European Neuropsychopharmacology (2015) 25, 889–901

http://dx.doi.org/10924-977X/& 2015 E

nCorrespondence tTel.: +39 0 64990210

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www.elsevier.com/locate/euroneuro

Modulation of Rho GTPases rescues brainmitochondrial dysfunction, cognitive deficitsand aberrant synaptic plasticity in femalemice modeling Rett syndrome

Bianca De Filippisa,n, Daniela Valentib, Valentina Chiodic,Antonella Ferrantec, Lidia de Barib, Carla Fiorentinic,Maria Rosaria Domenicic, Laura Ricceria, Rosa Anna Vaccab,Alessia Fabbric, Giovanni Laviolaa

aDepartment of Cell Biology and Neuroscience, Istituto Superiore di Sanità, Roma, ItalybInstitute of Biomembranes and Bioenergetics, National Council of Research, Bari, ItalycDepartment of Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanità, Roma, Italy

Received 16 October 2014; received in revised form 3 February 2015; accepted 22 March 2015

KEYWORDSIntellectual disabil-ities;NMDA receptors;Transgenic mice;Neural plasticity;Energy metabolism;Behavioralphenotyping

0.1016/j.euroneurlsevier B.V. and E

o: Department of7; fax: +39 0 649ianca.defilippis@i

AbstractRho GTPases are molecules critically involved in neuronal plasticity and cognition. We havepreviously reported that modulation of brain Rho GTPases by the bacterial toxin CNF1rescues the neurobehavioral phenotype in MeCP2-308 male mice, a model of Rett syndrome(RTT). RTT is a rare X-linked neurodevelopmental disorder and a genetic cause of intellectualdisability, for which no effective therapy is available. Mitochondrial dysfunction has beenproposed to be involved in the mechanism of the disease pathogenesis. Here we demonstratethat modulation of Rho GTPases by CNF1 rescues the reduced mitochondrial ATP productionvia oxidative phosphorylation in the brain of MeCP2-308 heterozygous female mice, thecondition which more closely recapitulates that of RTT patients. In RTT mouse brain, CNF1also restores the alterations in the activity of the mitochondrial respiratory chain (MRC)complexes and of ATP synthase, the molecular machinery responsible for the majority of cellenergy production. Such effects were achieved through the upregulation of the proteincontent of those MRC complexes subunits, which were defective in RTT mouse brain.Restored mitochondrial functionality was accompanied by the rescue of deficits in cognitivefunction (spatial reference memory in the Barnes maze), synaptic plasticity (long-termpotentiation) and Tyr1472 phosphorylation of GluN2B, which was abnormally enhanced in thehippocampus of RTT mice. Present findings bring into light previously unknown functional

o.2015.03.012CNP. All rights reserved.

Cell Biology and Neuroscience, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161 Roma, Italy.57821.ss.it (B. De Filippis).

B. De Filippis et al.890

mitochondrial alterations in the brain of female mice modeling RTT and provide the firstevidence that RTT brain mitochondrial dysfunction can be rescued by modulation of RhoGTPases.& 2015 Elsevier B.V. and ECNP. All rights reserved.

1. Introduction

Rett syndrome (RTT) is a severe neurodevelopmentaldisorder affecting primarily girls, with an incidence of1:10,000 born females (Hagberg et al., 2002). Mutationsin the X-linked gene encoding for methyl-CpG-bindingprotein 2 (MeCP2), the founding member of proteinsbinding methylated DNA (Guy et al., 2011), have beenidentified as the main genetic cause of RTT and accountfor more than 95% of classic cases. How mutations inMeCP2 lead to the symptomatology and the neuropatho-logical signs typical of RTT is however still unknown andno effective therapy is currently available for thisdisabling syndrome (Chahrour and Zoghbi, 2007).

We have demonstrated that treatment with the bacterialprotein CNF1 rescues the neurobehavioral abnormalities inMeCP2-308 male mice, a RTT mouse model carrying a trun-cating mutation of the MeCP2 gene (De Filippis et al., 2012).CNF1, a protein toxin produced by several strains ofEscherichia coli, exerts its enzymatic activity on proteinsbelonging to the Rho GTPases' family (Fabbri et al., 2013),low-molecular-weight guanine nucleotide binding proteinscrucially involved in neuronal plasticity and cognition, asconfirmed by their involvement in disorders associated withintellectual disability (De Filippis et al., 2014b). CNF1 istaken up into mammalian cells by receptor-mediated endo-cytosis and is delivered from late endosomes into thecytosol where it locks the Rho GTPases Rho, Rac andCdc42 in their GTP-bound activated state (Fabbri et al.,2013). The availability of the recombinant protein CNF1C866S, in which the enzymatic activity of the bacterialprotein on Rho GTPases is selectively abrogated by changeof cystein with serine at position 866 (Schmidt et al., 1998),allowed us to unequivocally verify the specific involvementof Rho GTPases in the rescue of RTT symptoms.

Recently, we have extended these findings by demon-strating that modulation of Rho GTPases also reverses thealterations in Rho GTPases-dependent signaling pathwayscritically involved in two key biological processes in RTTmouse brain: the regulation of activity-dependent cytos-keletal remodeling and protein translation (De Filippiset al., 2014a).

As a whole these data suggest a crucial role for RhoGTPases as therapeutic targets for RTT and their criticalinvolvement in the pathophysiology of the syndrome.

Several lines of evidence point to mitochondria dysfunc-tion as putative pathogenic mechanisms in RTT (Coker andMelnyk, 1991; Eeg-Olofsson et al., 1990; Valenti et al.,2014a). Evidence of mitochondrial impairment in peripheralorgans has been described in both patients and RTT mousemodels (Gold et al., 2014; Valenti et al., 2014a). Dysfunc-tional mitochondrial energy metabolism in the brain ofRTT patients and mouse models has been also suggested

(Gibson et al., 2010; Grosser et al., 2012; Li et al., 2014).Although these data indicate that mitochondrial dysfunctionmay occur in the brain of RTT patients, little is still knownabout alterations at the functional level in brain mitochon-dria and the cause of defective mitochondria in RTT brain isstill unclear.

To better understand how mitochondrial dysfunction maycontribute to RTT pathogenesis, we have carried out in thebrain of a RTT mouse model (MeCP2-308 mice) a detailedfunctional study of the oxidative phosphorylation (OXPHOS)apparatus, the mitochondrial molecular machinery respon-sible for the majority of cell energy production. Given thatRTT is an X-linked disorder primarily affecting females, wefocused on MeCP2-308 heterozygous female mice (Het)(Katz et al., 2012).

Based on previous in vivo evidence demonstrating thatCNF1 increases bioenergetic markers in RTT mouse brain (DeFilippis et al., 2012) and enhances ATP content in braintissue of an Alzheimer's disease mouse model (Loizzo et al.,2013), and on in vitro evidence of CNF1 effects uponstructure and functionality of the mitochondrial network(Fiorentini et al., 1998; Miraglia et al., 2007; Travaglioneet al., 2014), we have also explored whether and howmodulation of brain Rho GTPases by CNF1 rescues mitochon-drial dysfunction in RTT mouse brain.

To substantiate the efficacy of modulation of Rho GTPasesin reversing RTT-related neurobehavioural abnormalitiesand extend our previous findings (De Filippis et al., 2012),we also explored whether CNF1 has beneficial effects onspatial memory and synaptic plasticity deficits (long-termpotentiation, LTP) in heterozygous female mice.

2. Experimental procedures

2.1. Animals

The experimental subjects were MeCP2-308 heterozygous (Het)female mice [B6.129S-MeCP2tm1Hzo/J, stock number: 005439] andwild-type (wt) littermates. The MeCP2-308 model bears a truncat-ing mutation, leading to the expression of a protein truncated ataminoacid 308 (Shahbazian et al., 2002; Ricceri et al., 2008).

