Systemic delivery of MeCP2 rescues behavioral and cellular deficits in female mouse models of Rett syndrome

Abstract

De novo mutations in the X-linked gene encoding the transcription factor methyl-CpG binding protein 2 (MECP2) are the most frequent cause of the neurological disorder Rett syndrome (RTT). Hemizygous males usually die of neonatal encephalopathy. Heterozygous females survive into adulthood but exhibit severe symptoms including microcephaly, loss of purposeful hand motions and speech, and motor abnormalities, which appear after a period of apparently normal development. Most studies have focused on male mouse models because of the shorter latency to and severity in symptoms, yet how well these mice mimic the disease in affected females is not clear. Very few therapeutic treatments have been proposed for females, the more gender-appropriate model. Here, we show that self-complementary AAV9, bearing MeCP2 cDNA under control of a fragment of its own promoter (scAAV9/MeCP2), is capable of significantly stabilizing or reversing symptoms when administered systemically into female RTT mice. To our knowledge, this is the first potential gene therapy for females afflicted with RTT.

Figures

Figure 1.

MeCP2 expression is restored throughout the brain after systemic delivery of scAAV9/cre into Mecp2stop/y mice. A, Schematic of the scAAV9/cre viral construct. ITR, inverted terminal repeat; CMV, cytomegalovirus enhancer; CBA, chicken-β-actin promoter; CRE, cre recombinase; BGH, bovine growth hormone. B, Representative sagittal brain sections from 10-week-old WT or virally injected mice. Scale bar, 1 mm. C, Efficiency of virally mediated MeCP2 expression in specified brain regions relative to total DAPI-positive cells (18 sections, n = 3 mice). D, Left: Western blot of MeCP2 protein levels in injected mice. Lane 1, Mecp2+/y; Lane 2, Mecp2stop/y-AAV9/cre. Right: Quantified data of Western blot (n = 3). E, Immunostaining of representative cortical section showing MeCP2 expression in neurons (NeuN+) and astrocytes (GFAP+). Arrow and arrowhead indicate a MeCP2+ astrocyte and neuron, respectively. Scale bar, 10 μm. F, Immunostaining for MeCP2 restoration in Mecp2stop/y mice after scAAV9/cre injection. Cell counts are relative to indicated cell type-specific markers (18 sections, n = 3 mice). G, Western blot (left) of MeCP2 protein levels in different peripheral tissues. Lane 1, Mecp2+/y; Lane 2, Mecp2stop/y-AAV9/cre. Right: Quantified data of Western blot (n = 3). Data are means ± SEM. In C and F, *p < 0.05, **p < 0.01, ***p < 0.001, and NS by one-way ANOVA (Newman–Keuls multiple-comparisons test). Data are means ± SEM.

Figure 2.

Systemic delivery of scAAV9/cre into Mecp2stop/y mice rescues abnormal respiration and open field motor activities. A, Experimental paradigm. Mice were analyzed 4–5 weeks after injection. B, Kaplan-Meier survival curve shows significant extension in longevity in scAAV9/cre mice. ***p < 0.001, *p < 0.05, log-rank test. C, Observational scores show stabilization in Mecp2stop/y mice injected with scAAV9/cre (n = 7), scAAV9/Control (n = 5), and noninjected Mecp2+/y mice (n = 12). D, E, Open field activity in scAAV9/cre (n = 3), scAAV9/Control (n = 3), and Mecp2+/y (n = 4) mice. F, Representative plethysmographic recordings from an age-matched Mecp2+/y mouse (top), Mecp2Stop/y mouse before (middle), and 5 weeks post scAAV9/cre injection (bottom). Scale shows time and tidal volume. G, Respiratory pattern returns to normal in scAAV9/cre (n = 6), scAAV9/Control (n = 3), and Mecp2+/y (n = 12) mice. In D, E, and G, *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA (Newman–Keuls multiple-comparison test). Data are means ± SEM.

Figure 3.

Systemic delivery of scAAV9/cre virus into Mecp2stop/+ mice prevents progression of behavioral abnormalities. A, Experimental paradigm for injection and scoring. B, Observational scores of mice (n = 10) injected with indicated virus or control, noninjected Mecp2+/+ mice (n = 10). Arrow indicates time of behavioral analysis. C, Rotarod analysis on third day of test. D, Platform test for balance. E, Inverted grid test for strength (n = 9). F, Nesting ability (n = 9) with scoring guide at right. G, Novel object recognition analysis showing discrimination index on last day of test (n = 9). H, Brain weights analyzed 40 weeks after injection or in age-matched noninjected mice (n = 4). *p < 0.05, **p < 0.01, ***p < 0.001, NS by one-way ANOVA (Newman–Keuls multiple-comparison test for C and H) and one-way ANOVA (Dunn's multiple-comparison test for D–G). Data are means ± SEM.

Figure 4.

