Effects of early-life exposure to THIP on phenotype development in a mouse model of Rett syndrome

Abstract

Background: Rett syndrome (RTT) is a neurodevelopmental disorder caused mostly by disruptions in the MECP2 gene. MECP2-null mice show imbalances in neuronal excitability and synaptic communications. Several previous studies indicate that augmenting synaptic GABA receptors (GABA_ARs) can alleviate RTT-like symptoms in mice. In addition to the synaptic GABA_ARs, there is a group of GABA_ARs found outside the synaptic cleft with the capability to produce sustained inhibition, which may be potential therapeutic targets for the control of neuronal excitability in RTT.

Methods: Wild-type and MECP2-null mice were randomly divided into four groups, receiving the extrasynaptic GABA_AR agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol hydrochloride (THIP) and vehicle control, respectively. Low-dose THIP was administered to neonatal mice through lactation. RTT-like symptoms including lifespan, breathing, motor function, and social behaviors were studied when mice became mature. Changes in neuronal excitability and norepinephrine biosynthesis enzyme expression were studied in electrophysiology and molecular biology.

Results: With no evident sedation and other adverse side effects, early-life exposure to THIP extended the lifespan, alleviated breathing abnormalities, enhanced motor function, and improved social behaviors of MECP2-null mice. Such beneficial effects were associated with stabilization of locus coeruleus neuronal excitability and improvement of norepinephrine biosynthesis enzyme expression.

Conclusions: THIP treatment in early lives might be a therapeutic approach to RTT-like symptoms in MECP2-null mice and perhaps in people with RTT as well.

Keywords: Behavior; Gaboxadol; Locus coeruleus; MECP2; Rett syndrome; THIP.

Fig. 1

THIP administration extended the lifespan of MECP2-null mice. a Twenty-nine MECP2-null mice were used in the survival experiment and 14 of them were delivered THIP orally (solid line) and 13 without THIP treatment (dashed line). (**P < 0.01; Mantel-Cox test). b Percentage of survival in the tested mice. In the vehicle group, 50 % MECP2-null mice died within 52 days, while THIP treatment expanded the 50 % lifespan to 82 days

Fig. 2

THIP administration alleviated the breathing abnormalities in MECP2-null mice. A ₁, A ₂ Typical records of breathing activity from both WT and MECP2-null mice with and without THIP administration. B Distributions of apnea count in different aged MECP2-null mice with and without THIP treatment. C In MECP2-null mice, THIP administration significantly reduced the apnea count at ages of 4–6 weeks (vehicle: n = 26, THIP: n = 8, P = 0.002) and 6–8 weeks (vehicle: n = 19, THIP: n = 8, P = 0.021), although the significance was not found in 2–4 weeks (vehicle: n = 45, THIP: n = 7, P = 0.081; ^### P < 0.001 in Kruskal-Wallis test; *P < 0.05, **P < 0.01 in Mann-Whitney post hoc comparison). D, E Similar effects of THIP treatment on breathing frequency variation was observed in these mice (2–4 weeks: P = 0.037; 4–6 weeks: P = 0.004; 6–8 weeks: P < 0.001; *P < 0.05, **P < 0.01, ***P < 0.001; one-way ANOVA and Tukey’s post hoc)

Fig. 3

THIP administration improved motor function of MECP2-null mice. a Significant main effects of THIP treatment (F = 23.74, df = 1, P < 0.001) and genotype (F = 147.85, df = 1, P < 0.001) were observed, as well as a significant interaction (F = 12.04, df = 1, P < 0.001). ^(### P < 0.001, two-way ANOVA). The grip strength of MECP2-null mice was significantly increased with THIP treatment (WT: n = 18 and n = 18 mice; MECP2-null: n = 23 and n = 22; vehicle and THIP, respectively; ***P < 0.001, Tukey’s post hoc). b Significant main effects of THIP treatment (F = 5.26, df = 1, P < 0.05) and genotype (F = 30.4, df = 1, P < 0.001) were observed, as well as a significant interaction (F = 13.25, df = 1, P < 0.001) (^# P < 0.05, two-way ANOVA). THIP administration significantly reduced the footfault ratio (including both hindlimb and forelimb) of MECP2-null mice (WT: n = 22 and n = 23 mice; MECP2-null: n = 20 and n = 25; vehicle and THIP, respectively; ***P < 0.001, Tukey’s post hoc). c The spontaneous locomotion of WT and MECP2-null mice was not significantly affected by THIP treatment. The main effect of THIP treatment was not significant (F = 0.26, df = 1, P = 0.614), as the main effect of genotype (F = 3.00, df = 1, P = 0.095). The interaction of these two factors was not significant (F = 0.99, df = 1, P = 0.329) (WT: n = 8 and n = 7 mice; MECP2-null: n = 9 and n = 6; vehicle and THIP, respectively; two-way ANOVA)

