Functional recovery with recombinant human IGF1 treatment in a mouse model of Rett Syndrome

Abstract

Rett Syndrome is a neurodevelopmental disorder that arises from mutations in the X-linked gene methyl-CpG binding protein 2 (MeCP2). MeCP2 has a large number of targets and a wide range of functions, suggesting the hypothesis that functional signaling mechanisms upstream of synaptic and circuit maturation may contribute to our understanding of the disorder and provide insight into potential treatment. Here, we show that insulin-like growth factor-1 (IGF1) levels are reduced in young male Mecp2-null (Mecp2(-/y)) mice, and systemic treatment with recombinant human IGF1 (rhIGF1) improves lifespan, locomotor activity, heart rate, respiration patterns, and social and anxiety behavior. Furthermore, Mecp2-null mice treated with rhIGF1 show increased synaptic and activated signaling pathway proteins, enhanced cortical excitatory synaptic transmission, and restored dendritic spine densities. IGF1 levels are also reduced in older, fully symptomatic heterozygous (Mecp2(-/+)) female mice, and short-term treatment with rhIGF1 in these animals improves respiratory patterns, reduces anxiety levels, and increases exploratory behavior. In addition, rhIGF1 treatment normalizes abnormally prolonged plasticity in visual cortex circuits of adult Mecp2(-/+) female mice. Our results provide characterization of the phenotypic development of Rett Syndrome in a mouse model at the molecular, circuit, and organismal levels and demonstrate a mechanism-based therapeutic role for rhIGF1 in treating Rett Syndrome.

Keywords: female mice; male mice; molecular therapeutic; respiration; synaptic function.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

rhIGF1 promotes survival, improves physiological condition, and restores normal levels of social and anxiety-related behavior of Mecp2−/y mice. (A) Serum levels of endogenous IGF1 measured in P28 and P56 mice relative to WT P28 controls [statistical comparisons used unpaired t test for equal variances; WT (P28 and P56), n = 12 and 10; Mecp2−/y (P28 and P56), n = 10 and 9]. (B) Mean survival age of Mecp2−/y animals treated with vehicle or rhIGF1 (unpaired t test; Mecp2−/y vehicle, n = 27; rhIGF1, n = 29). (C) Weight variation from P14 (starting day of injection) to P56. Comparisons between KO vehicle and treated were done week by week with an unpaired t test for equal variances (WT vehicle, n = 26; rhIGF1, n = 22; Mecp2−/y vehicle, n = 27; rhIGF1, n = 29). (D and E) Scatterplot of average breathing (D) and cardiac (E) rates. Each dot represents a single animal; lines indicate population average (ANOVA with Newman–Keuls post hoc analysis for multiple comparison). (F) Nocturnal locomotor activity expressed as average number of beam crossings per minute (ANOVA with Newman–Keuls post hoc analysis for multiple comparisons performed at each separate time point; WT vehicle, n = 25; rhIGF1, n = 28; Mecp2−/y vehicle, n = 21; rhIGF1, n = 26). (G) Three-chamber test measurements showing the percentage of time the animals spent socializing with a stranger mouse during the first contact (solid bars) and 30 min after the first contact (hatched bars) at P28–35 (paired t test). (H) Percentage of time spent in the open arms of a plus maze as measurement of anxiety-related behavior at P28–35 (ANOVA with Newman–Keuls post hoc analysis for multiple comparisons; WT vehicle, n = 18; rhIGF1, n = 16; Mecp2−/y vehicle, n = 21; rhIGF1, n = 23). Error bars represent SEM. *P < 0.05; **P < 0.01; ***P < 0.001. See also Fig. S1.

Fig. 2.

Curtailment of abnormal visual cortical plasticity by rhIGF1 treatment. (A) Schematic showing MD paradigm. Black and red lines, respectively, depict the contralateral and ipsilateral projections that connect the deprived (contralateral) and nondeprived (ipsilateral) eye to V1. (B) Experimental timeline for treatment, eyelid suture, and optical imaging. (C) Average response amplitudes measured in V1 from the deprived contralateral (C) and nondeprived ipsilateral (I) eye in P28 female mice (Mann–Whitney test; WT and WT+MD, n = 3 each; Mecp2−/+, n = 4; Mecp2−/++MD, n = 4 each). (D) Average ODI values in P28 females (Mann–Whitney test). (E) Response amplitudes observed in P60 females (Mann–Whitney test; n = 5 for each group). (F) ODI representation for P60 females for each condition (ANOVA with Newman–Keuls post hoc analysis for multiple comparisons). Error bars represent SEM. †P < 0.10; *P < 0.05; **P < 0.01.

