Mecp2-null Mice Research Articles

Human microglial cells as a therapeutic target in a neurodevelopmental disease model

Although microglia are macrophages of the central nervous system, their involvement is not limited to immune functions. The roles of microglia during development in humans remain poorly understood due to limited access to fetal tissue. To understand how microglia can impact human neurodevelopment, the methyl-CpG binding protein 2 (MECP2) gene was knocked out in human microglia-like cells (MGLs). Disruption of the MECP2 in MGLs led to transcriptional and functional perturbations, including impaired phagocytosis. The co-culture of healthy MGLs with MECP2-knockout (KO) neurons rescued synaptogenesis defects, suggesting a microglial role in synapse formation. A targeted drug screening identified ADH-503, a CD11b agonist, restored phagocytosis and synapse formation in spheroid-MGL co-cultures, significantly improved disease progression, and increased survival in MeCP2-null mice. These results unveil a MECP2-specific regulation of human microglial phagocytosis and identify a novel therapeutic treatment for MECP2-related conditions.

A281 CASE REPORT: STEATOTIC LIVER DISEASE DIAGNOSED IN A 24-YEAR-OLD FEMALE WITH RETT SYNDROME

Abstract Background Rett syndrome (RTT) is a rare, severe neurodevelopmental disorder seen almost exclusively in females that is caused by spontaneous loss-of-function mutations in the methyl CpG binding protein 2 (MECP2) gene. Girls with RTT typically demonstrate normal development for the first 6-18 months of life before displaying evidence of variable levels of developmental milestone regression and physical disability. Although perturbed lipid metabolism in the nervous system is the main contributor to the pathophysiology of RTT, mutations to the MECP2 gene also contribute to disease burden by causing perturbations to peripheral systems, leading to consequences such as dyslipidemia (DLD) and steatotic liver disease (SLD). Although DLD has been described in human cell lines and girls with RTT, SLD has only been described in RTT mouse models. Aims This report aims to present a case of a patient with RTT who was diagnosed with SLD on liver biopsy in the context of DLD but without insulin resistance or other comorbidities associated with metabolic syndrome. Methods Retrospective review of one patient. Results A 24-year-old woman with genetically proven RTT (T158M point mutation to MECP2) complicated by several physical disabilities was seen at a tertiary care center clinic for evaluation of abnormal liver enzymes (ALT 163, AST 51, ALP 258, total bilirubin 6). Abdominal ultrasound demonstrated moderately echogenic liver parenchyma consistent with hepatic steatosis. Additional investigations for an etiology were unremarkable and the patient had no history of alcohol misuse or the use of new medications or supplements. Regarding risk factors for SLD, her homeostatic model assessment for insulin resistance (HOMA-IR) score was 1.8, hemoglobin A1c was 4.8%, and her lipid profile demonstrated elevated triglycerides and LDL, in keeping with DLD. The decision was made to proceed with a liver biopsy. This demonstrated moderate steatosis and no ballooning, categorized as grade A1 and stage F1. Conclusions This case demonstrates that RTT patients have similar lipid metabolism perturbations to those seen in MECP2-null mouse models, resulting in similar metabolic abnormalities such as DLD and SLD. The absence of insulin resistance and other comorbidities associated with metabolic syndrome further support these perturbations are caused by a pathophysiologic mechanism specific to RTT. This demonstrates the importance of screening RTT patients for DLD and SLD despite their young age to detect these conditions before complications arise. It also demonstrates that MECP2-null mice can be used as reliable models to identify future therapies for RTT, such as ketogenic diet, ω-3 PUFAs supplementation, and lipid-lowering statin therapy. Funding Agencies None

RIPK1 activation in Mecp2-deficient microglia promotes inflammation and glutamate release in RTT

