Persistent Expression of Serotonin Receptor 5b Alters Breathing Behavior in Male MeCP2 Knockout Mice.

Mutations in the transcription factor methyl-CpG-binding protein 2 (MeCP2) cause the neurodevelopmental disorder Rett syndrome (RTT). Besides many other neurological problems, RTT patients show irregular breathing with recurrent apneas or breath-holdings. MeCP2-deficient mice, which recapitulate this breathing phenotype, show a dysregulated, persistent expression of G-protein-coupled serotonin receptor 5-ht5b (Htr5b) in the brainstem. To investigate whether the persistence of 5-ht5b expression is contributing to the respiratory phenotype, we crossbred MeCP2-deficient mice with 5-ht5b-deficient mice to generate double knockout mice (Mecp2-/y ;Htr5b-/-). To compare respiration between wild type (WT), Mecp2-/y and Mecp2-/y ;Htr5b-/- mice, we used unrestrained whole-body plethysmography. While the breathing of MeCP2-deficient male mice (Mecp2-/y ) at postnatal day 40 is characterized by a slow breathing rate and the occurrence of prolonged respiratory pauses, we found that in MeCP2-deficient mice, which also lacked the 5-ht5b receptor, the breathing rate and the number of pauses were indistinguishable from WT mice. To test for a potential mechanism, we also analyzed if the known coupling of 5-ht5b receptors to Gi proteins is altering second messenger signaling. Tissue cAMP levels in the medulla of Mecp2-/y mice were decreased as compared to WT mice. In contrast, cAMP levels in Mecp2-/y ;Htr5b-/- mice were indistinguishable from WT mice. Taken together, our data points towards a role of 5-ht5b receptors within the complex breathing phenotype of MeCP2-deficient mice.

The Serotonin Receptor Subtype 5b Specifically Interacts with Serotonin Receptor Subtype 1A.

Previously, we described the dysregulation of serotonin (5-HT) receptor subtype 5b (5-ht5b) in a mouse model of Rett syndrome (RTT). 5-ht5b has not been extensively studied, so we set out to characterize it in more detail. Unlike common cell surface receptors, 5-ht5b displays no membrane expression, while receptor clusters are located in endosomes. This unusual subcellular localization is at least in part controlled by glycosylation of the N-terminus, with 5-ht5b possessing fewer glycosylation sites than related receptors. We analyzed whether the localization to endosomes has any functional relevance and found that 5-ht5b receptors can specifically interact with 5-HT1A receptors and retain them in endosomal compartments. This interaction reduces 5-HT1A surface expression and is mediated by interactions between the fourth and fifth trans-membrane domain (TMD). This possibly represents a mechanism by which 5-ht5b receptors regulate the activity of other 5-HT receptor.

Analysis of the Serotonergic System in a Mouse Model of Rett Syndrome Reveals Unusual Upregulation of Serotonin Receptor 5b.

Mutations in the transcription factor methyl-CpG-binding-protein 2 (MeCP2) cause a delayed-onset neurodevelopmental disorder known as Rett syndrome (RTT). Although alteration in serotonin levels have been reported in RTT patients, the molecular mechanisms underlying these defects are not well understood. Therefore, we chose to investigate the serotonergic system in hippocampus and brainstem of male Mecp2-/y knock-out mice in the B6.129P2(C)-Mecp2(tm1.1Bird) mouse model of RTT. The serotonergic system in mouse is comprised of 16 genes, whose mRNA expression profile was analyzed by quantitative RT-PCR. Mecp2-/y mice are an established animal model for RTT displaying most of the cognitive and physical impairments of human patients and the selected areas receive significant modulation through serotonin. Using anatomically and functional characterized areas, we found region-specific differential expression between wild type and Mecp2-/y mice at post-natal day 40. In brainstem, we found five genes to be dysregulated, while in hippocampus, two genes were dysregulated. The one gene dysregulated in both brain regions was dopamine decarboxylase, but of special interest is the serotonin receptor 5b (5-ht5b), which showed 75-fold dysregulation in brainstem of Mecp2-/y mice. This dysregulation was not due to upregulation, but due to failure of down-regulation in Mecp2-/y mice during development. Detailed analysis of 5-ht5b revealed a receptor that localizes to endosomes and interacts with Gαi proteins.

Oxidative burden and mitochondrial dysfunction in a mouse model of Rett syndrome.

