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. 2009 Apr 1;18(7):1252-65.
doi: 10.1093/hmg/ddp025. Epub 2009 Jan 15.

Loss of Tsc2 in radial glia models the brain pathology of tuberous sclerosis complex in the mouse

Sharon W Way  1 , James McKenna 3rd, Ulrike Mietzsch, R Michelle Reith, Henry Cheng-Ju Wu, Michael J Gambello
Affiliations

Loss of Tsc2 in radial glia models the brain pathology of tuberous sclerosis complex in the mouse

Sharon W Way et al. Hum Mol Genet. .

Abstract

Tuberous sclerosis complex (TSC) is an autosomal dominant, tumor predisposition disorder characterized by significant neurodevelopmental brain lesions, such as tubers and subependymal nodules. The neuropathology of TSC is often associated with seizures and intellectual disability. To learn about the developmental perturbations that lead to these brain lesions, we created a mouse model that selectively deletes the Tsc2 gene from radial glial progenitor cells in the developing cerebral cortex and hippocampus. These Tsc2 mutant mice were severely runted, developed post-natal megalencephaly and died between 3 and 4 weeks of age. Analysis of brain pathology demonstrated cortical and hippocampal lamination defects, hippocampal heterotopias, enlarged dysplastic neurons and glia, abnormal myelination and an astrocytosis. These histologic abnormalities were accompanied by activation of the mTORC1 pathway as assessed by increased phosphorylated S6 in brain lysates and tissue sections. Developmental analysis demonstrated that loss of Tsc2 increased the subventricular Tbr2-positive basal cell progenitor pool at the expense of early born Tbr1-positive post-mitotic neurons. These results establish the novel concept that loss of function of Tsc2 in radial glial progenitors is one initiating event in the development of TSC brain lesions as well as underscore the importance of Tsc2 in the regulation of neural progenitor pools. Given the similarities between the mouse and the human TSC lesions, this model will be useful in further understanding TSC brain pathophysiology, testing potential therapies and identifying other genetic pathways that are altered in TSC.