Rett syndrome (RTT) is a devastating neurodevelopmental disorder primarily caused by mutations in the methyl-CpG binding protein 2 (Mecp2) gene. Here, we found that inhibition of Receptor-Interacting Serine/Threonine-Protein Kinase 1 (RIPK1) kinase ameliorated progression of motor dysfunction after onset and prolonged the survival of Mecp2-null mice. Microglia were activated early in myeloid Mecp2-deficient mice, which was inhibited upon inactivation of RIPK1 kinase. RIPK1 inhibition in Mecp2-deficient microglia reduced oxidative stress, cytokines production and induction of SLC7A11, SLC38A1, and GLS, which mediate the release of glutamate. Mecp2-deficient microglia release high levels of glutamate to impair glutamate-mediated excitatory neurotransmission and promote increased levels of GluA1 and GluA2/3 proteins in vivo, which was reduced upon RIPK1 inhibition. Thus, activation of RIPK1 kinase in Mecp2-deficient microglia may be involved both in the onset and progression of RTT.

Adenosinergic System and BDNF Signaling Changes as a Cross-Sectional Feature of RTT: Characterization of Mecp2 Heterozygous Mouse Females.

Rett Syndrome is an X-linked neurodevelopmental disorder (RTT; OMIM#312750) associated to MECP2 mutations. MeCP2 dysfunction is seen as one cause for the deficiencies found in brain-derived neurotrophic factor (BDNF) signaling, since BDNF is one of the genes under MeCP2 jurisdiction. BDNF signaling is also dependent on the proper function of the adenosinergic system. Indeed, both BDNF signaling and the adenosinergic system are altered in Mecp2-null mice (Mecp2-/y), a representative model of severe manifestation of RTT. Considering that symptoms severity largely differs among RTT patients, we set out to investigate the BDNF and ADO signaling modifications in Mecp2 heterozygous female mice (Mecp2+/-) presenting a less severe phenotype. Symptomatic Mecp2+/- mice have lower BDNF levels in the cortex and hippocampus. This is accompanied by a loss of BDNF-induced facilitation of hippocampal long-term potentiation (LTP), which could be restored upon selective activation of adenosine A2A receptors (A2AR). While no differences were observed in the amount of adenosine in the cortex and hippocampus of Mecp2+/- mice compared with healthy littermates, the density of the A1R and A2AR subtype receptors was, respectively, upregulated and downregulated in the hippocampus. Data suggest that significant changes in BDNF and adenosine signaling pathways are present in an RTT model with a milder disease phenotype: Mecp2+/- female animals. These features strengthen the theory that boosting adenosinergic activity may be a valid therapeutic strategy for RTT patients, regardless of their genetic penetrance.

Activity-induced MeCP2 phosphorylation regulates retinogeniculate synapse refinement.

Mutations in MECP2 give rise to Rett syndrome (RTT), an X-linked neurodevelopmental disorder that results in broad cognitive impairments in females. While the exact etiology of RTT symptoms remains unknown, one possible explanation for its clinical presentation is that loss of MECP2 causes miswiring of neural circuits due to defects in the brain's capacity to respond to changes in neuronal activity and sensory experience. Here, we show that MeCP2 is phosphorylated at four residues in the mouse brain (S86, S274, T308, and S421) in response to neuronal activity, and we generate a quadruple knock-in (QKI) mouse line in which all four activity-dependent sites are mutated to alanines to prevent phosphorylation. QKI mice do not display overt RTT phenotypes or detectable gene expression changes in two brain regions. However, electrophysiological recordings from the retinogeniculate synapse of QKI mice reveal that while synapse elimination is initially normal at P14, it is significantly compromised at P20. Notably, this phenotype is distinct from the synapse refinement defect previously reported for Mecp2 null mice, where synapses initially refine but then regress after the third postnatal week. We thus propose a model in which activity-induced phosphorylation of MeCP2 is critical for the proper timing of retinogeniculate synapse maturation specifically during the early postnatal period.

Systemic proteome phenotypes reveal defective metabolic flexibility in Mecp2 mutants.

