De novo variants in the Helicase-C domain of CHD8 are associated with severe phenotypes including autism, language disability and overgrowth.

CHD8, which encodes Chromodomain helicase DNA-binding protein 8, is one of a few well-established Autism Spectrum Disorder (ASD) genes. Over 60 mutations have been reported in subjects with variable phenotypes, but little is known concerning genotype-phenotype correlations. We have identified four novel de novo mutations in Chinese subjects: two nonsense variants (c.3562C>T/p.Arg1188X, c.2065C>A/p.Glu689X), a splice site variant (c.4818-1G>A) and a missense variant (c.3502T>A/p.Tyr1168Asn). Three of these were identified from a 445-member ASD cohort by ASD gene panel sequencing of the 96 subjects who remained negative after molecular testing for copy number variation, Rett syndrome, FragileX and tuberous sclerosis complex (TSC). The fourth (p.Glu689X) was detected separately by diagnostic trio exome sequencing. We used diagnostic instruments and a comprehensive review of phenotypes, including prenatal and postnatal growth parameters, developmental milestones, and dysmorphic features to compare these four subjects. In addition to autism, they also presented with prenatal onset macrocephaly, intellectual disability, overgrowth during puberty, sleep disorder, and dysmorphic features, including broad forehead with prominent supraorbital ridges, flat nasal bridge, telecanthus and large ears. For further comparison, we compiled a comprehensive list of CHD8 variants from the literature and databases, which revealed constitutive and somatic truncating variants in the HELIC (Helicase-C) domain in ASD and in cancer patients, respectively, but not in the general population. Furthermore, HELIC domain mutations were associated with a severe phenotype defined by a greater number of clinical features, lower verbal IQ, and a prominent, consistent pattern of overgrowth as measured by weight, height and head circumference. Overall, this study adds to the ASD-associated loss-of-function mutations in CHD8 and highlights the clinical importance of the HELIC domain of CHD8.

CDKL5 influences RNA splicing activity by its association to the nuclear speckle molecular machinery.

Mutations in the human X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been shown to cause severe neurodevelopmental disorders including infantile spasms, encephalopathy, West-syndrome and an early-onset variant of Rett syndrome. CDKL5 is a serine/threonine kinase whose involvement in Rett syndrome can be inferred by its ability to directly bind and mediate phosphorylation of MeCP2. However, it remains to be elucidated how CDKL5 exerts its function. Here, we report that CDKL5 localizes to specific nuclear foci referred to as nuclear speckles in both cell lines and tissues. These sub-nuclear structures are traditionally considered as storage/modification sites of pre-mRNA splicing factors. Interestingly, we provide evidence that CDKL5 regulates the dynamic behaviour of nuclear speckles. Indeed, CDKL5 overexpression leads to nuclear speckle disassembly, and this event is strictly dependent on its kinase activity. Conversely, its down-regulation affects nuclear speckle morphology leading to abnormally large and uneven speckles. Similar results were obtained for primary adult fibroblasts isolated from CDKL5-mutated patients. Altogether, these findings indicate that CDKL5 controls nuclear speckle morphology probably by regulating the phosphorylation state of splicing regulatory proteins. Nuclear speckles are dynamic sites that can continuously supply splicing factors to active transcription sites, where splicing occurs. Notably, we proved that CDKL5 influences alternative splicing, at least as proved in heterologous minigene assays. In conclusion, we provide evidence that CDKL5 is involved indirectly in pre-mRNA processing, by controlling splicing factor dynamics. These findings identify a biological process whose disregulation might affect neuronal maturation and activity in CDKL5-related disorders.

Pontine norepinephrine defects in Mecp2-null mice involve deficient expression of dopamine beta-hydroxylase but not a loss of catecholaminergic neurons.

