Mecp2-deficient Mice Research Articles

Setdb1-mediated histone H3K9 hypermethylation in neurons worsens the neurological phenotype of Mecp2-deficient mice

Rett syndrome (RTT, OMIM # 312750), a neurodevelopmental disorder of early childhood, is primarily caused by mutations in the gene encoding methyl-CpG-binding protein 2 ( MECP2). Various molecular functions have been ascribed to MECP2, including the regulation of histone modifications associated with repressive chromatin remodeling, but the role of these mechanisms for the pathophysiology of RTT remains unclear. Here, we explore whether or not neuronal expression of the histone H3-lysine 9 specific methyl-transferase, Setdb1 (Set domain, bifurcated 1)/Eset/Kmt1e, which is normally present only at low levels in differentiated neurons, rescues the RTT-like phenotype of Mecp2-deficient mice. A myc-tagged Setdb1 cDNA was expressed through the tau locus for ubiquitous expression in CNS neurons, or under control of the calcium/calmodulin-dependent protein kinase II (CK) promoter to selectively target postmitotic neurons in forebrain. However, the CK-Setdb1 transgene lead to an enhanced neurological deficit, and the tauSetdb1 allele further shortened life span of mice with a brain-wide deletion of Mecp2 during prenatal development. In contrast, no neurological deficits or premature death was observed in CK-Setdb1 and tauSetdb1 mice expressing wildtype Mecp2. However, levels of trimethylated H3K9 at pericentromeric repeats were fully maintained in differentiated neurons from symptomatic Mecp2 null mutant mice. Based on these results, we draw two conclusions: First, neuronal chromatin in RTT brain is not affected by a generalized deficit in H3K9 trimethylation. Second, artificial up-regulation of this repressive chromatin mark via Setdb1 gene delivery specifically to neurons is harmful for the Mecp2-deficient brain. This article is part of a Special Issue entitled ‘Trends in Neuropharmacology: In Memory of Erminio Costa’.

Impaired hippocampal Ca 2+ homeostasis and concomitant K + channel dysfunction in a mouse model of rett syndrome during anoxia

Methyl-CpG-binding protein 2 (MeCP2) deficiency causes Rett syndrome (RTT), a neurodevelopmental disorder characterized by severe cognitive impairment, synaptic dysfunction, and hyperexcitability. Previously we reported that the hippocampus of MeCP2-deficient mice ( Mecp2 −/y ), a mouse model for RTT, is more susceptible to hypoxia. To identify the underlying mechanisms we now focused on the anoxic responses of wildtype (WT) and Mecp2 −/y CA1 neurons in acute hippocampal slices. Intracellular recordings revealed that Mecp2 −/y neurons show only reduced or no hyperpolarizations early during cyanide-induced anoxia, suggesting potassium channel (K + channel) dysfunction. Blocking adenosine-5′-triphosphate-sensitive K + channels (K ATP-) and big-conductance Ca 2+-activated K + channels (BK-channels) did not affect the early anoxic hyperpolarization in either genotype. However, blocking Ca 2+ release from the endoplasmic reticulum almost abolished the anoxic hyperpolarizations in Mecp2 −/y neurons. Single-channel recordings confirmed that neither K ATP- nor BK-channels are the sole mediators of the early anoxic hyperpolarization. Instead, anoxia Ca 2+-dependently activated various small/intermediate-conductance K + channels in WT neurons, which was less evident in Mecp2 −/y neurons. Yet, pharmacologically increasing the Ca 2+ sensitivity of small/intermediate-conductance K Ca channels fully restored the anoxic hyperpolarization in Mecp2 −/y neurons. Furthermore, Ca 2+ imaging unveiled lower intracellular Ca 2+ levels in resting Mecp2 −/y neurons and reduced anoxic Ca 2+ transients with diminished Ca 2+ release from intracellular stores. In conclusion, the enhanced hypoxia susceptibility of Mecp2 −/y hippocampus is primarily associated with disturbed Ca 2+ homeostasis and diminished Ca 2+ rises during anoxia. This secondarily attenuates the activation of K Ca channels and thereby increases the hypoxia susceptibility of Mecp2 −/y neuronal networks. Since cytosolic Ca 2+ levels also determine neuronal excitability and synaptic plasticity, Ca 2+ homeostasis may constitute a promising target for pharmacotherapy in RTT.

