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

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

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

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