Rett syndrome is a neurodevelopmental disorder caused by Mecp2 gene mutations. In RTT patients and Mecp2-null (Mecp2(-/Y)) mice, norepinephrine (NE) content drops significantly, which may play a role in breathing arrhythmia, sleep disorders and sudden death. However, the underlying mechanisms for the NE defect are not fully understood. The NE defect may result from decreased NE biosynthesis, loss of catecholaminergic neurons or both. Although deficiency in tyrosine hydroxylase (TH) has been demonstrated, it is possible that dopamine beta-hydroxylase (DBH), the critical enzyme converting dopamine to NE, is also affected. To test these possibilities, we studied DBH expressions in pontine catecholaminergic neurons of Mecp2(-/Y) mice identified with breathing abnormalities. In comparison to the wild type, Mecp2(-/Y) mice at 2months of age showed approximately 50% decrease in the expressions of DBH and TH, at both protein and mRNA levels in the locus coeruleus (LC) region. Consistently, DBH and TH immunoreactivity was markedly decreased in LC neurons of Mecp2(-/Y) mice. No evidence was found for selective deficiency in TH- or DBH-containing neurons in Mecp2(-/Y) mice, as almost all TH-positive cells expressed DBH. By counting TH-immunoreactive cells in the LC, we found that the Mecp2(-/Y) mice lost only approximately 5% of the catecholaminergic neurons as compared to wild-type, although their LC volume shrank by approximately 15%. These results strongly suggest that the NE defect in Mecp2(-/Y) mice is likely to result from deficient expression of not only TH but also DBH without significant loss of catecholaminergic neurons in the LC.

Cyclin-dependent kinase-like 5 (CDKL5) mutation screening in Rett syndrome and related disorders.

Rett syndrome (RTT) is a severe neurodevelopmental disorder affecting females almost exclusively and is characterized by a wide spectrum of clinical manifestations. Mutations in the X-linked methyl-CpG-binding protein 2 (MECP2) gene have been found in up to 95% of classical RTT cases and a lesser proportion of atypical cases. Recently, mutations in another X-linked gene, CDKL5 (cyclin-dependent kinase-like 5) have been found to cause atypical RTT, in particular the early onset seizure (Hanefeld variant) and one female with autism. In this study we screened several cohorts of children for CDKL5 mutations, totaling 316 patients, including individuals with a clinical diagnosis of RTT but who were negative for MECP2 mutations (n=102), males with X-linked mental retardation (n=9), patients with West syndrome (n=52), patients with autism (n=59), patients with epileptic encephalopathy (n=33), patients with Aicardi syndrome (n=7) and other patients with intellectual disability with or without seizures (n=54). In all, seven polymorphic variations and four de novo mutations (c.586C>T [p.S196L]; c.58G>C [p.G20R]; c.2504delC [p.P835fs]; deletion of exons 1-3) were identified, and in all instances of the latter the clinical phenotype was that of an epileptic encephalopathy. These results suggest that pathogenic CDKL5 mutations are unlikely to be identified in the absence of severe early-onset seizures and highlight the importance of screening for large intragenic and whole gene deletions.

A novel mutation in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene associated with a severe Rett phenotype.

Mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have recently been reported in patients with severe neurodevelopmental disorder characterized by early-onset seizures, infantile spasms, severe psychomotor impairment and very recently, in patients with Rett syndrome (RTT)-like phenotype. Although the involvement of CDKL5 in specific biological pathways and its neurodevelopmental role have not been completely elucidated, the CDKL5 appears to be physiologically related to the MECP2 gene. Here we report on the clinical and CDKL5 molecular investigation in a very unusual RTT case, with severe, early-neurological involvement in which we have shown in a previous report, a novel P388S MECP2 mutation [Conforti et al. (2003); Am J Med Genet A 117A: 184-187]. The patient has had severe psychomotor delay since the first month of life and infantile spasms since age 5 months. Moreover, at age 5 years the patient suddenly presented with renal failure. The severe pattern of symptoms in our patient, similar to a CDKL5 phenotype, prompted us to perform an analysis of the CDKL5, which revealed a novel missense mutation never previously described. The X-inactivation assay was non-informative. In conclusion, this report reinforces the observation that the CDKL5 phenotype overlaps with RTT and that CDKL5 analysis is recommended in patients with a seizure disorder commencing during the first months of life.

Altered Gene Expression of Thyroid Hormone Transporters and Deiodinases in iPS MeCP2-Knockout Cells-Derived Neurons.

