The enhancement of activity rescues the establishment of Mecp2 null neuronal phenotypes (2021)
Figure 5

Figure 5: Early treatment with Ampakine ameliorates behavioral deficits of Mecp2 null mice

A: Schematic representation of the in vivo CX546 administration protocol.

B: Kaplan Mayer test shows an improved lifespan of Mecp2 null CX546 treated animals compared to Mecp2 null control animals. Sample size: wt=11, ko=16, ko treated with CX546=11. Gehan-Breslow-Wilcoxon test was used to compare the two groups. **: p-value<0.01.

C: The body weight of mice was assessed at four different time points (P30, P50, P70 and P90). Two-way ANOVA indicated a significant effect for both time (F(1,813,47,13)=421,1 and p<0,0001) and treatment (F(2,33=7,540 and p<0,0001). Tukey's multiple comparison test was used to compare within each time point the effect of the CX546 treatment on Mecp2 null mice. **: p-value<0.01; ****: p-value<0.0001. Values are represented as average ± SEM. Sample size: n=10 wt and n= 27 ko (16 treated with vehicle and 11 treated with CX546). Each dot represents a single animal.

D,E: Severity scores were calculated at different time points (P55, P70, P80 in D). The score for hind limbs clasping was assessed twice a week starting from P30 (E). Sample size for D and E: n=10 wt treated with vehicle and n= 20 ko (9 treated with vehicle and 11 treated with CX546). Data are represented as mean ± SEM.

F,G: Line graph (F) shows mice motor learning assessed on the accelerating rotarod during three consecutive trials. Two-way ANOVA indicated a significant effect of time (F(2,21)=5,490 and p=0,0121) and genotype (F(2,33)=16,33 and p<0.0001). Bonferroni multiple comparison test highlighted a significant increase in the latency to fall between trial 1 and trial 3 only for null mice treated with CX546. *: p-value<0.05; **: p-value<0.01. The scatter plot (G) shows latency to fall (in seconds) on the accelerating rotarod in the last trial. One-way ANOVA was used to compare ko mice and ko treated mice to the wt group. The Tukey's multiple comparison test highlighted a significant reduction in the latency to fall between wt and ko but not between wt and ko treated with Ampakine; **: p-value<0.01. Values are represented as average ± SEM. Sample size: n=6 wt treated with vehicle and n=14 ko (6 treated with vehicle and 8 treated with CX546). Each dot represents a single animal.

H: The graph shows the assessment of the discrimination index (DI) between two different objects for each mouse during the last trial of a Novel Object Recognition test. One-way ANOVA was used to compare ko mice and ko treated mice to the wt group. The Tukey's multiple comparison test highlighted a p-value of 0,07 between wt and ko treated with vehicle but not between wt and ko treated with CX546. Data are represented as mean ± SEM. Sample size: n=8 wt treated with vehicle n= 12 ko (6 treated with vehicle and 6 treated with CX546). Each dot represents a single animal.

I,J: The graphs show the expression level of Bdnf (I) and Kcc2 (J) in cortical samples obtained from P45 animals. One-way ANOVA was used to assess difference in the expression of the two selected genes between wt, ko e CX546 treated ko animals. For Bdnf expression (I) Tukey's multiple comparison test highlighted significant differences between both wt and ko and ko and ko treated with CX546 but no difference between wt and ko treated with CX546 (*: p-value<0.05; **: p-value<0.01). For Kcc2 expression (J) Tukey's multiple comparison test highlighted significant differences between wt and ko but no difference between wt and ko treated (*: p-value<0.05). Data are represented as values ± SEM. Sample size for I: n=10 wt, n=9 ko and n=6 ko treated with CX546. Sample size for J: n=11 wt, n=12 ko and n=7 ko treated with CX546. Each dot represents a single animal.

Expanded figure 1: Lack of Mecp2 does not influence the NPC fate commitment

A-D: DIV8 and DIV22 cultures were stained with Nestin (to mark neuroprogenitors; green) and Map2 (to mark neurons; red) (panels A and B). Scatter plot in C represents the number of Nestin positive cells counted in both wt and null cultures at DIV8 (panel C). Scatter plot in D reports Ki67 (marker of cell cycle) expression levels (2-∆ct) at different DIVs. Panel C: Student's t-test, Panel D: Two-Way ANOVA and Bonferroni post-hoc test. *: p-value<0.05; **: p-value<0.01; °°°°: p-value<0.001. Scatter plots show the mean ± SEM from at least 6 wells obtained from two independent preparations. Scale bars A, B: 20 µm.

