin polymerization, meshwork formation and bundling, promote spine formation and maturation [11], whereas RhoA activation, which promotes actin contraction, results in spine retraction [12]. Dendritic spines are small, actinrich protrusions, and actin dynamics regulates their shape and morphological plasticity. Importantly, activation of NMDA receptors (as occurs in LTP) affects dendritic spine morphogenesis by activation of Rac1 and actin remodeling [13], linking activitydependent synaptic plasticity to Rho GTPases. In the nervous system, the Rho GTPases play a key role in several processes, and mutations in proteins involved in Rho GTPase signaling may be causative in some forms of mental retardation. We have found that a bacterial protein toxin from Escherichia coli, which activates the Rho GTPases, can improve learning and memory in mice [14,15]. This toxin, named cytotoxic necrotizing factor 1 (CNF1), acts by blocking the Rho GTPases in their activated, GTP-bound state by catalyzing the deamidation of a single glutamine residue of the Rho molecules, thus impeding GTP hydrolysis and leading to their persistent activation [16,17]. CNF1 modulation of cerebral RhoA and Rac1 activity in mice rearranges cerebral actin cytoskeleton, enhances neurotransmission and synaptic plasticity and improves cognitive performances [14,15]. Also, CNF1 counteracts the formalin-induced inflammatory pain in mice, after both peripheral and central administration, further sustaining its ability in modulating CNS pathophysiology [18]. Very recently, we have demonstrated that CNF1, by directly modulating the brain Rho GTPases, triggers structural remodeling and functional plasticity into the adult rat visual cortex [19] and improves the behavioral phenotype in a mouse model of Rett syndrome [20]. Therefore, CNF1 can be viewed as a new pharmacological agent able to enhance the changes in neuronal connectivity associated with memory for by which the toxin can ameliorate the neuronal function. To address this question, we have performed a study on primary neuronal cultures of rat embryonic brain with the aim of investigating the effects of CNF1 on in vitro neuronal growth and differentiation, focusing on the development of dendritic tree and synapse formation. Our data show that while direct administration of CNF1 to neuronal cultures has a harmful effect on neuronal maturation, hippocampal neurons conditioned by CNF1-treated mation and to improve neuronal plasticity. It remains, however, to define the mechanisms astrocytes show an increase in dendrite growth and synapse formation, sustaining a role for CNF1 in improving astrocytic neurosupportive activity. CNF1 modifies neuritic tree and synapse development in neurons during differentiation To analyze the effects of CNF1 on neuronal differentiation, hippocampal cultures were treated at day-in-vitro (DIV) 2 with CNF1 and fixed at DIV 14. At this stage, staining with the nuclear dye Hoechst 33342 showed that, although ce (...truncated)