MeCP2 is an X-linked gene; its mutation causes Rett Syndrome (RTT), a severe neurodevelopmental disability that affects mainly girls. Acting as a transcription factor, the MeCP2 protein is able to regulate several hormone-related genes, such as the thyroid hormones (TH), which are known to play an important role in the development of the central nervous system (CNS). Although only a few studies have associated RTT and TH, TH deficit can lead to neurological deregulation by triggering functional deficiencies during adulthood. Here, we used human-induced pluripotent stem cell (iPSC) to generate MeCP2-knockout neuronal progenitor cells and adult neurons. Using this cellular model, we then investigated the expression of genes associated with TH homeostasis, such as the TH transporters (LAT1, LAT2, MCT8, MCT10, and OATP4A1) and deiodinases (DIO1, 2, and 3). Then, we treated the neural cells with THs and analyzed the expression of several genes related to neurodevelopment and functional maintenance. Our results showed that several TH-related genes, such as deiodinases, are altered in RTT samples when compared to WT cells. Moreover, the treatment of the neural cells with THs increased the amount of MAP2 and synapsin-1 expression in RTT cells. Our work provided evidences that TH homeostasis is compromised in RTT-derived neural cells, which could be an important factor to contribute to the imbalance in the neurodevelopmental phenotype presented in this syndrome and can lead us to better understand other neurodevelopmental diseases.

Cyclin-dependent kinase-like 5 binds and phosphorylates DNA methyltransferase 1.

DNA methyltransferase 1 (Dnmt1) is an enzyme that recognizes and methylates hemimethylated CpG after DNA replication to maintain methylation patterns. Although the N-terminal region of Dnmt1 is known to interact with various proteins, such as methyl-CpG-binding protein 2 (MeCP2), the associations of protein kinases with this region have not been reported. In the present study, we found that a 110-kDa protein kinase in mouse brain could bind to the N-terminal domain of Dnmt1. This 110-kDa kinase was identified as cyclin-dependent kinase-like 5 (CDKL5) by LC-MS/MS analysis. CDKL5 and Dnmt1 were found to colocalize in nuclei and appeared to interact with each other. Catalytically active CDKL5, CDKL5(1-352), phosphorylated the N-terminal region of Dnmt1 in the presence of DNA. Considering that defects in the MeCP2 or CDKL5 genes cause Rett syndrome, we propose that the interaction between Dnmt1 and CDKL5 may contribute to the pathogenic processes of Rett syndrome.

Autonomic cardiovascular control in methyl-CpG-binding protein 2 (Mecp2) deficient mice.

Methyl-CpG-binding protein 2 is a transcription factor that is involved in gene silencing. It is mutated in the majority of cases of Rett syndrome. This X-linked neurodevelopmental disorder is reported to involve abnormalities in autonomic cardiovascular regulation. As an initial step in understanding the basis for these abnormalities we have characterized autonomic cardiovascular function in Mecp2 deficient mice. Arterial pressure waves were recorded in freely moving animals using telemetry. Baseline blood pressure and pulse interval (PI) as well as indices of heart rate variability (HRV): standard deviation of PI (SDNN), range encompassing 90% of PIs (PI90) and standard deviation of adjacent PIs (SDSD) were similar in Mecp2(+/+) and Mecp2(+/-) animals. Spectral analysis of mean arterial pressure (MAP) and PI in the frequency domain showed similar relative power in low frequency 1 (LF1, 08-0.4 Hz), low frequency 2 (LF2, 0.4-1.0 Hz), middle frequency (MF, 1-3 Hz) and high frequency (HF, 3.0-10.0 Hz) bands. Autonomic blockade with atropine or propranolol as well as elevation in ambient temperature to 32 degrees C resulted in changes in blood pressure, PI and HRV that did not differ between the strains. Atropine, propranolol and elevated temperature resulted in similar changes in both MAP and PI spectral power. Baroreceptor function was tested using intravenous injections of nitroprusside followed by phenylephrine. Maximum gain was not different. These results do reveal any disturbance of autonomic cardiovascular regulation in the Mecp2 deficient mouse genotype.

Deletion of protein tyrosine phosphatase, non-receptor type 4 (PTPN4) in twins with a Rett syndrome-like phenotype.

