Correction of respiratory disorders in a mouse model of Rett syndrome

Abstract

Rett syndrome (RTT) is an autism spectrum disorder caused by mutations in the X-linked gene that encodes the transcription factor methyl-CpG-binding protein 2 (MeCP2). A major debilitating phenotype in affected females is frequent apneas, and heterozygous Mecp2-deficient female mice mimic the human respiratory disorder. GABA defects have been demonstrated in the brainstem of Mecp2-deficient mice. Here, using an intact respiratory network, we show that apnea in RTT mice is characterized by excessive excitatory activity in expiratory cranial and spinal nerves. Augmenting GABA markedly improves the respiratory phenotype. In addition, a serotonin 1a receptor agonist that depresses expiratory neuron activity also reduces apnea, corrects the irregular breathing pattern, and prolongs survival in MeCP2 null males. Combining a GABA reuptake blocker with a serotonin 1a agonist in heterozygous females completely corrects their respiratory defects. The results indicate that GABA and serotonin 1a receptor activity are candidates for treatment of the respiratory disorders in Rett syndrome.

Fig. 1.

Characterization of apnea in Mecp2^−/+ mice studied in situ. (A) Representative traces show increased post-I activity in cVN that terminates before end of PN apnea and marked increase in HN that persists throughout PN apnea. (B) Example of tonic HN accompanied by tonic AbN during PN apnea. Note expiratory activity in one HN burst before the apnea (arrowed) and in several after PN apnea. (C) Incidence of apneas in Mecp2^+/+ (n = 16) and Mecp2^−/+ (n = 17) mice studied in situ. *P ≤ 0.0001 (unpaired t test). (D) Cycle triggered average of 20 consecutive cycles for integrated HN activity superimposed on integrated PN activity recorded in situ. There is significant HN activity during postinspiration.

Fig. 2.

Effect of augmenting GABA on respiratory disorders in conscious, freely moving Mecp2^−/+ mice. (A) Representative pleural pressure records by telemetry for WT and Mecp2^−/+ showing isolated apnea (breath interval ≥ 1.0 s), periodic breathing, and effect of the GABA transporter blocker NO-711; inspiration is downward. (B) Effect of NO-711, diazepam (Diaz, a broad-spectrum GABA_A receptor allosteric modulator), and L-838,417 (L-838, a derivative that is relatively specific for receptors that contain α2, α3, and α5 receptor subunits). (B.1) Apnea. WT. *P = 0.002 vs. Mecp2^−/+ vehicle; **P = 0.008 vs., Mecp2^−/+ vehicle; ***P = 0.015 vs. Mecp2^−/+ vehicle; ****P = 0.028 vs. Mecp2^−/+ vehicle (n = 9 for WT, n = 8 for vehicle, n = 5 for treatments) (two-way repeated-measures ANOVA with strain and treatment as the two factors). (B.2) Periodic breathing. *P = <0.001 vs. Mecp2^−/+ vehicle. (B.3) Irregularity score. *P = <0.001 vs. Mecp2^−/+ vehicle; **P = 0.022 vs. Mecp2^−/+ vehicle.

Fig. 4.

Effect of blocking GABA reuptake and of the serotonin 1a receptor agonist 8-OH-DPAT on respiratory disorders in Mecp2 null male mice. (A) Dose-dependent effect of 8-OH-DPAT on apnea, periodic breathing, and irregularity score. (A.1) Apnea. *P = <0.001 vs. Mecp2^-/y 0 mg/kg; **P = 0.046 and 0.02 vs. Mecp2^-/y 0 mg/kg (n = 9 for WT, 10 for Mecp2^-/y 0 and 300 μg/kg, and 4 for Mecp2^-/y 75 and 150 μg/kg) (two-way repeated-measures ANOVA with strain and treatment as the two factors). (A.2) Periodic breathing. *P ≤ 0.001 vs. Mecp2^-/y 0 mg/kg; **P = 0.003 and <0.001 vs. Mecp2^-/y 0 mg/kg. (A.3) Irregularity score. *P = 0.001 vs. Mecp2^-/y 0 mg/kg; **P = 0.048 vs. Mecp2^-/y 0 mg/kg; ***P = 0.009 vs. Mecp2^-/y 0 mg/kg. (A.4) Box plot for untreated (n = 22) and Mecp2 null males treated with 8-OH-DPAT osmotic pump. P = 0.035 (n = 9) (Kaplan–Meier log–rank test). (B) Effect of NO-711 (5 mg/kg i.p.) on apnea and irregularity score. (B.1) Apnea. *P = 0.001 (n = 4) (paired t test). (B.2) Irregularity score. *P = 0.004.

Fig. 3.

Effect of NO-711 on residual apneas in Mecp2^−/+ mice studied in situ. (A) HN, AbN, and PN traces before (control) and after adding NO-711 (5.2 μM) to the perfusate. (B.1–B.3) Effect of the GABA reuptake blocker on (B.1) the incidence of apnea (*P = 0.009, n = 5, paired t test), (B.2) the HN burst envelope in residual PN apneas (*P = 0.005, n = 5), and (B.3) the incidence of apneas that contained tonic AbN activity (*P = 0.04, n = 4).

Fig. 5.

Effect of combined GABA reuptake block and serotonin 1a agonist on apnea and irregularity score in Mecp2^−/+ studied in situ and conscious Mecp2^-/y mice. (A.1) Apnea in Mecp2^−/+ female mice treated with combined NO-711 (5.2 μM) and 8-OH-DPAT (0.1 μM). *P = 0.01 (n = 6) (ANOVA). (A.2) Irregularity score. *P = 0.001. (B.1) Apnea in Mecp2^-/y males treated with combined NO-711 (1.0 mg/kg) and 8-OH-DPAT (150 μg/kg). *P = 0.016 (n = 5) (paired t test). (B.2) Irregularity score. *P = 0.05.