All procedures were carried out in accordance with the EuropeanCommunities Council Directive (86/609/EEC) and formally approvedby Italian Ministry of Health.

2.2. CNF1 preparation

CNF1 was obtained from the 392 pISS strain kindly provided by V.Falbo (Rome, Italy) and purified as previously described (Falzanoet al., 1993). The recombinant protein CNF1 C866S, in which theenzymatic activity on Rho GTPases is abrogated by change ofcystein with serine at position 866 (Schmidt et al., 1998), was usedas a control in all experiments. The plasmid coding for CNF1 C866S,purified as previously described (Falzano et al., 1993), was kindly

891Brain Rho GTPases and mitochondria in Rett syndrome

provided by E. Lemichez (U627 INSERM, Nice, France). All dilutionswere prepared with 20 mM Tris, pH 7.4.

2.3. Intracerebroventricular (icv) injections of CNF1

MeCP2-308 female mice were icv injected at about one year of age,an age at which abnormalities have been reported to start appear-ing in MeCP2-308 heterozygous females (Shahbazian et al., 2002).

Mice were anaesthetized with sevoflurane and randomly assignedto receive an icv injection (10�10 M; 2 μl) of either CNF1 solution orthe recombinant protein CNF1 C866S as a control as in De Filippiset al. (2012).

2.4. Experiment 1

2.4.1. Mitochondrial analysisOne month after the single icv injection of either CNF1 or control,the brains of MeCP2-308 females and their wt littermates werecryopreserved in a ice-cold solution consisting of 50 mM K-MES (pH7.1), 3 mM K2HPO4, 9.5 mM MgCl2, 3 mM ATP plus 20% glycerol and10 mg/ml BSA and stored at �80 1C until assay. Mitochondriaisolated from these cryopreserved tissues showed to preserve thecapability to generate mitochondrial membrane potential, as freshisolated mitochondria, and to maintain their integrity (Valentiet al., 2014b). Levels of mitochondrial membrane integrity, assayedbefore each experiment, were about 98% and 85% for the outer andinner membranes, respectively, as measured by polarographicascorbate test and photometric assay of citrate synthase activity(Valenti et al., 2014b). For each experiment, 5 mice per experi-mental group were used.2.4.1.1. Measurements of mitochondrial ATP production via oxi-dative phosphorylation. Mitochondria were isolated from cryopre-served whole brain tissues and the rate of ATP production byOXPHOS was determined essentially as previously described (Valentiet al., 2010). Mitochondria (0.5 mg of mitochondrial protein) wereincubated at 37 1C in 2 ml of respiratory medium consisting of210 mM mannitol, 70 mM sucrose, 20 mM Tris/HCl, 5 mM KH2PO4/K2HPO4, (pH 7.4), 3 mM MgCl2, and 5 mg/ml BSA in the presence ofthe ATP detecting system (ATP ds) consisting of glucose (2.5 mM),hexokinase (HK, 2 e.u.), glucose 6-phosphate dehydrogenase (G6P-DH, 1 e.u.) and NADP+ (0.25 mM) in the presence of glutamate plusmalate (GLU/MAL, 5 mM each) or succinate (SUCC, 5 mM) plusrotenone (ROT, 3 mM), as energy sources. The reduction of NADP+ inthe extramitochondrial phase, which reveals ATP formation fromexternally added ADP (0.5 mM), was monitored as an increase inabsorbance at 340 nm. Care was taken to use enough HK/G6P-DHcoupled enzymes to ensure a non-limiting ADP-regenerating systemfor the measurement of ATP production. Given the limited amountof isolated mitochondria obtained with the isolation procedure used(about 1–2 mg of mitochondrial protein per gram of wet weight; seeValenti et al. (2014b)), measurements of ATP production werecarried out in mitochondria from brain tissues not less than of0.2–0.3 g of weight.2.4.1.2. Measurement of mitochondrial respiratory chain (MRC)complex activities. Measurements of MRC complex activities werecarried out in mitochondrial membrane-enriched fractions obtainedfrom mitochondria isolated in hippocampus and the cortex, two brainareas critically involved in cognitive function. For isolation of mito-chondrial membrane-enriched fractions, mitochondrial pellets werefirst frozen at �80 1C, then thawed at 2–4 1C, suspended in 1 ml of10 mM Tris–HCl (pH 7.5) plus 1 mg/ml BSA and exposed to ultrasoundenergy for 8 s at 0 1C (11 pulse 0.7 s on, 0.7 s off) at 20 kHz, intensity2. The ultrasound-treated mitochondria were centrifuged at 600g for10 min, 4 1C. The supernatant was centrifuged again at 14,000g for10 min, 4 1C and the resulting pellet was kept at �80 1C until use.Measurement of MRC complex activities were performed with 0.05 mgproteins of mitochondrial membrane-enriched fractions essentially as

in Valenti et al. (2013), by three assays which rely on the sequentialaddition of reagents to measure the activities of (i) NADH:ubiquinoneoxidoreductase (complex I) followed by ATP synthase (complex V),(ii) succinate:ubiquinone oxidoreductase (complex II) and (iii) cyto-chrome c oxidase (complex IV) followed by cytochrome c oxidoreduc-tase (complex III).

2.4.1.3. Immunoblot analysis. Isolated mitochondria were lysedwith 0.1% Triton in PBS in the presence of a protease inhibitorcocktail (Sigma-Aldrich). Mitochondrial lysate (0.01 mg protein) wasresolved by a 4–12% SDS-NuPAGE Bis Tris gel (Life Technologies) andtransferred to a polyvinylidene difluoride membrane (Millipore).Membranes were blocked by TBS-T (50 mM Tris, 150 mM NaCl, 0.02%Tween 20, pH 7.5) containing 3% BSA and probed with theMitoProfile OXPHOS cocktail (1:1000 dilution, MitoSciences). Immu-noblot analysis was performed, using horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibodies and enhancedchemiluminescence Western blotting reagents (Amersham, Pharma-cia Biotech). Membranes were also probed with anti-porin antibody(1:1000 dilution, MitoSciences) as internal loading control anddensitometry value of immunoreactive bands for each sample wasnormalized vs. the corresponding densitometry value of porin.

2.5. Experiment 2

In a separate cohort of mice we explored whether CNF1 hasbeneficial effects on RTT-related deficits in spatial memory andsynaptic plasticity.

2.5.1. Behavioral analyses2.5.1.1. Visual acuity test. Given that spatial memory testsnecessitate a properly functioning visual system to be accom-plished, preliminary to the CNF1 experiments, a cohort MeCP2-308 heterozygous females and wt littermates of about 1 year of age(N=20–21 per experimental group) was tested in the visual acuitytest as previously described with minor modifications (Crawley,1999). For further details see Supplementary Methods.

2.5.1.2. Barnes maze test. After 1 month from the single CNF1 icvtreatment, MeCP2-308 mice and wt littermates were tested in theBarnes Maze test (N=11–12 per experimental group). Referencememory deficits have been already reported in MeCP2-308 malemice in the Morris water maze (MWM) task (Moretti et al., 2006).Even though MeCP2-308 mice are characterized by a mild pheno-type (De Filippis et al., 2010), to overcome the possibility of acontribution of swimming difficulties in the worse cognitive perfor-mance of RTT mice, we adopted the Barnes maze test, a dry-landversion of the MWM, which has been specifically developed toevaluate spatial memory in mice (Barnes, 1979). Mice were testedas previously described with minor modifications (Sunyer et al.,2007). For further details see Supplementary Methods.

2.5.2. Neurobiological analysesThe brains of the experimental mice were processed for neurobio-logical analyses at least 10 days after behavioral testing. Half of themice were used for electrophysiology experiments (N=6 perexperimental group). The brains of the remaining subjects weredissected and rapidly frozen for biochemical analyses (N=5–6 perexperimental group).