Fig. 4

THIP administration alleviated the defects of social behaviors in MECP2-null mice. During the habituation period in the three chamber test, both WT and null mice took similar amount of times (a) and transitions (b) in either side of the chambers indicating no preference. The main effect preference was not significant. (a F = 1.72, df = 1, P = 0.195; b F = 0.20, df = 1, P = 0.656; three-way ANOVA). The transitions between the chambers also suggested that the tested animals are not in a sedative state. c In the sociability test, a significant difference was detected within the main factor of preference (F = 23.31, df = 1, ^### P < 0.001, three-way ANOVA). WT mice spent significantly more time in the chamber containing an animal than the empty one, whereas the MECP2-null mice lost such a preference. THIP administration increased the time expenditure of MECP2-null mice in interacting with another mouse (*P < 0.05; Tukey’s post hoc). No significant differences were found in the main factor of genotype (F = 1.20, df = 1, P = 0.278) or THIP treatment (F = 0.03, df = 1, P = 0.863). The interactions of genotype × treatment (F = 0.46, df = 1, P = 0.500), genotype × preference (F = 1.41, df = 1, P = 0.239), treatment × preference (F = 3.31, df = 1, P = 0.074), or genotype × THIP treatment × preference (F = 2.08, df = 1, P = 0.155) were not significant as well (three-way ANOVA). d In the social novelty test, the main factor of preference showed a significant difference (F = 54.48, df = 1, ^### P < 0.001, three-way ANOVA). WT mice spent significantly more time in the chamber with a novel animal than the chamber with a familiar one, whereas the MECP2-null mice did not show the preference to either chamber. With THIP treatment the novelty preference was improved in the MECP2-null mice (**P < 0.01, ***P < 0.001; Tukey’s post hoc). No significant differences were found in the main factor of genotype (F = 0.57, df = 1, P = 0.453) or THIP treatment (F = 0.17, df = 1, P = 0.681). The interactions of genotype × treatment (F = 0.02, df = 1, P = 0.888), genotype × preference (F = 0.49, df = 1, P = 0.487), treatment × preference (F = 1.58, df = 1, P = 0.213), or genotype × THIP treatment × preference (F = 1.35, df = 1, P = 0.250) were not significant as well (vehicle: n = 12 and n = 9; THIP: n = 8 and n = 6; WT and MECP2-null, respectively; three-way ANOVA)

Fig. 5

THIP administration suppressed the hyperexcitability of LC neurons in MECP2-null mice. A ₁, A ₂ Typical recordings of spontaneous firing of LC neurons in WT and MECP2-null mice at 1 month of age without THIP treatment. B ₁, B ₂ Spontaneous firing of LC neurons in WT and MECP2-null mice of the same age with THIP pretreatment. C–F THIP administration did not significantly change membrane potentials, input resistance, action potential overshoot, and action potential threshold in both WT and MECP2-null mice. No significant main effect of THIP treatment (F = 0.09, df = 1, P = 0.765; F = 1.15, df = 1, P = 0.289; F = 0.27, df = 1, P = 0.606; F = 0.76, df = 1, P = 0.387; C, D, E, F, respectively) and genotype (F = 1.45, df = 1, P = 0.234; F = 0.09, df = 1, P = 0.765; F = 0.01, df = 1, P = 0.921; F = 0.99, df = 1, P = 0.324; C, D, E, F, respectively) were observed, either the interaction (F = 0, df = 1, P = 1.000; F = 1.46, df = 1, P = 0.232; F = 0.03, df = 1, P = 0.863; F = 3.58, df = 1, P = 0.064; C, D, E, F, respectively). G The main effect of genotype was significant (F = 10.06, df = 1, P < 0.01), whereas the main effect of THIP treatment was not (F = 1.72, df = 1, P = 0.196). The interaction of these two factors was significant (F = 8.6, df = 1, P < 0.01) as well (^## P < 0.01; two-way ANOVA). The firing activity of LC neurons in MECP2-null mice is significantly increased compared to the WT and chronic treatment with THIP abolished the hyperexcitability (vehicle: n = 14 and n = 13; THIP: n = 13 and n = 16; in WT and MECP2-null, respectively; ***P < 0.001; Tukey’s post hoc)

Fig. 6

Improvement of TH and DBH expressions with THIP administration in MECP2-null mice. a-c, qPCR analysis showed that during THIP treatment (P37), both TH and DBH transcript levels were significantly increased (vehicle: n = 4 and n = 4 animals; THIP: n = 5 and n = 5 animals; WT and MECP2-null, respectively). d-f, The Western analysis also indicated that THIP treatment significantly increased the protein expressions of both TH and DBH (vehicle: n = 4 and n = 4 animals; THIP: n = 4 and n = 4 animals; WT and MECP2-null, respectively; *P < 0.05, **P < 0.01, ***P < 0.001; one-tailed Student’s t test)

Fig. 7

Alteration of GABA_AR subunits in the LC area of MECP2-null mice. qPCR analysis indicated that the mRNA levels of δ and α6 subunits were 2.0 and 3.5 times higher than the WT levels, while THIP treatment significantly reduced the expression level of α6 subunit, without alteration of δ, β1, and β2 subunits (vehicle: n = 4 and n = 4 animals; THIP: n = 5 and n = 5 animals; WT and MECP2-null, respectively; **P < 0.01; Student’s t test)