Abstract
The deletion of Mecp2, the gene encoding methyl-CpG-binding protein 2, causes severe breathing defects and developmental anomalies in mammals. In Mecp2-null mice, impaired GABAergic neurotransmission is demonstrated at the early stage of life. GABAergic dysfunction in neurons in the rostral ventrolateral medulla (RVLM) is considered as a primary cause of breathing abnormality in Mecp2-null mice, but its molecular mechanism is unclear. Here, we report that mRNA expression levels of Gad1, which encodes glutamate decarboxylase 67 (GAD67), in the RVLM of Mecp2-null (Mecp2-/y, B6.129P2(C)-Mecp2tm1.1Bird/J) mice is closely related to the methylation status of its promoter, and valproate (VPA) can upregulate transcription from Gad1 through epigenetic mechanisms. The administration of VPA (300 mg/kg/day) together with L-carnitine (30 mg/kg/day) from day 8 to day 14 after birth increased Gad1 mRNA expression in the RVLM and reduced apnea counts in Mecp2-/y mice on postnatal day 15. Cytosine methylation levels in the Gad1 promoter were higher in the RVLM of Mecp2-/y mice compared to wild-type mice born to C57BL/6J females, while VPA treatment decreased the methylation levels in Mecp2-/y mice. Chromatin immunoprecipitation assay revealed that the VPA treatment reduced the binding of methyl-CpG binding domain protein 1 (MBD1) to the Gad1 promoter in Mecp2-/y mice. These results suggest that VPA improves breathing of Mecp2-/y mice by reducing the Gad1 promoter methylation, which potentially leads to the enhancement of GABAergic neurotransmission in the RVLM.
Highlights
Mutations in the gene encoding methyl-CpG-binding protein 2 (MeCP2) cause Rett syndrome (RTT), a severe neurodevelopmental disorder that affects mostly girls [1,2,3]
We examined the breathing patterns of mice on PND15 using whole-body plethysmography and found that the Mecp2-/y mice injected with saline as the vehicle from day 8 to day 14 after birth displayed an increased number of apnea (>1.0 s) episodes compared to the saline-injected WT mice (p < 0.05) (Figure 1) the 15-day old Mecp2-/y mice did not show long-lasting apneas which emerge during the symptomatic period [10,11]
We demonstrated for the first time that VPA treatment reduced apnea episodes and reduced methylation levels of Gad1 promoter in 15-day-old Mecp2-/y mice
Summary
Mutations in the gene encoding methyl-CpG-binding protein 2 (MeCP2) cause Rett syndrome (RTT), a severe neurodevelopmental disorder that affects mostly girls [1,2,3]. It has been reported that Mecp2-null mice breathe normally at birth and start to show erratic rhythm and overt neurological symptoms in the post-weaning period (3–5 weeks) [8,9,10]. Reduced inhibitory synaptic inputs to neurons in the rostral ventrolateral medulla (RVLM), a site critical for respiratory rhythmogenesis, were demonstrated as early as PND7 in Mecp2-null mice compared to WT controls [14]. These knowledges triggered our interest in the early manifestation of breathing abnormalities and underlying molecular events in Mecp2-null mice, which might not be detected with previous approaches
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