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Rett syndrome (RTT) is a rare neurological disorder caused by mutations in the X-linked MECP2 gene and a major cause of intellectual disability in females. No cure exists for RTT. We previously reported that the behavioural phenotype and brain mitochondria dysfunction are widely rescued by a single intracerebroventricular injection of the bacterial toxin CNF1 in a RTT mouse model carrying a truncating mutation of the MeCP2 gene (MeCP2-308 mice). Given the heterogeneity of MECP2 mutations in RTT patients, we tested the CNF1 therapeutic efficacy in a mouse model carrying a null mutation (MeCP2-Bird mice). CNF1 selectively rescued cognitive defects, without improving other RTT-related behavioural alterations, and restored brain mitochondrial respiratory chain complex activity in MeCP2-Bird mice. To shed light on the molecular mechanisms underlying the differential CNF1 effects on the behavioural phenotype, we compared treatment effects on relevant signalling cascades in the brain of the two RTT models. CNF1 provided a significant boost of the mTOR activation in MeCP2-308 hippocampus, which was not observed in the MeCP2-Bird model, possibly explaining the differential effects of CNF1. These results demonstrate that CNF1 efficacy depends on the mutation beared by MeCP2-mutated mice, stressing the need of testing potential therapeutic approaches across RTT models.

Stress vulnerability is a critical factor for the development of trauma-related disorders; however, its biological underpinnings are not clear. We demonstrated that dysfunctions in the X-linked epigenetic factor methyl-CpG binding protein 2 (MeCP2) provide trauma vulnerability in male mice. Given the prominent role of sex in stress outcomes, we explored the effects of MeCP2 hypofunctionality in females. Female mice carrying truncated MeCP2 (heterozygous and homozygous) and wild type controls (wt) were tested for fear memory. Stress-induced corticosterone release and brain expression of hypothalamic-pituitary-adrenal (HPA) axis regulatory genes were also evaluated in wt and mutant mice of both sexes. Although heterozygous females displayed a normal stress-related behavioural profile, homozygous mice showed enhanced memory recall for the threatening context compared to wt, thus recapitulating the phenotype previously evidenced in hemizygous males. Interestingly, MeCP2 truncation abolished the sex differences in stress-induced corticosterone release, which was found increased in mutant males, whereas blunted in mutant females in a zygosity-independent manner. Although heterozygous mice did not differ from controls, homozygous females and hemizygous males showed increased hypotalamic Crh and Avp mRNAs and a differentially altered expression of Fkbp5 in cortical areas. Present results demonstrate that in female mice carrying truncated MeCP2, altered stress responsivity is driven by homozygosity, whereas heterozygosity does not lead to maladaptive stress outcomes. MeCP2 dysfunctions thus provide stress vulnerability in mice with sex- and zygosity-dependent outcomes.

Mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene cause CDKL5 Deficiency Disorder (CDD), a rare neurodevelopmental syndrome characterized by severe behavioural and physiological symptoms. No cure is available for CDD. CDKL5 is a kinase that is abundantly expressed in the brain and plays a critical role in neurodevelopmental processes, such as neuronal morphogenesis and plasticity. This study provides the first characterization of the neurobehavioural phenotype of 1 year old Cdkl5-null mice and demonstrates that stimulation of the serotonin receptor 7 (5-HT7R) with the agonist molecule LP-211 (0.25 mg/kg once/day for 7 days) partially rescues the abnormal phenotype and brain molecular alterations in Cdkl5-null male mice. In particular, LP-211 treatment completely normalizes the prepulse inhibition defects observed in Cdkl5-null mice and, at a molecular level, restores the abnormal cortical phosphorylation of rpS6, a downstream target of mTOR and S6 kinase, which plays a direct role in regulating protein synthesis. Moreover, we demonstrate for the first time that mitochondria show prominent functional abnormalities in Cdkl5-null mouse brains that can be restored by pharmacological stimulation of brain 5-HT7R.