Physiological definition of upper airway obstructions in mouse model for Rett syndrome

Rett syndrome is a neuro-developmental disease accompanied by breathing symptoms including breath-hold events, and is caused by mutation of the transcriptional repressor methyl-CpG-binding protein 2 (MeCP2). Males of Mecp2-deficient mice (Mecp2(-/y)) also develop breathing symptoms, with erratic rhythm and life-threatening apnoeas from postnatal day 30 (P30), leading to respiratory distress and premature death at around P60. We investigated the respiratory function of conscious Mecp2(-/y) mice at P40-P60 using conventional whole-body plethysmography, double-chamber plethysmography and chest EMG recordings. Double-chamber plethysmography revealed a persistent increase in respiratory work-load with enlarged chest movements, but no subsequent increase of tidal volume thus revealing a mismatch between airflow and muscle work-load. Apnoeas occurred with cessation of both chest movements and ventilation, but some (40%) developed with persisting rhythmic chest EMG discharges or chest movements without respiratory airflow, suggesting respiratory efforts against obstructed airways. Airway obstruction was maintained even when the respiratory drive increased significantly, triggering large chest EMG discharges and movements. Whole-body plethysmography of Mecp2(-/y) mice revealed significant increases of spirograms, reflecting forced chest movements against partially obstructed airways. The persisting chest EMG discharges and rhythmic chest movements without respiratory airflow suggest that Mecp2 inactivation alters neural circuits controlling the upper airway dilator muscles. The observed breath-hold events in Mecp2(-/y) mice might imply disturbance of neural circuits attached to voluntary control of breathing.

Synaptic Maturation at Cortical Projections to the Lateral Amygdala in a Mouse Model of Rett Syndrome

Rett syndrome (RTT) is a neuro-developmental disorder caused by loss of function of Mecp2 - methyl-CpG-binding protein 2 - an epigenetic factor controlling DNA transcription. In mice, removal of Mecp2 in the forebrain recapitulates most of behavioral deficits found in global Mecp2 deficient mice, including amygdala-related hyper-anxiety and lack of social interaction, pointing a role of Mecp2 in emotional learning. Yet very little is known about the establishment and maintenance of synaptic function in the adult amygdala and the role of Mecp2 in these processes. Here, we performed a longitudinal examination of synaptic properties at excitatory projections to principal cells of the lateral nucleus of the amygdala (LA) in Mecp2 mutant mice and their wild-type littermates. We first show that during animal life, Cortico-LA projections switch from a tonic to a phasic mode, whereas Thalamo-LA synapses are phasic at all ages. In parallel, we observed a specific elimination of Cortico-LA synapses and a decrease in their ability of generating presynaptic long term potentiation. In absence of Mecp2, both synaptic maturation and synaptic elimination were exaggerated albeit still specific to cortical projections. Surprisingly, associative LTP was unaffected at Mecp2 deficient synapses suggesting that synaptic maintenance rather than activity-dependent synaptic learning may be causal in RTT physiopathology. Finally, because the timing of synaptic evolution was preserved, we propose that some of the developmental effects of Mecp2 may be exerted within an endogenous program and restricted to synapses which maturate during animal life.

Ocular MECP2 Protein Expression in Patients With and Without Rett Syndrome

Rett syndrome is a neurodevelopmental disorder caused by mutations in the methyl CpG binding protein 2 gene (MECP2). The MECP2 protein is expressed primarily in neurons, and mutations in the gene lead to the clinical features of Rett syndrome in human patients and neurologic deficits in murine models. Visual function is relatively preserved in Rett syndrome patients, but the cause is unknown. The eyes of two Rett syndrome patients who died of the disease were analyzed; no gross or microscopic changes were found. MECP2 expression was examined using immunohistochemistry; nuclear protein expression was largely limited to ganglion cells and the portion of the inner nuclear layer populated by amacrine cells. No significant differences in MECP2 protein level or distribution were identified in the two eyes from the Rett syndrome patients, compared with 11 controls. The findings were compared with MECP2 expression in the brain of these two subjects and in MECP2-deficient mice. The findings suggest that the normally limited expression of MECP2 in visual pathway neurons may underlie the intact vision observed in Rett syndrome.

