Article Figures & Data
Figures
- Figure 1.
Relative levels of tuberin in WT and Tsc2Mjg+/− mice in lysates of hippocampus and frontal cortex. Relative levels were measured by means of Western blots (A) with two treatments: control and 24 h after a 1-h exposure to isoflurane anesthesia. Data (normalized to WT control mice) were analyzed by means of ANOVA with genotype and treatment as between subject variables. In hippocampus (B) neither the genotype × treatment interaction (F(1,19) = 0.013, p = 0.912) nor the main effect of treatment (F(1,19) = 0.758, p = 0.395) was statistically significant, but the main effect of genotype (F(1,19) = 20.454, p < 0.001) was. In frontal cortex (C) neither the genotype × treatment interaction (F(1,19) = 0.265, p = 0.612) nor the main effect of treatment (F(1,19) = 1.189, p = 0.289) was statistically significant, but the main effect of genotype (F(1,19) = 20.115, p < 0.001) was. Bars represent the means ± SEM for six mice per group except for the isoflurane-exposed WT which had five mice. Regardless of treatment, tuberin levels were 25% and 24% lower in Tsc2Mjg+/− mice in hippocampus and frontal cortex, respectively.
- Figure 2.
Representative digitized parametric images of rCPS from WT (A, C) and Tsc2Mjg+/− (B, D) mice. Images illustrate the patterns of effects of the mutation on rCPS at the level of the frontal association cortex (A, B) and dorsal hippocampus (C, D). The colorbars on the right define the color scales for the images (upper and lower colorbars pertain to A, B and C, D, respectively). At both levels, images show that rCPS is decreased in the Tsc2Mjg+/− mouse compared with WT. Scale bar under D pertains to all images.
- Figure 3.
Relative levels of signaling proteins known to affect protein synthesis in lysates of hippocampus from WT and Tsc2Mjg+/− mice. Relative levels were measured by means of Western blots (A) with two treatments: control and 24 h after a 1-h exposure to isoflurane anesthesia. Data were analyzed by means of ANOVA with genotype and treatment as between subject variables (Table 3). Aligned dot plots (B–M) indicate protein expression (normalized to WT control mice). Solid horizontal lines represent means ± SEM for six WT control, five WT isoflurane-treated, six Tsc2Mjg+/− control, and six Tsc2Mjg+/− isoflurane-treated mice. Horizontal dashed line represents a relative expression of 1.0. For Akt Thr 308 (J) the genotype × treatment interaction was statistically significant and results of post hoc Bonferroni corrected t tests are shown on the figure; *0.01 ≤ p ≤ 0.05, **0.001 ≤ p ≤ 0.01, ***p ≤ 0.001.
- Figure 4.
Relative levels of signaling proteins known to affect protein synthesis in lysates of frontal cortex from WT and Tsc2Mjg+/− mice. Relative levels were measured by means of Western blots (A) with two treatments: control and 24 h after a 1-h exposure to isoflurane anesthesia. Data were analyzed by means of ANOVA with genotype and treatment as between subject variables (Table 3). Aligned dot plots (B–M) indicate protein expression (normalized to WT control mice). Solid horizontal lines represent means ± SEM for six WT control, five WT isoflurane-treated, six Tsc2Mjg+/− control, and six Tsc2Mjg+/− isoflurane-treated mice. Horizontal dashed line represents a relative expression of 1.0.
Tables
Variable WT (8) Tsc2 Mjg+/− (5) Age (d) 96 ± 2 95 ± 2 Body weight (g) 41.0 ± 2.0 41.2 ± 3.2 Hematocrit (%) 49.5 ± 1.7 50.2 ± 1.8 Mean arterial blood pressure (mmHg) 112 ± 4 113 ± 2 Arterial blood glucose (mm) 6.3 ± 0.3 6.2 ± 0.6 Arterial plasma leucine (μm) 137 ± 5 127 ± 6 Values are the means ± SEM for the number of mice indicated in parentheses.