Rett syndrome (RTT), a neurodevelopmental disorder that predominantly affects females, is primarily caused by variants in MECP2. Variants in other genes such as CDKL5 and FOXG1 are usually associated with individuals who manifest distinct phenotypes that may overlap with RTT. Individuals with phenotypes suggestive of RTT are typically screened for variants in MECP2 and then subsequently the other genes dependent on the specific phenotype. Even with this screening strategy, there are individuals in whom no causative variant can be identified, suggesting that there are other novel genes that contribute to the RTT phenotype. Here we report a de novo deletion of protein tyrosine phosphatase, non-receptor type 4 (PTPN4) in identical twins with a RTT-like phenotype. We also demonstrate the reduced expression of Ptpn4 in a Mecp2 null mouse model of RTT, as well as the activation of the PTPN4 promoter by MeCP2. Our findings suggest that PTPN4 should be considered for addition to the growing list of genes that warrant screening in individuals with a RTT-like phenotype.

PTP1B inhibition suggests a therapeutic strategy for Rett syndrome.

The X-linked neurological disorder Rett syndrome (RTT) presents with autistic features and is caused primarily by mutations in a transcriptional regulator, methyl CpG-binding protein 2 (MECP2). Current treatment options for RTT are limited to alleviating some neurological symptoms; hence, more effective therapeutic strategies are needed. We identified the protein tyrosine phosphatase PTP1B as a therapeutic candidate for treatment of RTT. We demonstrated that the PTPN1 gene, which encodes PTP1B, was a target of MECP2 and that disruption of MECP2 function was associated with increased levels of PTP1B in RTT models. Pharmacological inhibition of PTP1B ameliorated the effects of MECP2 disruption in mouse models of RTT, including improved survival in young male (Mecp2-/y) mice and improved behavior in female heterozygous (Mecp2-/+) mice. We demonstrated that PTP1B was a negative regulator of tyrosine phosphorylation of the tyrosine kinase TRKB, the receptor for brain-derived neurotrophic factor (BDNF). Therefore, the elevated PTP1B that accompanies disruption of MECP2 function in RTT represents a barrier to BDNF signaling. Inhibition of PTP1B led to increased tyrosine phosphorylation of TRKB in the brain, which would augment BDNF signaling. This study presents PTP1B as a mechanism-based therapeutic target for RTT, validating a unique strategy for treating the disease by modifying signal transduction pathways with small-molecule drugs.

Choline acetyltransferase activity and vesamicol binding in Rett syndrome and in rats with nucleus basalis lesions.

The decline in choline acetyltransferase activity has been identified previously within the brains of patients with Rett syndrome and Alzheimer's disease. The level of [3H]vesamicol binding to a terminal vesicular acetylcholine transporter is inversely related to the decline in cortical choline acetyltransferase activity in Alzheimer's disease, which may be due to compensatory processes within surviving cholinergic terminals. In order to investigate whether similar cholinergic compensatory processes are present in the Rett syndrome brain and are altered by normal aging, we investigated the density of cholinergic vesicular transporters in (i) the brains of Rett syndrome patients, and (ii) young and old rats with experimentally-induced cholinergic cell loss. In Rett syndrome, a significant decline in choline acetyltransferase activity within the putamen and thalamus was directly correlated with a decline in [3H]vesamicol binding. In both young and old rats, basal forebrain lesions decreased cortical choline acetyltransferase activity significantly, while [3H]vesamicol binding was unchanged. In contrast to young and old lesioned rats and patients with Alzheimer's disease, cholinergic cells in the brains of patients with Rett syndrome do not compensate for the loss of cholinergic cells by increasing acetylcholine vesicular storage.

Genomic structure of a human holocytochrome c-type synthetase gene in Xp22.3 and mutation analysis in patients with Rett syndrome.

The human holocytochrome c-type synthetase (HCCS) gene is located on Xp22.3 and is one of the genes identified in a 450-Kb region deleted in the neurodevelopmental disorder microphthalmia with linear skin defects. Several other developmental disorders with or without a neurological phenotype have been linked to Xp22.3. This region of the X chromosome was also found to be concordant in patients with Rett syndrome (RTT)in previously performed exclusion mapping. Based on its chromosomal location and its role in the mitochondrial respiratory chain, we analyzed HCCS as a candidate gene for RTT. The genomic structure of this gene, which occupies an 11-Kb region and consists of seven exons, was determined. All intron-exon boundaries were sequenced and primers were designed for polymerase chain reaction (PCR) amplification of each coding exon. PCR-amplified products from genomic DNA isolated from 20 RTT patients were screened for mutations using heteroduplex analysis. No mutations were detected. The genomic characterization of this gene will allow us to perform mutation analysis for other inherited disorders linked to this region.

