Female MeCP2-deficient Mice Research Articles

Modulation of Brain Cholesterol Metabolism through CYP46A1 Overexpression for Rett Syndrome.

Rett syndrome (RTT) is a rare neurodevelopmental disorder caused by mutation in the X-linked gene methyl-CpG-binding protein 2 (Mecp2), a ubiquitously expressed transcriptional regulator. RTT results in mental retardation and developmental regression that affects approximately 1 in 10,000 females. Currently, there is no curative treatment for RTT. Thus, it is crucial to develop new therapeutic approaches for children suffering from RTT. Several studies suggested that RTT is linked with defects in cholesterol homeostasis, but for the first time, therapeutic evaluation is carried out by modulating this pathway. Moreover, AAV-based CYP46A1 overexpression, the enzyme involved in cholesterol pathway, has been demonstrated to be efficient in several neurodegenerative diseases. Based on these data, we strongly believe that CYP46A1 could be a relevant therapeutic target for RTT. Herein, we evaluated the effects of intravenous AAVPHP.eB-hCYP46A1-HA delivery in male and female Mecp2-deficient mice. The applied AAVPHP.eB-hCYP46A1 transduced essential neurons of the central nervous system (CNS). CYP46A1 overexpression alleviates behavioral alterations in both male and female Mecp2 knockout mice and extends the lifespan in Mecp2-deficient males. Several parameters related to cholesterol pathway are improved and correction of mitochondrial activity is demonstrated in treated mice, which highlighted the clear therapeutic benefit of CYP46A1 through the neuroprotection effect. IV delivery of AAVPHP.eB-CYP46A1 is perfectly well tolerated with no inflammation observed in the CNS of the treated mice. Altogether, our results strongly suggest that CYP46A1 is a relevant target and overexpression could alleviate the phenotype of Rett patients.

Exploring sex differences in inhibitory synaptic transmission in the kolliker-fuse of rett syndrome mice

Rett Syndrome (RTT) is a rare neurodevelopmental disorder characterized by severe motor impairments including breathing problems. RTT is caused by mutations in the methyl CpG binding protein 2 (MeCP2) gene located on the X chromosome. Accordingly, girls and women account for the majority of RTT patients. The genetic nature of RTT lends itself to study in mice with loss of function mutations in the MeCP2 gene. Importantly, RTT mice display breathing irregularities similar to human patients. Insufficient inhibitory neurotransmission in the brainstem respiratory network is thought to contribute to the breathing problems in RTT. In the pontine Kölliker-Fuse (KF) area of RTT mice, there are fewer GABAergic neurites and injection of a GABA reuptake antagonist alleviates breathing irregularity in vivo. However, no one has examined the physiology of inhibitory synaptic transmission in the KF of RTT mice. In the present work, we tested the hypothesis that inhibitory synaptic transmission was deficient in the respiratory-controlling KF of MeCP2Bird mice. Although girls and women make up the majority of RTT patients, most studies examining synaptic physiology in the brainstem have been performed using male MeCP2-deficient mice. Hemizygous male MeCP2Bird mice lack MeCP2 protein and exhibit rapid onset of severe RTT symptoms which leads to short life spans. Due to partial X-inactivation, MeCP2 expression in heterozygous female MeCP2-deficient mice is mosaic, leading to variable symptom severity and delayed onset, which more closely resembles the human condition. In the present study, we examined inhibitory synaptic transmission in the KF of female heterozygous MeCP2Bird mice compared to male hemizygous MeCP2Bird mice and wild type littermates. Whole-cell voltage-clamp recordings from KF neurons contained in acute brain slices were used to measure inhibitory currents. Most notably, the frequency of spontaneous inhibitory postsynaptic currents is reduced in the KF of male, but not female, MeCP2Bird mice compared to wild type littermates. To better understand this sex difference in inhibitory neurotransmission, we assessed the influence of estrous cycle stage and MeCP2 expression status in the recorded neuron. The outcomes of this research will increase our understanding of synaptic function in respiratory control neurons in female Rett Syndrome mice. This research was supported by the International Rett Syndrome Foundation Basic Research Grant 3608. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Electrographic and pharmacological characterization of a progressive epilepsy phenotype in female MeCP2-deficient mice