E-H: Nuclei deriving from heterozygous cortices were stained for Mecp2 (green) and DAPI; neurons and astrocytes were distinguished using antibodies against Tuj1 and Gfap respectively (red; E, G). White arrows indicate two representative cells that are positive or negative for Mecp2. Scatter plots report the percentage of cells positive or negative for Mecp2 counted at DIV22 over the total of Tuj1 (F) and Gfap (H) positive cells. No difference between the two groups was highlighted (Student's t-test). Graphs show the mean ± SEM from at least 3 wells obtained from two independent preparations; at least 10 fields were measured for each well. Scale bars: 30 µm (E, G); 10 µm (e', e'', g', g'').

Expanded figure 2: Transcriptional differences between wt and Mecp2 null samples in basal condition

A,B: PCA analyses show the segregation of wt and null samples based on the expression of different sets of genes (see Appendix Table S1). 16 genes typically expressed by cortical neuroprogenitors were considered in A, while 51 genes typically expressed by neurons were considered in B. A' and B' are close ups at different DIVs. At least 5 samples derived from three independent preparations were used to produce the points composing the wt and null cluster. Each point represents a single well.

C: The expression of 10 neuronal genes was assessed by qRT-PCR at DIV 21 in wt and Mecp2 null cultures. Significant differences in gene expression between wt and null cultures were screened through Student's t-test. Histograms show the mean ± SEM from at least 3 samples obtained from two independent preparations. *: p-value<0.05; **: p-value<0.01; ***: p-value<0.001.

Expanded figure 3: Experimental setting of the in vitro Ampakine treatment

A: Scatter plot shows the expression of the AMPA receptor subunit Gria2 mRNA in cultures differentiated from NPCs at three different time points expressed as 2-∆ct (DIV3, DIV7, and DIV14). Two-way ANOVA followed by Bonferroni post hoc test indicated a reduced Gria2 transcription in null neurons only at DIV14. **: p-value<0.01. Values represent the mean ± SEM. Sample size: 4 wells obtained from two independent preparations.

B: Scatter plot shows the levels of Akt phosphorylation in wt cells at DIV9 treated acutely with vehicle, CX546 or CX546 and NBQX. One-way ANOVA was used to assess difference between groups. Tukey's multiple comparison test highlighted significant differences between vehicle and CX546 treated cells and between CX546 treated cells and CX-NBQX treated cells. Values represent the mean ± SEM. *: p-value<0.05. Sample size: at least 6 samples obtained from two independent preparations.

C: To assess CX546 toxicity we measured cell viability through MTT assay. Cells were collected at DIV14 after Ampakine exposure (10 and 20 µM) from DIV7 to DIV10 and at DIV10 after daily or every other day exposure to 20 µM CX546. Two way ANOVA indicated a significant effect of both the 20 µM exposures (F(2,17)=8,913 and p=0,0023), thus indicating the toxicity of this protocol. Sample size: n≥3 wells deriving from two independent preparations. Values represent the mean ± SEM.

Expanded figure 4: KCl neuronal activity enhancement rescues Mecp2 null defective morphology

A-D: Scatter plots showing the effects of adding 4 mM KCl on 4 morphological parameters assessed in wt and ko cultures at DIV 22. One-way ANOVA followed by Tukey's multiple comparison test was used to assess differences between groups (*: p-value<0.05; **: p-value<0.01; ***: p-value<0.001). Values represent the mean ± SEM. Sample size: n≥7 wells from three independent preparations, at least 105 cells were measured in each well.

E: Schematic representation of KCl exposure.

F,G: Scatter plots showing the effects of an early (DIV0-7) and late (DIV7-14) treatment with 4 mM KCl on 2 morphological parameters assessed at DIV14 in wt and ko cultures. Sample size: n≥40 neurons from two independent preparations. Two-way ANOVA followed by Bonferroni multiple comparison test was used to assess differences between groups. (*: p-value<0.05; **: p-value<0.01; ***: p-value<0.001). Values represent the mean ± SEM.