Genes mutated in monogenic neurodevelopmental disorders are broadly expressed. This observation supports the concept that monogenic neurodevelopmental disorders are systemic diseases that profoundly impact neurodevelopment. We tested the systemic disease model focusing on Rett syndrome, which is caused by mutations in MECP2. Transcriptomes and proteomes of organs and brain regions from Mecp2-null mice as well as diverse MECP2-null male and female human cells were assessed. Widespread changes in the steady-state transcriptome and proteome were identified in brain regions and organs of presymptomatic Mecp2-null male mice as well as mutant human cell lines. The extent of these transcriptome and proteome modifications was similar in cortex, liver, kidney, and skeletal muscle and more pronounced than in the hippocampus and striatum. In particular, Mecp2- and MECP2-sensitive proteomes were enriched in synaptic and metabolic annotated gene products, the latter encompassing lipid metabolism and mitochondrial pathways. MECP2 mutations altered pyruvate-dependent mitochondrial respiration while maintaining the capacity to use glutamine as a mitochondrial carbon source. We conclude that mutations in Mecp2/MECP2 perturb lipid and mitochondrial metabolism systemically limiting cellular flexibility to utilize mitochondrial fuels.

Blood–Brain Barrier Integrity Is Perturbed in a Mecp2-Null Mouse Model of Rett Syndrome

Rett syndrome (RTT, online MIM 312750) is a devastating neurodevelopmental disorder characterized by motor and cognitive disabilities. It is mainly caused by pathogenetic variants in the X-linked MECP2 gene, encoding an epigenetic factor crucial for brain functioning. Despite intensive studies, the RTT pathogenetic mechanism remains to be fully elucidated. Impaired vascular function has been previously reported in RTT mouse models; however, whether an altered brain vascular homeostasis and the subsequent blood-brain barrier (BBB) breakdown occur in RTT and contribute to the disease-related cognitive impairment is still unknown. Interestingly, in symptomatic Mecp2-null (Mecp2-/y, Mecp2tm1.1Bird) mice, we found enhanced BBB permeability associated with an aberrant expression of the tight junction proteins Ocln and Cldn-5 in different brain areas, in terms of both transcript and protein levels. Additionally, Mecp2-null mice showed an altered expression of different genes encoding factors with a role in the BBB structure and function, such as Cldn3, Cldn12, Mpdz, Jam2, and Aqp4. With this study, we provide the first evidence of impaired BBB integrity in RTT and highlight a potential new molecular hallmark of the disease that might open new perspectives for the setting-up of novel therapeutic strategies.

A single-cell atlas reveals the heterogeneity of meningeal immunity in a mouse model of Methyl CpG binding protein 2 deficiency.

Methyl CpG binding protein 2 (MeCP2) is a DNA methylation reader protein. Mutations in MeCP2 are the major cause of Rett syndrome (RTT). Increasing evidence has shown that dysregulated immunity and chronic subclinical inflammation are linked to MeCP2 deficiency and contribute to RTT development and deterioration. The meninges surrounding the central nervous system (CNS) contain a wide repertoire of immune cells that participate in immune surveillance within the CNS and influence various brain functions; however, the characterization and role of meningeal immunity in CNS with MeCP2 deficiency remain poorly addressed. Here, we used single-cell sequencing to profile Mecp2-deficient meningeal immune cells from the dura mater, which has been reported to contain the most meningeal immune cells during homeostasis. Data showed that the meninges of Mecp2-null mice contained the same diverse immune cell populations as control mice and showed an up-regulation of immune-related processes. B cell populations were greater in Mecp2-null mice than in control mice, and the expression of genes encoding for immunoglobulins was remarkably higher. Mecp2-deficient meninges also contained more cytotoxic CD8+ T cells than control meninges. With increased interferon-γ transcription in T and natural killer cells, meningeal macrophages showed decreased suppression and increased activity in Mecp2-deficienct mice. Together, these findings provide novel insights into meningeal immunity, which is a less studied aspect of neuroimmune interactions in Mecp2-mutated diseases, and offer an essential resource for comparative analyses and data exploration to better understand the functional role of meningeal immunity in RTT.