Rett Syndrome is a neurodevelopmental disorder caused primarily by mutations in the gene encoding Methyl-CpG-binding protein 2 (MECP2). Spontaneous epileptiform activity is a common co-morbidity present in Rett syndrome, and hyper-excitable neural networks are present in MeCP2-deficient mouse models of Rett syndrome. In this study we conducted a longitudinal assessment of spontaneous cortical electrographic discharges in female MeCP2-deficient mice and defined the pharmacological responsiveness of these discharges to anti-convulsant drugs. Our data show that cortical discharge activity in female MeCP2-deficient mice progressively increases in severity as the mice age, with discharges being more frequent and of longer durations at 19–24 months of age compared to 3 months of age. Semiologically and pharmacologically, this basal discharge activity in female MeCP2-deficient mice displayed electroclinical properties consistent with absence epilepsy. Only rarely were convulsive seizures observed in these mice at any age. Since absence epilepsy is infrequently observed in Rett syndrome patients, these results indicate that the predominant spontaneous electroclinical phenotype of MeCP2-deficient mice we examined does not faithfully recapitulate the most prevalent seizure types observed in affected patients.

Disruption of MeCP2 attenuates circadian rhythm in CRISPR/Cas9-based Rett syndrome model mouse.

Methyl-CpG-binding protein 2 (Mecp2) is an X-linked gene encoding a methylated DNA-binding nuclear protein which regulates transcriptional activity. The mutation of MECP2 in humans is associated with Rett syndrome (RTT), a neurodevelopmental disorder. Patients with RTT frequently show abnormal sleep patterns and sleep-associated problems, in addition to autistic symptoms, raising the possibility of circadian clock dysfunction in RTT. In this study, we investigated circadian clock function in Mecp2-deficient mice. We successfully generated both male and female Mecp2-deficient mice on the wild-type C57BL/6 background and PER2(Luciferase) (PER2(Luc)) knock-in background using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system. Generated Mecp2-deficient mice recapitulated reduced activity in mouse models of RTT, and their activity rhythms were diminished in constant dark conditions. Furthermore, real-time bioluminescence imaging showed that the amplitude of PER2(Luc)-driven circadian oscillation was significantly attenuated in Mecp2-deficient SCN neurons. On the other hand, in vitro circadian rhythm development assay using Mecp2-deficient mouse embryonic stem cells (ESCs) did not show amplitude changes of PER2(Luc) bioluminescence rhythms. Together, these results show that Mecp2 deficiency abrogates the circadian pacemaking ability of the SCN, which may be a therapeutic target to treat the sleep problems of patients with RTT.

Abstract 19072: Cardiac Autonomic Dysfunction in Mice Lacking Methyl CpG Binding Protein 2

