Neuronal morphology in MeCP2 mouse models is intrinsically variable and depends on age, cell type, and Mecp2 mutation

Abstract

Rett Syndrome (RTT), a progressive neurological disorder characterized by developmental regression and loss of motor and language skills, is caused by mutations in the X-linked gene encoding methyl-CpG binding protein 2 (MECP2). Neurostructural phenotypes including decreased neuronal size, dendritic complexity, and spine density have been reported in postmortem RTT brain tissue and in Mecp2 animal models. How these changes in neuronal morphology are related to RTT-like phenotype and MeCP2 function, and the extent to which restoration of neuronal morphology can be used as a cellular readout in therapeutic studies, however, remain unclear. Here, we systematically examined neuronal morphology in vivo across three Mecp2 mouse models representing Mecp2 loss-of-function, partial loss-of-function, and gain-of-function mutations, at developmental time points corresponding to early- and late-symptomatic RTT-like behavioral phenotypes. We found that in Mecp2 loss-of-function mouse models, dendritic complexity is reduced in a mild, age-dependent, and brain region-specific manner, whereas soma size is reduced consistently throughout development. Neither phenotype, however, is altered in Mecp2 gain-of-function mice. Our results suggest that, in the cell types we examined, the use of dendritic morphology as a cellular readout of RTT phenotype and therapeutic efficacy should be cautioned, as it is intrinsically variable. In contrast, soma size may be a robust and reliable marker for evaluation of MeCP2 function in Mecp2 loss-of-function studies.

Keywords: Dendritic branching; MECP2; Mouse model; Neuronal morphology; Rett syndrome; Soma size.

Figure 1. Thy1-GFP/Mreporter imaging strategy

(a) Somatosensory cortex layer V pyramidal neurons are labeled with GFP throughout the cell body and dendritic tree in Thy1-GFP/M reporter mice. Scale bar = 250 µm. (b) Hippocampus CA1 pyramidal neurons are labeled with GFP throughout the cell body and dendritic tree in Thy1-GFP/M reporter mice. Scale bar = 250 µm. (c) Somatosensory cortex layer V pyramidal neurons extend their apical dendrites to the pial surface (arrow) and their basal dendrites deep into layer VI. Scale bar = 50 µm. (d) Basal dendrites of somatosensory cortex layer V pyramidal neurons, quantified by Sholl analysis, measuring number of intersections between concentric circles drawn around the cell soma (Sholl radii) and dendritic branches. Scale bar = 50 µm. (e) Apical dendrites of somatosensory cortex layer V pyramidal neurons, quantified by Sholl analysis. Scale bar = 50 µm.

Figure 2. Dendritic complexity in somatosensory cortex of Mecp2^−/ymice

(a) Sholl analysis of somatosensory cortex layer V pyramidal neurons in P30 WT (n=24 neurons from 4 mice) and Mecp2^−/y mice (n=19 neurons from 4 mice) shows reduced dendritic complexity in Mecp2^−/y mice relative to WT. Basal dendritic arbor is denoted b negative distance from soma, 0 urn marks soma position within cortex, and apical dendritic arbor is denoted by positive distance from soma. Two-way ANOVA with Bonferroni correction, p<0.0001 (interaction); *p<0.05, **p<0.01, ***p<0.001. Bars represent mean ± sem. (b) Sholl analysis of somatosensory cortex layer V pyramidal neurons in P60 WT (n=36 neurons from 4 mice) and Mecp2^−/y mice (n=31 neurons from 4 mice) show more widespread reduction in dendritic complexity in Mecp2^−/y mice relative to WT than seen in P30 animals. Two-way ANOVA with Bonferroni correction, p<0.0001 (interaction); *p<0.05, ***p<0.001.

