Modulation of RhoGTPases Improves the Behavioral Phenotype and Reverses Astrocytic Deficits in a Mouse Model of Rett Syndrome

Abstract RhoGTPases are crucial molecules in neuronal plasticity and cognition, as confirmed by their role in non-syndromic mental retardation. Activation of brain RhoGTPases by the bacterial cytotoxic necrotizing factor 1 (CNF1) reshapes the actin cytoskeleton and enhances neurotransmission and synaptic plasticity in mouse brains. We evaluated the effects of a single CNF1 intracerebroventricular inoculation in a mouse model of Rett syndrome (RTT), a rare neurodevelopmental disorder and a genetic cause of mental retardation, for which no effective therapy is available. Fully symptomatic MeCP2-308 male mice were evaluated in a battery of tests specifically tailored to detect RTT-related impairments. At the end of behavioral testing, brain sections were immunohistochemically characterized. Magnetic resonance imaging and spectroscopy (MRS) were also applied to assess morphological and metabolic brain changes. The CNF1 administration markedly improved the behavioral phenotype of MeCP2-308 mice. CNF1 also dramatically reversed the evident signs of atrophy in astrocytes of mutant mice and restored wt-like levels of this cell population. A partial rescue of the overexpression of IL-6 cytokine was also observed in RTT brains. CNF1-induced brain metabolic changes detected by MRS analysis involved markers of glial integrity and bioenergetics, and point to improved mitochondria functionality in CNF1-treated mice. These results clearly indicate that modulation of brain RhoGTPases by CNF1 may constitute a totally innovative therapeutic approach for RTT and, possibly, for other disorders associated with mental retardation. INTRODUCTION Proteins belonging to the RhoGTPases’ family, including Rho, Rac, and Cdc42 subfamilies, have a crucial role in neural plasticity and act as molecular switches (Etienne-Manneville and Hall, 2002) that respond to extracellular stimuli and induce dynamic changes in neuronal and glial morphology and functionality (Feltri et al, 2008; Hall, 2005; Luo, 2000; Nakayama et al, 2000; Tashiro et al, 2000). In line with their central role in controlling structural plasticity and actin cytoskeleton dynamics (Etienne-Manneville and Hall, 2002; Hall, 2005), aberrant Rho signaling has been reported to be associated with abnormalities in dendrites and spines in non-syndromic mental retardation, and to be responsible for cognitive impairments (Ramakers, 2002; van Galen and Ramakers, 2005). In line with these observations, activation of brain RhoGTPases by intracerebroventricular (icv) inoculation of the cytotoxic necrotizing factor 1 (CNF1), an Escherichia coli toxin, which catalyzes the deamidation of a single glutamine residue of the RhoGTPases (Lemichez et al, 1997; Schmidt et al, 1997), enhances learning and memory performances, induces a re-arrangement of cerebral actin cytoskeleton and enhances neurotransmission and synaptic plasticity in mice. All these effects persist for weeks and are strictly dependent on RhoGTPases activation (De Viti et al, 2010; Diana et al, 2007; Pavone et al, 2009). Recently, Rho GTPases signaling pathways have been suggested to be involved in the pathophysiology of a clinical variant of Rett syndrome (RTT) (Chen et al, 2011b), a rare and severe neurodevelopmental disorder and a genetic cause of mental retardation. About 90% of classic RTT cases are caused by mutations in the methyl-CpG-binding protein 2 (MeCP2) gene (Amir et al, 1999; Chahrour and Zoghbi, 2007). MeCP2 has multifunctional roles at the cellular level, mainly acting as a regulator of transcription (Chahrour et al, 2008; Hite et al, 2009; Ogier and Katz, 2008). Anatomical features in brains of RTT patients and MeCP2-null mice include reduced dendritic arborization and dendritic spine formation as well as reduced synaptic plasticity (Belichenko et al, 2009; Gonzales and LaSalle, 2010; Moretti et al, 2006). Recently, functional abnormalities in cultured astrocytes have been ascribed to MeCP2 deficiency, which may impact neuronal dendritic maturation (Ballas et al, 2009; Cahoy et al, 2008; Maezawa et al, 2009; Nagai et al, 2005). No cure currently exists for this disabling disorder. Notably, among the extracellular stimuli affecting RhoGTPases activity, well-established MeCP2 gene targets have been reported, such as brain-derived neurotrophic factor and corticotropin-releasing hormone (Chang et al, 2006; McGill et al, 2006; Miyamoto et al, 2006; Swinny and Valentino, 2006). The MeCP2-deficient state in RTT brains could therefore prevent RhoGTPases from properly carrying out their functions. To assess whether pharmacological interventions targeting RhoGTPases may be an effective therapeutic strategy for RTT, we evaluated the effects of a single icv injection of CNF1 at an advanced stage of the disease on RTT-like symptomatology and pathophysiological features in MeCP2-308 hemizygous (hz) male mice, a model of RTT that expresses a truncated form of the MeCP2 gene (De Filippis et al, 2010; Moretti et al, 2005, 2006; Shahbazian et al, 2002). Magnetic resonance imaging (MRI) and spectroscopy (MRS), powerful non-invasive techniques, also allowed detailed in vivo detection of morphological, functional and metabolic features of the disease in selected brain areas as well as the effects of CNF1 treatment thereon. MATERIALS AND METHODS CNF1 Preparation CNF1 was obtained from the 392 ISS strain kindly provided by V Falbo (Rome, Italy) and purified as previously described (Falzano et al, 1993). In all experiments, the recombinant protein CNF1 C866S, in which the enzymatic activity on RhoGTPases is abrogated by change of cystein with serine at position 866 (Schmidt et al, 1998), was used as a control. The plasmid coding for CNF1 C866S, purified as previously described (Falzano et al, 1993), was kindly provided by E Lemichez (U627 INSERM, Nice, France). For further details, see Supplementary Methods. Animals To evaluate the efficacy of CNF1 on advanced RTT symptomatology, the experimental subjects were 8 months old hz male MeCP2-308 mice (B6.129S-MeCP2tm1Hzo/J, stock number: 005439; backcrossed to C57BL/6J mice for at least 12 generations from the Jackson Laboratories (USA)) and wt littermates. All procedures were carried out in accordance with the European Communities Council Directive (86/609/EEC) and formally approved by Italian Ministry of Health. For further details, see Supplementary Methods. Icv Injections of CNF1 Mice were icv injected with 2 μl of either the CNF1 solution (10−10 M) or the recombinant protein CNF1 C886S (10−10 M) (as in De Viti et al, 2010; Diana et al, 2007; Pavone et al, 2009) over 1 min at the following coordinates: AP=0.0;. ML=+0.72 DV=−2.0 relative to bregma to target lateral ventricles. For further details, see Supplementary Methods. Behavioral Testing A battery of behavioral tests was carried out to determine CNF1 effects on cognition and motor function, the most affected behavioral domains in RTT. Contextual and cued fear-conditioning test (...truncated)