Figure 1

<p>Typical high-resolution ¹H NMR spectrum of the brain extract from a Mecp2-deficient mouse (water-soluble metabolites). The entire spectral region of interest (upper left spectrum) as well as magnified subregions are shown. Creatinine (Crn) was not quantified since the weak singlet was not sufficiently separated from other peaks forming a broad hump between 3.01 and 3.06 ppm. Abbreviations: ac, acetate; ala, alanine; asp, aspartate; Cho, choline; Cr, creatine; GABA, γ-aminobutyrate; gln, glutamine; glu, glutamate; gly, glycine; GPC, glycerophosphocholine; lac, lactate; <i>myo</i>-Ins, myo-inositol; NAA, <i>N</i>-acetylaspartate; NANA, <i>N</i>-acetylneuraminate; PC, phosphocholine; <i>scyllo</i>-Ins, <i>scyllo</i>-inositol; suc, succinate; tau, taurine.</p

Chronic treatment with the GABA reuptake inhibitor tiagabine significantly extends the lifespan of <i>Mecp2</i>-deficient mice.

<p>The lifespan was measured in animals treated with the vehicle (drinking water) or tiagabine. The vehicle group lived 67.1±3.3 days (n = 13) while the tiagabine-treated group lived for 79.5±4.7 days (n = 10). The survival analysis was performed using a Kaplan-Meir log-rank test (<i>P</i><0.05).</p

Protein changes during development in <i>Mecp2</i>-deficient and wild type mice.

<p><i>Data for each group (P35 or P55) was statistically analyzed by a one-way ANOVA (Mecp2^-/y vs WT) and P-values of the ANOVA during developmental changes are shown in parenthesis.</i></p><p>n.s, non-significant.</p

Impact of the oral tiagabine treatment on the motor performances of <i>Mecp2</i>^–/y mice (Tigabine treated n = 10 and untreated n = 13 at P30).

<p>Histograms showing the body weight and the behavioral performances in the tiagabine-treated (<i>Mecp2</i>^–/y Tiagabine, gray) and the vehicle group (<i>Mecp2</i>^–/y vehicle, black) animals. The body weight curve starts from P30 to P100, and there were no significant differences between treated and untreated <i>Mecp2</i>^-/y. Rotarod: <i>Mecp2</i>^–/y tiagabine (n = 10) and <i>Mecp2</i>^–/y vehicle (n = 13) performed similarly at each developmental stages. Open field test: The total distance traveled by the <i>Mecp2</i>^–/y mice and their average velocity were not affected by tiagabine treatment at any developmental stage. Grip strength test: The forelimb and forelimb+hind limb grip strength measurements were unaffected by the tiagabine treatment at all ages. Data are shown as mean±SEM.</p

Alteration of the GABA/glutamate pathway in the <i>Mecp2</i> brain, at both late and early stages of the disease.

<p>A) Examples of western blots showing significant differences between <i>Mecp2^-/y</i> and WT samples. On the left: Western blot analysis of GAD enzymes (GAD1, GAD2) in the caudate-putamen and hippocampus at an early stage (P35) and in the ventral midbrain at a late stage of the disease (P55). Each lane represents a sample from a different animal. On the right: Western blot analysis of the potassium-chloride transporter member 5 (Kcc2 or Slc12a5 in the hippocampus where a reduction of this channel is significant at an early (P35) and late stage. Each lane represents a different animal B) Quantification of all western blots carried out in the study, at both stages (P35: <i>Mecp2^-/y</i> dashed/white bars and WT dashed/grey bars and P55: <i>Mecp2^-/y</i> white bars and WT grey bars) in three structures of the brain (hippocampus, Caudate-putamen and Ventral Midbrain). Levels of the three proteins of interests (GAD2, Kcc2, Nkcc1) were normalized to GAPDH protein level. For all panels, n = 4–6 animals/group. Results are expressed in percentage relative to wild type animals arbitrarily set to 100%. (*<i>P</i><0.05, **<i>P</i><0.01).</p

Relative expression level of genes involved in the GABA and glutamate metabolism in three brain areas, using quantitative RT-PCR.