2.5.2.1. Hippocampal slice electrophysiology. To evaluate theinduction of LTP in hippocampal slices in wt and MeCP2-308 femalemice, brains were isolated, immersed in ice-cold artificial cere-brospinal fluid (ACSF) (containing, in mM, 126 NaCl, 3.5 KCl,1.2 NaH2PO4, 1.2 MgCl2, 2 CaCl2, 25 NaHCO3 and 11 glucose, pH7.3, saturated with 95% O2 and 5% CO2), and parasagittal slices(400 mm) containing the hippocampus were prepared with a vibra-tome. Slices were incubated for at least 1 h in ACSF at roomtemperature, after which a single slice was transferred to a

Figure 1 Modulation of Rho GTPases by CNF1 rescues the rateof mitochondrial ATP synthesis in RTT mouse brain.(a) Schematic representation of the ATP detecting system(ds). IMS, intermembrane space; MIM, mitochondrial innermembrane: OM, outer membrane space. (b) Representativespectrophotometric traces. Isolated mitochondria from cryo-preserved MeCP2-308 female (Het) or wt mouse brains (0.3 mgof protein each) were incubated at 37 1C in 2 ml of respirationmedium in the presence of the ATP ds in the presence ofrespiratory substrate succinate (SUCC; 5 mM) plus 3 mM rote-none. Where indicated, ADP (0.5 mM) was added. The numbersalong curves are the rates of the increase in absorbance at340 nm measured as tangents to the initial slopes and expressedas nmol of NADP+ reduced/min per mg of mitochondrialprotein. Control mice (ctrl) received the recombinant proteinCNF1 C866S.

B. De Filippis et al.892

submerged recording chamber and continuously superfused at 32–33 1C with ACSF (2.7–3 ml/min).Field excitatory post-synaptic potentials (fEPSPs) were recorded in

stratum radiatum with a glass recording electrode (filled with 2 MNaCl, 1–3 MΩ resistance) after stimulation of the Schaffer collat-erals through a Teflon-coated, bipolar concentric platinum electro-des (FHC) and a stimulus isolator (Isostim A320, WPI Instruments).Traces were acquired, amplified and analyzed with DAM-80 ACdifferential amplifier (WPI Instruments) and with the WinLTP soft-ware (Anderson and Collingridge, 2007). One stimulus (100 msduration) was delivered every 20 s and three consecutive responseswere averaged. Stimuli were set to an intensity that evokes a fEPSPwith a slope of 60% of the maximum fEPSP slope, and were between0.1 and 1 mA. LTP was induced by a theta-burst stimulation (TBS)

consisting in 2 trains of 5 sets of bursts (four stimuli, 100 Hz) with aninterburst interval of 200 ms and a 20 s interval between each train.

2.5.2.2. Immunoblotting analysis. The expression of GluN1,GluN2A, GluN2B, vGLUT 1, GLT-1 and glutamine synthetase wasevaluated in the hippocampus. Hippocampi were homogenized onice in Ripa buffer [phosphate-buffered saline, 1% NP-40, 0.5%sodium deoxycholate, 0.1% sodium dodecyl sulfate, phosphataseand protease inhibitors] and centrifuged (12,000g for 20 min, 4 1C).Supernatants were used for protein determination (Chiodi et al.,2012). 50-mg (for NMDA receptor) or 5-mg (for vGLUT1, GLT-1 andglutamine synthetase) of protein were separated by 8% sodiumdodecyl sulfate–polyacrylamide electrophoresis gels. Proteins weretransferred to polyvinylidene difluoride membranes by electroblot-ting overnight at 4 1C. To avoid non-specific immunodetection,membranes were incubated for 1 h in T-TBS (50 mM Tris–HCl,150 mM NaCl, 0.05% Tween-20, pH 7.4) containing 5% non-fat milk.Blots were incubated overnight at 4 1C with the following anti-bodies: a rabbit anti-GluN1 (1:1000 dilution Chemicon, Millipore,DBA, Italy), a mouse anti-GluN2A (1:500, Chemicon), a mouse anti-GluN2B (1:1000, Chemicon), a rabbit anti-GluN2B phosphorTyr1472(1:1000, Millipore), a guinea pig anti-Glial Glutamate Transporter 1(GLT1, 1:8000, Millipore), a rabbit anti-Vesicular Glutamate Trans-porter 1 (vGLUT1,1:8000, Synaptic Systems, Germany), a mouseanti-glutamine synthetase (1:2000, Millipore), a rabbit anti-ERK1/2(1:4000, Cell Signaling,Euroclone, Italy). After incubation with HRP-conjugated secondary antibodies (Jackson Immunoresearch, LiStarFish, Italy) immunoreactive bands were revealed by enhancedchemiluminescent substrate onto X-ray films. Densitometric analy-sis was conducted using the ImageJ64 program. Immunoreactivebands were normalized with respect to ERK.

2.6. Data analysis

Data were analyzed with the ANOVA model, including genotype andtreatment as between-subject factors, or with a repeated measuresANOVA if there was a within-subjects factor. If data violated theassumptions of normality or equality of variance, non-parametricanalyses of variance were performed. The Levene test was appliedto confirm that variance did not differ between groups. To unravelthe presence of outliers, the Grubbs' test was applied. Posthoccomparisons were performed by Tukey HSD. All experiments wereperformed by experimenters blind to genotype and treatmentassignment.

3. Results

3.1. Modulation of Rho GTPases by CNF1 rescuesdefective mitochondrial ATP production in RTTmouse brain

To verify whether mitochondrial bioenergetic function isdefective in RTT mouse brain and CNF1 effect thereon, weevaluated the ATP production via OXPHOS by spectroscopicmeasurements in mitochondria isolated from the wholebrain of MeCP2-308 female mice and wt littermates, treatedwith either CNF1 or the mutant CNF1 C866S as a control. Tothis aim, brain mitochondria in which membrane integrityhas been first tested, were incubated with the ATP detect-ing system (Figure 1a) under conditions in which OXPHOScan occur (Valenti et al., 2010).

The appearance of ATP outside mitochondria arisingfrom externally added ADP was monitored as an increasein absorbance at 340 nm due to NADPH formation, andwas considered as an index of mitochondrial ATP

Table 1 CNF1 treatment restored ATP production viaoxidative phosphorylation in mitochondria isolated fromRTT mouse brain.

Mitochondrial ATP synthesis rate (nmol/min/mg protein)

SUCC GLU/MAL

wt, ctrl 8672 9572wt, CNF1 8472 9872Het, ctrl 5372αα 9372Het, CNF1 8471** 9771

The rate of mitochondrial ATP production was measured inmitochondria isolated from cryopreserved mouse brains ofeither MeCP2-308 female mice (Het) or wt littermate con-trols in the presence of the ATP detecting system and of therespiratory substrates of either complex I (glutamate plusmalate, GLU/MAL) or complex II (succinate SUCC plusrotenone). The reaction started by adding ADP. Values aremeans7S.E.M. of 5 experiments. Control mice (ctrl)received the recombinant protein CNF1 C866S.

ααwt ctrl vs. Het ctrl: po0.01.nnHet ctrl vs. Het CNF1: po0.01.

893Brain Rho GTPases and mitochondria in Rett syndrome

synthesis (Figure 1a). To provide more complete infor-mation on the efficiency of the OXPHOS system anduncover the relative contribution of the individual MRCcomplexes which form the OXPHOS apparatus, the eva-luation of mitochondrial ATP production was carried outfollowing the addition, as energy sources, of the respira-tory substrates of either complex I (glutamate plusmalate) or complex II (succinate).