Background: Rett Syndrome (RTT) is an X-linked dominant neurodevelopmental disorder primarily caused by mutations in Methyl-CpG-binding protein ( MECP2 ), a transcriptional regulator. Twenty-five percent of all deaths in RTT are sudden and unexpected, and autonomic nervous system (ANS) dysfunction is hypothesized to be the cause of sudden deaths in RTT. However, the role of MeCP2 in cardiac autonomic function has not been investigated in depth. Design/Methods: Male (2 months) and female (9 months) Mecp2 deficient and wildtype mice were implanted with ETA-F10 telemeters and 24h recordings were taken to calculate heart rate, heart rate variability, and incidence of arrhythmic events. Effects of pharmacological stimulants or inhibitors on heart rate were normalized to saline injection response. Results: Male Mecp2 deficient mice (NULL) have a decreased heart rate (WT = 634 ±3, NULL = 518 ±8, p<0.001), while female deficient mice (NULL/+) do not present with a decreased heart rate. Interestingly, NULL and NULL/+ mice both have a high incidence of sinus pauses and AV block (WT 1/5, NULL 6/6, NULL /+ 4/4, p<0.05). Additionally, both NULL and NULL/+ mice have increased bradycardia events defined as a heart rate below 200 bpm, (WT = 17 ±6, NULL = 307 ±94, NULL/+ = 198 ±67, p<0.05) and an increased heart rate variability. The normalized heart rate response to atropine, a parasympathetic blocker, of NULL mice was above wildtype levels (p<0.05) Chemical denervation by combined antagonism of parasympathetic (atropine) and sympathetic (propranolol) resulted in NULL mice having an increased normalized heart rate response compared to wildtype (p<0.05). Finally, the incidence of AVB/sinus pauses counts per 30 minutes post-acute treatment with atropine and chemical denervation (NULL saline 49 ±10, NULL atropine 8 ±5, NULL atropine/propranolol 0.5 ±0.5 p<0.05) decreased the severity of the cardiac phenotype in NULL mice. Conclusions: In summary, Mecp2 is required for a normal heart rhythm. Loss of Mecp2 causes bradycardia, sinus pauses, AV block, and increased heart rate variability that may be attributed by aberrant innervation. Acute atropine and chemical denervation was therapeutic and ameliorated the cardiac phenotype observed in Mecp2 deficient mice.

Rescue of behavioral and EEG deficits in male and female Mecp2-deficient mice by delayed Mecp2 gene reactivation

Mutations of the X-linked gene encoding methyl CpG binding protein type 2 (MECP2) are the predominant cause of Rett syndrome, a severe neurodevelopmental condition that affects primarily females. Previous studies have shown that major phenotypic deficits arising from MeCP2-deficiency may be reversible, as the delayed reactivation of the Mecp2 gene in Mecp2-deficient mice improved aspects of their Rett-like phenotype. While encouraging for prospective gene replacement treatments, it remains unclear whether additional Rett syndrome co-morbidities recapitulated in Mecp2-deficient mice will be similarly responsive to the delayed reintroduction of functional Mecp2. Here, we show that the delayed reactivation of Mecp2 in both male and female Mecp2-deficient mice rescues established deficits in motor and anxiety-like behavior, epileptiform activity, cortical and hippocampal electroencephalogram patterning and thermoregulation. These findings indicate that neural circuitry deficits arising from the deficiency in Mecp2 are not engrained, and provide further evidence that delayed restoration of Mecp2 function can improve a wide spectrum of the Rett-like deficits recapitulated by Mecp2-deficient mice.

Characterization of seizure-like events recorded in vivo in a mouse model of Rett syndrome

Rett syndrome is a neurodevelopmental disorder caused by mutations in the X-linked gene encoding methyl-CpG-binding protein 2 (MECP2). Spontaneous recurrent discharge episodes are displayed in Rett-related seizures as in other types of epilepsies. The aim of this paper is to investigate the seizure-like event (SLE) and inter-SLE states in a female MeCP2-deficient mouse model of Rett syndrome and compare them to those found in other spontaneous recurrent epilepsy models. The study was performed on a small population of female MeCP2-deficient mice using telemetric local field potential (LFP) recordings over a 24 h period. Durations of SLEs and inter-SLEs were extracted using a rule-based automated SLE detection system for both daytime and nighttime, as well as high and low power levels of the delta frequency range (0.5–4 Hz) of the recorded LFPs. The results suggest SLE occurrences are not influenced by circadian rhythms, but had a significantly greater association with delta power. Investigating inter-SLE and SLE states by fitting duration histograms to the gamma distribution showed that SLE initiation and termination were associated with random and deterministic mechanisms, respectively. These findings when compared to reported studies on epilepsy suggest that Rett-related seizures share many similarities with absence epilepsy.

