CNF1 Increases Brain Energy Level, Counteracts Neuroinflammatory Markers and Rescues Cognitive Deficits in a Murine Model of Alzheimer's Disease

Counteracts Neuroinflammatory Markers and Rescues Cognitive Deficits in a Murine Model of Alzheimer's Disease. PLoS ONE 8(5): e65898. doi:10.1371/journal.pone.0065898 CNF1 Increases Brain Energy Level, Counteracts Neuroinflammatory Markers and Rescues Cognitive Deficits in a Murine Model of Alzheimer's Disease Stefano Loizzo 0 Roberto Rimondini 0 Sara Travaglione 0 Alessia Fabbri 0 Marco Guidotti 0 Alberto Ferri 0 Gabriele Campana 0 Carla Fiorentini 0 Stephen D. Ginsberg, Nathan Kline Institute and New York University School of Medicine, United States of America 0 1 Department of Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanita` , Rome , Italy , 2 Department of Pharmacology, University of Bologna , Bologna , Italy , 3 Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanita` , Rome , Italy , 4 Institute of Cellular Biology and Neurobiology, CNR , Roma , Italy Overexpression of pro-inflammatory cytokines and cellular energy failure are associated with neuroinflammatory disorders, such as Alzheimer's 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-1b 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 Alzheimer's disease via a control of neuroinflammation and a rescue of systemic energy homeostasis. - . These authors contributed equally to this work. " GC and CF are joint senior authors. The apolipoproteins E (apoE) are cholesterol transporters of high importance for neuronal plasticity, glucose utilization, mitochondrial functions and modulation of inflammation [1,2]. However, individuals homozygous for one dysfunctional variant of apoE, the apoE4 allele, are known to be at major genetic risk for developing atherosclerosis [3] and sporadic Alzheimers Disease (AD) [4,5]. ApoE4 allele is also associated with an enhanced risk or severity of several other neurodegenerative disorders [6], with a faster progression of certain neuromuscular diseases, including diabetic neuropathy and human immunodeficiency viral neuropathy [7], and with neuroinflammation, in pathologies like multiple sclerosis [8], Parkinsons disease [9,10] and those neurological diseases involving excitotoxic mechanisms [11]. In fact, apoE4 genotype has a profound influence on the extent of disease-related synaptic deterioration due to its dumping effects on dendritic growth [12], and therefore, on neuronal plasticity. In addition, apoE4 uniquely undergoes neuron-specific proteolysis, resulting in bioactive toxic fragments that enter the cytosol, alter the cytoskeleton and disrupt mitochondrial energy balance [13]. Transgenic mice homozygous for human apoE4, show mitochondrial dysfunction, with decreased production of metabolic energy in terms of decreased levels of adenosine triphosphate (ATP) and show accumulation of beta amyloid (Ab or Abeta) in the brain [14,15,16]. Moreover, pro-inflammatory cytokines, important markers of the inflammatory component of apoE4 [17] and, in general, of AD [18], are strictly connected with brain energy metabolism [19]. Recently, we have demonstrated that an Escherichia coli protein toxin, named Cytotoxic Necrotizing Factor 1 (CNF1), improves the mitochondrial activity and regulates pro-inflammatory cytokines levels in a mouse model of Retts syndrome [20]. These effects, accompained by a long-lasting amelioration of cognitive performances, were strictly Rho GTPases-dependent. The Rho GTPases, ubiquitously expressed molecular switches that cycle between a GDP-bound inactive and a GTP-bound active state in eukaryotic cells, encompass the three subfamilies Rho, Rac and Cdc42 that control different signalling pathways. All of them are constitutively activated by CNF1 through deamidation of a critical glutamine residue that lock them in their activated, GTP-bound state [21,22]. The threeshold of this activation is subsequently attenuated because high levels of activated Rho GTPases are recognized by cells that ubiquitinate and degrade them to more physiological levels [23]. The ability of Rho GTPases to control actin polymerization [24], plays important roles in the morphogenesis of the dendritic spines in the brain [25,26,27] as well as in the synaptic plasticity [28,29,30,31,32,33]. Our previous studies showed the ability of CNF1 to trigger structural remodelling and functional plasticity in rodents [34,35]. Deficits in neuronal plasticity have been reported in several pathologies of the central nervous system (CNS) characterized by energy and cognitive deficiencies, including Rett syndrome [36] and AD [37]. Very recently, it has been reported that CNF1 can ameliorate cognitive performances in four-month old TgCRND8 mice, an AD model with early-onset Ab deposits [38], thus confirming our previous hypothesis [39]. It remains totally unexplored, however, the mechanism by which CNF1 can improve the AD-linked behavioural deficits, and whether CNF1 can counteract the presence of Ab tangles that are considered the main cause of cognitive impairment. To address these questions, we used clearly symptomatic (12 months old) apoE4 hemizygous (hz) male mice that show, on a normal diet, altered relative quantities of different plasma lipoprotein particles, and delayed clearance of very low density lipoprotein (vLDL) particles, with only half the clearance rate observed in the apoE3 targeted replacement mice [40]. Furthermore, apoE4 mice, if compared to apoE3, are characterized by a more rapid, age-related cognitive decline associated with neuroinflammatory responses [41]. Finally, apoE4 mouse model is considered useful for studying the role of human apoE polymorphism in atherosclerosis, lipid metabolism [3] and Alzheimers disease [2]. Using this animal model, we found that a single dose of intracerebroventricular (icv) administration of CNF1 improved spatial and emotional memory and modified the cell energy, in terms of ATP content, as well as the levels of Ab and of the proinflammatory cytokine IL-1b. It is noteworthy, that all these aspects are directly or indirectly regulated by Rho GTPases [41,43] and are considered crucial markers in AD mouse models [14,15,16,17,18]. Taken altogether, we can speculate that the striking improvement o (...truncated)