As shown by the representative spectrophotometrictraces (Figure 1b), a decrease of about 35% in the rateof mitochondrial ATP synthesis was found in RTT mousebrain compared to wt controls when succinate, a complexII respiratory substrate, was used (po0.01 after posthoccomparisons on the significant genotype*treatment inter-actions: F(1,16)=87.83; po0.001; Table 1). We did notfind any significant difference in mitochondrial ATP pro-duction when complex I substrates (glutamate plusmalate) were added to mitochondria isolated from bothHet and wt brain, suggesting that complex I activity is notimpaired in RTT mouse brain (Table 1). Notably, a com-plete normalization in the rate of mitochondrial ATPsynthesis following the addition of succinate was foundin mitochondria isolated from CNF1-treated RTT mousebrains (po0.01, Figure 1b and Table 1).

3.2. Modulation of Rho GTPases normalizes theactivity of the MRC complexes in RTT mouse cortexand hippocampus

The analysis of the activity of the MRC complexes was carried outin mitochondria isolated from the hippocampus and cortex, twobrain areas known to be critically involved in cognitive function.In line with the functional results, no significant differences incomplex I activity were found between wt and Het mice and theCNF1 treatment did not affect it (Figure 2a). Conversely, asignificant reduction (ranging between 30% and 40%) of theactivity of all MRC complexes downstream to complex I, i.e.complexes II–-V, was found in both RTT mouse hippocampus andcortex compared to wt controls (Figure 2b–e) (po0.01 afterposthoc comparisons on the significant genotypentreatmentinteractions: Complex II: hippocampus: F(1,20)=6.26; p=0.021,cortex: F(1,20)=2.42; p=0.135, Figure 2b; Complex III: hippo-campus: F(1,16)=18.56; po0.001, cortex: F(1,16)=38.23;po0.001, Figure 2c; Complex IV: hippocampus: F(1,16)=11.28;p=0.004, cortex: F(1,16)=34.33; po0.001, Figure 2d; ComplexV: hippocampus: F(1,16)=10.58; p=0.005, cortex: F(1,16)=11.41; p=0.004, Figure 2e).

CNF1 increased the activity of all the defective MRCcomplexes in both brain areas, thus restoring wt-like levels(po0.05 after posthoc comparisons on the significantgenotypentreatment interactions, Figure 2).

3.3. Defective protein content of MRC complexessubunits is restored by CNF1 treatment in RTTmouse cortex and hippocampus

To verify whether the impairment in the activity of complexes II–Vin RTT mouse brain and the CNF1 promoting effect on theactivity of these complexes were associated with changesin their protein content, the levels of some MRC complexessubunits were analyzed, as shown in a representative

immunoblot (Figure 3a) and in the statistical analysis ofdensitometry values (Figure 3b and c). No differences werefound as for protein content of complex I between RTT andwt controls, thus providing support to our functional data.On the other hand, a reduction in the levels of the subunitsof all the others OXPHOS complexes was observed inmitochondria isolated from both hippocampus and cortexof RTT mice (po0.05 after posthoc comparisons on thesignificant genotypentreatment interactions: Complex II(SDHB complex II subunit): hippocampus: F(1,16)=73.57;po0.001, cortex: F(1,16)=88.68; po0.001; Complex III(core 2 subunit): hippocampus: F(1,16)=99.70; po0.001,cortex: F(1,16)=144.16; po0.001; Complex IV (cyto-chrome c oxidase): hippocampus: F(1,16)=193.70;po0.001, cortex: F(1,16)=51.70; po0.001) (Figure 3band c).

CNF1 significantly upregulated the amount of all theanalyzed OXPHOS subunits selectively in RTT mouse brain(po0.01 after posthoc comparisons on the significant geno-typentreatment interactions, Figure 3b and c). A significantrescue of the general reduction in the protein content of MRCcomplexes subunits in RTT mouse mitochondria was thusachieved by the CNF1 treatment in both the analyzed brainareas. Interestingly, the CNF1 treatment also increased com-plex I subunit, NDFUS8, which was not defective in RTT mousebrain (po0.01 after posthoc comparisons on the significantgenotypentreatment interactions: hippocampus: F(1,16)=150.00; po0.001, cortex: F(1,16)=70.55; po0.01). Asshown in Figure 2a, such effect did not significantly impactcomplex I activity, confirming that this specific MRC complex,which presented activity levels comparable to wt controls inRTT mouse brain, was already operating at its maximalefficiency.

Figure 2 The reduced functionality of the mitochondrial respiratory chain complexes in the hippocampus and cortex of RTT mice iscounteracted by CNF1. The activities of CI (complex I, NADH:ubiquinone oxidoreductase) (a), CII (complex II, succinate:ubiquinoneoxidoreductase) (b), CIII (complex III, cytochrome c reductase) (c), CIV (complex IV, cytochrome c oxidase) (d) and CV (complex V,ATP synthase) (e) were measured spectrophotometrically in mitochondrial membrane-enriched fractions from hippocampus andcortex brain areas MeCP2-308 female mice (Het) and wt controls. Data are means7SEMs of 5 experiments. Control mice (ctrl)received the recombinant protein CNF1 C866S. wt ctrl vs. Het ctrl: αpo0.05, ααpo0.01; Het ctrl vs. Het CNF1, *po 0.05, **po0.01.

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3.4. CNF1 treatment rescues reference memorydeficits in the Barnes maze test in RTT female mice

Learning and memory deficits have been reported in anumber of paradigms in RTT mouse models (Katz et al.,

2012; Schaevitz et al., 2013). It is renowned that cognitiveabilities strictly depend on mitochondrial energy metabo-lism (Valenti et al., 2014a). In a separate cohort of mice, wehave therefore investigated whether CNF1 could rescuereference memory deficits in MeCP2-308 female mice.

Figure 3 CNF1 increases the protein content of mitochondrial respiratory chain complexes subunits which are defective in RTTmouse hippocampus and cortex. (a) Representative immunoblotting and (b) and (c) densitomentric analysis of 20-kDa subunit ofcomplex I (CI), 30-kDa subunit of complex II (CII), core 2 protein of complex III (CIII), COX I of complex IV (CIV) and α subunit of F1ATPase (CV) in mitochondria extract (0.01 mg protein) from both hippocampus and cortex of untreated and CNF1-treated wt andMeCP2-308 heterozygous (Het). Protein levels of porin, a mitochondrial protein marker, was also analyzed. The levels of theanalyzed proteins normalized vs. porin, are expressed as the percentage of untreated WT. Data are means7SEMs of 5–6experiments. wt ctrl vs. Het ctrl: α=po0.05, αα=po0.01; Het ctrl vs. Het CNF1, *po0.05,**po0.01.

895Brain Rho GTPases and mitochondria in Rett syndrome

Figure 4 A single administration of CNF1 reverses RTT-related cognitive impairments in the Barnes maze test. MeCP2-308 femalemice (Het, ctrl) displayed longer primary latencies (a) and primary path lengths (b) to locate the virtual target hole (first nose pokein the virtual target hole) during the Probe test (24 h after the last training trial). RTT mice (Het, ctrl) also performed a lowernumber of nose pokes (c) in the target hole compared to wt controls (wt, ctrl), thus confirming a poorer performance in thiscognitive test. CNF1 restored wt-like levels in all these parameters in Het mice (a-c). Control mice (ctrl) received the recombinantprotein CNF1 C866S. Data are means7SEMs. αwt ctrl vs. Het ctrl, po0.05 and *Het ctrl vs. Het CNF1, po0.05.

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3.4.1. Visual acuity testThe state of visual acuity in RTT mice was first verified. Nosignificant genotype effects on latency to step over thedrop-off (two paws criterion) were found [wt: 44.975.6;Het: 57.976.5; F(1, 39)=2.37; p=0.135], thus confirmingthat visual acuity is not impaired in MeCP2-308 female micecompared to wt controls. Latency to first head dipping fromthe ridge toward the visual cliff was also comparablebetween mutant and wt mice [wt: 31.575.0; Het:31.975.2; F(1, 39)o1; p40.90].