An HPLC-linked assay of phosphoribosylpyrophosphate synthetase activity in the erythrocytes of adults and children with neurological disorders.

A two-step non-radioactive method that uses reverse-phase high-performance liquid chromatography (RP-HPLC) is described for the determination of phosphoribosylpyrophosphate synthetase (EC 2.7.6.1) activity in human erythrocytes. The method is accurate and easily reproducible in different chromatographic systems; it is based on the quantification of phosphoribosylpyrophosphate by conversion into orotidine monophosphate and uridine monophosphate. Phosphoribosylpyrophosphate synthetase activity was determined in the erythrocytes of healthy adults and children, the latter showing significantly higher activity than the former. The enzyme activity assayed in children with different neurological disorders was significantly lower in patients with Rett syndrome than in control children or in autistic or mentally retarded patients.

Rett syndrome and mitochondrial enzyme deficiencies.

The etiology of Rett syndrome is unknown. Structural mitochondrial abnormalities have been described in muscle in patients with Rett syndrome. We report three children with Rett syndrome and normal muscle mitochondrial structure on light and electron microscopy. However, all had abnormalities in mitochondrial respiratory chain enzymes.

Abnormal carbohydrate metabolism in cerebrospinal fluid in Rett syndrome.

We analyzed lactate, pyruvate, and citric acid cycle intermediates in cerebrospinal fluid by high-performance liquid chromatography in Rett syndrome patients (n = 27; mean age, 5.7 +/- 3.4 years) and age-matched female controls (n = 12; mean age, 7.0 +/- 3.3 years). The lactate, pyruvate, alpha-ketoglutarate, and malate were significantly elevated in Rett syndrome compared to the controls. The lactate/pyruvate ratio was not different. On the other hand, cerebrospinal fluid citrate, cis-aconitate, succinate, fumarate, and oxaloacetate were not significantly different in Rett syndrome patients than in the controls. We also evaluated the correlation between these acids and clinical symptoms and signs, including clinical stage, seizures medications (anticonvulsants or naltrexone), developmental quotient, self-abuse, and hyperventilation or apnea or both. The concentrations of all these acids did not differ significantly with clinical stage. Lactate elevation significantly correlated with apnea. Lactate and pyruvate elevation significantly correlated with hyperventilation or with both breathing abnormalities. Our observations in this sample of patients with Rett syndrome led us to speculate that patients with the Rett syndrome may have defective carbohydrate metabolism. Elevated mitochondrial reduced nicotinamide-adenine dinucleotide-linked substrates suggest that reduced nicotinamide-adenine dinucleotide-ubiquinone oxidoreductase may be deficient in the brain in Rett syndrome patients.

Neurobiology and neurochemistry of Rett syndrome.

The current status of neurobiological and neurochemical research on Rett syndrome is reviewed, and correlations are developed with previously described neurophysiological, neuroimaging, neuropathological, and immunohistochemical changes. We review the abnormalities reported in the biogenic amine neurotransmitters/receptor systems, and of beta-phenylethylamine, an endogenous amine synthesized by the decarboxylation of phenylalanine in dopaminergic neurons of the nigrostriatal system. We also discuss the roles of other neurotransmitters, including beta-endorphin and substance P, and neurotrophic factors, including nerve growth factors. Recently, DNA mutations in the methyl-CpG binding protein 2, mapped to Xq28, have been identified in some patients with Rett syndrome. The multiple abnormalities in the various neurotransmitters/receptor systems explain the pervasive effects of Rett syndrome.

Reduced expression of neuropeptides can be related to respiratory disturbances in Rett syndrome.

We immunohistochemically examined neurotransmitter systems, which function in the brainstem and are involved in neuronal organization of respiration, in an autopsy brain from a patient with Rett syndrome (RS). Immunoreactivity (IR) for tyrosine hydroxylase, a functional marker for catecholaminergic neurons, was severely reduced in the locus ceruleus, while that for tryptophan hydroxylase involved in serotonin synthesis was spared in the raphe nuclei. In the brainstem, IR for substance P (SP) was reduced in the parabrachial complex and that for methionine-enkephalin (met-enk) was affected in the parabrachial, hypoglossal, dorsal vagal and solitary nuclei. In addition, expressions of these neuropeptides were also disturbed in the basal ganglia. A widespread altered expression of antagonistic neuropeptides, SP and met-enk, may be involved in the pathogenesis of RS, especially in its respiratory manifestation.