Alterations of cortical and hippocampal EEG activity in MeCP2-deficient mice

Rett syndrome is a pediatric neurological condition caused by mutations of the gene encoding the transcriptional regulator MECP2. In this study, we examined cortical and hippocampal electroencephalographic (EEG) activity in male and female MeCP2-deficient mice at symptomatic stages during different behavioral states. During acute sleep, MeCP2-deficient mice displayed normal delta-like activity in cortex and sharp-wave activity in hippocampus. However, when the mice were awake but immobile, abnormal spontaneous, rhythmic EEG discharges of 6–9 Hz were readily detected in the somatosensory cortex. During exploratory activity, MeCP2-deficient mice displayed clear theta rhythm activity in hippocampus, but its peak frequency was significantly attenuated compared to wild type. Collectively, these findings indicate that a deficiency in MeCP2 function in mice leads to alterations in EEG activity with similarities to what has been observed clinically in Rett syndrome patients.

MeCP2 deficiency down-regulates specific nuclear proteins that could be partially recovered by valproic acid in vitro

MeCP2, the major causative factor of Rett syndrome and related phenotypes including autism, is a two-face nuclear modulator acting via transcriptional and chromatin remodeling mechanisms. This study investigated the expression of several nuclear proteins and their dependence on MeCP2 dose and presence of the Rett causative R306C mutation. To this end, we developed in vitro models representing MeCP2 deficiency induced by siRNAs, and cells expressing the R306C mutation. Using an extended antibody microarray validated by specific assays, revealed that MeCP2 dose was correlated with specific nuclear proteins profiles including the BRM/SNF2 component of SWI/SNF complex, PRMT1 methyl transferase and HDAC2. Furthermore, while exposing the MeCP2 knock-down system to therapeutic concentrations of valproic acid (VPA), a known HDACs inhibitor, we observed a partial restoration of MeCP2 expression levels. Exposure to VPA also increased the levels of BRM, as well as of BDNF, an important co-factor in MeCP2-mediated pathway. Our findings provide additional evidence of diverse mechanisms of MeCP2 function as transcriptional repressor and activator of specific genes. As it has been recently demonstrated that post-natal restoration of MeCP2 deficiency may reverse neurological defects in a mouse model of Rett syndrome, we suggest to study the restorative effect of HDAC inhibitors in MeCP2-deficient mouse model.

Early abnormalities of post-sigh breathing in a mouse model of Rett syndrome

Rett syndrome is a neurodevelopmental disease accompanied by complex, disabling symptoms, including breathing symptoms. Because Rett syndrome is caused by mutations in the transcriptional repressor methyl-CpG-binding protein 2 (MeCP2), Mecp2-deficient mice have been generated as experimental model. Males of Mecp2-deficient mice (Mecp2(-/y)) breathe normally at birth but show abnormal respiratory responses to hypoxia and hypercapnia from postnatal day 25 (P25). After P30, Mecp2(-/y) mice develop breathing symptoms reminiscent of Rett syndrome, aggravating until premature death at around P60. Using plethysmography, we analyzed the sighs and the post-sigh breathing pattern of unrestrained wild type male mice (WT) and Mecp2(-/y) mice from P15 to P60. Sighs are spontaneous large inspirations known to prevent lung atelectasis and to improve alveolar oxygenation. However, Mecp2(-/y) mice show early abnormalities of post-sigh breathing, with long-lasting post-sigh apnoeas, reduced tidal volume when eupnoea resumes and lack of post-sigh bradypnoea which develop from P15, aggravate with age and possibly contribute to breathing symptoms to come.

