Evidence for both neuronal cell autonomous and nonautonomous effects of methyl-CpG-binding protein 2 in the cerebral cortex of female mice with Mecp2 mutation

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Abstract

Rett syndrome (RTT) is an X-linked neurodevelopmental disorder caused by mutations in the gene MECP2, encoding methyl-CpG-binding protein 2 (MeCP2). Few studies have explored dendritic morphology phenotypes in mouse models of RTT and none have determined whether these phenotypes in affected females are cell autonomous or nonautonomous. Using confocal microscopy analysis we have examined the structure of dendrites and spines in the motor cortex of wild-type (WT) and Mecp2-mutant mice expressing green fluorescent protein (GFP). In Mecp2 GFP female mice age 6–7 months we found significant decreases in the density of spines, width of dendrites, size of spine heads, while increases were found in the length of spine necks, dendritic irregularities, spineless spots, and long spines. We show for the first time that a lower density of spines and smaller spine head area are phenotypes that distinguish MeCP2+ from MeCP2− dendrites in female Mecp2 GFP mice. In Mecp2 GFP male mice at three weeks of age, we found reduced spine density, thinner apical oblique dendrites and increased dendritic irregularities and long spines. Significantly, the changes affected both MeCP2− and MeCP2+ neurons, pointing to the ability of MeCP2− to impact the structure of MeCP2+ neurons. Our findings are evidence that MeCP2 deficiency results in both cell autonomous and nonautonomous changes.

Introduction

Rett syndrome (RTT) results from the presence of mutations in the gene encoding methyl-CpG-binding protein 2 (MeCP2) on the X chromosome (Amir et al., 1999). The disorder affects primarily young girls with a prevalence of 1 in 10,000–15,000 (Hagberg, 1995, Hagberg, 1989). The brain is severely affected. Girls with RTT show developmental regression, diminished cognitive ability, loss of acquired speech skills, stereotypic hand movements, seizures, breathing irregularities and autonomic dysfunction (Hagberg, 1995, Hagberg, 1989, Nomura and Segawa, 2005, Percy, 2002, Rett, 1966). Studies performed on the brains of subjects with RTT and on mouse models of RTT show that the Mecp2 mutation has relatively little influence on neurogenesis and neuronal migration. The organization of cortical layers and the structure of the hippocampus are largely unaffected (Belichenko et al., 2008, Belichenko et al., 1997, Belichenko et al., 1994, Guy et al., 2001, Leontovich et al., 1999; see review in Armstrong, 2005). In contrast, striking changes in neuronal maturation are seen including: 1) significant reductions in the number and length of dendrites; 2) decreased dendritic spine number and regional loss of spines in the cortex; and 3) disorganization of afferent fibers to the spines of pyramidal neurons and in the shape of the axonal bundles in which they travel (Armstrong et al., 1995, Armstrong, 1992, Belichenko et al., 1994, Belichenko and Dahlström, 1995). The evidence is thus consistent that RTT exerts pathogenetic events that impact the maturation of neurons and the circuits in which they participate (Belichenko et al., 2008, Belichenko et al., 1994).

Because the MECP2 gene is located on the X chromosome (Amir et al., 1999), random inactivation of this chromosome (see review in Payer and Lee, 2008) in RTT females renders them mosaic for neurons expressing normal MeCP2 activity (MeCP2+), and neurons expressing abnormal MeCP2 or neurons completely lacking MeCP2 (MeCP2−). An interesting question is whether disease manifestations are due to abnormalities only in neurons that fail to express normal MeCP2 or, alternatively are linked to changes in all neurons. Three possibilities related to cell autonomous and nonautonomous effects of the Mecp2-mutation can be envisioned: i) MeCP2− neurons are severely damaged and impact MeCP2+ neurons structure and function through their linkage in circuits; ii) MeCP2+ neurons substantially improve aspects of the abnormal phenotypes in MeCP2− neurons through linkage in circuits; and iii) MeCP2− neurons and MeCP2+ neurons are independent in their manifestations of disease pathology. In this case only MeCP2− neurons are responsible for neurological symptoms of RTT. In the present study we ask whether or not abnormal phenotypes affect only MeCP2− neurons or extend to MeCP2+ neurons. We chose to investigate whether or not the MeCP2 mutation displays cell autonomous or non-cell autonomous effects in female Mecp2 mutant mice. To carry out this work we crossed WT male mice expressing green fluorescent protein (GFP; GFP-M line; Feng et al., 2000) with female Mecp2-mutant mice (Dr. A. Bird strain, Mecp2B, Guy et al., 2001). Combining GFP with MeCP2 immunoreactivity (IR) and high resolution confocal microscopy, we examined dendrite and dendritic spine morphology in the motor cortex of heterozygous female Mecp2B GFP mice, defining these structures in MeCP2+ or MeCP2− neurons. To compare the severity of dendritic phenotypes in females with brains in which all neurons are MeCP2−, we examined the same phenotypes in male mice produced by the same mating. Comparing MeCP2− neurons in Mecp2B males to neurons in WT males, we discovered significant decreases in the density of spines, width of dendrites, and increases in dendritic irregularities (i.e. swellings or narrowings) and long spines. When we compared MeCP2− neurons in Mecp2B females to neurons in WT female mice, significant decreases in the density of spines, width of dendrites, size of spine heads, and increases in the length of spine necks, dendritic irregularities, long spines and spineless spots were found. The same phenotypes were evident comparing MeCP2+ neurons in Mecp2B females with neurons in WT females with the exception of dendritic irregularities. The changes in MeCP2− neurons in females were in general less severe than in neurons in Mecp2B males. Remarkably, changes in spine density and in the area of spine heads were phenotypes that clearly distinguish MeCP2+ from MeCP2− dendrites in female Mecp2B GFP mice. These data are evidence for both cell autonomous and nonautonomous changes in neurons due to MeCP2 deficiency.

Section snippets

Materials and methods

All experiments were conducted in accordance with the National Institutes of Health guidelines for the care and use of animals and with an approved animal protocol from the Stanford University Institutional Animal Care and Use Committee.

Results

Mecp2B heterozygous female mice of age 6 to 7 months uniformly showed RTT-related neurological symptoms. They were ataxic, developed a stiff and uncoordinated gait, and demonstrated the hindlimb-clasping phenotype and irregular breathing. At 3 weeks of age, Mecp2B male mice were also ataxic and showed hindlimb-clasping. These phenotypes are identical to those previously described in Mecp2B mutant mice (Guy et al., 2001).

Discussion

Studies to understand the neurobiology of RTT have benefited from the ability to examine mouse models of the disorder (Chahrour et al., 2008, Chen et al., 2001, Guy et al., 2001; reviewed in Percy, 2008). Especially relevant to linking neurobiological phenotypes to clinical manifestations of RTT are studies in female mice harboring a mutation of one copy of Mecp2. On the basis of X chromosome inactivation, the brains of these mice are mosaic for MeCP2 deficiency. A reasonable expectation is

Acknowledgments

We thank Janice Valletta, Ryoko Takimoto-Kimura, Jackie Rodriguez, and Hong-Hua Li for technical assistance with mice husbandry, Dr. Uta Francke for providing the Mecp2B mice for breeding, and Michael Maloney for critical reading of the manuscript. This work was supported by the Larry L Hillblom Foundation, Down Syndrome Research and Treatment Foundation, and by the Natalie Foundation.

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