Direct brain injections of scAAV9/cre virus into Mecp2stop mice result in predominantly neuronal-specific MeCP2 expression and a mild improvement of behavioral phenotypes. A, Schematic showing site of injections (*). B, Experimental paradigm for males. C, Immunostaining of representative striatal section from a Mecp2stop/y mouse injected with scAAV9/cre shows MeCP2 expression in neurons (NeuN+). Scale bar, 200 μm. D–G, Quantification of immunostaining data (n = 3). Data are means ± SEM. Cell counts are relative to total DAPI-positive cells (D), total NeuN-positive cells (E), total NeuN-negative cells (F), and total MeCP2-positive cells (G). H, Experimental paradigm for females. I, Observational scores for female Mecp2Stop/+ mice injected with scAAV9/cre (n = 5), scAAV9/Control (n = 5), and noninjected Mecp2+/+ mice (n = 12).

Figure 5.

Systemic injection of Mecp2Bnull/y mice with scAAV9/MeCP2 virus results in MeCP2 expression in different cell types in brain. A, Schematic of scAAV9/MeCP2 vector. Mouse Mecp2-e1 is cloned downstream of 730 bp fragment of Mecp2 promoter. Other abbreviations are as noted in the legend to Figure 1. B, Experimental paradigm. C, Immunostaining of MeCP2 expression in different brain regions. Cell counts are relative to total DAPI-positive cells (36 sections, n = 2 mice). D, Expression of MeCP2 in neurons and non-neuronal cells varies with brain region. Cell counts are relative to indicated cell-specific marker (12 sections, n = 2 mice). *p < 0.05, **p < 0.01, ***p < 0.001, NS by one-way ANOVA (Newman–Keuls multiple-comparison test). Data are means ± SEM. E, Western blot of MeCP2 protein in different brain regions. Lane 1, Mecp2+/y; Lane 2, Mecp2Bnull/y; and Lane 3, Mecp2Bnull/y-AAV9/MeCP2.

Figure 6.

MeCP2 expressed from virus binds to DNA, restores normal neuronal somal size, and improves survival. A, Ectopic MeCP2 localizes to DAPI+ heterochromatin puncta in Mecp2Bnull/y-scAAV9/MeCP2–injected mice. Shown is colocalization of DAPI and MeCP2 in olfactory neuron (top), CA3 pyramidal neuron (middle), and dentate gyrus astrocyte (bottom). Pearson's correlation coefficient = 0.943, 0.932, and 0.985, respectively. Scale bar, 5 μm. B, Image (left) shows representative MeCP2-positive (arrow) and MeCP2-negative (arrowhead) CA3 pyramidal neurons. Scale bar, 10 μm. Average somal diameters (right) of MeCP2-positive (purple) and MeCP2-negative (red) CA3 pyramidal neurons and olfactory bulb mitral cells from Mecp2Bnull/y-scAAV9/MeCP2–injected mice (n = 2). Also shown are measurements from WT mice (black bars; n = 2). The number of cells analyzed is indicated above each bar. ***p < 0.001 by one-way ANOVA (Newman–Keuls multiple-comparison test). Data are means ± SEM. C, Kaplan-Meier survival curve. D, Observational scores. Mecp2Bnull/y-scAAV9/MeCP2 (n = 5), Mecp2Bnull/y-AAV9/Control (n = 6), and Mecp2+/y (n = 6). Data are means ± SEM. E, Field pixel intensities of MeCP2-Cy3 immunofluorescence measured from brainstem sections of Mecp2+/y and Mecp2Bnull/y-scAAV9/MeCP2 mice. Traces represent pixel intensities from individual fields and each field is indicated by a differently colored trace, n = 2 mice per genotype, 5 fields per mouse.

Figure 7.

Systemic delivery of scAAV9/MeCP2 virus into Mecp2Bnull/+ mice prevents progression of aberrant behaviors. A, Experimental paradigm. Mice were analyzed 5 months after injection. B, Field pixel intensities of MeCP2-Cy3 immunofluorescence measured from brainstem sections of WT and scAAV9/MeCP2–injected females. Traces represent pixel intensities from individual fields and each field is indicated by a differently colored trace. n = 2 mice per genotype, 5 fields per mouse. C, Observational scores of Mecp2Bnull/+ mice injected with scAAV9/MeCP2 (n = 8), scAAV9/Control (n = 5), and noninjected (Mecp2+/+) mice (n = 8). Arrow indicates time of behavioral analysis. D, Rotarod activity on third day of test. E, Inverted grid test. F, Platform test for scAAV9/MeCP2 (n = 8), scAAV9/Control (n = 5), and Mecp2+/+ (n = 8) mice. G, Nesting ability of scAAV9/MeCP2 (n = 8), scAAV9/Control (n = 5), and Mecp2+/+ (n = 8) mice. *p < 0.05, **p < 0.01, ***p < 0.001, NS by one-way ANOVA (Newman–Keuls multiple-comparison test for D and one-way ANOVA with Dunn's multiple-comparison test for E–G). Data are means ± SEM.