Rett syndrome (RTT) is a rare neurodevelopmental disorder usually caused by mutations in the X-linked gene methyl-CpG-binding protein 2 (MECP2). Several Mecp2 mutant mouse lines have been developed recapitulating part of the clinical features. In particular, Mecp2-308 female heterozygous mice, bearing a truncating mutation, are a validated model of the disease. While recent data suggest a role for inflammation in RTT, little information on the inflammatory status in murine models of the disease is available. Here, we investigated the inflammatory status by proteomic 2-DE/MALDI-ToF/ToF analyses in symptomatic Mecp2-308 female mice. Ten differentially expressed proteins were evidenced in the Mecp2-308 mutated plasma proteome. In particular, 5 positive acute-phase response (APR) proteins increased (i.e., kininogen-1, alpha-fetoprotein, mannose-binding protein C, alpha-1-antitrypsin, and alpha-2-macroglobulin), and 3 negative APR reactants were decreased (i.e., serotransferrin, albumin, and apolipoprotein A1). CD5 antigen-like and vitamin D-binding protein, two proteins strictly related to inflammation, were also changed. These results indicate for the first time a persistent unresolved inflammation of unknown origin in the Mecp2-308 mouse model.

Rett syndrome (RTT) is a rare neurodevelopmental disorder, characterized by severe behavioral and physiological symptoms. Mutations in the methyl CpG binding protein 2 gene (MECP2) cause more than 95% of classic cases, and currently there is no cure for this devastating disorder. Recently we have demonstrated that neurobehavioral and brain molecular alterations can be rescued in a RTT mouse model, by pharmacological stimulation of the brain serotonin receptor 7 (5-HT7R). This member of the serotonin receptor family, crucially involved in the regulation of brain structural plasticity and cognitive processes, can be stimulated by systemic repeated treatment with LP-211, a brain-penetrant selective agonist. The present study extends previous findings by demonstrating that LP-211 treatment (0.25 mg/kg, once per day for 7 days) rescues mitochondrial respiratory chain impairment, oxidative phosphorylation deficiency and the reduced energy status in the brain of heterozygous female mice from two highly validated mouse models of RTT (MeCP2-308 and MeCP2-Bird mice). Moreover, LP-211 treatment completely restored the radical species overproduction by brain mitochondria in the MeCP2-308 model and partially recovered the oxidative imbalance in the more severely affected MeCP2-Bird model. These results provide the first evidence that RTT brain mitochondrial dysfunction can be rescued targeting the brain 5-HT7R and add compelling preclinical evidence of the potential therapeutic value of LP-211 as a pharmacological approach for this devastating neurodevelopmental disorder.

Rett syndrome (RTT) is a rare neurodevelopmental disorder affecting almost exclusively females, caused in the overwhelming majority of the cases by loss-of-function mutations in the gene encoding methyl-CpG binding protein 2 (MECP2). High circulating levels of oxidative stress (OS) markers in patients suggest the involvement of OS in the RTT pathogenesis. To investigate the occurrence of oxidative brain damage in Mecp2 mutant mouse models, several OS markers were evaluated in whole brains of Mecp2-null (pre-symptomatic, symptomatic, and rescued) and Mecp2-308 mutated (pre-symptomatic and symptomatic) mice, and compared to those of wild type littermates. Selected OS markers included non-protein-bound iron, isoprostanes (F2-isoprostanes, F4-neuroprostanes, F2-dihomo-isoprostanes) and 4-hydroxy-2-nonenal protein adducts. Our findings indicate that oxidative brain damage 1) occurs in both Mecp2-null (both −/y and stop/y) and Mecp2-308 (both 308/y males and 308/+ females) mouse models of RTT; 2) precedes the onset of symptoms in both Mecp2-null and Mecp2-308 models; and 3) is rescued by Mecp2 brain specific gene reactivation. Our data provide direct evidence of the link between Mecp2 deficiency, oxidative stress and RTT pathology, as demonstrated by the rescue of the brain oxidative homeostasis following brain-specifically Mecp2-reactivated mice. The present study indicates that oxidative brain damage is a previously unrecognized hallmark feature of murine RTT, and suggests that Mecp2 is involved in the protection of the brain from oxidative stress., Highlights • Oxidative damage is demonstrated in the brain, and more specifically in the neurons, of Mecp2 mutant mouse models. • A direct evidence between enhanced oxidative stress and Mecp2 deficiency is provided. • Oxidative damage precedes the behavioral abnormalities in Mecp2 mutant mice. • Mecp2 is likely involved in the protection of the brain from oxidative stress.