Figure 3. Dendritic complexity in somatosensory cortex of Mecp2^T158A/y mice

(a) Sholl analysis of somatosensory cortex layer V pyramidal neurons in P30 WT (n=40 neurons from 4 animals) and Mecp2^T158A/y mice (n=34 neurons from 4 animals) show no change dendritic complexity in Mecp2^T158A/y mice relative to WT. Two-way ANOVA, p>0.05. Bars represent mean ± sem. (b) Sholl analysis of somatosensory cortex layer V pyramidal neurons in P90 WT (n=36 neurons from 4 mice) and Mecp2^T158A/y mice (n=39 neurons from 4 mice) show reduced dendritic complexity in Mecp2^T158A/y mice relative to WT specifically in the basal dendritic arbor. Two-way ANOVA with Bonferroni correction, p<0.0001 (interaction); **p<0.01.

Figure 4. Dendritic complexity in hippocampus CA1 of Mecp2^T158A/y mice

(a) Sholl analysis of hippocampal CA1 pyramidal neurons in P30 WT (27 neurons from 5 mice) and Mecp2^T158A/y mice (n=33 neurons from 5 mice) show no change dendritic complexity in Mecp2^T158A/y mice relative to WT. Two-way ANOVA, p>0.05. Bars represent mean ± sem. (b) Sholl analysis of hippocampal CA1 pyramidal neurons in P90 WT (n=32 neurons from 5 mice) and Mecp2^T158A/y mice (n=40 neurons from 5 mice) show altered dendritic complexity in Mecp2^T158A/y mice relative to WT specifically in the basal dendritic arbor. Two-way ANOVA with Bonferroni correction, p<0.0001 (interaction); *p<0.05, **p<0.01. (c) Sholl analysis of basal dendritic complexity of hippocampal CA1 pyramidal neurons in P90 WT and Mecp2^T158A/y mice show no difference in peak number of crossings in Mecp2^T158A/y mice relative to WT.

Figure 5. Soma size is regulated throughout development by MeCP2 function

(a) Somatosensory cortex layer V pyramidal neuron soma size is reduced in Mecp2^−/y mice relative to WT at both P30 (WT: n=124 neurons from 4 mice; Mecp2^−/y: n=103 neurons from 4 mice) and P60 (WT: n=126 neurons from 4 mice; Mecp2^−/y: n=104 neurons from 4 mice). **p<0.01, unpaired two-tailed Student t-test with Bonferroni correction. Bars represent mean ± SEM. (b) Somatosensory cortex layer V pyramidal neuron soma size is reduced in Mecp2^T158A/y mice relative to WT at both P30 (WT: n=115 neurons from 4 mice; Mecp2^T158A/y: n=121 neurons from 4 mice) and P90 (WT: n=81 neurons from 4 mice; Mecp2^T158A/y: n=106 neurons from 4 mice). **p<0.01, unpaired two-tailed Student t-test with Bonferroni correction.

Figure 6. Dendritic complexity in Mecp2^Tg1 mice

(a) Sholl analysis of somatosensory cortex layer V pyramidal neurons in P30 WT (n=16 neurons from 2 mice) and Mecp2^Tg1 mice (n=19 neurons from 2 mice) show no change dendritic complexity in Mecp2^Tg1 mice relative to WT. Two-way ANOVA, p>0.05. Bars represent mean ± sem. (b) Sholl analysis of somatosensory cortex layer V pyramidal neurons in P140 WT (n=23 neurons from 2 mice) and Mecp2^Tg1 mice (n=23 neurons from 2 mice) show no change dendritic complexity in Mecp2^Tg1 mice relative to WT. Two-way ANOVA, p>0.05. (c) Somatosensory cortex layer V pyramidal neuron soma size is not affected in Mecp2^Tg1mice relative to WT at both P30 (WT: n=101 neurons from 2 mice; Mecp2^Tg1: n=110 neurons from 2 mice) and P140 (P140; n=110 neurons from 2 mice; Mecp2^Tg1: n=113 neurons from 2 mice). p>0.05, unpaired two-tailed Student t-test.