Region WT Tsc2 Mjg+/− Difference (%) p value Cortex Frontal 6.99 ± 0.48 (n = 8) 4.82 ± 0.32 (n = 5) −31 0.008 Parietal 8.73 ± 0.50 (n = 7) 5.50 ± 0.27 (n = 5) −37 <0.001 Auditory 8.33 ± 0.53 (n = 5) 5.22 ± 0.30 (n = 5) −37 <0.001 Visual 8.85 ± 0.75 (n = 6) 5.37 ± 0.34 (n = 4) −39 0.001 Corpus callosum Medial 2.94 ± 0.14 (n = 8) 2.07 ± 0.12 (n = 5) −30 0.001 Lateral 3.31 ± 0.18 (n = 8) 2.22 ± 0.14 (n = 5) −33 0.001 Thalamus Anterodorsal nucleus 14.67 ± 1.13 (n = 5) 8.44 ± 0.86 (n = 4) −42 <0.001 Paraventricular nucleus 13.94 ± 1.59 (n = 6) 9.15 ± 0.73 (n = 4) −34 0.011 Dorsomedial 7.65 ± 0.39 (n = 6) 4.64 ± 0.40 (n = 3) −39 <0.001 Lateral dorsal nucleus 7.39 ± 0.48 (n = 6) 4.50 ± 0.32 (n = 4) −39 <0.001 Medial geniculate nucleus 7.87 ± 0.60 (n = 4) 4.21 ± 0.53 (n = 5) −47 <0.001 Ventral posterior 8.37 ± 0.62 (n = 8) 5.41 ± 0.36 (n = 5) −35 0.005 Hypothalamus Suprachiasmatic nucleus 10.25 ± 0.82 (n = 5) 6.96 ± 1.29 (n = 4) −32 0.007 Paraventricular nucleus 15.65 ± 0.85 (n = 8) 9.97 ± 1.09 (n = 5) −36 0.002 Supraoptic nucleus 18.09 ± 1.67 (n = 6) 9.52 ± 0.90 (n = 4) −47 <0.001 Basolateral amygdala 8.87 ± 0.60 (n = 8) 5.66 ± 0.36 (n = 5) −36 0.002 Hippocampus Dorsal 7.08 ± 0.39 (n = 8) 4.81 ± 0.38 (n = 5) −32 0.002 Ventral 7.02 ± 0.43 (n = 6) 4.18 ± 0.27 (n = 5) −41 <0.001 Cerebellum Interpeduncular nucleus 9.05 ± 0.62 (n = 3) 4.89 ± 0.56 (n = 3) −46 <0.001 Flocculus 9.93 ± 0.88 (n = 4) 4.01 ± 0.44 (n = 2) −60 <0.001 Arbor vitae 3.00 ± 0.21 (n = 5) 1.32 ± 0.14 (n = 4) −56 <0.001 Simple lobule 10.13 ± 0.86 (n = 5) 5.43 ± 0.64 (n = 3) −46 <0.001 Culmen 3.63 ± 0.23 (n = 5) 2.25 ± 0.09 (n = 3) −38 <0.001 There were no statistically significant genotype differences.
Protein Interaction Main effect F(df,error) value p value pAKT Ser473 Treatment × Genotype F(1,19) = 0.190 0.668 Treatment F(1,19) = 0.097 0.759 Genotype F(1,19) = 1.820 0.193 pAKT Thr308 Treatment × Genotype F(1,19) = 6.766 0.018* Treatment F(1,19) = 30.807 <0.001* Genotype F(1,19) = 5.334 0.032* pAMPK Treatment × Genotype F(1,19) = 1.856 0.189 Treatment F(1,19) = 0.018 0.895 Genotype F(1,19) = 0.019 0.893 pCREB Treatment × Genotype F(1,19) = 0.170 0.684 Treatment F(1,19) = 0.195 0.663 Genotype F(1,19) = 0.540 0.471 peIF2α Treatment × Genotype F(1,19) = 0.177 0.678 Treatment F(1,19) = 0.837 0.372 Genotype F(1,19) = 0.651 0.430 pERK Treatment × Genotype × Band F(1,19) = 0.155 0.699 Treatment × Band F(1,19) = 0.069 0.796 Genotype × Band F(1,19) = 0.328 0.574 Treatment × Genotype F(1,19) = 0.652 0.429 Treatment F(1,19) = 0.001 0.971 Genotype F(1,19) = 0.248 0.624 Band F(1,19) = 127.210 <0.001* pGSK3α/β Treatment × Genotype × Band F(1,19) = 0.661 0.426 Treatment × Band F(1,19) = 0.227 0.639 Genotype × Band F(1,19) = 1.259 0.276 Treatment × Genotype F(1,19) = 0.692 0.416 Treatment F(1,19) = 0.388 0.541 Genotype F(1,19) = 0.262 0.614 Band F(1,19) = 265.256 <0.001* pmTOR Treatment × Genotype F(1,19) = 0.032 0.861 Treatment F(1,19) = 4.789 0.041* Genotype F(1,19) = 0.135 0.717 p-p70 S6K Thr389 Treatment × Genotype × Band F(1,19) = 0.191 0.667 Treatment × Band F(1,19) = 0.096 0.760 Genotype × Band F(1,19) = 1.127 0.302 Treatment × Genotype F(1,19) = 0.020 0.889 Treatment F(1,19) = 0.046 0.832 Genotype F(1,19) = 0.003 0.955 Band F(1,19) = 325.069 <0.001* p-p70 S6K Thr421/Ser424 Treatment × Genotype × Band F(1,19) = 0.231 0.636 Treatment × Band F(1,19) = 0.463 0.504 Genotype × Band F(1,19) = 0.075 0.788 Treatment × Genotype F(1,19) <0.001 0.998 Treatment F(1,19) = 0.762 0.394 Genotype F(1,19) = 0.942 0.344 Band F(1,19) = 140.106 <0.001* pS6 Treatment × Genotype × Site F(1,19) = 1.343 0.261 Treatment × Site F(1,19) = 0.406 0.532 Genotype × Site F(1,19) = 0.506 0.485 Treatment × Genotype F(1,19) = 0.337 0.569 Treatment F(1,19) = 6.396 0.020* Genotype F(1,19) = 0.092 0.765 Site F(1,19) = 38.962 <0.001* Values are the mean ± SEM for the number of mice indicated in parentheses. Whereas experiments were completed in eight WT and five Tsc2Mjg+/− mice, the number of mice analyzed depended on the quality of the autoradiograms at the level of each ROI. Bonferroni-corrected post hoc tests showed that Tsc2Mjg+/− mice had statistically significantly lower rCPS in all brain regions analyzed.