Progressive noradrenergic deficits in the locus coeruleus of Mecp2 deficient mice

Methyl-CpG binding protein 2 (MeCP2) is a transcriptional regulator. Mutations in this gene cause a wide range of neurological disorders. Mecp2 deficiency has been previously associated to catecholaminergic dysfunctions leading to autonomic defects in the brainstem and the sympathoadrenergic system of the mouse. The present study was undertaken to determine if the locus coeruleus (LC), the main noradrenergic cell group of the brain, is affected. Using real type PCR, we found a reduction of the tyrosine hydroxylase (Th) mRNA level, the rate-limiting enzyme in catecholamine synthesis, in the whole pons of P15 (-36%), P30 (-47%) and P50 (-42%) Mecp2 null male as well as in adult heterozygous female (-44%) mice. Using immunoquantification we did not observe any difference of the Th staining level in P30 null male mice. However at P50, we demonstrated a significant decrease in both the Th staining level (-24%), and the number of Th-positive neurons (-23%). We subsequently characterized a reduction (-28%) of the dendritic density of the Th-positive fibers surrounding the LC in P50 null male mice. In heterozygous female mice immunoquantification did not revealed significant modifications, but only a tendency towards reduction. Finally, we did not found any apoptotic neurons in the pons indicating that LC neurons are not dying but are more likely loosing their catecholaminergic phenotype. In conclusion, our results showing a progressive catecholaminergic deficit in the LC of Mecp2 deficient null male mice could open new perspectives to better understand the autonomic and cognitive deficits due to the lack of Mecp2.

Early breathing defects after moderate hypoxia or hypercapnia in a mouse model of Rett syndrome

Rett syndrome (RTT) is a rare neurodevelopmental disease caused by mutations in the transcriptional repressor methyl-CpG-binding protein 2 (MeCP2) and accompanied by complex symptoms, including erratic breathing and life-threatening apnoeas. In Mecp2-deficient male mice (Mecp2(-/y)), breathing is normal at birth but becomes altered after postnatal day 30 (P30), with erratic rhythm and apnoeas aggravating until death at around P60. Using plethysmography, we analyzed breathing of unrestrained wild type mice and Mecp2(-/y) at P15, P25 and P30 under air and under short-lasting exposure to moderate hypoxia or hypercapnia. In Mecp2(-/y) with normal resting ventilation, we report exacerbated respiratory responses to hypoxia at P30 and transient apnoeas with erratic rhythm after hypoxia and hypercapnia at P30, P25 and occasionally P15. Then environmental factors may induce breathing defects well before than expected in Mecp2(-/y) and possibly in RTT patients. We therefore suggest avoiding exposure of young RTT patients to environmental situations where they may encounter moderate hypoxia or hypercapnia.

Dendritic spine pathologies in hippocampal pyramidal neurons from Rett syndrome brain and after expression of Rett-associated MECP2 mutations

Rett syndrome (RTT) is an X chromosome-linked neurodevelopmental disorder associated with the characteristic neuropathology of dendritic spines common in diseases presenting with mental retardation (MR). Here, we present the first quantitative analyses of dendritic spine density in postmortem brain tissue from female RTT individuals, which revealed that hippocampal CA1 pyramidal neurons have lower spine density than age-matched non-MR female control individuals. The majority of RTT individuals carry mutations in MECP2, the gene coding for a methylated DNA-binding transcriptional regulator. While altered synaptic transmission and plasticity has been demonstrated in Mecp2-deficient mouse models of RTT, observations regarding dendritic spine density and morphology have produced varied results. We investigated the consequences of MeCP2 dysfunction on dendritic spine structure by overexpressing (∼twofold) MeCP2-GFP constructs encoding either the wildtype (WT) protein, or missense mutations commonly found in RTT individuals. Pyramidal neurons within hippocampal slice cultures transfected with either WT or mutant MECP2 (either R106W or T158M) showed a significant reduction in total spine density after 48 h of expression. Interestingly, spine density in neurons expressing WT MECP2 for 96 h was comparable to that in control neurons, while neurons expressing mutant MECP2 continued to have lower spine density than controls after 96 h of expression. Knockdown of endogenous Mecp2 with a specific small hairpin interference RNA (shRNA) also reduced dendritic spine density, but only after 96 h of expression. On the other hand, the consequences of manipulating MeCP2 levels for dendritic complexity in CA3 pyramidal neurons were only minor. Together, these results demonstrate reduced dendritic spine density in hippocampal pyramidal neurons from RTT patients, a distinct dendritic phenotype also found in neurons expressing RTT-associated MECP2 mutations or after shRNA-mediated endogenous Mecp2 knockdown, suggesting that this phenotype represent a cell-autonomous consequence of MeCP2 dysfunction.