3.4.2. Barnes maze3.4.2.1. Training. Time needed to enter the target holedecreased across the adaptation (AD, days 1–2) and acquisi-tion (AC, days 3–6) trials [repeated measures: F(5, 210)=11.79; po0.001], confirming that all the mice learned tolocate the target hole across the training sessions. MeCP2-308 female mice did not differ from wt controls during thetraining trials (no genotype nor genotypentreatment inter-action were found for those parameters).3.4.2.2. Probe test. After 24 h from the last training trial,the target hole was closed to assess spatial reference memoryretention in the probe test. MeCP2-308 female mice showed apoorer performance compared to wt controls: a 2.5-foldincrease in latency to the first nose poke in the virtual targethole (primary latency), path length to reach the virtual targethole (primary path length) and number of primary errors (nosepokes in holes other that the target) was evident in MeCP2-308females compared to control wt mice [primary latency: wt ctrlvs. Het ctrl, po0.05 after posthoc comparisons on thegenotypentreatment interaction: F(1,42)=5.65; p=0.022;Figure 4a; primary path length: wt ctrl vs. Het ctrl, po0.05after posthoc comparisons on the genotypentreatment inter-action: F(1,42)=6.55; p=0.014; Figure 4b; primary errors: wtctrl vs. Het ctrl, po0.05 after posthoc comparisons on thegenotypentreatment interaction: F(1,42)=5.01; p=0.030,data not shown]. MeCP2-308 mice also performed a lowernumber of nose pokes in the target hole than wt littermates[wt ctrl vs. Het ctrl, po 0.05 after posthoc comparisons on thegenotypentreatment interaction: F(1,42)=4.92; p=0.032;

Figure 4c]. These results confirm that MeCP2-308 female miceshow a deficit in spatial reference memory retention (Morettiet al., 2006). CNF1 was able to reverse this impairment andrestored wt-like levels in all these parameters (Het ctrl vs. HetCNF1, po0.05 after posthoc comparisons on the genotypen-treatment interactions, Figure 4a–c).

3.5. CNF1 treatment rescues synaptic plasticitydeficits in the hippocampus of RTT female mice

To unravel whether modulation of brain Rho GTPasesimpacts RTT-related synaptic plasticity deficits, werecorded extracellular fEPSPs in CA1 area of hippocampalslices after stimulation of Schaffer collaterals in wt andMeCP2-308 female mice, treated with either CNF1 orcontrol, as previously described (Moretti et al., 2006). Asexpected, TBS induced a long-lasting increase of fEPSP slopein wt mice. In MeCP2-308 female mice, however, the fEPSPslope almost returned to the pre-tetanus values at 60 min ofrecording (see Figure 5a). Posthoc comparisons on thegenotypentreatmentnrepeated measures interaction [F(56,1120)=1.47; p=0.014] confirmed that MeCP2-308 femalesshow a reduced post tetanic potentiation (PTP, min 1–3 afterTBS: po0.01) and a reduced synaptic potentiation (min 30–50 after TBS: po0.05) compared to aged-matched wtcontrols (Figure 5a). This impairment in synaptic plasticitywas counteracted by the CNF1 treatment which significantlyincreased PTP and elicited LTP in mutant RTT mice (PTP, min1–3: po0.01; LTP, min 30–50 after TBS: po0.01; Figure 5b),without affecting synaptic plasticity in wt mice. Whentreated with CNF1, RTT mice thus showed values notdifferent from those of wt controls (Figure 5c).

3.6. CNF1 treatment restores wt-like levels ofTyr1472 phosphorylation of GluN2B subunit in thehippocampus of RTT mice

Based on previous findings suggesting increased basaltonic activation of NMDA receptors in RTT mouse brain

Figure 5 CNF1 completely and persistently reverses long-termpotentiation (LTP) deficits in RTT mice. Field EPSP (fEPSPs)were recorded in the CA1 area of hippocampal slices derivedfrom wt and MeCP2-308 female mice (Het, ctrl); LTP wasinduced by theta burst stimulation (TBS, indicated by thearrows) of Schaffer collaterals. (a) Hippocampal PTP and LTP(1–3 min and 30–50 min after TBS, respectively) were signifi-cantly impaired in slices from Het mice compared to wtcontrols. (b) The treatment of Het mice with CNF1 (Het,CNF1) significantly improved PTP and LTP deficits with respectto Het controls. (c) Comparison of the time course of fEPSPslope after TBS did not show significant differences betweenCNF1-treated Het and wt mice. Each point represents themean7S.E.M. of 6 experiments. CNF1 C866S was used as acontrol. Insets in (a) and (b) show representative fEPSPsrecorded at the time indicated by the letters (A: Basal; B:PTP; C: LTP). Each trace is the average of three successivefEPSPs (artifacts of stimulation have been truncated). Calibra-tion bars: 0.5 mV, 10 ms.

Figure 6 CNF1 treatment restores wt-like levels of Tyr1472phosphorylation of GluN2B subunit in the hippocampus of RTTmice. Top: Representative Western blot analysis of GluN2Bsubunit and of its 1472-tyrosine phosphorylation status (�180kDa) in the hippocampus of wt and MeCP2-308 female mice(Het) treated with CNF1 or the recombinant protein CNF1C866S (ctrl). Bottom: densitometric analysis is expressed asthe ratio between Phospho-GluN2B and total GluN2B. Data aremeans7SEMs. αwt ctrl vs. Het ctrl, po0.05 and *Het ctrl vs.Het CNF1, po0.05.

897Brain Rho GTPases and mitochondria in Rett syndrome

(Kron et al., 2012), we also investigated whether LTPimpairment is associated with alterations in NMDA recep-tor expression or glutamate turnover in MeCP2-308 femalemouse brains and CNF1 effects thereon. The expressionlevel of GluN1, GluN2A, GluN2B was thus evaluated in thehippocampus of MeCP2-308 female mice. No significantchanges were evidenced in RTT females vs. wt littermates(see Supplementary Figure 1). However, the phosphoryla-tion level of GluN2B subunit onto 1472-tyrosine residueresulted significantly increased in mutants compared to wtlittermates (po0.05 after posthoc comparisons on thegenotypentreatment interaction: F(1,8)=23,06; p=0.001,Figure 6). Interestingly, in mutant mice CNF1 treatmentinduced a decrease in GluN2B-tyrosine phosphorylation(po0.05), which reached wt-like levels.

In order to evaluate if alterations in glutamate trans-port, re-uptake and synthesis are evident in RTT mice thatmight explain synaptic plasticity deficits, the expression ofvGLUT 1, GLT-1 and glutamine synthetase were also

B. De Filippis et al.898

evaluated. No differences in the expression of vGLUT1,GLT-1 and glutamine synthetase were observed betweenmutant and wt mice; CNF1 treatment did not influence theexpression of these proteins (see Supplementary Figure 1).

4. Discussion

This study demonstrates that mitochondria, the power-houses of the cells, show prominent abnormalities at thefunctional level in RTT mouse brain. Furthermore, evi-dence is provided that such functional alterations can berestored by modulation of brain Rho GTPases by CNF1.Such effects were achieved through upregulation of theprotein content of some MRC complexes subunits, whichwere defective in RTT mouse brain. Restoration of mito-chondrial functionality came with the rescue of RTT-related deficits in cognitive function (spatial referencememory in the Barnes maze test), synaptic plasticity(long-term potentiation) and Tyr1472 phosphorylation ofGluN2B, which was abnormally enhanced in the hippo-campus of RTT female mice.

One major conclusion of the present study concerns theprevalent functional alterations of mitochondria we uncov-ered in RTT mouse brain. Given the absence of well-developed methodologies to study brain mitochondrialfunctions directly in brain tissues, we analyzed mitochon-dria isolated from brain tissue, an experimental approachknown to reflect mitochondrial function in vivo (Frezzaet al., 2007). Despite potential problems, this representsat the moment, the main tool for studying mitochondrialbioenergetics in neurological disorders.

