CNF1 Enhances Brain Energy Content and Counteracts Spontaneous Epileptiform Phenomena in Aged DBA/2J Mice

RESEARCH ARTICLE CNF1 Enhances Brain Energy Content and Counteracts Spontaneous Epileptiform Phenomena in Aged DBA/2J Mice Sara Travaglione1☯, Giulia Ballan1☯, Andrea Fortuna1, Alberto Ferri2, Marco Guidotti3, Gabriele Campana4, Carla Fiorentini1‡, Stefano Loizzo1‡* 1 Department of Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161, Roma, Italy, 2 Institute of Cellular Biology and Neurobiology, CNR, Via del Fosso di Fiorano 64/65, 00143, Roma, Italy, 3 Department of Veterinary Public Health and Food Safety, Viale Regina Elena 299, 00161, Roma, Italy, 4 Department of Pharmacy and Biotechnology, University of Bologna, Via Irnerio 48, 40126, Bologna, Italy ☯ These authors contributed equally to this work. ‡ These authors also contributed equally to this work. * OPEN ACCESS Citation: Travaglione S, Ballan G, Fortuna A, Ferri A, Guidotti M, Campana G, et al. (2015) CNF1 Enhances Brain Energy Content and Counteracts Spontaneous Epileptiform Phenomena in Aged DBA/ 2J Mice. PLoS ONE 10(10): e0140495. doi:10.1371/ journal.pone.0140495 Editor: Giuseppe Biagini, University of Modena and Reggio Emilia, ITALY Received: June 18, 2015 Accepted: September 25, 2015 Published: October 12, 2015 Copyright: © 2015 Travaglione et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper. Abstract Epilepsy, one of the most common conditions affecting the brain, is characterized by neuroplasticity and brain cell energy defects. In this work, we demonstrate the ability of the Escherichia coli protein toxin cytotoxic necrotizing factor 1 (CNF1) to counteract epileptiform phenomena in inbred DBA/2J mice, an animal model displaying genetic background with an high susceptibility to induced- and spontaneous seizures. Via modulation of the Rho GTPases, CNF1 regulates actin dynamics with a consequent increase in spine density and length in pyramidal neurons of rat visual cortex, and influences the mitochondrial homeostasis with remarkable changes in the mitochondrial network architecture. In addition, CNF1 improves cognitive performances and increases ATP brain content in mouse models of Rett syndrome and Alzheimer's disease. The results herein reported show that a single dose of CNF1 induces a remarkable amelioration of the seizure phenotype, with a significant augmentation in neuroplasticity markers and in cortex mitochondrial ATP content. This latter effect is accompanied by a decrease in the expression of mitochondrial fission proteins, suggesting a role of mitochondrial dynamics in the CNF1-induced beneficial effects on this epileptiform phenotype. Our results strongly support the crucial role of brain energy homeostasis in the pathogenesis of certain neurological diseases, and suggest that CNF1 could represent a putative new therapeutic tool for epilepsy. Funding: The authors received no specific funding for this work. Competing Interests: The authors have read the journal's policy and the authors of this manuscript have the following competing interests: The authors declare the patent PCT/EP2013/051061 (2013): "Use of CNF1 to treat neuroinflammation and astrogliosis in CNS diseases." Carla Fiorentini, Roberto Rimondini-Giorgini, Fiorella Malchiodi-Albedi, Stefano Introduction Epilepsy is caused by a variety of factors and is characterized by the abnormal firing of neurons, and by frequent seizures that bring about progressive damage to the brain. The abnormal synchronized discharge of a large number of neurons leads to a great consumption of bio-energy PLOS ONE | DOI:10.1371/journal.pone.0140495 October 12, 2015 1 / 17 CNF1 Counteracts Epileptiform Phenomena Loizzo, Sara Travaglione, Gabriele Campana, that is related to material pertinent to this article. The authors confirm that this does not alter their adherence to all the PLOS ONE policies. in the brain. [1,2]. On the other hand, mutations affecting genes encoding for proteins that maintain energy homeostasis within the cell, often result in an epileptiform phenotype [3,4]. This implies that energy failure can strongly affect neuronal excitability and synaptic transmission, thus contributing to epileptogenesis [5,6]. Furthermore, one of most deleterious seizure effect is the loss of dendritic spines. This latter feature was evidenced in both pathological specimens from animal seizure models [7,8], and human epilepsy patients [9,10], and was connected to depolymerization of actin [11]. Actin dynamic is a key regulator of the shape and morphological plasticity of axons, dendrites, and dendritic spines, and is controlled by the Rho family of small GTPases [12,13]. Interestingly, the Rho GTPases also orchestrate the close relationship between the actin cytoskeleton and mitochondrial shape and function [14]. In this context, we propose, as a therapeutic tool against seizures, a bacterial protein toxin from Escherichia coli, named cytotoxic necrotizing factor 1 (CNF1) that is able to 'modulate' the Rho GTPases’ activation/degradation process [15,16]. Recently, we have demonstrated that CNF1, boosts in vitro the mitochondrial ATP production and promotes mitochondria elongation by phosphorylating Drp1, a protein member of the dynamin family of large GTPases that controls mitochondrial fission [17]. Interestingly, the recruitment of Drp1 to mitochondria is facilitated by the actin cytoskeleton activity [18,19]. All these results are in line with what we have demonstrated using CNF1 in vivo. In fact, a single intacerebroventricular (icv) injection of the purified bacterial toxin can increase spine density and length in pyramidal neurons [20], lower the levels of neuroinflammation markers, and improve cognitive performances in Rett syndrome (RTT) [21] and Alzheimer’s disease (AD) [22] mouse models. In particular, in the RTT pathological model, we observed a rescue of brain mitochondrial electron transport chain activity [23] and an augmented expression of proteins involved in ATP regeneration [21]. In the AD mouse model, CNF1 promotes a systemic energy homeostasis rescue, with an increase of hippocampal and cortex tissue ATP content [22]. All these CNF1-induced effects are long lasting. Possibly, the toxin can act by engaging pathways that control the actin cytoskeleton organization, thus increasing in neuroplasticity, and mitochondrial activity as well. Therefore, CNF1 may represent a therapeutic tool also for counteracting epilepsy or to preserve the brain from seizure-induced damage. To evaluate the CNF1 ability to counteract seizures generation, we have studied CNF1 effects in an inbred strain of mice, the DBA/2J (D2). D2 is a multipurpose neurological disease model because of its susceptibility to disorders that may involve neuronal (...truncated)