β2-Adrenergic receptor agonist ameliorates phenotypes and corrects microRNA-mediated IGF1 deficits in a mouse model of Rett syndrome

Abstract

Rett syndrome is a severe childhood onset neurodevelopmental disorder caused by mutations in methyl-CpG-binding protein 2 (MECP2), with known disturbances in catecholamine synthesis. Here, we show that treatment with the β2-adrenergic receptor agonist clenbuterol increases survival, rescues abnormalities in respiratory function and social recognition, and improves motor coordination in young male Mecp2-null (Mecp2(-/y)) mice. Importantly, we demonstrate that short-term treatment with clenbuterol in older symptomatic female heterozygous (Mecp2(-/+)) mice rescues respiratory, cognitive, and motor coordination deficits, and induces an anxiolytic effect. In addition, we reveal abnormalities in a microRNA-mediated pathway, downstream of brain-derived neurotrophic factor that affects insulin-like growth factor 1 (IGF1) expression in Mecp2(-/y) mice, and show that treatment with clenbuterol restores the observed molecular alterations. Finally, cotreatment with clenbuterol and recombinant human IGF1 results in additional increases in survival in male null mice. Collectively, our data support a role for IGF1 and other growth factor deficits as an underlying mechanism of Rett syndrome and introduce β2-adrenergic receptor agonists as potential therapeutic agents for the treatment of the disorder.

Keywords: LIN28A; let-7f.

Fig. 1.

Treatment with clenbuterol substantially increases survival and improves behavior of Mecp2 KO mice. (A) Schematic showing the duration of clenbuterol treatment and time of behavioral assays in Mecp2^−/y mice. (B) Graph showing cumulative survival distributions for KO vehicle [KO (v), red circles], and KO clenbuterol-treated [KO (t), green circles] mice. P value shown in graph is based on Gehan–Breslow–Wilcoxon test. (C) Graph showing mean ± SEM latency to fall rotarod motor coordination test values (in seconds) in WT vehicle [WT (v), black circles], KO vehicle [KO (v), red circles], and clenbuterol-treated [KO (t), green circles] mice during the second experimental day. (D) Graph showing mean ± SEM breath rate values (breaths per minute) for the three experimental groups. Stars in C and D depict statistical significance based on ANOVA with Newman–Keuls test for multiple comparisons (***P < 0.001, **P < 0.01, *P < 0.05). (E) Graph showing mean ± SEM percentage of time spent in the chamber with the Stimulus mouse for WT (v) (n = 29), KO (v) (n = 9), and KO (v) (n = 11) during the first (social approach or SA, filled bars) and second day (social recognition or SR, bars with diagonal lines) of the three-chamber test. The asterisks denote statistical significance based on two-tailed paired t test (***P < 0.001, **P < 0.01, with the exact P values depicted in the figure).

Fig. 2.

Treatment with clenbuterol improves multiple behavioral deficits in symptomatic Mecp2^−/+ mice. (A) Schematic showing the duration of clenbuterol treatment and time of behavioral assays in Mecp2^−/+ mice. (B, Upper) Representative 5-s trace from a Mecp2^−/+ mouse before clenbuterol treatment showing characteristic (>0.5 s, black arrows) episodes of apnea. (Scale bar, 0.5 s.) (Lower) Graph showing the total number of apneas during 12-min plethysmography sessions in Mecp2^−/+ mice before (filled red circles) and after (filled green circles) 3 wk of clenbuterol treatment. (C) Representative 5-s respiratory traces pre- and post-clenbuterol treatment of the same Mecp2^−/+ mouse, as well as a WT control. (D) Graph showing mean ± SEM inspiratory (Ti) and expiratory (Te) times in milliseconds following plethysmography in Mecp2^−/+ mice before and after 3 wk of clenbuterol treatment, as well as in WT female mice. Stars depict statistical significance based on Kruskal–Wallis ANOVA with Dunn's multiple-comparison test (***P < 0.001, **P < 0.01, *P < 0.05). (E) Graph showing the object recognition performance ratio (SI Materials and Methods) before and after 3–4 wk of clenbuterol treatment. (F) Graph showing latency to fall rotarod motor coordination test values (in seconds) before and after 4 wk of clenbuterol treatment. Results from the first experimental day are shown. (G, Upper) Representative traces from the open field assay for the same female Mecp2^−/+ mouse pre- and post-clenbuterol treatment and for a WT female control mouse. (Lower) Graph showing the percentage of time spent in the center of the arena before (pre, red filled circles) and after (post, green filled circles) 3 wk of daily clenbuterol treatment in female Mecp2^+/− mice. In all graphs but D, stars between treatments depict statistical significance (**P < 0.01, *P < 0.05) based on two-tailed paired Student t test (E and F) or Wilcoxon matched-pairs signed rank test (B and G), and stars over data points depict significance relative to WT female mice based on two-tailed one-sample Student t test (E and F), or two-tailed Wilcoxon signed rank test (B and G) (***P < 0.001, *P < 0.05). In graphs B, E, F, and G, the dotted lines and blue shading depict the mean ± SEM values of female WT control mice.