Modulating GABAA receptors that contain alpha 2/3 subunits corrects respiratory disturbances in a mouse model of Rett syndrome

We have recently shown that augmenting γ‐aminobutyric acid (GABA) A inhibition in a mouse model of Rett syndrome corrects periodic apneas and restores cycle regularity to that of wild type (2008 EB abstracts [on CD‐ROM], Abstract#1232.12). These mice are deficient in α2 subunits of GABAA receptors (J Neurophysiol 99:112, 2008). Here we show that allosteric modulation of GABAA receptors with a compound that is relatively specific for receptors that contain α2/3 subunits (L‐838,417, Nature Neurosci 3:587, 2000) is equally efficient as a broad‐spectrum benzodiazepine in correcting the respiratory disorder. Studies were performed in heterozygous methyl‐CpG‐binding protein 2 (Mecp2) deficient female mice with continuous monitoring of pleural pressure by telemetry. Following ip injection of L‐838,417 (20 mg/Kg) or vehicle respiratory pattern was examined for 3 hrs.Adjusting for differences in wake/sleep pattern from animal activity (Physiol Genomics 28:232, 2006) did not alter the effect of L‐838,417.Support: MOD #6‐FY‐314 and IRSF #0802

Reciprocal co-regulation of EGR2 and MECP2 is disrupted in Rett syndrome and autism

Mutations in MECP2, encoding methyl-CpG-binding protein 2 (MeCP2), cause the neurodevelopmental disorder Rett syndrome (RTT). Although MECP2 mutations are rare in idiopathic autism, reduced MeCP2 levels are common in autism cortex. MeCP2 is critical for postnatal neuronal maturation and a modulator of activity-dependent genes such as Bdnf (brain-derived neurotropic factor) and JUNB. The activity-dependent early growth response gene 2 (EGR2), required for both early hindbrain development and mature neuronal function, has predicted binding sites in the promoters of several neurologically relevant genes including MECP2. Conversely, MeCP2 family members MBD1, MBD2 and MBD4 bind a methylated CpG island in an enhancer region located in EGR2 intron 1. This study was designed to test the hypothesis that MECP2 and EGR2 regulate each other’s expression during neuronal maturation in postnatal brain development. Chromatin immunoprecipitation analysis showed EGR2 binding to the MECP2 promoter and MeCP2 binding to the enhancer region in EGR2 intron 1. Reduction in EGR2 and MeCP2 levels in cultured human neuroblastoma cells by RNA interference reciprocally reduced expression of both EGR2 and MECP2 and their protein products. Consistent with a role of MeCP2 in enhancing EGR2, Mecp2-deficient mouse cortex samples showed significantly reduced EGR2 by quantitative immunofluorescence. Furthermore, MeCP2 and EGR2 show coordinately increased levels during postnatal development of both mouse and human cortex. In contrast to age-matched Controls, RTT and autism postmortem cortex samples showed significant reduction in EGR2. Together, these data support a role of dysregulation of an activity-dependent EGR2/MeCP2 pathway in RTT and autism.