5-HT1A Receptor Agonist Treatment Partially Ameliorates Rett Syndrome Phenotypes in mecp2-Null Mice by Rescuing Impairment of Neuron Transmission and the CREB/BDNF Signaling Pathway.

Rett syndrome (RTT) is an X-linked neurodevelopmental disorder caused by mutations in the gene that encodes methyl CpG-binding protein 2 (MECP2) and is characterized by the loss of acquired motor and language skills, stereotypic movements, respiratory abnormalities and autistic features. There has been no effective treatment for this disorder until now. In this study, we used a Mecp2-null (KO) mouse model of RTT to investigate whether repeated intraperitoneal treatment with the 5-HT1A receptor agonist tandospirone could improve the RTT phenotype. The results showed that administration of tandospirone significantly extended the lifespan of Mecp2-KO mice and obviously ameliorated RTT phenotypes, including general condition, hindlimb clasping, gait, tremor and breathing in Mecp2-KO mice. Tandospirone treatment significantly improved the impairment in GABAergic, glutaminergic, dopaminergic and serotoninergic neurotransmission in the brainstem of Mecp2-KO mice. Decreased dopaminergic neurotransmission in the cerebellum of Mecp2-KO mice was also significantly increased by tandospirone treatment. Moreover, RNA-sequencing analysis found that tandospirone modulates the RTT phenotype, partially through the CREB1/BDNF signaling pathway in Mecp2-KO mice. These findings provide a new option for clinical treatment.

Rett syndrome with atrial tachycardia in a girl.

Rett syndrome with atrial tachycardia in a girl.

MeCP2 deficiency impairs motor cortical circuit flexibility associated with motor learning

Loss of function mutations in the X-linked gene encoding methyl-CpG binding protein 2 (MECP2) cause Rett syndrome (RTT), a postnatal neurological disorder. The loss of motor function is an important clinical feature of RTT that manifests early during the course of the disease. RTT mouse models with mutations in the murine orthologous Mecp2 gene replicate many human phenotypes, including progressive motor impairments. However, relatively little is known about the changes in circuit function during the progression of motor deficit in this model. As the motor cortex is the key node in the motor system for the control of voluntary movement, we measured firing activity in populations of motor cortical neurons during locomotion on a motorized wheel-treadmill. Different populations of neurons intermingled in the motor cortex signal different aspects of the locomotor state of the animal. The proportion of running selective neurons whose activity positively correlates with locomotion speed gradually decreases with weekly training in wild-type mice, but not in Mecp2-null mice. The fraction of rest-selective neurons whose activity negatively correlates with locomotion speed does not change with training in wild-type mice, but is higher and increases with the progression of locomotion deficit in mutant mice. The synchronization of population activity that occurs in WT mice with training did not occur in Mecp2-null mice, a phenotype most clear during locomotion and observable across all functional cell types. Our results could represent circuit-level biomarkers for motor regression in Rett syndrome.

Early life stress exacerbates behavioural and neuronal alterations in adolescent male mice lacking methyl-CpG binding protein 2 (Mecp2).

The methyl-CpG binding protein 2 gene (MECP2) encodes an epigenetic transcriptional regulator implicated in neuronal plasticity. Loss-of-function mutations in this gene are the primary cause of Rett syndrome and, to a lesser degree, of other neurodevelopmental disorders. Recently, we demonstrated that both Mecp2 haploinsuficiency and mild early life stress decrease anxiety-like behaviours and neuronal activation in brain areas controlling these responses in adolescent female mice. Here, we extend this work to males by using Mecp2-null and wild type adolescent mice subjected to maternal separation and their non-stressed controls. We assessed their behavioural responses in a battery of anxiety-provoking tests. Upon exposure to an elevated plus maze in aversive conditions, we evaluated changes in c-FOS expression in stress- and anxiety-related brain regions. In addition, we assessed the impact of maternal separation in neuronal maturation using doublecortin and reelin as surrogate markers. Mutant males showed reduced motor abilities, increased activation of the olfactory bulbs, probably due to breathing abnormalities, and decreased activation of the paraventricular thalamic nucleus, when compared to wild type mice. In addition, maternal separation increased the number of immature doublecortin-like neurons found in Mecp2-null animals. Moreover, this work shows for the first time that reelin is decreased in the mutant animals at the olfactory tubercle, piriform cortex and hippocampal dentate gyrus, an effect also associated to maternal separation. Taken together, our results suggest that maternal separation exacerbates some phenotypical alterations associated with lack of MeCP2 in adolescent males.

