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. 2012 Oct 1;21(19):4286-300.
doi: 10.1093/hmg/dds262. Epub 2012 Jun 29.

Graded loss of tuberin in an allelic series of brain models of TSC correlates with survival, and biochemical, histological and behavioral features

Elizabeth Yuan  1 , Peter T Tsai, Emily Greene-Colozzi, Mustafa Sahin, David J Kwiatkowski, Izabela A Malinowska
Affiliations

Graded loss of tuberin in an allelic series of brain models of TSC correlates with survival, and biochemical, histological and behavioral features

Elizabeth Yuan et al. Hum Mol Genet. .

Abstract

Tuberous sclerosis complex (TSC) is a neurodevelopmental disorder with prominent brain manifestations due to mutations in either TSC1 or TSC2. Here, we describe novel mouse brain models of TSC generated using conditional hypomorphic and null alleles of Tsc2 combined with the neuron-specific synapsin I cre (SynIcre) allele. This allelic series of homozygous conditional hypomorphic alleles (Tsc2(c-del3/c-del3)SynICre(+)) and heterozygote null/conditional hypomorphic alleles (Tsc2(k/c-del3)SynICre(+)) achieves a graded reduction in expression of Tsc2 in neurons in vivo. The mice demonstrate a progressive neurologic phenotype including hunchback, hind limb clasp, reduced survival and brain and cortical neuron enlargement that correlates with a graded reduction in expression of Tsc2 in the two sets of mice. Both models also showed behavioral abnormalities in anxiety, social interaction and learning assays, which correlated with Tsc2 protein levels as well. The observations demonstrate that there are graded biochemical, cellular and clinical/behavioral effects that are proportional to the extent of reduction in Tsc2 expression in neurons. Further, they suggest that some patients with milder manifestations of TSC may be due to persistent low-level expression of functional protein from their mutant allele. In addition, they point to the potential clinical benefit of strategies to raise TSC2 protein expression from the wild-type allele by even modest amounts.

