Gene expression analysis exposes mitochondrial abnormalities in a mouse model of Rett syndrome

Abstract

Rett syndrome (RTT) is a severe neurological disorder caused by mutations in the X-linked MECP2 gene, which encodes a methyl-CpG binding transcriptional repressor. Using the Mecp2-null mouse (an animal model for RTT) and differential display, we found that mice with neurological symptoms overexpress the nuclear gene for ubiquinol-cytochrome c reductase core protein 1 (Uqcrc1). Chromatin immunoprecipitation demonstrated that MeCP2 interacts with the Uqcrc1 promoter. Uqcrc1 encodes a subunit of mitochondrial respiratory complex III, and isolated mitochondria from the Mecp2-null brain showed elevated respiration rates associated with respiratory complex III and an overall reduction in coupling. A causal link between Uqcrc1 gene overexpression and enhanced complex III activity was established in neuroblastoma cells. Our findings raise the possibility that mitochondrial dysfunction contributes to pathology of the Mecp2-null mouse and may contribute to the long-known resemblance between Rett syndrome and certain mitochondrial disorders.

FIG. 1.

Global analysis of gene expression by ADDER reveals misexpressed genes in Mecp2-null mouse brain. (a) Top panel: primers used for differential display PCR. Shaded bars represent constant regions with 3′-terminal varied regions. Variable cDNA sequences are represented by lines. Bottom panel: differential display gels with two bands with altered intensity that were subsequently recovered and identified as Uqcrc1 (asterisk in center panel) and mt-Nd2 (asterisk in right panel). (b) Real-time PCR data showing Uqcrc1 up-regulation in early- and late-symptomatic mice but not in presymptomatic mice. Three pools, each containing RNA from three brains, were analyzed in quadruplicate to generate these data. The horizontal lines represent the means, the boxes delineate the standard errors of the means, and whiskers extend the standard deviations. ***, significant difference (t test, P < 0.001).

FIG. 2.

MeCP2 binds the promoter region of the Uqcrc1 gene. (a) Uqcrc1 promoter map showing CpG frequency, methylation status, and the region used for PCR amplification of immunoprecipitated DNA. (b) Chromatin immunoprecipitation reveals MeCP2 bound to the Uqcrc1 promoter in wt mouse brain but not in Mecp2-null mouse brain. Bdnf promoter III is a positive control. DNA is specifically immunoprecipitated with MeCP2 antibody but not with rabbit serum. No DNA is precipitated from Mecp2-null mouse brain by anti-MeCP2 antibody. Chromatin quality is similar in wt and Mecp2-null mouse brains, because anti-dimethyl H3 K9 antibody is able to precipitate similar amounts of DNA.

FIG. 3.

Investigation of the respiratory chain in mitochondria isolated from whole brains of wt and Mecp2-null mice. (a) Electron micrographs showing isolated mitochondria from a symptomatic Mecp2-null (KO) mouse and an age-matched wt littermate. Bar, 2 μm. (b) Typical output traces from a polarographic oxygen electrode. Initially mitochondria consume very little oxygen, but following the addition of substrates, oxygen consumption is moderately increased (state 2). Addition of ADP (state 3) permits rapid respiration, during which the proton gradient is relieved via ATP synthase. Following phosphorylation of all the ADP present, respiration slows again (state 4). At this point, mitochondria were directly uncoupled via the addition of the protonophore FCCP, allowing the proton pumps to run freely. (c to h) Each pair of bars shows respiration rates using one of three different substrates: pyruvate plus malate (PM), succinate (Succ.), or TMPD (see Materials and Methods). Panels c, d, e, and f compare respiration rates during the different respiratory states from brain mitochondria isolated from presymptomatic Mecp2-null animals (light bars; n = 8 runs with separate mitochondrial preparations derived from four animals) and age-matched wt littermate controls (dark bars). Data from symptomatic Mecp2-null mouse mitochondria (n = 16 runs with preparations from eight animals) compared to wt controls are shown at the right in each panel. Significantly increased respiration rates were observed in mitochondria from symptomatic Mecp2-null animals with complex I substrates (PM) and the complex II substrate (Succ.) for all respiratory states. All comparisons were tested with the t test. *, P < 0.05; **, P < 0.01; ***, represent P < 0.001. (g) Calculated ATP/O ratios for presymptomatic and postsymptomatic Mecp2-null animals and wt controls. No significant differences were observed with any of the substrates. (h) Calculated respiratory control (RC) ratios for presymptomatic and postsymptomatic Mecp2-null animals and wt controls. Significant decreases were observed in symptomatic Mecp2-null animals compared to wt controls for complex I (P = 0.05) and complex II (P < 0.001) substrates only. Error bars indicate standard deviations.

FIG. 4.

Analysis of the respiratory complexes by blue native electrophoresis. (a) Coomassie blue-stained blue native electrophoresis gel shows resolved mitochondrial respiratory complexes. (b and c) Enzymatic staining of complexes I and IV, respectively. Samples from littermates are beside each other. The stained band is highlighted with thicker arrows. (d) Statistical analysis of the enzymatic staining. Three experiments were done with three pairs of mice, and data were evaluated using the t test. Complex IV staining in Mecp2-null mice is significantly lower (P < 0.001), while complex I staining is the same (P = 0.38). Error bars show standard deviations.

FIG. 5.

Overexpression of Uqcrc1 in an N2A cell line causes increased mitochondrial respiration. (a) Real-time PCR analysis of Uqcrc1 mRNA expression relative to GAPDH. Uqcrc1 expression in control cell line was normalized to 1. (b) Representative Western blot of whole-cell lysates from control and Uqcrc1-overexpressing N2A cells. Porin serves as a loading control. Statistical analysis of duplicate experiments indicates 1.6-fold overexpression of Uqcrc1 protein. (c) Oxygen electrode data for the ADP-uncoupled respiration rates of permeabilized control and Uqcrc1-overexpressing cells in the presence of pyruvate/malate, succinate, or TMPD/ascorbate as respiratory substrates. Significance was tested using the t test. **, P < 0.01; *, P < 0.05. Error bars indicate standard deviations.