Vitamin D modulates cortical transcriptome and behavioral phenotypes in an Mecp2 heterozygous Rett syndrome mouse model

Rett syndrome (RTT) is an X-linked neurological disorder caused by mutations in the transcriptional regulator MECP2. Mecp2 loss-of-function leads to the disruption of many cellular pathways, including aberrant activation of the NF-κB pathway. Genetically attenuating the NF-κB pathway in Mecp2-null mice ameliorates hallmark phenotypes of RTT, including reduced dendritic complexity, raising the question of whether NF-κB pathway inhibitors could provide a therapeutic avenue for RTT. Vitamin D is a known inhibitor of NF-κB signaling; further, vitamin D deficiency is prevalent in RTT patients and male Mecp2-null mice. We previously demonstrated that vitamin D rescues the aberrant NF-κB activity and reduced neurite outgrowth of Mecp2-knockdown cortical neurons in vitro, and that dietary vitamin D supplementation rescues decreased dendritic complexity and soma size of neocortical projection neurons in both male hemizygous Mecp2-null and female heterozygous mice in vivo. Here, we have identified over 200 genes whose dysregulated expression in the Mecp2+/− cortex is modulated by dietary vitamin D. Genes normalized with vitamin D supplementation are involved in dendritic complexity, synapses, and neuronal projections, suggesting that the rescue of their expression could underpin the rescue of neuronal morphology. Further, there is a disruption in the homeostasis of the vitamin D synthesis pathway in Mecp2+/− mice, and motor and anxiety-like behavioral phenotypes in Mecp2+/− mice correlate with circulating vitamin D levels. Thus, our data indicate that vitamin D modulates RTT pathology and its supplementation could provide a simple and cost-effective partial therapeutic for RTT.

The Chloride Homeostasis of CA3 Hippocampal Neurons Is Not Altered in Fully Symptomatic Mepc2-null Mice.

Rett syndrome (RTT) is an X-linked neurodevelopmental disorder caused mainly by mutations in the MECP2 gene. Mouse models of RTT show reduced expression of the cation-chloride cotransporter KCC2 and altered chloride homeostasis at presymptomatic stages. However, whether these alterations persist to late symptomatic stages has not been studied. Here we assess KCC2 and NKCC1 expressions and chloride homeostasis in the hippocampus of early [postnatal (P) day 30–35] and late (P50–60) symptomatic male Mecp2-null (Mecp2–/y) mice. We found (i) no difference in the relative amount, but an over-phosphorylation, of KCC2 and NKCC1 between wild-type (WT) and Mecp2–/y hippocampi and (ii) no difference in the inhibitory strength, nor reversal potential, of GABAA-receptor-mediated responses in Mecp2–/y CA3 pyramidal neurons compared to WT at any stages studied. Altogether, these data indicate the presence of a functional chloride extrusion mechanism in Mecp2–/y CA3 pyramidal neurons at symptomatic stages.

Perineuronal net degradation rescues CA2 plasticity in a mouse model of Rett syndrome.

Perineuronal nets (PNNs), a specialized form of extracellular matrix, are abnormal in the brains of people with Rett syndrome (RTT). We previously reported that PNNs function to restrict synaptic plasticity in hippocampal area CA2, which is unusually resistant to long-term potentiation (LTP) and has been linked to social learning in mice. Here we report that PNNs appear elevated in area CA2 of the hippocampus of an individual with RTT and that PNNs develop precociously and remain elevated in area CA2 of a mouse model of RTT (Mecp2-null). Further, we provide evidence that LTP could be induced at CA2 synapses prior to PNN maturation (postnatal day 8-11) in wild-type mice and that this window of plasticity was prematurely restricted at CA2 synapses in Mecp2-null mice. Degrading PNNs in Mecp2-null hippocampus was sufficient to rescue the premature disruption of CA2 plasticity. We identified several molecular targets that were altered in the developing Mecp2-null hippocampus that may explain aberrant PNNs and CA2 plasticity, and we discovered that CA2 PNNs are negatively regulated by neuronal activity. Collectively, our findings demonstrate that CA2 PNN development is regulated by Mecp2 and identify a window of hippocampal plasticity that is disrupted in a mouse model of RTT.