Specialization of the left and right hemispheres to control behavioural responses may represent one of the mechanisms underlying individual differences in personality structure, as well as the preferential use of one hand. The present study investigated the relationship between personality and hand preference in common marmosets (Callithrix jacchus), a little New World monkey that presents highly consistent and stable individual hand preferences for simple reaching. To address this issue, data on 56 different behaviours from the species' behavioural repertoire were measured in 10 different laboratory tests and during observations under social conditions on 16 adult common marmosets. Stable behavioural variables were aggregated a priori into 13 personality traits. Exploratory Factor Analysis (EFA) on personality traits was carried out to verify the presence of major personality factors, and their relationship with direction and strength of individual hand preferences was assessed by multiple regression, taking into account sex and age of the subjects. The largest number of species-specific behaviours so far investigated in this species was taken into account and robust temporal stability between two testing periods was verified. We confirm that common marmosets are characterized by specific and stable personality profiles. A single personality factor, accounting for about 38% of the total variance, was found by EFA, that describes the interest towards unusual and new experiences and resembles the human Openness domain. The strength of the hand preference was found to be predicted by this personality factor, that we named Inquisitiveness. Present results highlight common marmoset as a useful primate model for the study of the relationship between personality and lateralization.

Rett syndrome (RTT) is a pervasive developmental disorder, primarily affecting girls with a prevalence of 1 in every 10,000 births; it represents the second most common cause of intellectual disability in females. Mutations in the gene encoding methyl-CpG-binding protein 2 (MECP2) have been identified as clear etiological factors in more than 90% of classical RTT cases. Whereas the mechanisms leading to the severe, progressive and specific neurological dysfunctions when this gene is mutated still remain to be elucidated, a series of different mouse models have been generated, bearing different Mecp2 mutation. Neurobehavioural analysis in these mouse lines have been carried out and phenotyping analysis can be now utilised to preclinically evaluate the effects of potential RTT treatments. This review summarizes the different results achieved in this research field taking into account different key targets identified to ameliorate RTT phenotype in mouse models, including those not directly downstream of MeCP2 and those limited to the early phases of postnatal development. This article is part of the Special Issue entitled 'Neurodevelopmental Disorders'.

Rett syndrome (RTT) is a pervasive neurodevelopmental disorder mainly caused by mutations in the X-linked MECP2 gene associated with severe intellectual disability, movement disorders, and autistic-like behaviors. Its pathogenesis remains mostly not understood and no effective therapy is available. High circulating levels of oxidative stress markers in patients and the occurrence of oxidative brain damage in MeCP2-deficient mouse models suggest the involvement of oxidative stress in RTT pathogenesis. However, the molecular mechanism and the origin of the oxidative stress have not been elucidated. Here we demonstrate that a redox imbalance arises from aberrant mitochondrial functionality in the brain of MeCP2-308 heterozygous female mice, a condition that more closely recapitulates that of RTT patients. The marked increase in the rate of hydrogen peroxide generation in the brain of RTT mice seems mainly produced by the dysfunctional complex II of the mitochondrial respiratory chain. In addition, both membrane potential generation and mitochondrial ATP synthesis are decreased in RTT mouse brains when succinate, the complex II respiratory substrate, is used as an energy source. Respiratory chain impairment is brain area specific, owing to a decrease in either cAMP-dependent phosphorylation or protein levels of specific complex subunits. Further, we investigated whether the treatment of RTT mice with the bacterial protein CNF1, previously reported to ameliorate the neurobehavioral phenotype and brain bioenergetic markers in an RTT mouse model, exerts specific effects on brain mitochondrial function and consequently on hydrogen peroxide production. In RTT brains treated with CNF1, we observed the reactivation of respiratory chain complexes, the rescue of mitochondrial functionality, and the prevention of brain hydrogen peroxide overproduction. These results provide definitive evidence of mitochondrial reactive oxygen species overproduction in RTT mouse brain and highlight CNF1 efficacy in counteracting RTT-related mitochondrial defects.