Consistent with previous studies (Kriaucionis et al.,2006), we found that all the MRC complex activities down-stream of complex I (complex II-V) are highly compromised.In this line, a reduction in mitochondrial ATP production viaOXPHOS was evident only when complex I was bypassed andthe complex II substrate succinate was used as respiratorysource.

A widespread down-regulation of MRC complexes subunitswas also found in RTT mouse brain cortex and hippocampus,which possibly underlines the functional mitochondrialdeficits. These results are consistent with previous studiesdemonstrating reduced expression of proteins related tomitochondrial functions in lymphomonocytes (Pecorelliet al., 2013), embryonic stem cell-derived neurons (Liet al., 2014) and brain cortex (Gibson et al., 2010) of RTTpatients.

Present results demonstrate that functional alterationsare prevalent in RTT mitochondria and might account forthe perturbed whole cell energy metabolism (Toloe et al.,2014) and the abnormal cell oxidative stress level in RTTmouse brain (De Felice et al., 2013; Grosser et al., 2012).Further studies aimed at evaluating the possibility thatmitochondrial dysfunction may precede the establishmentof behavioral and respiratory symptoms would certainlyhelp shedding further light on this interesting topic. Brainoxidative damage as well as deficits in neural activity havebeen in fact reported before the onset of neurologicalsymptoms in RTT (Dani et al., 2005; De Felice et al.,2013; Grosser et al., 2012).

Of note, the detailed functional characterization ofmitochondria here provided was carried out in the brainof RTT heterozygous female mice, the genetic and hormonalcondition which more closely recapitulate that of RTTpatients. This, together with the demonstration thatMeCP2-308 heterozygous female mice show RTT-relatedimpairment in spatial reference memory and in Schaffer-collateral LTP (Moretti et al., 2006; Na et al., 2013),confirms that heterozygosity and the phenotypic variabilitydue to X chromosome inactivation do not preclude the useof RTT female mice in neurobehavioral analyses (Katz et al.,2012).

Interestingly, in spite of the mild phenotype shown by theMeCP2-308 mouse model (De Filippis et al., 2010; Woodset al., 2012), we here report a quite pronounced alterationin the expression pattern and in the activity of theindividual mitochondrial complexes. Present results providesupport to the hypothesis that mitochondrial alterationsmay be well tolerated by the brain tissue, thus causingneuronal dysfunction, in the absence of neurodegenerationin RTT mouse brain (Muller and Can, 2014).

Another key conclusion from our study is that the CNF1treatment completely restored normal mitochondrial func-tionality in RTT mouse brain. Of note, such CNF1-inducedrescue of mitochondrial abnormalities in RTT mouse hippo-campus and cortex was accompanied by the reversal ofcognitive and synaptic plasticity deficits. These resultsconfirm the therapeutic efficacy of the CNF1 treatment inthe genetic and the hormonal milieu which is more relevantfor translational purposes (i.e. females). Long-lastingeffects of a single icv treatment with CNF1 are also demo-nstrated, which are consistent with previous data demon-strating that brain Rho GTPases are still slightly activated 28days after a single icv treatment with CNF1 (Cerri et al.,2011; Diana et al., 2007).

Interestingly, CNF1-induced reversal of LTP deficits wasalso accompanied by restoration of wt-like levels of Tyr1472phosphorylation of GluN2B, which was abnormally enhancedin the hippocampus of RTT mice. NMDAR tyrosine phosphor-ylation has been reported to regulate the trafficking ofNMDA receptors (Goebel-Goody et al., 2009). In particular,enhanced Tyr1472 phosphorylation of the GluN2B is posi-tively associated with both its surface expression andfunctionality (Gladding and Raymond, 2011). The increasewe found in the phosphorylation level of GluN2B in RTTmouse hippocampus may thus represent an attempt tocompensate for RTT-related defects in synaptic plasticity.

Even though we cannot at the moment exclude that othermechanisms may be involved, present results argue for acausal link between brain mitochondrial dysfunction and themanifestation of RTT-related symptomatology and suggestthat improved cerebral metabolism and cell energy produc-tion might explain CNF1 beneficial effects on cognitivebehavior and synaptic plasticity, processes which strictlydepend on mitochondrial energy metabolism and oxygensupply (Valenti et al., 2014a).

We have recently demonstrated that in normal epithe-lial cells, the bacterial protein CNF1 affects mitochon-drial morphology, thus increasing the ATP production(Travaglione et al., 2014). Such an effect is achievedthrough the inhibition of the pro-fission protein Drp1 andthe subsequent shift towards enhanced fusion of the

899Brain Rho GTPases and mitochondria in Rett syndrome

functional organelles. In RTT mouse brain, we found thatthe CNF1-mediated reversal of the functional mitochon-drial deficits comes along with the upregulation of theprotein content of the MRC complexes subunits whichwere defective in RTT mouse brain. A different mechan-ism, affecting either transcription or translation, both ofwhich are known to be deranged in RTT (Guy et al., 2011;Ricciardi et al., 2011), of proteins related to mitochon-dria functionality, might thus explain CNF1 effects onmitochondria functionality in RTT mouse brain.

Even though further studies are needed to dissect themolecular mechanisms underlying such beneficial effects,the use as a control treatment of a mutant CNF1 proteinwhose Rho enzymatic activity was abrogated by the changeof the cystein at position 866 with a serine, allows us tounequivocally confirm the pivotal role of Rho GTPases in therescue of mitochondrial dysfunction in CNF1-treated RTTmouse brain. Moreover, the icv route of administration ofthe CNF1 treatment provides unambiguous evidence thatthe CNF1-dependent rescue of RTT symptoms is centrallymediated (De Filippis et al., 2012).

Brain Rho GTPases are best known as molecular hubsgoverning both activity-dependent cytoskeletal remodelingand protein synthesis, two biological processes which playkey roles in intellectual disabilities disorders (Ba et al.,2013; De Filippis et al., 2014b). Present results extend thesefindings by demonstrating that this family of key intracel-lular signaling molecules is also critically involved in regula-tion of brain mitochondrial homeostasis in RTT.

Increasing evidence suggest that mitochondrial dysfunction(Valenti et al., 2014a) and deviations from normal RhoGTPases activation state (De Filippis et al., 2014b) disruptcognition and synaptic plasticity and represent central actorsin a range of intellectual disability disorders. The presentstudy provides the first evidence that these brain alterationsmay be intimately interconnected, thus providing support tothe therapeutic potential of drugs targeting Rho GTPases andtheir downstream effectors (De Filippis et al., 2014a) fordisorders associated with cognitive dysfunction.

Role of the funding source

This research was supported by Jerome Lejeune Foundation(France), AIRETT (Italy) and IRE-IFO (RF2008) “MECP2phosphorilation and related kinase in Rett syndrome” toG.L.; European Research Projects on Rare Diseases to C.F;and IRSF HeARTAward (3107) to B.D.F; and Research (MIUR)-Program FIRB-MERIT (1-RBNE08HWLZ-012). The fundingbodies had no further roles.

Contributors

Design of the work: All authors; Data acquisition: BDF, DV, VC, AFe,LdB; Interpretation of data: All authors; Data analysis and manu-script preparation: BDF; Revision of the manuscript: All authors.

Conflict of interest

None of the authors declare financial interests or potential conflictof interests.

Acknowledgments

The authors are grateful to Stella Falsini, Luisa Chessa, MattiaMusto, Oriane Blanquie and Massimo Giambenedetti for technicalassistance and to Luigia Cancemi and Giovanni Dominici foranimal care.

Appendix A. Supplementary information

Supplementary data associated with this article can befound in the online version at http://dx.doi.org/10.1016/j.euroneuro.2015.03.012.