Fig. 3.

A miRNA-mediated pathway controlling IGF1 expression is altered in Mecp2 male null mice and restored by clenbuterol treatment. (A, Upper) Representative blot showing levels of phosphorylated CREB (pCREB) normalized to total CREB (tCREB) WT vehicle [WT (v), black bars; n = 7], KO vehicle [KO (v), red bars; n = 6], and clenbuterol-treated [KO (t), green bars; n = 5] mice. (Lower) Graph showing mean ± SEM cerebellar pCREB/tCREB ratio based on Western blotting. (B and C) Graphs showing mean ± SEM cerebellar BDNF mRNA levels based on quantitative real-time PCR (B), and BDNF protein levels based on ELISA (C). Number of samples for WT (v), KO (v), and KO (t) were 12, 9, and 8, respectively for mRNA and 9, 8, and 8 for protein levels. (D, Upper) Representative blot showing levels of LIN28A normalized to α-tubulin for the three groups of mice described in A. (Lower) Graph showing mean ± SEM normalized cerebellar LIN28A protein levels for WT (v) (n = 7), KO (v) (n = 5), and KO (v) (n = 5). (E and F) Graphs showing mean ± SEM cerebellar mature let-7f miRNA (E) and IGF1 mRNA (F) levels in WT (v) (n = 12), KO (v) (n = 9), and KO (t) (n = 8) mice. The stars in graphs A–F depict statistical significance based on ANOVA with Newman–Keuls test for multiple comparisons (**P < 0.01, *P < 0.05, comparing WTv vs. KOv and KOt vs. KOv). (G) Graph showing mean ± SEM serum IGF1 protein levels (in nanograms per milliliter) based on ELISA in female WT mice (FWT, filled black bars; n = 10) and Mecp2^+/− mice before (HET pre, filled red bars; n = 9) and after (HET post, filled green bars; n = 9) clenbuterol treatment for 4 wk. The stars (*P < 0.05) depict significance based on ANOVA with Newman–Keuls multiple-comparison test. (H, Upper) Predicted interaction between let-7f miRNA (red) and mouse IGF1-1 3′-UTR sequences (black). The bars represent canonical Watson–Crick base pair and the double dots depict G-U wobble base pairing. (Lower) Graph showing mean ± SEM relative luminescence in let-7f and negative control miRNA (NC-miR) precursor transfected HEK293 cells expressing luciferase fused to mouse IGF1 3′-UTR. Relative luminescence was calculated by dividing firefly to Renilla luciferase, and all values were transformed to ratios relative to NC-miR. (I) Graph showing mean ± SEM relative to negative controls (anti-NC for miRNA inhibitor and sh-NC for shRNA control) or sIGF1 mRNA levels in Hep G2 cells after let-7f inhibition (anti-let-7f; n = 5) and shRNA-mediated knockdown of LIN28A (sh-LIN28A; n = 5). The stars in H and I depict statistical significance (**P < 0.01, *P < 0.05) based on two-tailed Student t test.

Fig. 4.

Clenbuterol-mediated restoration of brain growth factor expression and synergistic effects with rhIGF1 in male Mecp2-null mice. (A–C) Graphs showing sample-by-sample correlation between (A) BDNF mRNA and LIN28A protein levels, (B) mature let-7f miRNA levels with LIN28A protein, as well as (C) IGF1 mRNA expression. Colors depict sample identity [blue for WT (v), green for KO (t), and red for KO (v) mice]. (D) Schematic depicting the proposed molecular mechanism linking Mecp2-regulated BDNF to IGF1 through LIN28A and IGF1. The effect of clenbuterol treatment in restoring NGF and BDNF levels through CREB activation and indirectly affecting IGF1 expression, thus regulating growth factor-mediated functions, is also shown. (E) Graph showing cumulative survival distributions for Mecp2 KO mice receiving clenbuterol and rhIGF1 cotreatment [KO (Clen + IGF1), blue circles]. Survival of KO vehicle [KO (v), open red circles] and clenbuterol-treated [KO (clen), open green circles] mice is also depicted for comparison (from Fig. 1A). The stars represent statistical significance based on Gehan–Breslow–Wilcoxon test (***P < 0.001, *P < 0.05).