Tyrosine hydroxylase deficit in the chemoafferent and the sympathoadrenergic pathways of the Mecp2 deficient mouse

Mutations in the gene encoding the transcriptional methyl-CpG binding protein 2 (Mecp2) cause a wide range of neurological disorders and the better known of these diseases is Rett syndrome (RS). Mecp2 deficiency has been previously associated to catecholaminergic dysfunction in the mouse brainstem. Here we report a catecholaminergic deficit in the peripheral nervous system of the Mecp2−/ y males and heterozygous Mecp2+/− female mice. We used immunoquantification associated to densitometry to evaluate the amount of tyrosine hydroxylase, the rate-limiting enzyme in catecholamine synthesis, in the key organs of the chemoafferent and sympathoadrenergic pathways: the carotid body (CB), the petrosal ganglion (PG), the superior cervical ganglion (SCG) and the adrenal medulla (AM). Our results show that the TH staining level is weaker in the CB (−15%), PG (−26%), SCG (−34%), AM (−35%) of Mecp2−/ y mice and to a lesser extent in the PG (−11%) and AM (−18%) in Mecp2+/− mice. We evaluated in vivo the chemoreflex sensitivity of Mecp2−/ y mice using whole-body plethysmography to record the breathing of Mecp2−/ y mice in normoxia and in response to acute hypoxia (10% O 2). Our results show that the hypoxic ventilatory response is significantly increased in Mecp2−/ y mice (+50%) demonstrating in vivo disturbances of the chemoafferent pathway. In conclusion, our results offer new insights to better understand the mechanisms leading to autonomic dysfunction in RS.

Insufficient GABA inhibition underlies periodic and irregular breathing in methyl‐CpG‐binding protein 2 (Mecp2) deficient mice

Mutations in the MECP2 gene cause Rett syndrome (RTT) whose phenotype includes periodic (PB) and irregular breathing. Using telemetry to record pleural pressure we have found that heterozygous Mecp2 deficient female mice have frequent episodes of PB. The amount of PB was augmented by breathing 40 % oxygen and decreased in 12% oxygen, indicating that peripheral chemoreception is not responsible for the increased controller gain (J Appl Physiol in Press). Medullary respiratory neurons (Brain Research 710:150,1996; J Neurophysiol 80:2368,1998) and phrenic motoneuons (J Neurosci 19:2368,1999) receive concurrent inhibition with excitation during inspiration. Mecp2+/− are relatively deficient in the β3 subunit of GABAA receptors (Hum Mol Genet 14:483,2005). We hypothesized that insufficient GABA inhibition underlies the increased controller gain in Mecp2 deficient animals. NO‐711 a GABA transport inhibitor reduced the number of PB episodes from 18.1±4.9 to 3.5±1.8/hr. and extended the longest period without PB from 26.5±6 to 74.5±23.7 min. Similarly the GABA receptor modulator diazepam reduced the incidence to 3.2±0.7/hr and extended PB free to 96.7±35.9 min. NO‐711 reduced the coefficient of variation for TTOT from 35±3.4 to 22.6±3.8%. These data indicate that augmenting GABA reduces PB and respiratory irregularity seen in Mecp2 deficient mice to the levels observed in wild type mice. MOD 6‐FY06‐314

Targeted delivery of an Mecp2 transgene to forebrain neurons improves the behavior of female Mecp2-deficient mice

Rett syndrome is an X-linked neurological condition affecting almost exclusively girls that is caused by mutations of the MECP2 gene. Recent studies have shown that transgenic delivery of MeCP2 function to Mecp2-deficient male mice can improve their Rett-like behavior. However, as the brain of a Rett girl contains a mosaic of MeCP2 expressing and non-expressing neurons, and the over-expression of MeCP2 in neurons can induce a severe progressive neurological phenotype, testing whether functional rescue can be achieved by gene re-introduction strategies in a female model of Rett syndrome is warranted. To address this, we generated transgenic mice expressing an epitope-tagged Mecp2 transgene in forebrain neurons. These mice over-express MeCP2 protein at about 1.6 times normal levels in cortex and develop impaired motor behavior by 9-12 months of age. To test whether forebrain-targeted MeCP2 restoration would improve behavior in female Mecp2(-/+) mice, we crossed these transgenics with Mecp2(-/+) mice and examined the behavioral properties of the female rescue mice for 1 year. These assessments revealed that the diminished rearing activity, impaired mobility and the diminished locomotive activity of female Mecp2(-/+) mice were restored to wild-type levels in the rescue mice. These results show that improvement of Rett-like behavior can be achieved in Mecp2(-/+) females by targeted gene re-introduction without inducing deficits relating to MeCP2 over-expression.