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Figures

Figure 1.
Figure 1.
Survival, weight gain and brain weights of Tsc2kc+ and Tsc2cc+ mice. (A) The cumulative survival curve of Tsc2kc+ (n = 97), Tsc2cc+ (n = 60) and control (n = 62) mice. Median survival of Tsc2kc+ and Tsc2cc+ mice is 89 and 137 days, respectively. Pairwise comparison of each survival curve is significantly different (P < 0.0001, Mantel–Haenszel test). (B) Weight gain in Tsc2cc+ (top) and Tsc2kc+ (bottom) mice. Mean and standard deviation are shown. *P< 0.05; **P < 0.001; by the Mann–Whitney test. n > 3 for each cohort at each time point. Equal numbers of male and female mice were used for each genotype. (C) Brain weight of Tsc2kc+ and Tsc2cc+ mice, and controls. Mean and standard deviation are shown. *P< 0.05; **P < 0.001; by the Mann–Whitney test in comparison with control mice. n ≥ 3 for each cohort at each time point, except n = 2 for Tsc2kc+ at 90 days.
Figure 2.
Figure 2.
Phenotype scores for Tsc2kc+ and Tsc2cc+ mice. Average phenotype severity scores with standard deviations are shown according to age for each type of mutant. n ≥ 3 for each cohort at each time point, except n = 2 for Tsc2kc+ at age 120 days. (A) Severity of Hunchback, scored as: 1, normal; 2, present, mild to moderate; 3, severe. (B) Severity of hind limb strain, scored as: 1, normal lateral spread of hind limbs; 2, hind limb shaking and out-of-plane movement; 3, hind limb movement toward midline; 4, clasping of hind limbs.
Figure 3.
Figure 3.
Cortical immunostaining for pS6-S235/236 expression in Tsc2kc+ and Tsc2cc+ mice. Coronal sections of the M1 region of cerebral cortex are shown for mice of age 10 days (A), 30 days (B), 60 days (C) and 90 days (D). Progressively stronger pS6-S235/236 staining is seen predominantly in layers IV and V of both Tsc2kc+ and Tsc2cc+ mice. At all ages, cell staining and size are greater in Tsc2kc+ than in Tsc2cc+. Insets show the largest cells seen in these fields at higher magnification. Scale bars in large images are all 100 µm; in small insets are 20 µm.
Figure 4.
Figure 4.
Neuron soma size in Tsc2kc+ and Tsc2cc+ mice. Average neuron soma size with standard deviations are shown according to age for each type of mutant. Neurons were measured in layers IV–V of the M1 region of cerebral cortex. *P< 0.05; **P < 0.001; by the Mann–Whitney test. Asterisks above a data set indicate comparison with control mice; asterisks above the bar (at top) indicate comparison between Tsc2kc+ and Tsc2cc+. n ≥ 2 mice for each cohort at each time point.
Figure 5.
Figure 5.
Expression of pS6-S235/236 in the hippocampus of Tsc2cc+ and Tsc2kc+ mice. Immunohistochemistry images are shown for control, Tsc2cc+ and Tsc2kc+ mice at 60 days of age. pS6-S235/236 strongly positive neurons are seen in both Tsc2cc+ and Tsc2kc+ sections. Note the presence of ectopic pS6-S235/236+ cells in the stratum oriens (arrows between cortex and CA1) of both mutants. The scale bar is 200 µm for all images. Hippocampus regions: cornu ammonis 1 (CA1), CA3, dentate gyrus (DG).
Figure 6.
Figure 6.
Reduced Tsc2 expression and markers of mTORC1 activation in brain lysates of Tsc2kc+ and Tsc2cc+ mice. (A) Immunoblot analysis of brain lysates from Tsc2kc+ and Tsc2cc+ mice. Proteins probed are indicated at left; blots on left are from 30-day-old mice; blots on right are from 90-day-old mice. Genotype of controls (from left): 30 days: Tsc2ww+, Tsc2cw−; 90 days: Tsc2cc−, Tsc2cw−. (B) Immunoblot and quantitative analysis of brain lysates from 90-day-old Tsc2kc+ and Tsc2cc+ mice. Immunoblots are shown as in (A). Expression levels of Tsc2 relative to GAPDH, pAkt(S473) relative to Akt, pS6-S235/236 relative to S6 and pS6-S240/244 relative to S6 are shown at right. All ratios for the control samples are normalized to 1. Note that Tsc2:GAPDH levels are markedly reduced, more so in Tsc2kc+ lysates, compared with controls, as are pAkt-S473:Akt levels, while pS6-S235/236:S6 and pS6-S240/244:S6 levels are markedly increased. Note that one column of the blot was removed as it was not relevant; all others are from the same blot and exposure. *P < 0.05. ns, not significant. All P-values by the t-test.
Figure 7.
Figure 7.
Locomotor activity in Tsc2 mutant mice. (A) Tsc2 mutants display increased distance traveled (cm) in the open field. Time in minutes is shown on the x-axis. *P < 0.05 mutant versus control. Total n = 40; minimum n per genotype = 8. (B) Tsc2 mutants display increased time on an accelerating rotarod. *P < 0.05 mutant versus control, trials 3–5. Total n = 25; minimum n per genotype = 6. *P < 0.05 mutant versus control. (C and D) Representative open-field (C) and elevated plus maze (D) track plots. Diagram illustrates locations of open and closed arms of the maze. (E) Tsc2kc+ mice display increased time spent in the center quadrant of the open field. Same n as (A). (F and G) Tsc2kc+ mice spend more time (F) and enter into the closed arms of the elevated plus maze more frequently (F) in comparison with control littermates. Total n = 41; minimum n per genotype = 10. *P < 0.05. ns, not significant. All P-values by the t-test, except open field by ANOVA.
Figure 8.
Figure 8.
Abnormal social behavior and impaired water T-maze performance in Tsc2 mutants. (A and C) In the three-chambered social apparatus, both controls and Tsc2 mutants spend a greater amount of time in the chamber with the novel animal (A), and more time interacting with the novel animal (C) in comparison with a novel object. Total n = 39; minimum n per genotype = 9. (B and D) Tsc2kc+ mice show no preference for the chamber with the novel animal in comparison with that with a familiar animal in an assay of social novelty (B), and spend similar time interacting with both the novel and familiar animals (D). This is in distinct contrast to both control and Tsc2cc+ mice. Same n as (A) and (C). (E and F) Water T-maze assay. (E) Number of correct trials (out of 15). (F) Number of trials prior to five consecutive correct responses. Tsc2kc+ mice have significantly fewer correct trials on day 1, and take significantly more trials to achieve five consecutive correct responses on day 1, in comparison with controls. Similarly, they have fewer correct trials after reversal and take longer to achieve five consecutive correct responses on reversal day 1. Tsc2cc+ mice displayed normal results on day 1, but had significantly fewer correct trials on reversal day 1, and took longer to achieve five consecutive correct responses on reversal day 1. Total n = 39; minimum n per genotype = 9. * P < 0.05. ns, not significant. All P-values by the t-test.
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