Mitochondrial dysfunction in the skeletal muscle of a mouse model of Rett syndrome (RTT): implications for the disease phenotype.

Rett syndrome (RTT) is a severe neurodevelopmental disorder, predominantly caused by mutations in the X-linked Methyl-CpG-binding protein 2 (MECP2) gene. Patients present with numerous functional deficits including intellectual disability and abnormalities of movement. Clinical and biochemical features may overlap with those seen in patients with primary mitochondrial respiratory chain disorders. In the late stages of the disorder, patients suffer from motor deterioration and usually require assisted mobility. Using a mouse model of RTT (Mecp2(tm1Tam)), we studied the mitochondrial function in the hind-limb skeletal muscle of these mice. We identified a reduction in cytochrome c oxidase subunit I (MTCO1) at both the transcript and protein level, in accordance with our previous findings in RTT patient brain studies. Mitochondrial respiratory chain (MRC) enzyme activity of complexes II+III (COII+III) and complex IV (COIV), and glutathione (GSH) levels were significantly reduced in symptomatic mice, but not in the pre-symptomatic mice. Our findings suggest that mitochondrial abnormalities in the skeletal muscle may contribute to the progressive deterioration in mobility in RTT through the accumulation of free radicals, as evidenced by the decrease in reduced glutathione (GSH). We hypothesise that a diminution in GSH leads to an accumulation of free radicals and an increase in oxidative stress. This may impact on respiratory chain function and contribute in part to the progressive neurological and motor deterioration seen in the Mecp2-mutant mouse. Treatment strategies aimed at restoring cellular GSH levels may prove to be a novel target area to consider in future approaches to RTT therapies.

Expression and functional analysis of Dkk1 during early gonadal development.

WNT signalling plays a central role in mammalian sex determination by promoting ovarian development and repressing aspects of testis development in the early gonad. Dickkopf homolog 1 (DKK1) is a WNT signalling antagonist that plays critical roles in multiple developmental systems by modulating WNT activity. Here, we examined the role of DKK1 in mouse sex determination and early gonadal development. Dkk1 mRNA was upregulated sex-specifically during testis differentiation, suggesting that DKK1 could repress WNT signalling in the developing testis. However, we observed overtly normal testis development in Dkk1-null XY gonads, and found no significant upregulation of Axin2 or Sp5 that would indicate increased canonical WNT signalling. Nor did we find significant differences in expression of key markers of testis and ovarian development. We propose that DKK1 may play a protective role that is not unmasked by loss-of-function in the absence of other stressors.

Fate-Mapping Technique: Targeted Whole-Embryo Electroporation of DNA Constructs into the Germ Layers of Mouse Embryos 7-7.5 Days Post-coitum.

INTRODUCTIONFate maps reveal body plan organization and presage the expression of molecular characteristics of cell lineages and formation of body parts. This protocol targets DNA expression constructs into the germ layers of gastrula-stage mouse embryos by focal electroporation. Plasmids utilizing a promoter that drives widespread, non-lineage-restricted expression of transgenes are introduced to cells in defined germ layer regions by whole-embryo electroporation. Germ-layer cells are exposed to the DNA by microinjecting the plasmids into the proamniotic cavity (ectoderm) or directly into the intercellular space of the mesenchyme (mesoderm), or by incubating the embryo in the DNA solution (endoderm). Electroporation is performed on whole embryos in vitro by electric current-mediated permeation of the cell membrane, which allows DNA adsorbed to cell surfaces to enter the cells. A point electrode is used to focus the electric field to the intended site of electroporation and a plate electrode is used to generate the current at an effective voltage low enough to minimize damage to the embryonic tissue. Expression of the transgene can be used to track the fate and movement of cells and the cDNA to study the functional consequences of overexpression of genes during embryonic development in vitro.