Article Figures & Data
Figures
- Figure 1.
Vitamin D rescues aberrant NF-κB activation in Mecp2 knock-down cortical neurons. A, Mecp2-null mice have reduced serum vitamin D levels [25(OH)D] compared with the wild-type littermates at eight weeks of age (N = 4 mice/genotype). B, C, E15.5 cortical neurons were nucleofected with a construct expressing GFP as reporter and either a control shRNA (shScram) or an shRNA targeting Mecp2 (shMecp2). shMecp2 nucleofection visibly reduced the expression of MeCP2 protein at 7 DIV (B) and downregulated the overall expression of Mecp2 ∼50% after 14 DIV, in cultures in which the transfection efficiency was ∼60% (C). Arrowheads indicate nucleofected GFP-positive neurons; arrows indicate neighboring non-nucleofected GFP-negative neurons. N = 4 experimental replicates. D, Dissociated P1 cortical neurons were nucleofected with shScram or shMecp2, then were cultured for 2 d. Addition of calcitriol, the activated form of vitamin D (VitD), to culture medium for 24 h rescues the ∼1.75-fold increase in NF-κB-dependent transcription that occurs with knock-down of Mecp2 in cortical neurons in vitro. However, calcitriol has no effect on shScram control nucleofected neurons (N = 4 biological replicates). E, F, Mecp2 knock-down results in increased nuclear p65 localization in cortical neurons, which is indicative of NF-κB activation. Addition of calcitriol to the culture medium reduces p65 protein expression in the nucleus of Mecp2 knock-down cortical neurons, but not control (shScram) neurons. C, D, N = shScram no treatment: 33 neurons, vehicle: 30 neurons; 100 nm VitD: 33 neurons; shMecp2 no treatment: 23 neurons, vehicle: 22 neurons; 100 nm VitD: 22 neurons from three independent experiments. Expression of GFP was employed to identify transfected neurons. AU = relative luminescence units. A, B, D, Two-tailed t test. E, One-way ANOVA with Tukey’s multiple comparison test; *p < 0.05, **p < 0.01, NS = not significant. Scale bar = 20 μm. Error bar: ±SEM.
- Figure 2.
Vitamin D rescues reduced neurite outgrowth of Mecp2 knock-down cortical neurons. A–C, Dissociated E15.5 cortical neurons were nucleofected with a construct expressing a GFP reporter and either a control shRNA (shScram) or an shRNA targeting Mecp2 (shMecp2), then were plated and cultured for 7 d. Neurons were either maintained in standard culture medium, or were supplemented with vehicle (EtOH) or 100 nm calcitriol (VitD) from 2 to 7 DIV. A, Representative images of GFP+ cortical neurons at 7 DIV under each condition. B, Representative traces of GFP+ cortical neurons under each condition. C, Total neurite outgrowth of GFP+ neurons was quantified from randomly selected neurons, from each of three independent experiments (N = shScram no treatment: 30 neurons, vehicle: 26 neurons; 100 nm VitD: 27 neurons; shMecp2 no treatment: 28 neurons, vehicle: 26 neurons; 100 nm VitD: 27 neurons). Supplementation with calcitriol rescues the reduced neurite outgrowth of Mecp2 knock-down neurons relative to EtOH vehicle control but does not have a significant effect on control cortical neurons. Thus, shMecp2 neurons with calcitriol are not significantly different from shScram. C, One-way ANOVA with Tukey’s multiple comparison test; *p < 0.05, NS = not significant. Scale bar = 50 μm. Error bar: ±SEM.
- Figure 3.