References

Anderson, W.W., Collingridge, G.L., 2007. Capabilities of theWinLTP data acquisition program extending beyond basic LTPexperimental functions. J. Neurosci. Methods 162, 346–356.

Ba, W., van der Raadt, J., Nadif Kasri, N., 2013. Rho GTPasesignaling at the synapse: Implications for intellectual disability.Exp. Cell Res. 319, 2368–2374.

Barnes, C.A., 1979. Memory deficits associated with senescence: aneurophysiological and behavioral study in the rat. J. Comp.Physiol. Psychol. 93, 74–104.

Cerri, C., Fabbri, A., Vannini, E., Spolidoro, M., Costa, M., Maffei, L.,Fiorentini, C., Caleo, M., 2011. Activation of Rho GTPases triggersstructural remodeling and functional plasticity in the adult rat visualcortex. J. Neurosci. 31, 15163–15172.

Chahrour, M., Zoghbi, H.Y., 2007. The story of Rett syndrome: fromclinic to neurobiology. Neuron 56, 422–437.

Chiodi, V., Uchigashima, M., Beggiato, S., Ferrante, A., Armida, M.,Martire, A., Potenza, R.L., Ferraro, L., Tanganelli, S., Wata-nabe, M., Domenici, M.R., Popoli, P., 2012. Unbalance of CB1receptors expressed in GABAergic and glutamatergic neurons ina transgenic mouse model of Huntington's disease. Neurobiol.Dis. 45, 983–991.

Coker, S.B., Melnyk, A.R., 1991. Rett syndrome and mitochondrialenzyme deficiencies. J. Child. Neurol. 6, 164–166.

Crawley, J.N., 1999. Behavioral phenotyping of transgenic andknockout mice: experimental design and evaluation of generalhealth, sensory functions, motor abilities, and specific beha-vioral tests. Brain Res. 835, 18–26.

Dani, V.S., Chang, Q., Maffei, A., Turrigiano, G.G., Jaenisch, R.,Nelson, S.B., 2005. Reduced cortical activity due to a shift in thebalance between excitation and inhibition in a mouse model ofRett syndrome. Proc. Natl. Acad. Sci. U S A. 102 (35),12560–12565.

De Felice, C., Della Ragione, F., Signorini, C., Leoncini, S., Pecorelli, A.,Ciccoli, L., Scalabri, F., Marracino, F., Madonna, M., Belmonte, G.,Ricceri, L., De Filippis, B., Laviola, G., Valacchi, G., Durand, T.,Galano, J.M., Oger, C., Guy, A., Bultel-Ponce, V., Guy, J., Filosa, S.,Hayek, J., D’Esposito, M., 2013. Oxidative brain damage in Mecp2-mutant murine models of Rett syndrome. Neurobiol. Dis. 68, 66–77.

De Filippis, B., Fabbri, A., Simone, D., Canese, R., Ricceri, L.,Malchiodi-Albedi, F., Laviola, G., Fiorentini, C., 2012. Modula-tion of RhoGTPases improves the behavioral phenotype andreverses astrocytic deficits in a mouse model of Rett syndrome.Neuropsychopharmacology 37, 1152–1163.

De Filippis, B., Nativio, P., Fabbri, A., Ricceri, L., Adriani, W.,Lacivita, E., Leopoldo, M., Passarelli, F., Fuso, A., Laviola, G.,2014. Pharmacological stimulation of the brain serotonin recep-tor 7 as a novel therapeutic approach for Rett syndrome.Neuropsychopharmacology 39 (11), 2506–2518.

B. De Filippis et al.900

De Filippis, B., Ricceri, L., Laviola, G., 2010. Early postnatalbehavioral changes in the Mecp2-308 truncation mouse modelof Rett syndrome. Genes Brain Behav. 9, 213–223.

De Filippis, B., Romano, E., Laviola, G., 2014. Aberrant Rho GTPasessignaling and cognitive dysfunction: in vivo evidence for acompelling molecular relationship. Neurosci. Biobehav. Rev. 46(2), 285–301.

Diana, G., Valentini, G., Travaglione, S., Falzano, L., Pieri, M.,Zona, C., Meschini, S., Fabbri, A., Fiorentini, C., 2007. Enhance-ment of learning and memory after activation of cerebral RhoGTPases. Proc. Natl. Acad. Sci. USA 104, 636–641.

Eeg-Olofsson, O., al-Zuhair, A.G., Teebi, A.S., Daoud, A.S., Zaki, M.,Besisso, M.S., Al-Essa, M.M., 1990. Rett syndrome: a mitochondrialdisease? J. Child. Neurol. 5, 210–214.

Fabbri, A., Travaglione, S., Fiorentini, C., 2013. Escherichia colicytotoxic necrotizing factor 1 (CNF1): toxin biology, in vivoapplications and therapeutic potential. Toxins (Basel) 2,283–296.

Falzano, L., Fiorentini, C., Donelli, G., Michel, E., Kocks, C.,Cossart, P., Cabanie, L., Oswald, E., Boquet, P., 1993. Inductionof phagocytic behaviour in human epithelial cells by Escherichiacoli cytotoxic necrotizing factor type 1. Mol. Microbiol. 9,1247–1254.

Fiorentini, C., Matarrese, P., Straface, E., Falzano, L., Fabbri, A.,Donelli, G., Cossarizza, A., Boquet, P., Malorni, W., 1998. Toxin-induced activation of Rho GTP-binding protein increases Bcl-2expression and influences mitochondrial homeostasis. Exp. CellRes. 242, 341–350.

Frezza, C., Cipolat, S., Scorrano, L., 2007. Organelle isolation:functional mitochondria from mouse liver, muscle and culturedfibroblasts. Nat. Protoc. 2, 287–295.

Gibson, J.H., Slobedman, B., Harikrishnan, K.N., Williamson, S.L.,Minchenko, D., El-Osta, A., Stern, J.L., Christodoulou, J., 2010.Downstream targets of methyl CpG binding protein 2 and theirabnormal expression in the frontal cortex of the human Rettsyndrome brain. BMC Neurosci. 11, 53.

Gladding, C.M., Raymond, L.A., 2011. Mechanisms underlying NMDAreceptor synaptic/extrasynaptic distribution and function. Mol.Cell. Neurosci. 48, 308–320.

Goebel-Goody, S.M., Davies, K.D., Alvestad Linger, R.M., Freund, R.K.,Browning, M.D., 2009. Phospho-regulation of synaptic and extrasy-naptic N-methyl-D-aspartate receptors in adult hippocampal slices.Neuroscience 158, 1446–1459.

Gold, W.A., Williamson, S.L., Kaur, S., Hargreaves, I.P., Land, J.M.,Pelka, G.J., Tam, P.P., Christodoulou, J., 2014. Mitochondrialdysfunction in the skeletal muscle of a mouse model of Rettsyndrome (RTT): implications for the disease phenotype. Mito-chondrion 15, 10–17.

Grosser, E., Hirt, U., Janc, O.A., Menzfeld, C., Fischer, M.,Kempkes, B., Vogelgesang, S., Manzke, T.U., Opitz, L., Salinas-Riester, G., Muller, M., 2012. Oxidative burden and mitochon-drial dysfunction in a mouse model of Rett syndrome. Neurobiol.Dis. 48, 102–114.

Guy, J., Cheval, H., Selfridge, J., Bird, A., 2011. The role of MeCP2in the brain. Annu. Rev. Cell. Dev. Biol. 27, 631–652.

Hagberg, B., Hanefeld, F., Percy, A., Skjeldal, O., 2002. An updateon clinically applicable diagnostic criteria in Rett syndrome.Comments to Rett Syndrome Clinical Criteria Consensus PanelSatellite to European Paediatric Neurology Society Meeting,Baden Baden, Germany, 11 September 2001. Eur. J. Paediatr.Neurol. 6, 293–297.