Rho GTPases are molecules critically involved in neuronal plasticity and cognition. We have previously reported that modulation of brain Rho GTPases by the bacterial toxin CNF1 rescues 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 intellectual disability, for which no effective therapy is available. Mitochondrial dysfunction has been proposed to be involved in the mechanism of the disease pathogenesis. Here we demonstrate that modulation of Rho GTPases by CNF1 rescues the reduced mitochondrial ATP production via oxidative phosphorylation in the brain of MeCP2-308 heterozygous female mice, the condition which more closely recapitulates that of RTT patients. In RTT mouse brain, CNF1 also 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 cell energy production. Such effects were achieved through the upregulation of the protein content of those MRC complexes subunits, which were defective in RTT mouse brain. Restored mitochondrial functionality was accompanied by the rescue of deficits in cognitive function (spatial reference memory in the Barnes maze), synaptic plasticity (long-term potentiation) and Tyr1472 phosphorylation of GluN2B, which was abnormally enhanced in the hippocampus of RTT mice. Present findings bring into light previously unknown functional mitochondrial alterations in the brain of female mice modeling RTT and provide the first evidence that RTT brain mitochondrial dysfunction can be rescued by modulation of Rho GTPases.

Rett syndrome (RTT) is a rare neurodevelopmental disorder, characterized by severe behavioral and physiological symptoms. Mutations in the methyl CpG-binding protein 2 gene (MECP2) cause >95% of classic cases, and currently there is no cure for this devastating disorder. The serotonin receptor 7 (5-HT7R) is linked to neuro-physiological regulation of circadian rhythm, mood, cognition, and synaptic plasticity. We presently report that 5-HT7R density is consistently reduced in cortical and hippocampal brain areas of symptomatic MeCP2–308 male mice, a RTT model. Systemic repeated treatment with LP-211 (0.25 mg/kg once/day for 7 days), a brain-penetrant selective 5-HT7R agonist, was able to rescue RTT-related defective performance: anxiety-related profiles in a Light/Dark test, motor abilities in a Dowel test, the exploratory behavior in the Marble Burying test, as well as memory in the Novelty Preference task. In the brain of RTT mice, LP-211 also reversed the abnormal activation of PAK and cofilin (key regulators of actin cytoskeleton dynamics) and of the ribosomal protein (rp) S6, whose reduced activation in MECP2 mutant neurons by mTOR is responsible for the altered protein translational control. Present findings indicate that pharmacological targeting of 5-HT7R improves specific behavioral and molecular manifestations of RTT, thus representing a first step toward the validation of an innovative systemic treatment. Beyond RTT, the latter might be extended to other disorders associated with intellectual disability.

Clinical manifestations typical of mitochondrial diseases are often present in various genetic syndromes associated with intellectual disability, a condition leading to deficit in cognitive functions and adaptive behaviors. Until now, the causative mechanism leading to intellectual disability is unknown and the progression of the condition is poorly understood. We first report latest advances on genetic and environmental regulation of mitochondrial function and its role in brain development. Starting from the structure, function and regulation of the oxidative phosphorylation apparatus, we review how mitochondrial biogenesis and dynamics play a central role in neurogenesis and neuroplasticity. We then discuss how dysfunctional mitochondria and alterations in reactive oxygen species homeostasis are potentially involved in the pathogenesis of various neurodevelopmental syndromes with a special focus on Down, Rett, Fragile X syndromes and autism spectrum disorders. Finally, we review and suggest novel therapeutic approaches aimed at improving intellectual disability by activating mitochondrial function and reducing oxidative stress to amiliorate the quality of life in the subjects affected. © 2014 Elsevier Ltd. All rights reserved.