Effect of inspired oxygen on periodic breathing in methy-CpG-binding protein 2 (Mecp2) deficient mice

Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the X-linked gene methyl-CpG-binding protein 2 (Mecp2) that encodes a DNA binding protein involved in gene silencing. Periodic breathing (Cheyne-Stokes respiration) is commonly seen in RTT. Freely moving mice were studied with continuous recording of pleural pressure by telemetry. Episodes of periodic breathing in heterozygous Mecp2 deficient (Mecp2(+/-)) female mice (9.4 +/- 2.2 h(-1)) exceeded those in wild-type (Mecp2(+/+)) animals (2.5 +/- 0.4 h(-1)) (P = 0.010). Exposing Mecp2(+/-) animals to 40% oxygen increased the amount of periodic breathing from 118 +/- 25 s/30 min in air to 242 +/- 57 s/30 min (P = 0.001), and 12% oxygen tended to decrease it (67 +/- 29 s/30 min, P = 0.14). Relative hyperoxia and hypoxia did not affect the incidence of periodic breathing in Mecp2(+/+) animals. The ventilation/apnea ratio (V/A) was less at all levels of oxygen in heterozygous Mecp2(+/-) females compare with wild type (P = 0.003 to P < 0.001), indicating that their loop gain is larger. V/A in Mecp2(+/-) fell from 2.42 +/- 0.18 in normoxia to 1.82 +/- 0.17 in hyperoxia (P = 0.05) indicating an increase in loop gain with increased oxygen. Hyperoxia did not affect V/A in Mecp2(+/+) mice (3.73 +/- 0.28 vs. 3.5 +/- 0.28). These results show that periodic breathing in this mouse model of RTT is not dependent on enhanced peripheral chemoreceptor oxygen sensitivity. Rather, the breathing instability is of central origin.

MeCP2 Deficiency in the Brain Decreases BDNF Levels by REST/CoREST-Mediated Repression and Increases TRKB Production

Disruptions in the expression of the Bdnf gene that encodes a neurotrophic factor involved in neuronal survival, differentiation and synaptic plasticity has been proposed to contribute to the molecular pathogenesis of Rett syndrome. Rett syndrome (RTT) is a neurodevelopmental disorder, caused by mutations in the X-linked methyl CpG binding protein 2 gene (MeCP2). MeCP2 deficiency in the brain has been shown to decrease overall expression of Bdnf in spite of an observed increase in the activity of promoter III that appears to be controlled directly by MeCP2. Therefore, how MeCP2 deficiency causes an overall down regulation of Bdnf expression was an enigma. Here we report that MeCP2 deficiency in human and mouse brain causes an increase in expression of two neuronal gene transcriptional repressors REST (RE1 silencing transcription factor), and CoREST. MeCP2 binds to and is involved in repression of Rest and CoRest promoters despite their unmethylated state. MeCP2 depletion is associated with a change in the histone modification profile to a more active conformation. The elevated levels of REST and CoREST in the brain of RTT patients and MeCP2 deficient mice result in down regulation of Bdnf, apparently by their binding to the RE1 (element) located between the first two promoters of the Bdnf gene. Interestingly, the Trk2 gene that encodes the BDNF receptor, TRKB, was overexpressed in MeCP2 deficient human and mouse brains either directly or as an attempt to compensate for BDNF deficiency.