Katz, D.M., Berger-Sweeney, J.E., Eubanks, J.H., Justice, M.J., Neul, J.L., Pozzo-Miller, L., Blue, M.E., Christian, D., Crawley, J.N.,Giustetto, M., Guy, J., Howell, C.J., Kron, M., Nelson, S.B., Samaco,R.C., Schaevitz St, L.R., Hillaire-Clarke, C., Young, J.L., Zoghbi, H.Y.,Mamounas, L.A., 2012. Preclinical research in Rett syndrome: settingthe foundation for translational success. Dis. Model. Mech. 5,733–745.

Kriaucionis, S., Paterson, A., Curtis, J., Guy, J., Macleod, N., Bird, A.,2006. Gene expression analysis exposes mitochondrial abnormalitiesin a mouse model of Rett syndrome. Mol. Cell. Biol. 26, 5033–5042.

Kron, M., Howell, C.J., Adams, I.T., Ransbottom, M., Christian, D.,Ogier, M., Katz, D.M., 2012. Brain activity mapping in Mecp2 mutantmice reveals functional deficits in forebrain circuits, including keynodes in the default mode network, that are reversed with ketaminetreatment. J. Neurosci. 32, 13860–13872.

Li, Y., Wang, H., Muffat, J., Cheng, A.W., Orlando, D.A., Loven, J.,Kwok, S.M., Feldman, D.A., Bateup, H.S., Gao, Q., Hockemeyer, D.,Mitalipova, M., Lewis, C.A., Vander Heiden, M.G., Sur, M., Young, R.A., Jaenisch, R., 2014. Global transcriptional and translationalrepression in human-embryonic-stem-cell-derived Rett syndromeneurons. Cell Stem Cell 13, 446–458.

Loizzo, S., Rimondini, R., Travaglione, S., Fabbri, A., Guidotti, M.,Ferri, A., Campana, G., Fiorentini, C., 2013. CNF1 increasesbrain energy level, counteracts neuroinflammatory markers andrescues cognitive deficits in a murine model of Alzheimer'sdisease. PLoS One 8, e65898.

Miraglia, A.G., Travaglione, S., Meschini, S., Falzano, L., Matarrese, P.,Quaranta, M.G., Viora, M., Fiorentini, C., Fabbri, A., 2007. Cytotoxicnecrotizing factor 1 prevents apoptosis via the Akt/IkappaB kinasepathway: role of nuclear factor-kappaB and Bcl-2. Mol. Biol. Cell 18,2735–2744.

Moretti, P., Levenson, J.M., Battaglia, F., Atkinson, R., Antalffy, B.,Amstrong, D., Arancio, O., Sweatt, J.D., Zoghbi, H.Y., 2006.Learning and memory and synaptic plasticity are impaired in amouse model of Rett syndrome. J. Neurosci. 26, 319–327.

Muller, M., Can, K., 2014. Aberrant redox homoeostasis andmitochondrial dysfunction in Rett syndrome. Biochem. Soc.Trans. 42, 959–964.

Na, E.S., Nelson, E.D., Kavalali, E.T., Monteggia, L.M., 2013. Theimpact of MeCP2 loss- or gain-of-function on synaptic plasticity.Neuropsychopharmacology 38, 212–219.

Pecorelli, A., Leoni, G., Cervellati, F., Canali, R., Signorini, C.,Leoncini, S., Cortelazzo, A., De Felice, C., Ciccoli, L., Hayek, J.,Valacchi, G., 2013. Genes related to mitochondrial functions,protein degradation, and chromatin folding are differentiallyexpressed in lymphomonocytes of Rett syndrome patients.Mediat. Inflamm. 2013, 137629.

Ricceri, L., De Filippis, B., Laviola, G., 2008. Mouse models of Rettsyndrome: from behavioural phenotyping to preclinical evalua-tion of new therapeutic approaches. Behav. Pharmacol. 19,501–517.

Ricciardi, S., Boggio, E.M., Grosso, S., Lonetti, G., Forlani, G., Stefanelli,G., Calcagno, E., Morello, N., Landsberger, N., Biffo, S., Pizzorusso,T., Giustetto, M., Broccoli, V., 2011. Reduced AKT/mTOR signalingand protein synthesis dysregulation in a Rett syndrome animal model.Hum. Mol. Genet. 20, 1182–1196.

Schaevitz, L.R., Gomez, N.B., Zhen, D.P., Berger-Sweeney, J.E.,2013. MeCP2 R168X male and female mutant mice exhibit Rett-like behavioral deficits. Genes Brain Behav. 12, 732–740.

Schmidt, G., Selzer, J., Lerm, M., Aktories, K., 1998. The Rho-deamidating cytotoxic necrotizing factor 1 from Escherichia colipossesses transglutaminase activity. Cysteine 866 and histidine881 are essential for enzyme activity. J. Biol. Chem. 273,13669–13674.

Shahbazian, M., Young, J., Yuva-Paylor, L., Spencer, C., Antalffy, B.,Noebels, J., Armstrong, D., Paylor, R., Zoghbi, H., 2002. Mice withtruncated MeCP2 recapitulate many Rett syndrome features anddisplay hyperacetylation of histone H3. Neuron 35, 243–254.

Sunyer, B., Gert, L., Harald, H.g., Sudarshan, P., 2007. Barnesmaze, a useful task to assess spatial reference memory in themice. http://dx.doi.org/10.1038/nprot.2007.390.

Toloe, J., Mollajew, R., Kugler, S., Mironov, S.L., 2014. Metabolicdifferences in hippocampal ‘Rett’ neurons revealed by ATPimaging. Mol. Cell. Neurosci. 59, 47–56.

901Brain Rho GTPases and mitochondria in Rett syndrome

Travaglione, S., Loizzo, S., Rizza, T., Brocco, A.D., Ballan, G.,Guidotti, M., Vona, R., Nottia, M.D., Torraco, A., Carrozzo, R.,Fiorentini, C., Fabbri, A., 2014. Enhancement of mitochondrialATP production by the E. coli cytotoxic necrotizing factor 1.FEBS J. 15, 3473–3488.

Valenti, D., de Bari, L., De Filippis, B., Henrion-Caude, A., Vacca, R.A.,2014a. Mitochondrial dysfunction as a central actor in intellectualdisability-related diseases: an overview of Down syndrome, autism,Fragile X and Rett syndrome. Neurosci. Biobehav. Rev. http://dx.doi.org/10.1016/j.neubiorev.2014.01.012.

Valenti, D., de Bari, L., De Filippis, B., Ricceri, L., Vacca, R.A.,2014b. Preservation of mitochondrial functional integrity inmitochondria isolated from small cryopreserved mouse brainareas. Anal. Biochem. 444, 25–31.

Valenti, D., Manente, G.A., Moro, L., Marra, E., Vacca, R.A., 2013.Deficit of complex I activity in human skin fibroblasts withchromosome 21 trisomy and overproduction of reactive oxygenspecies by mitochondria: involvement of the cAMP/PKA signal-ling pathway. Biochem. J. 435, 679–688.

Valenti, D., Tullo, A., Caratozzolo, M.F., Merafina, R.S., Scartezzini, P.,Marra, E., Vacca, R.A., 2010. Impairment of F1F0-ATPase, adeninenucleotide translocator and adenylate kinase causes mitochondrialenergy deficit in human skin fibroblasts with chromosome 21 trisomy.Biochem. J. 431, 299–310.

Woods, R., Vallero, R.O., Golub, M.S., Suarez, J.K., Ta, T.A., Yasui, D.H.,Chi, L.H., Kostyniak, P.J., Pessah, I.N., Berman, R.F., LaSalle, J.M.,2012. Long-lived epigenetic interactions between perinatal PBDEexposure and Mecp2308 mutation. Hum. Mol. Genet. 21, 2399–2411.