Epigenetic modifications in the nervous system and their impact upon cognitive impairments.

Epigenetic regulation has been long considered to be a critical mechanism in the control of key aspects of cellular functions such as cell division, growth, and cell fate determination. Exciting recent developments have demonstrated that epigenetic mechanisms can also play necessary roles in the nervous system by regulating, for example, neuronal gene expression, DNA damage, and genome stability. Despite the fact that postmitotic neurons are developmentally less active then dividing cells, epigenetic regulation appears to provide means of both long-lasting and very dynamic regulation of neuronal function. Growing evidence indicates that epigenetic mechanisms in the central nervous system (CNS) are important for regulating not only specific aspects of individual neuronal metabolism but also for maintaining function of neuronal circuits and regulating their behavioral outputs. Multiple reports demonstrated that higher-level cognitive behaviors, such as learning and memory, are subject to a sophisticated epigenetic control, which includes interplay between multiple mechanisms of neuronal chromatin modification. Experiments with animal models have demonstrated that various epigenetic manipulations can affect cognition in different ways, from severe dysfunction to substantial improvement. In humans, epigenetic dysregulation has been known to underlie a number of disorders that are accompanied by mental impairment. Here, we review some of the epigenetic mechanisms that regulate cognition and how their disruption may contribute to cognitive dysfunctions. Due to the fact that histone acetylation and DNA methylation are some of the best-studied and critically important epigenomic modifications our research team has particularly strong expertise in, in this review, we are going to concentrate on histone acetylation, as well as DNA methylation/hydroxymethylation, in the mammalian CNS. Additional epigenetic modifications, not surveyed here, are being discussed in depth in the other review articles in this issue of Neuropharmacology.

Novel mutations in cyclin-dependent kinase-like 5 (CDKL5) gene in Indian cases of Rett syndrome.

Rett syndrome is a severe neurodevelopmental disorder, almost exclusively affecting females and characterized by a wide spectrum of clinical manifestations. Both the classic and atypical forms of Rett syndrome are primarily due to mutations in the methyl-CpG-binding protein 2 (MECP2) gene. Mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been identified in patients with atypical Rett syndrome, X-linked infantile spasms sharing common features of generally early-onset seizures and mental retardation. CDKL5 is known as serine/threonine protein kinase 9 (STK9) and is mapped to the Xp22 region. It has a conserved serine/threonine kinase domain within its amino terminus and a large C-terminal region. Disease-causing mutations are distributed in both the amino terminal domain and in the large C-terminal domain. We have screened the CDKL5 gene in 44 patients with atypical Rett syndrome who had tested negative for MECP2 gene mutations and have identified 6 sequence variants, out of which three were novel and three known mutations. Two of these novel mutations p.V966I and p.A1011V were missense and p.H589H a silent mutation. Other known mutations identified were p.V999M, p.Q791P and p.T734A. Sequence homology for all the mutations revealed that the two mutations (p.Q791P and p.T734A) were conserved across species. This indicated the importance of these residues in structure and function of the protein. The damaging effects of these mutations were analysed in silico using PolyPhen-2 online software. The PolyPhen-2 scores of p.Q791P and p.T734A were 0.998 and 0.48, revealing that these mutations could be deleterious and might have potential functional effect. All other mutations had a low score suggesting that they might not alter the activity of CDKL5. We have also analysed the position of the mutations in the CDKL5 protein and found that all the mutations were present in the C-terminal domain of the protein. The C-terminal domain is required for cellular localization through protein-protein interaction; any mutations in this domain might alter this function of the protein. This is the first report from India showing the mutation in CDKL5 gene in Indian cases of Rett syndrome. Our study emphasizes the role of CDKL5 mutation screening in cases of atypical Rett syndrome with congenital seizure variant.

Modulation of RhoGTPases improves the behavioral phenotype and reverses astrocytic deficits in a mouse model of Rett syndrome.

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.