Vitamin D supplementation modestly improves Mecp2-null phenotypes and increases their reduced lifespan. A, Experimental plan for in vivo vitamin D treatment of Mecp2−/y and Mecp2+/y littermates. B, Supplementing the diet of the mice with vitamin D (VitD) significantly increases their total serum levels of 25(OH)D, regardless of genotype, which is most apparent with 50 IU/g supplemented chow. C, Mecp2−/y on 50 IU/g VitD have a small, but significant, reduction in total phenotypic score at eight weeks of age compared with Mecp2−/y on control 1 IU/g VitD. D, Kaplan–Meier survival curves. Mecp2−/y mice on 50 IU/g VitD chow survive significantly longer than Mecp2−/y mice on control chow, while Mecp2−/y mice on 10 IU/g VitD display a trend toward increased median lifespan (p = 0.04; log-rank test). The median lifespan of Mecp2−/y on 1 IU/g is 68.5 d, 81 d on 10 IU/g, and 83 d on 50 IU/g. E, The mean age of death of Mecp2-/y mice on the control chow is significantly lower than the animals on 50 IU/g VitD. B, C, E, One-way ANOVA with Tukey’s multiple comparison test; *p < 0.05, **p < 0.01, ***p < 0.001, NS = not significant. B, N = 4 mice per condition. C–E, N = 16 Mecp2+/y 1 IU, 17 Mecp2-/y 1 IU, 15 Mecp2-/y 10 IU, 14 Mecp2-/y 50 IU. Error bar: ±SEM.
- Figure 4.
Dietary vitamin D supplementation does not significantly alter neuronal morphology or health in wild-type (Mecp2+/y) mice. Treatment of Mecp2+/y mice with vitamin D supplemented chow between four and eight weeks of age does not alter (A) soma size (p = 0.67, one-way ANOVA; 1 IU/g n = 76, 10 IU/g n = 103, 50 IU/g n = 84) or (B–D) dendritic complexity of Layer II/III pyramidal neurons, as measured by Golgi staining and (B) Sholl analysis, (C) quantification of the number of dendritic branches, or (D) quantification of total dendritic length (1 IU/g n = 20 neurons, 10 IU/g n = 29, 50 IU/g n = 18). In addition, vitamin D supplementation does not alter the (E) total phenotypic score (p = 0.34, one-way ANOVA) or (F) weight of Mecp2+/y mice (p = 0.66, one-way ANOVA). B, E, F, Two-way ANOVA with Bonferroni post hoc tests. A, C, D, One-way ANOVA with Tukey’s post hoc tests. NS = not significant. Error bar: ±SEM.
- Figure 5.
Vitamin D supplementation rescues reduced cortical dendritic complexity and soma size phenotypes in Mecp2-null mice. A, Representative traces of Layer II/III cortical CPN following Golgi staining. B–F, Dendritic complexity of CPN, as measured by (B) Sholl analysis, (C) number of branch points, and (D) total dendritic length, is significantly reduced in Mecp2−/y mice on both control 1 IU/g and 10 IU/g VitD chow, compared with Mecp2+/y on control 1 IU/g chow. Dendritic complexity of Mecp2-/y mice on 50 IU/g VitD, however, is essentially indistinguishable from wild type (Mecp2+/y). E, Mecp2−/y mice on both control 1 IU/g and 10 IU/g VitD chow have reduced secondary and tertiary dendrite lengths, which are rescued in Mecp2−/y mice on 50 IU/g VitD. F, The length of apical dendrites is also significantly lower in Mecp2-nulls on all chows, compared with wild-type mice. However, the length of basal dendrites of Mecp2−/y on 10 IU/g VitD and 50 IU/g VitD chow is rescued, and it is not significantly different from Mecp2+/y mice. G, Soma area of Layer II/III CPN is significantly reduced in Mecp2−/y cortex on both control chow and 10 IU/g VitD chow, relative to Mecp2+/y on control chow, but is rescued with 50 IU/g VitD. B, Two-way ANOVA, Bonferroni post hoc test. C–F, One-way ANOVA with Tukey’s multiple comparison test; *p < 0.05, **p < 0.01, ***p < 0.001, NS = not significant. * Compared with Mecp2+/y. # Compared with Mecp2−/y 1 IU/g VitD. B–F, N: Mecp2+/y IU = 21 neurons from three brains, Mecp2-/y 1 IU = 28 neurons from four brains, 10 IU = 19 neurons from three brains, 50 IU = 35 neurons from five brains. G, N: Mecp2+/y IU = 228 neurons from three brains, Mecp2-/y 1 IU = 263 neurons from four brains, 10 IU = 193 neurons from three brains, 50 IU = 204 neurons from five brains. Error bar: ±SEM.
