Abnormalities of cell packing density and dendritic complexity in the MeCP2 A140V mouse model of Rett syndrome/X-linked mental retardation

Abstract

Background: Rett syndrome (RTT), a common cause of mental retardation in girls, is associated with mutations in the MECP2 gene. Most human cases of MECP2 mutation in girls result in classical or variant forms of RTT. When these same mutations occur in males, they often present as severe neonatal encephalopathy. However, some MECP2 mutations can also lead to diseases characterized as mental retardation syndromes, particularly in boys. One of these mutations, A140V, is a common, recurring missense mutation accounting for about 0.6% of all MeCP2 mutations and ranking 21st by frequency. It has been described in familial X-linked mental retardation (XLMR), PPM- X syndrome (Parkinsonism, Pyramidal signs, Macroorchidism, X-linked mental retardation) and in other neuropsychiatric syndromes. Interestingly, this mutation has been reported to preserve the methyl-CpG binding function of the MeCP2 protein while compromising its ability to bind to the mental retardation associated protein ATRX.

Results: We report the construction and initial characterization of a mouse model expressing the A140V MeCP2 mutation. These initial descriptive studies in male hemizygous mice have revealed brain abnormalities seen in both RTT and mental retardation. The abnormalities found include increases in cell packing density in the brain and a significant reduction in the complexity of neuronal dendritic branching. In contrast to some MeCP2 mutation mouse models, the A140V mouse has an apparently normal lifespan and normal weight gain patterns with no obvious seizures, tremors, breathing difficulties or kyphosis.

Conclusion: We have identified various neurological abnormalities in this mouse model of Rett syndrome/X-linked mental retardation which may help to elucidate the manner in which MECP2 mutations cause neuronal changes resulting in mental retardation without the confounding effects of seizures, chronic hypoventilation, or other Rett syndrome associated symptoms.

Figure 1

Generation of the MeCP2 A140V "knock-in" mouse. (1A) Diagram showing the WT Mecp2 locus, the A140V targeting vector, and the recombined MeCP2 A140V locus used to create the knock-in mouse. (1B) PCR demonstrating ES cell DNA positive (177U9, W7, and A6) and negative (G3) for homologous recombination at the 3' end of the targeting vector. Lanes "WT" and "Mut" are plasmid DNA controls demonstrating the WT and A140V mutant PCR fragments (Upper Panel). The PCR fragments were digested with Acl I which cuts only the mutant PCR fragments, confirming the presence of the A140V mutation and producing a double band (1B Lower Panel). (1C) Southern blot analysis Bam HI digested ES cell DNA. A 5.5 kilobase pair (kbp) band was detected confirming homologous recombination at the 5' end of the targeting vector. The ladder on the left is in kbp. (1D) PCR genotyping results of F1 generation females. DNA from a WT female produces a single band while DNA from a heterozygous female (Het) produces two bands. The WT and Mut lanes show control plasmid reactions.

Figure 2

Mecp2 mRNA and protein are present in the brains of A140V mutant male mice. (2A) Total RNA was extracted from the cerebella of mice and used for cDNA synthesis. RT-PCR confirmed the presence of Mecp2 mRNA in the cerebella of 2 WT and 2 A140V (Mut) mice (Left Panel). The mutant mRNA species results in a larger fragment by PCR than the WT mRNA species. A second RT-PCR reaction amplifying a different segment of the cDNA containing the A140V mutation site confirmed that there was no contamination of the total RNA used for the cDNA synthesis with genomic DNA. WT and A140V (Mut) cDNA showed only the presence of a band of the correct size for cDNA PCR product (Right Panel). (2B) Immunohistochemistry on frozen brain slices demonstrates the presence of MeCP2 protein in the brain of an A140V hemizygous mouse. Staining in the hippocampus and the cerebellum are similar in the WT and A140V mice using an antibody recognizing the C-terminus of MeCP2. Scale bars are 200 μm.