Rett syndrome is an X chromosome-linked neurodevelopmental disorder associated with cognitive impairment, motor dysfunction and breathing irregularities causing intermittent hypoxia. Evidence for impaired mitochondrial function is also accumulating. A subunit of complex III is among the potentially dys-regulated genes, the inner mitochondrial membrane is leaking protons, brain ATP levels seem reduced, and Rett patient blood samples confirm increased oxidative damage. We therefore screened for mitochondrial dysfunction and impaired redox balance. In hippocampal slices of a Rett mouse model (Mecp2(-/y)) we detected an increased FAD/NADH baseline-ratio indicating intensified oxidization. Cyanide-induced anoxia caused similar decreases in FAD/NADH ratio and mitochondrial membrane potential in both genotypes, but Mecp2(-/y) mitochondria seemed less polarized. Quantifying cytosolic redox balance with the genetically-encoded optical probe roGFP1 confirmed more oxidized baseline conditions, a more vulnerable redox-balance, and more intense responses of Mecp2(-/y) hippocampus to oxidative challenge and mitochondrial impairment. Trolox treatment improved the redox baseline of Mecp2(-/y) hippocampus and dampened its exaggerated responses to oxidative challenge. Microarray analysis of the hippocampal CA1 subfield did not detect alterations of key mitochondrial enzymes or scavenging systems. Yet, quantitative PCR confirmed a moderate upregulation of superoxide dismutase 1 in Mecp2(-/y) hippocampus, which might be a compensatory response to the increased oxidative burden. Since several receptors and ion-channels are redox-modulated, the mitochondrial and redox changes which already manifest in neonates could contribute to the hyperexcitability and diminished synaptic plasticity in MeCP2 deficiency. Therefore, targeting cellular redox balance might qualify as a potential pharmacotherapeutic approach to improve neuronal network function in Rett syndrome.

Expression and function of serotonin 2A and 2B receptors in the mammalian respiratory network.

Neurons of the respiratory network in the lower brainstem express a variety of serotonin receptors (5-HTRs) that act primarily through adenylyl cyclase. However, there is one receptor family including 5-HT(2A), 5-HT(2B), and 5-HT(2C) receptors that are directed towards protein kinase C (PKC). In contrast to 5-HT(2A)Rs, expression and function of 5-HT(2B)Rs within the respiratory network are still unclear. 5-HT(2B)R utilizes a Gq-mediated signaling cascade involving calcium and leading to activation of phospholipase C and IP3/DAG pathways. Based on previous studies, this signal pathway appears to mediate excitatory actions on respiration. In the present study, we analyzed receptor expression in pontine and medullary regions of the respiratory network both at the transcriptional and translational level using quantitative RT-PCR and self-made as well as commercially available antibodies, respectively. In addition we measured effects of selective agonists and antagonists for 5-HT(2A)Rs and 5-HT(2B)Rs given intra-arterially on phrenic nerve discharges in juvenile rats using the perfused brainstem preparation. The drugs caused significant changes in discharge activity. Co-administration of both agonists revealed a dominance of the 5-HT(2B)R. Given the nature of the signaling pathways, we investigated whether intracellular calcium may explain effects observed in the respiratory network. Taken together, the results of this study suggest a significant role of both receptors in respiratory network modulation.

Serotonin receptor 1A-modulated phosphorylation of glycine receptor α3 controls breathing in mice.

Rhythmic breathing movements originate from a dispersed neuronal network in the medulla and pons. Here, we demonstrate that rhythmic activity of this respiratory network is affected by the phosphorylation status of the inhibitory glycine receptor α3 subtype (GlyRα3), which controls glutamatergic and glycinergic neuronal discharges, subject to serotonergic modulation. Serotonin receptor type 1A-specific (5-HTR1A-specific) modulation directly induced dephosphorylation of GlyRα3 receptors, which augmented inhibitory glycine-activated chloride currents in HEK293 cells coexpressing 5-HTR1A and GlyRα3. The 5-HTR1A-GlyRα3 signaling pathway was distinct from opioid receptor signaling and efficiently counteracted opioid-induced depression of breathing and consequential apnea in mice. Paradoxically, this rescue of breathing originated from enhanced glycinergic synaptic inhibition of glutamatergic and glycinergic neurons and caused disinhibition of their target neurons. Together, these effects changed respiratory phase alternations and ensured rhythmic breathing in vivo. GlyRα3-deficient mice had an irregular respiratory rhythm under baseline conditions, and systemic 5-HTR1A activation failed to remedy opioid-induced respiratory depression in these mice. Delineation of this 5-HTR1A-GlyRα3 signaling pathway offers a mechanistic basis for pharmacological treatment of opioid-induced apnea and other breathing disturbances caused by disorders of inhibitory synaptic transmission, such as hyperekplexia, hypoxia/ischemia, and brainstem infarction.