The enhancement of activity rescues the establishment of Mecp2 null neuronal phenotypes

MECP2 mutations cause Rett syndrome (RTT), a severe and progressive neurodevelopmental disorder mainly affecting females. Although RTT patients exhibit delayed onset of symptoms, several evidences demonstrate that MeCP2 deficiency alters early development of the brain. Indeed, during early maturation, Mecp2 null cortical neurons display widespread transcriptional changes, reduced activity, and defective morphology. It has been proposed that during brain development these elements are linked in a feed‐forward cycle where neuronal activity drives transcriptional and morphological changes that further increase network maturity. We hypothesized that the enhancement of neuronal activity during early maturation might prevent the onset of RTT‐typical molecular and cellular phenotypes. Accordingly, we show that the enhancement of excitability, obtained by adding to neuronal cultures Ampakine CX546, rescues transcription of several genes, neuronal morphology, and responsiveness to stimuli. Greater effects are achieved in response to earlier treatments. In vivo, short and early administration of CX546 to Mecp2 null mice prolongs lifespan, delays the disease progression, and rescues motor abilities and spatial memory, thus confirming the value for RTT of an early restoration of neuronal activity.

Neuronal non-CG methylation is an essential target for MeCP2 function

SummaryDNA methylation is implicated in neuronal biology via the protein MeCP2, the mutation of which causes Rett syndrome. MeCP2 recruits the NCOR1/2 co-repressor complexes to methylated cytosine in the CG dinucleotide, but also to sites of non-CG methylation, which are abundant in neurons. To test the biological significance of the dual-binding specificity of MeCP2, we replaced its DNA binding domain with an orthologous domain from MBD2, which can only bind mCG motifs. Knockin mice expressing the domain-swap protein displayed severe Rett-syndrome-like phenotypes, indicating that normal brain function requires the interaction of MeCP2 with sites of non-CG methylation, specifically mCAC. The results support the notion that the delayed onset of Rett syndrome is due to the simultaneous post-natal accumulation of mCAC and its reader MeCP2. Intriguingly, genes dysregulated in both Mecp2 null and domain-swap mice are implicated in other neurological disorders, potentially highlighting targets of relevance to the Rett syndrome phenotype.

Male-specific features are reduced in Mecp2-null mice: analyses of vasopressinergic innervation, pheromone production and social behaviour.

Deficits in arginine vasopressin (AVP) and oxytocin (OT), two neuropeptides closely implicated in the modulation of social behaviours, have been reported in some early developmental disorders and autism spectrum disorders. Mutations in the X-linked methyl-CpG-binding protein 2 (MECP2) gene are associated to Rett syndrome and other neuropsychiatric conditions. Thus, we first analysed AVP and OT expression in the brain of Mecp2-mutant mice by immunohistochemistry. Our results revealed no significant differences in these systems in young adult Mecp2-heterozygous females, as compared to WT littermates. By contrast, we found a significant reduction in the sexually dimorphic, testosterone-dependent, vasopressinergic innervation in several nuclei of the social brain network and oxytocinergic innervation in the lateral habenula of Mecp2-null males, as compared to WT littermates. Analysis of urinary production of pheromones shows that Mecp2-null males lack the testosterone-dependent pheromone darcin, strongly suggesting low levels of androgens in these males. In addition, resident-intruder tests revealed lack of aggressive behaviour in Mecp2-null males and decreased chemoinvestigation of the intruder. By contrast, Mecp2-null males exhibited enhanced social approach, as compared to WT animals, in a 3-chamber social interaction test. In summary, Mecp2-null males, which display internal testicles, display a significant reduction of some male-specific features, such as vasopressinergic innervation within the social brain network, male pheromone production and aggressive behaviour. Thus, atypical social behaviours in Mecp2-null males may be caused, at least in part, by the effect of lack of MeCP2 over sexual differentiation.