Rho GTPases are key intracellular signaling molecules that coordinate dynamic changes in the actin cytoskeleton, thereby stimulating a variety of processes, including morphogenesis, migration, neuronal development, cell division and adhesion. Deviations from normal Rho GTPases activation state have been proposed to disrupt cognition and synaptic plasticity. This review focuses on the functional consequences of genetic ablation of upstream and downstream Rho GTPases molecules on cognitive function and neuronal morphology and connectivity. Available information on this issue is described and compared to that gained from mice carrying mutations in the most studied Rho GTPases and from pharmacological in vivo studies in which brain Rho GTPases signaling was modulated. Results from reviewed literature provide definitive evidence of a compelling link between Rho GTPases signaling and cognitive function, thus supporting the notion that Rho GTPases and their downstream effectors may represent important therapeutic targets for disorders associated with cognitive dysfunction.

Studies of mitochondrial bioenergetics in brain pathophysiology are often precluded by the need to isolate mitochondria immediately after tissue dissection from a large number of brain biopsies for comparative studies. Here we present a procedure of cryopreservation of small brain areas from which mitochondrial enriched fractions (crude mitochondria) with high oxidative phosphorylation efficiency can be isolated. Small mouse brain areas were frozen and stored in a solution containing glycerol as cryoprotectant. Crude mitochondria were isolated by differential centrifugation from both cryopreserved and freshly explanted brain samples and compared with respect to their ability to generate membrane potential and to produce ATP. Intactness of outer and inner mitochondrial membranes was verified by polarographic ascorbate and cytochrome c tests and spectrophotometric assay of citrate synthase activity. Preservation of structural integrity and oxidative phosphorylation efficiency has been successfully obtained in crude mitochondria isolated from different areas of cryopreserved mouse brain samples. Long-term cryopreservation of small brain areas from which intact and phosphorylating mitochondria can be isolated for the study of mitochondrial bioenergetics, will significantly expand the study of mitochondrial defects in neurological pathologies allowing large comparative studies and favoring inter-laboratory and interdisciplinary analyses.

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.

We studied the long-term effects of a postnatal choline supplementation (from birth till weaning) in the truncated MeCP2-308 mouse model of Rett syndrome. Adult male mutant hemizygous (hz) mice showed a reduction of locomotor activity compared to wild type (wt) littermates. Early choline treatment restored wt-like locomotor activity levels in hz mice. Reduced striatal choline acetyl transferase (ChAT) activity and decreased levels of cortical mRNA NGF were found in hz mice. Choline supplementation increased striatal ChAT activity and also enhanced NGF and BDNF expression in cortical and hippocampal regions. As a whole, postnatal choline supplementation attenuates some of the behavioural and neurobiological abnormalities of the Mecp2-308 phenotype.

Members of captive colonies of common marmosets (Callithrix jacchus), a small New World primate, can occasionally be victims of repeated, and potentially fatal, attacks by a family-mate. This study examined the records of a colony, looking for past instances of such aggressions. The aim was to better understand the possible causes underlying this phenomenon and to identify variables that could minimize their occurrence. The results showed that both males and females behaved as aggressors at the same rate, but females attacked just females, whereas males attacked both males and females. Most aggressions involved siblings and occurred at a higher rate when the cages were occupied by more than 5 nonhuman animals. The influence of group size was significant only when females behaved as aggressors. This study suggests that several factors can determine the occurrence of these aggressions and that their relative importance depends on the sex of the aggressor. The results of this study highlight the importance of collecting further data to verify whether crowding in a confined space, rather than group size per se, affects the incidence of these aggressions.