Reply from M. Dutschmann and G. M. Stettner

We appreciate the interest and comments regarding our recently published article in The Journal of Physiology (Stettner et al. 2007a). The manuscript described disturbances in vagal-pontine interactions in the control of the postinspiratory activity as a potential cause of breathing disturbances in MECP2-deficient mice, an animal model for Rett syndrome (Guy et al. 2001). The Letter by Poon & Song addresses an aspect of the publication that is illustrated in Fig. 7, which contrasts the response to repetitive electrical stimulation of the vagal nerve of wild-type (C57BL/6J) and MECP2-deficient mice. In both groups of mice, the intensity of electrical stimulation of the central end of the vagus nerve was determined by the same procedure and the stimuli elicited a postinspiratory reflex apnoea (Hering–Breuer reflex). In particular, the intensity of vagal stimulation in our study was 1–2 × above threshold (low intensity stimulation), which was similar to that used to study plasticity following repetitive vagal stimulation (Siniaia et al. 2000). The data showed that the apnoea after cessation of the electrical stimulus was significantly longer in MECP2-deficient mice than in C57BL/6J mice. Furthermore, the reflex apnoea progressively decreased with repeated vagal stimuli in the wild-type but not MECP2-deficient mice. We interpreted our data as Mecp2−/y knockout mice lacked ‘desensitization’ to vagal stimulation. This terminology was criticised in the letter to the Editor. We want to emphasize that disturbed processing of vagal afferent input remains a part of the phenotype of the MECP2-deficient mice, whether they lack desensitization or ‘classic habituation’. We agree that a full description of the underlying plasticity phenomenon would require analysis of the poststimulus rebound activity. At this point, we cannot confirm the correspondent's insights regarding Fig. 7. Differences in the stimulus intensities between our and other studies relate to different experimental tools, for instance the central vagal nerve was stimulated via suction electrodes in our experiment. This may clarify confusion regarding a potential secondary or non-specific sensitization of the vagally evoked reflex response due to activation of rapidly adapting pulmonary stretch receptor afferent fibres or cardiopulmonary C-fibres. Even though group data for the poststimulus rebound were not analysed, Fig. 7 shows that expiration lengthened after the stimulus. Poststimulus rebound activity as described for rats (Siniaia et al. 2000), however, did not occur in C57BL/6J and MECP2-deficient mice. The C57BL/6J mouse strain itself may have an imbalance in ponto-medullary circuits. We have observed spontaneous postinspiratory apnoeas in the perfused brainstem preparation in situ (Stettner et al. 2007b) and others report severe periodic breathing patterns during the recovery from a hypoxic challenge in vivo (Han et al. 2002; Gonsenhauser et al. 2004). We speculate that the pontine Kölliker–Fuse nucleus (KF) may be a part of this imbalance, not only because neurones in the KF express postinspiratory activity in many species (Dick et al. 1994; Ezure, 2006; Dutschmann & Herbert, 2006), but also because KF neuronal activity determines the timing of postinspiratory activities of laryngeal muscles in the absence of vagal afferent feedback (Dutschmann & Herbert, 2006). If pontine mechanisms in the control of breathing are similar in rats and mice, then spontaneous postinspiratory apnoeas in C57BL/6J mice indicate a genetic imbalance in pontine mechanisms controlling postinspiratory activity in C57BL/6J mice that may be accentuated under the condition of MECP2 deficiency. This may cause the unusual poststimulus rebound illustrated in Fig. 7. Interestingly, another Mecp2−/y knockout model (Chen et al. 2001) has a different genetic background. If we are correct about the intrinsic imbalance prevalent in C57BL/6J mice, then these other MECP2-deficient mice should show distinct differences in their breathing phenotype. In our opinion, further experiments are required in MECP2-deficient mice with different genetic backgrounds to fully understand disturbed synaptic plasticity associated with the processing of afferent vagal feedback.

Oral treatment with desipramine improves breathing and life span in Rett syndrome mouse model

Rett syndrome is a neurodevelopmental disease due to Mecp2 gene mutations that is associated to complex neurological symptoms, with bioaminergic deficits and life-threatening apneas related to sudden and unexpected death. In male mice, Mecp2-deficiency similarly induces medullary bioaminergic deficits, severe apneas and short life span. Here, we show that long-term oral treatment of Mecp2-deficient male mice with desipramine, an old drug of clinical use known to block norepinephrine uptake and to strengthen its synaptic effects, significantly alleviates their breathing symptoms and prolongs their life span. Although these mouse results identify desipramine as the first oral pharmacological treatment potentially able to alleviate breathing symptoms of Rett syndrome, we recommend further studies of desipramine effects in Mecp2-deficient mice before attempting any clinical trials in Rett patients.