<p>RNA samples were obtained from either WT or <i>Mecp2</i>^-/y mice at two different ages (P35: <i>Mecp2^-/y</i> t dashed/white bars and WT dashed/grey bars and P55: <i>Mecp2</i>-<i>^-/y</i> t white bars and WT grey bars). The y-axis shows gene expression levels relative to wild type. Wild type level was arbitrarily set to 100%. Gene expression levels were normalized using GAPDH expression (*<i>P</i><0.05, **<i>P</i><0.01).</p

Summary of GABA (A) and glutamate (B) level measurements in the <i>Mecp2</i>^-/y brain at P35 and P55.

<p><i>Mecp2</i> deletion causes an age-dependent reduction of <b>A</b>) GABA levels in the hippocampus, the midbrain (SNpr, substantia nigra pars reticulata) and the cerebellum compared their WT littermates. The same trend was observed for <b>B</b>) the glutamate levels in the hippocampus and the spinal cord where the concentration was lower in <i>Mecp2^-/y</i> compared to WT at P55. It is worth noting that glutamate is reduced in the SNpr at P35, a time corresponding to the onset of the mouse pathology. The sagittal mouse brain drawing is adapted from <i>The Mouse Brain in Stereotaxic Coordinates atlas </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092169#pone.0092169-Paxinos1" target="_blank">[38]</a>. The gray area depicts the corpus callosum. CPu, Caudate-Putamen; SNpr, Substantia nigra pars reticulata.</p

Neurochemical analysis of the GABA levels in A, the motor cortex; B, caudate-putamen; C, hippocampus; D, hypothalamus; E, substantia nigra pars reticulata; F, brainstem; G, cerebellum; H, spinal cord in P35 <i>Mecp2-</i>deficient (dashed/white bars) and WT (dashed/grey bars) (n = 6 <i>Mecp2^-/y</i>, n = 9 WT for caudate-putamen, motor cortex, hypothalamus and brainstem/n = 6 <i>Mecp2^-/y</i>, n = 6 WT for hippocampus, substantia nigra pars reticulata, cerebelum and spinal cord) and P55 <i>Mecp2^-/y</i> (white bars) and WT (grey bars) mice dosage (n = 9 <i>Mecp2^-/y</i>, n = 8 WT for motor cortex, hypothalamus and brainstem/n = 9 <i>Mecp2^-/y</i>, n = 7 WT for caudate-putamen/n = 5 <i>Mecp2^-/y</i>, n = 6 WT for hippocampus, substantia nigra pars reticulata, cerebellum, spinal cord).

<p>Results are expressed as mean ± S.E.M., (*<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001).</p

Developmental changes (P55 vs P35) in GABA concentrations in <i>Mecp2</i>-deficient or wild type mice.

<p><i>Data for each group (WT or Mecp2^-/y) was statistically analyzed by a one-way</i> ANOVA (P55 vs P35) and <i>P</i>-values are shown is parenthesis.</p><p>n.s, non-significant.</p

Neurochemical analysis of glutamate levels in A, the motor cortex; B, caudate-putamen; C, hippocampus; D, hypothalamus; E, substantia nigra pars reticulata; F, brainstem; G, cerebellum; H, spinal cord in P35 <i>Mecp2^-/y</i> (dashed/white bars) and WT (dashed/grey bars) (n = 6 <i>Mecp2^-/y</i>, n = 9 WT for caudate-putamen, motor cortex, hypothalamus and brainstem/n = 6 <i>Mecp2^-/y</i>, n = 6 WT for hippocampus, substantia nigra pars reticulate, cerebellum and spinal cord) and P55 <i>Mecp2^-/y</i> (white bars) and WT (grey bars) mice dosage (n = 9 <i>Mecp2^-/y</i>, n = 8 WT for motor cortex, hypothalamus and brainstem/n = 9 <i>Mecp2^-/y</i>, n = 7 WT for caudate-putamen/n = 5 <i>Mecp2^-/y</i>, n = 6 WT for hippocampus, substantia nigra pars reticulata, cerebellum, spinal cord).

<p>Results are expressed as mean ± S.E.M., (*<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001).</p