Fluoxetine rescues rotarod motor deficits in Mecp2 heterozygous mouse model of Rett syndrome via brain serotonin

Motor skill is a specific area of disability of Rett syndrome (RTT), a rare disorder occurring almost exclusively in girls, caused by loss-of-function mutations of the X-linked methyl-CpG-binding protein2 (MECP2) gene, encoding the MECP2 protein, a member of the methyl-CpG-binding domain nuclear proteins family. Brain 5-HT, which is defective in RTT patients and Mecp2 mutant mice, regulates motor circuits and SSRIs enhance motor skill learning and plasticity.In the present study, we used heterozygous (Het) Mecp2 female and Mecp2-null male mice to investigate whether fluoxetine, a SSRI with pleiotropic effects on neuronal circuits, rescues motor coordination deficits. Repeated administration of 10 mg/kg fluoxetine fully rescued rotarod deficit in Mecp2 Het mice regardless of age, route of administration or pre-training to rotarod. The motor improvement was confirmed in the beam walking test while no effect was observed in the hanging-wire test, suggesting a preferential action of fluoxetine on motor coordination. Citalopram mimicked the effects of fluoxetine, while the inhibition of 5-HT synthesis abolished the fluoxetine-induced improvement of motor coordination. Mecp2 null mice, which responded poorly to fluoxetine in the rotarod, showed reduced 5-HT synthesis in the prefrontal cortex, hippocampus and striatum, and reduced efficacy of fluoxetine in raising extracellular 5-HT as compared to female mutants. No sex differences were observed in the ability of fluoxetine to desensitize 5-HT1A autoreceptors upon repeated administration. These findings indicate that fluoxetine rescues motor coordination in Mecp2 Het mice through its ability to enhance brain 5-HT and suggest that drugs enhancing 5-HT neurotransmission may have beneficial effects on motor symptoms of RTT.

The antitussive cloperastine improves breathing abnormalities in a Rett Syndrome mouse model by blocking presynaptic GIRK channels and enhancing GABA release

Rett Syndrome (RTT) is an X-linked neurodevelopmental disorder caused mainly by mutations in the MECP2 gene. One of the major RTT features is breathing dysfunction characterized by periodic hypo- and hyperventilation. The breathing disorders are associated with increased brainstem neuronal excitability, which can be alleviated with GABA agonists. Since neuronal hypoexcitability occurs in the forebrain of RTT models, it is necessary to find pharmacological agents with a relative preference to brainstem neurons. Here we show evidence for the improvement of breathing disorders of Mecp2-disrupted mice with the brainstem-acting drug cloperastine (CPS) and its likely neuronal targets. CPS is an over-the-counter cough medicine that has an inhibitory effect on brainstem neuronal networks. In Mecp2-disrupted mice, CPS (30 mg/kg, i.p.) decreased the occurrence of apneas/h and breath frequency variation. GIRK currents expressed in HEK cells were inhibited by CPS with IC50 1 μM. Whole-cell patch clamp recordings in locus coeruleus (LC) and dorsal tegmental nucleus (DTN) neurons revealed an overall inhibitory effect of CPS (10 μM) on neuronal firing activity. Such an effect was reversed by the GABAA receptor antagonist bicuculline (20 μM). Voltage clamp studies showed that CPS increased GABAergic sIPSCs in LC cells, which was blocked by the GABAB receptor antagonist phaclofen. Functional GABAergic connections of DTN neurons with LC cells were shown. These results suggest that CPS improves breathing dysfunction in Mecp2-null mice by blocking GIRK channels in synaptic terminals and enhancing GABA release.