- Figure 6.
Vitamin D supplementation rescues reduced dendritic spine density in Mecp2-/y Layer II/III CPN. A–D, Representative images of apical dendrites of Layer II/III CPN in somatosensory cortex following Golgi staining. Boxes indicate areas displayed at higher magnification in A’–D’. E, Spine density is significantly decreased in Mecp2-null neurons compared with wild-type littermates. This decrease is rescued with 50 IU/g vitamin D supplementation; *p < 0.05, one-way ANOVA with Tukey’s multiple comparison test. N = Mecp2+/y 1 IU: 43 dendrites from three brains, Mecp2−/y 1 IU: 54 dendrites from three brains, Mecp2+/y 50 IU: 33 dendrites from three brains, Mecp2-/y 50 IU: 64 dendrites from four brains. Scale bar = 200 μm (A–D) and 5 μm (A’–D’). ns = not significant. Error bar ±SEM.
- Figure 7.
Mecp2+/− female cortex has increased Irak1 expression, and displays partial rescue of reduced dendritic complexity and soma size phenotypes with vitamin D (VitD) supplementation. A, Female Mecp2+/− cortex also displays upregulation of Irak1 expression at five months, as previously determined in male Mecp2-/y cortex at eight weeks (two-tailed t test, p = 0.009; N: Mecp2+/+ = 8, Mecp2+/− = 7). B, Five-month-old Mecp2+/− mice show increased expression of the NF-κB downstream target CamkIId (two-tailed t test, p = 0.015; N: Mecp2+/+ = 8, Mecp2+/− = 7). C, Mecp2+/− females on control chow (1 IU) do not display lower levels of VitD at five months of age; however, supplementing the diet of the mice with 10 IU/g VitD from four weeks of age significantly increases total serum levels of 25(OH)D, independent of genotype. D, Representative traces of Layer II/III cortical CPNs. E, At five months of age, Mecp2+/− mice on both 10 IU/g and 50 IU/g VitD have increased dendritic complexity compared with Mecp2+/− on control 1 IU/g chow, as measured by Golgi staining and Sholl analysis, although it is not fully rescued to wild-type (Mecp2+/+) levels. Asterisks denote significant difference for Mecp2+/− on 1 IU (blue), 10 IU (red), and 50 IU/g VitD (green) compared with Mecp2+/+ on control chow. F, G, Mecp2+/− on all VitD chows show reduced number of branch points (F) and total dendritic length (G) compared with wild type, although there is a trend toward increased branch points and dendrite length with VitD supplementation. H, I, Mecp2+/− mice on 10 IU/g VitD demonstrate a significant increase in secondary dendrite length relative to control chow (H), and apical dendritic length that is not significantly different from wild type (I). J, Mecp2+/− mice on 10 IU/g VitD chow also show increased soma area, which is not significantly different from Mecp2+/+ mice on control chow. C, One-way ANOVA with Tukey’s multiple comparisons test. E, Two-way ANOVA with Bonferroni post hoc test. F–I, One-way ANOVA with Tukey’s post hoc test. C, N: Mecp2+/+ 1 IU, Mecp2+/− 1 IU and Mecp2+/− 10 IU = 4 animals, Mecp2+/+ 10 IU = 3 animals. E–I, N: Mecp2+/+ 1 IU = 46 neurons from five brains, Mecp2+/− 1 IU = 68 neurons from six brains, 10 IU = 62 neurons from six brains, 50 IU = 47 neurons from five brains. J, N = Mecp2+/+ 1 IU = 192 neurons from five brains, Mecp2+/− 1 IU = 366 neurons from six brains, 10 IU = 323 neurons from six brains, 50 IU = 234 neurons from five brains; *p < 0.05, **p < 0.01, ***p < 0.001. NS = not significant. Error bar: ±SEM.
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