Figure 3

Mecp2 mRNA levels are similar in WT and A140V mouse cerebellum by qRT-PCR. (3A) Quantitative real time PCR (qRT-PCR) reactions were performed using PCR assays for Mecp2 and Gapdh. qRT-PCR data graphs (Upper Panels) and melting curves (Lower Panels) for Mecp2 and Gapdh are shown. (3B) A table of C_t (cycle threshold) values is shown including mean and standard deviation (in parentheses) and ΔC_t used in the calculation of fold change (F) in mRNA levels. F (Mecp2:Gapdh) = 0.91.

Figure 4

MeCP2 A140V male mice show similar somatic growth patterns to WT controls. Male WT and MeCP2 A140V mice were weighed weekly between the ages of 6 and 18 weeks. No significant difference was found between the mean weights of the two groups. Error bars indicate one standard deviation.

Figure 5

Increases in cell density were found in the frontal cortex and olfactory bulbs of MeCP2 A140V mice. Brain slices from WT and A140V male mice were stained with cresyl violet. Shown are areas of the frontal cortex (A) and olfactory bulbs (B). Arrows indicate areas of increased cell density. Scale bars are 500 μm.

Figure 6

Increases in cell density were found in the hippocampus and dentate gyrus of MeCP2 A140V mice. Panel A shows cresyl violet stained hippocampi from WT and A140V mice. Staining is darker in the A140V mice due to an increase in cell density. Scale bar is 500 μm. Panel B contains enlargements of the CA1, dentate gyrus and CA2/CA3 regions of the hippocampi more clearly demonstrating the increase in cell density seen in Panel A. Scale bars in the enlarged images indicate 100 μm.

Figure 7

Increases in cell density were found in the cerebellum of MeCP2 A140V mice. Left panels show low power images of cresyl violet stained cerebelli from WT and A140V mice. The A140V cerebellum has noticeably darker staining due to an increase in cell density. Scale bar is 500 μm. Right panels show enlargement of these images demonstrating the differences in cell density. Scale bar is 100 μm. Arrows indicate areas of increased cell density.

Figure 8

Quantitative analysis of cell density in the cerebellum of MeCP2 A140V mice. Cresyl violet stained cell nuclei in cerebellar sections from WT and A140V mice were counted in two separate areas of the granule cell layer in the cerebellum. In each cerebellar area, three adjacent regions were counted and the mean taken. The means for each area for each genotype are shown on the bar graph. The differences between the means were significant, (*) p < 0.05, indicating an increase in cell density in the A140V cerebellum.

Figure 9

Qualitative abnormalities of neuronal dendritic branching were seen in the layer II/III pyramidal neurons of the visual cortex of MeCP2 A140V mice. Extended focus images of Golgi-Cox stained brain sections from WT and A140V mice are shown. Arrows indicate areas of apparent decreased dendritic branching in the A140V mouse in comparison to the WT mouse. Scale bar is 100 μm.

Figure 10

The Scholl analysis technique for assessing dendritic complexity was adapted for use in brightfield Z-stack images of Golgi-Cox stained neurons. The diagram shows an illustration of Scholl lines superimposed at 20 μm increments on a Z-stack of images of a stained neuron. This was done using Zeiss AxioVision software. Each Z-stack image is separated by only 0.280 μm allowing all dendritic intersections with Scholl lines to be clearly seen and counted. Drawing not to scale.

Figure 11

Dendritic complexity of pryamidal neurons in the somatosensory cortex is decreased in MeCP2 A140V mice. Scholl analysis was performed on Z-stack images of Golgi-Cox stained layer II-III pyramidal neurons from the somatosensory cortex of WT and A140V mice. Panel (A) shows the results of analysis of apical dendrites. Panel (B) shows the results of analysis of basal dendrites. Error bars indicate one standard deviation. (**) p < 0.01 (***) p < 0.001