Fig. 3.

Effects of rhIGF1 on excitatory synaptic transmission, spine density, and signaling pathways. (A) Averaged mEPSCs from V1 layer 2/3 cells comparing peak amplitudes (events: WT vehicle, n = 270; WT rhIGF1, n = 816; KO vehicle, n = 354; KO rhIGF1, n = 268). (B) Distribution of mEPSC amplitude across all cells. rhIGF1 treatment significantly shifted the population distribution toward higher peak amplitudes (Kolmogorov–Smirnov test between vehicle and treated Mecp2−/y with pooled data from five to eight cells from three to four animals in each group). (C) Representative micrographs of basal dendrites in supragranular pyramidal neurons at P42 stained with Golgi and their synaptic spine density measurements (one-way ANOVA with Newman–Keuls post hoc analysis for multiple comparisons). (Scale bar, 5 μm.) (D) Schematic of proposed signaling pathways downstream of MeCP2-mediated IGF1 activation. (E) Quantification of PSD95 (Left) Western blots in synaptoneurosomes as a percentage of WT littermate control levels and the ratios between phosphorylated and total Akt (Center) and ERK1/2 (Right) in whole-cell homogenates. Ratios are based on Western blot protein measurements at P28 (comparisons used a one-way ANOVA with Newman–Keuls post hoc analysis for multiple comparisons including WT controls). *P < 0.05; **P < 0.01. Error bars represent SEM. See also Fig. S2.

Fig. 4.

Measurement of rhIGF1 levels and IGF1 availability. (A) Levels of rhIGF1 in serum collected 2 and 24 h after i.p. injection of rhIGF1 (unpaired t test; n animals at P28, 2 and 24 h: WT, 14 and 7; Mecp2−/y, 22 and 7; n animals at P56, 2 and 24 h: WT, 18 and 5; Mecp2−/y, 18 and 6). See Fig. S3A. Hatched bars show measurements in P56 mice after 1 wk of treatment (n = 4 each). (B) Total IGF1 concentration in blood serum collected after 2 h calculated by addition of endogenous IGF1 (filled boxes) and rhIGF1 (hatched boxes) levels for each animal. Statistics were calculated with the summed values [one-way ANOVA with Newman–Keuls post hoc analysis for multiple comparisons of treatment effects, separate analysis for each age; WT (P28 and P56), n = 12 and 10; WT+rhIGF1 (P28 and P56), n = 10 and 15; Mecp2−/y (P28 and P56), n = 10 and 9; Mecp2−/y+rhIGF1 (P28 and P56), n = 11 and 12]. Error bars represent SEM. †P < 0.1; *P < 0.05; **P < 0.01; ****P < 0.0001. See also Fig. S3.

Fig. 5.

Effects of rhIGF1 on Mecp2−/+ breathing patterns, anxiety, and spatial recognition. (A) Representative breathing pattern traces of WT (Top) and Mecp2−/+ females before (Middle) and after (Bottom) treatment. Downward indicates inspiration (Fig. S4C). (B) Average inspiratory (Ti) and expiratory (Te) times were abnormal in Mecp2−/+ females, and treatment restored these to WT levels (paired t test for post/pre comparison and ordinary one-way ANOVA with Tukey’s post hoc analysis for multiple comparisons). Dots represent individual animals. (C) Total number of breath holds observed during 12-min recording sessions pre- and posttreatment (paired t test). Inset shows representative breath hold trace from a pretreatment female. (Scale bar, 500 ms.) (D) Performance ratio of time spent with displaced versus nondisplaced objects (see Materials and Methods for details of assay) pre- and posttreatment (paired t test). (E) Percent time spent in the open arms of elevated plus maze pre- and posttreatment (paired t test). (F) Relative serum levels of total IGF1 (murine and rhIGF1) measured pre- and posttreatment (one-way ANOVA with Tukey’s post hoc analysis for multiple comparison). Error bars represent S.E.M. *P < 0.05; **P < 0.01; ****P < 0.0001. In C–E, dashed line and gray shading represent mean and SEM of WT mice, respectively. See also Fig. S4.