Abstract
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.
Highlights
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
We have demonstrated that Cytotoxic Necrotizing Factor 1 (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]
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
Summary
Proteins belonging to the Rho GTPases’ family, including Rho, Rac and Cdc 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]. 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
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