Selective preservation of MeCP2 in catecholaminergic cells is sufficient to improve the behavioral phenotype of male and female Mecp2-deficient mice

Rett syndrome (RTT) is a neurodevelopmental disorder caused primarily by mutations of the X-linked MECP2 gene. Although the loss of MeCP2 function affects many neural systems, impairments of catecholaminergic function have been hypothesized to underlie several of the cardinal behavioral deficits of RTT patients and Mecp2-deficient mice. Although recent Mecp2 reactivation studies indicate that RTT may be a reversible condition, it remains unclear whether specifically preserving Mecp2 function within a specific system will be sufficient to convey beneficial effects. Here, we test whether the selective preservation of Mecp2 within catecholaminergic cells will improve the phenotype of Mecp2-deficient mice. Our results show that this targeted preservation of Mecp2 significantly improves the lifespan, phenotypic severity and cortical epileptiform discharge activity of both male and female Mecp2-deficient mice. Further, we found that the catecholaminergic preservation of Mecp2 also improves the ambulatory rate, rearing activity, motor coordination, anxiety and nest-building performances of Mecp2-deficient mice of each gender. Interestingly, our results also revealed a gender-specific improvement, as specific cortical and hippocampal electroencephalographic abnormalities were significantly improved in male, but not female, rescue mice. Collectively, these results support the role of the catecholaminergic system in the pathogenesis of RTT and provide proof-of-principle that restoring MeCP2 function within this specific system could represent a treatment strategy for RTT.

Regional MeCP2 expression levels in the female MeCP2-deficient mouse brain correlate with specific behavioral impairments

Using female MeCP2-deficient mice as a model, we tested whether MeCP2 expression levels would parallel one another in different regions of the brain and spinal cord, and/or whether the levels of MeCP2 protein in these specific neural regions would correlate with the degree of behavioral impairment seen in individual subjects. Our results show that MeCP2 protein levels are not uniform across different regions of the mosaic brain, and that the relative MeCP2 levels in one region did not necessarily predict its expression levels in other regions of the same mouse. Correlational analysis between region-specific MeCP2 expression levels and the degree of impairment exhibited by mice in specific behavioral tests revealed significant associations. Cortical MeCP2 expression was significantly correlated with general symptomatic severity and daily EEG delta power patterning. Hippocampal MeCP2 expression was significantly correlated with exploratory activity behaviors, anxiety-like behaviors, and epileptiform discharge rates, whereas cerebellar and spinal cord MeCP2 expression levels correlated only with daily EEG delta power patterning. Collectively, our results show that MeCP2 protein expression in female MeCP2-deficient mice is not uniform throughout specific brain regions, and indicate that specific behavioral deficits in these mice may correlate with the region-specific expression levels of the functional MeCP2 allele.

Daily Rhythmic Behaviors and Thermoregulatory Patterns Are Disrupted in Adult Female MeCP2-Deficient Mice

Mutations in the X-linked gene encoding Methyl-CpG-binding protein 2 (MECP2) have been associated with neurodevelopmental and neuropsychiatric disorders including Rett Syndrome, X-linked mental retardation syndrome, severe neonatal encephalopathy, and Angelman syndrome. Although alterations in the performance of MeCP2-deficient mice in specific behavioral tasks have been documented, it remains unclear whether or not MeCP2 dysfunction affects patterns of periodic behavioral and electroencephalographic (EEG) activity. The aim of the current study was therefore to determine whether a deficiency in MeCP2 is sufficient to alter the normal daily rhythmic patterns of core body temperature, gross motor activity and cortical delta power. To address this, we monitored individual wild-type and MeCP2-deficient mice in their home cage environment via telemetric recording over 24 hour cycles. Our results show that the normal daily rhythmic behavioral patterning of cortical delta wave activity, core body temperature and mobility are disrupted in one-year old female MeCP2-deficient mice. Moreover, female MeCP2-deficient mice display diminished overall motor activity, lower average core body temperature, and significantly greater body temperature fluctuation than wild-type mice in their home-cage environment. Finally, we show that the epileptiform discharge activity in female MeCP2-deficient mice is more predominant during times of behavioral activity compared to inactivity. Collectively, these results indicate that MeCP2 deficiency is sufficient to disrupt the normal patterning of daily biological rhythmic activities.

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.

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.