Protein Interaction Main effect F(df,error) value p value pAKT Ser473 Treatment × Genotype F(1,19) = 0.106 0.749 Treatment F(1,19) = 0.030 0.865 Genotype F(1,19) = 0.378 0.546 pAKT Thr308 Treatment × Genotype F(1,19) = 0.851 0.368 Treatment F(1,19) = 0.109 0.745 Genotype F(1,19) = 0.012 0.913 pAMPK Treatment × Genotype F(1,19) = 0.765 0.393 Treatment F(1,19) = 5.166 0.035* Genotype F(1,19) = 1.115 0.304 pCREB Treatment × Genotype F(1,19) = 0.043 0.838 Treatment F(1,19) = 2.655 0.120 Genotype F(1,19) = 0.033 0.859 peIF2α Treatment × Genotype F(1,19) = 0.235 0.634 Treatment F(1,19) = 0.370 0.550 Genotype F(1,19) = 0.265 0.613 pERK Treatment × Genotype × Band F(1,19) = 1.174 0.292 Treatment × Band F(1,19) = 0.560 0.464 Genotype × Band F(1,19) = 0.303 0.588 Treatment × Genotype F(1,19) = 2.099 0.164 Treatment F(1,19) = 0.350 0.561 Genotype F(1,19) = 0.402 0.533 Band F(1,19) = 145.346 <0.001* pGSK3α/β Treatment × Genotype × Band F(1,19) = 0.971 0.337 Treatment × Band F(1,19) = 1.758 0.201 Genotype × Band F(1,19) = 0.013 0.911 Treatment × Genotype F(1,19) = 0.240 0.630 Treatment F(1,19) = 0.568 0.460 Genotype F(1,19) = 0.004 0.953 Band F(1,19) = 260.590 <0.001* pmTOR Treatment × Genotype F(1,19) = 0.052 0.822 Treatment F(1,19) = 0.034 0.856 Genotype F(1,19) = 0.046 0.832 p-p70 S6K Thr389 Treatment × Genotype × Band F(1,19) = 2.292 0.146 Treatment × Band F(1,19) = 4.451 0.048* Genotype × Band F(1,19) = 0.001 0.975 Treatment × Genotype F(1,19) = 0.882 0.359 Treatment F(1,19) = 0.090 0.767 Genotype F(1,19) = 1.228 0.282 Band F(1,19) = 180.660 <0.001* p-p70 S6K Thr421/Ser424 Treatment × Genotype × Band F(1,19) = 0.085 0.773 Treatment × Band F(1,19) = 0.003 0.957 Genotype × Band F(1,19) = 0.279 0.603 Treatment × Genotype F(1,19) = 0.265 0.613 Treatment F(1,19) = 0.535 0.473 Genotype F(1,19) = 0.150 0.702 Band F(1,19) = 30.355 <0.001* pS6 Treatment × Genotype × Site F(1,19) < 0.001 0.985 Treatment × Site F(1,19) < 0.001 0.993 Genotype × Site F(1,19) = 0.049 0.826 Treatment × Genotype F(1,19) = 0.947 0.343 Treatment F(1,19) = 0.813 0.378 Genotype F(1,19) = 6.216 0.022* Site F(1,19) = 6.634 0.019* ↵* Denotes statistical significance p ≤ 0.05.
Variable Interaction Main effect F(df,error) value p value Body weight Strain × Genotype F(1,30) = 0.013 0.911 Strain F(1,30) = 103.935 <0.001* Genotype F(1,30) < 0.001 0.990 Arterial blood pressure Strain × Genotype F(1,30) = 0.001 0.981 Strain F(1,30) = 2.300 0.140 Genotype F(1,30) = 0.065 0.801 Arterial blood glucose concentration Strain × Genotype F(1,30) = 0.065 0.801 Strain F(1,30) < 0.001 0.983 Genotype F(1,30) = 0.0.439 0.513 Arterial plasma leucine concentration Strain × Genotype F(1,30) = 0.428 0.518 Strain F(1,30) = 14.494 <0.001* Genotype F(1,30) = 2.187 0.150 ↵*Denotes statistical significance p ≤ 0.05.
Comparison of physiological variables between the Tsc2Mjg+/− mice used in this study (values in Table 1) and Tsc2Djk+/− mice used in our previous study (Saré et al., 2018). Mean values in control and Tsc2Djk+/− mice, respectively, as follows: body weight: 98 ± 1 and 96 ± 2 g; arterial blood pressure: 109 ± 1 and 109 ± 2 mmHg; arterial blood glucose: 6.5 ± 0.4 and 6.1 ± 0.5 mm; arterial plasma leucine: 116 ± 4 and 111 ± 3 μm.
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