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Figures

Figure 1.
Figure 1.
Generation of Tsc2flox/ko;hGFAP-Cre mice. (A) Weight curves of mutant (Tsc2flox/ko;hGFAP-Cre) mice (red, n = 17) compared with the control (Tsc2+/flox) mice (blue, n = 15) demonstrating the retarded growth of the mutant animals. (B) A runted mutant, 21-day-old (P21) mouse compared with a control littermate. Note the domed head and splayed feet in the mutant animal. (C) The brain of the mutant mouse (right) was noticeably larger than the control (left). (D) Ventricles in the mutant mouse were dilated (right). (E) Immunoblot analyses of cortical and hippocampal lysates from mutant (‘Cre+’) mice demonstrated a marked decrease of tuberin, slightly decreased levels of hamartin and large increase in pS6 levels compared with the control (‘Cre-’). α-Tubulin was used as the loading control. (F and G) Pax6 and pS6 immunohistochemistry in E15.5 control (F) and mutant (G) mice showed increased activation of mTORC1 in the radial glial cells of the developing cortex. Scale bars, C, 1 mm; F and G, 10 µm.
Figure 2.
Figure 2.
Cerebral cortical defects and up-regulation of mTORC1 in cortical neurons and astrocytes in Tsc2flox/ko;hGFAP-Cre mice. All sections were taken from P21 mice. (A and B) The cortex of the mutant (B) was thicker than the control (A) and displayed lamination defects, blurring between the gray–white junction, and a much less-defined molecular layer (Layer I). (C and D) Higher magnification revealed enlarged cells in the cortex of the mutant (D) and more extracellular matrix between the cells compared with the control (C). (E) Comparison of the areas of NeuN-labeled neurons from mutant and control cortex revealed that mutant neurons are significantly larger (**P < 0.005, n = 6) than control neurons. (F and G) NeuN-labeled neurons in the mutant cortex (G) displayed substantial increase in pS6 expression compared with the control (F), indicating elevated mTORC1 activation. (H and I) S100-labeled astrocytes in the mutant cortex (I) also showed notable increase in pS6 expression compared with the control (H). Scale bars, A and B, 100 µm; C and D, 20 µm; F and G, 50 µm; H and I, 40 µm, inset 10 µm.
Figure 3.
Figure 3.
Hippocampal defects and up-regulation of mTORC1 in hippocampal neurons and astrocytes in Tsc2flox/ko;hGFAP-Cre mice. All sections were taken from P21 mice. (A and B) Severe lamination defects in the mutant mouse (B) were apparent in H&E-stained sections of the hippocampus, especially in the CA1 and CA3 regions. White arrows represent ring heterotopias that were evident throughout the SLM. (C and D) The DG was lined with large, ectopic granule cells in the mutant (D). (E–G) Numerous ectopic neurons (F, black arrows) and enlarged cells (G, black arrowhead) were also present in the SO of the mutant hippocampus (F), in contrast to the tight, well-organized pyramidal layer in the control (E). (H–J) High power magnification of a ring heterotopia demonstrated that they were mainly composed of NeuN-positive neurons (green cells) with some S100-positive astrocytes (I, J, red cells, white arrowheads), though some cells do not stain for either marker (I, J, black arrowheads). (K and L) NeuN-labeled neurons in the hippocampus of the mutant (L) displayed greater pS6 expression than the control (K). However, expression of pS6 in the DG of both the mutant and the control is reduced compared with their respective PL. (M) Despite low immunohistochemical pS6 expression in the mutant DG, average cell area of cells in the DG of the mutant was significantly larger (***P < 0.0005) than the control (n = 6). (N and O) S100-labeled astrocytes in the hippocampus of the mutant (O) had increased pS6 expression. The SLM region of the hippocampus is shown. Scale bars, A–F, 50 µm; G–J, 10 µm; K and L, 100 µm; M, 50 µm; N and O, 20 µm.
Figure 4.
Figure 4.
Post-natal developmental analysis of Tsc2flox/ko;hGFAP-Cre mice. H&E-stained sections from P0 to P15. (A–D) At P0, the size of the cerebral cortex was comparable between the control and the mutant. The ventricular zone (arrowheads) appeared thicker in the mutant and lamination abnormalities in CA1 and CA3 (arrows) were beginning to develop. (E–H) At P5, an increase in the cortical thickness of the mutant (F) was more apparent. The layering abnormalities in CA3 (arrow) were more pronounced. There were many ectopic cells in the SO of the mutant (H, white arrowheads) compared with the control. There was also the appearance of clusters of cells in the hippocampal fissure (black arrowhead). (I–M) By P10, the cerebral cortex of the mutant had continued to enlarge, and the layering was indistinct compared with the control. The ring heterotopias in the SLM were evident (inset J). The corpus callosum was also noticeably thicker. Sectioning through a heterotopia (K–M) demonstrated that they represent a nodule of ectopic cells. (N–Q) By P15, the same abnormalities were present as those seen at weaning. Scale bars, A, B, E–J, N, O, 100 µm; C, D, K–M, P, Q, 50 μm.
Figure 5.
Figure 5.
Lamination defects and astrogliosis in the Tsc2flox/ko;hGFAP-Cre mice. All sections and counts conducted in P21 mice. (A and b) Cux1 (A), a Layer II–IV marker, and FoxP2 (B), a Layer VI marker, were used to assess lamination defects (n = 6 and 4, respectively). The Cux1-labeled cells of the control resided mostly in their designated layers, whereas the labeled cells of the mutant were scattered throughout the cortex. FoxP2-labeled cells were generally in Layer VI for both control and mutant. (C–E) Although NeuN-labeled neurons (C) and Cux1-labeled cells (D) were not found to be significantly different in the cortex, there were significantly less FoxP2-labeled cells in the mutant (E, *P < 0.05). (F and H) Immunostaining of GFAP in the cortex (F) revealed substantial increase of GFAP-expressing astrocytes in the cortex compared with the control. Intense GFAP expression was also seen in the mutant ventricular zone of the lateral ventricle (white arrows). As quantification of GFAP+ cells in the cortex was difficult in the control, S100 (H) was used to label cortical astrocytes. No significant difference was found in S100+ cells between the mutant and the control. (G and I) GFAP expression was notably higher in the mutant hippocampus where astrocytes had larger cell bodies but shorter, thicker processes compared with the control. Significantly more GFAP-labeled cells (I) were present in the mutant hippocampus.
Figure 6.
Figure 6.
Cortical neural progenitor pool analysis at embryonic day E14.5. (A–C) BrdU pulse-labeling failed to detect any significant proliferative differences along the ventricles of the control and mutant embryos at E14.5 (n = 6). (D–F) Significantly more (F, ***P < 0.0005) Tbr2-labeled basal progenitor cells were found in the subventricular zone of the mutant (E) compared with the control (D) (n = 6). (G–I) The number of post-mitotic Tbr1-labeled neurons was significantly decreased (I, *P < 0.05) in the control (G) (n = 6). (J–L) Radial glia numbers (L), as labeled by pax6, were not found to be significantly different between the control and the mutant (J) (n = 4). CP, cortical plate, SVZ, subventricular zone, VZ, ventricular zone. A, B, G and H taken at 20× magnification, D, E, J and K taken at 40×.
Figure 7.
Figure 7.
Post-natal developmental analysis of hypomyelination and oligodendrocytes in the mutant. (A and B) Immunostaining of MBP at P10 (A) and P15 (B) demonstrated a defect in myelin formation in the mutant cortex (arrows). (C and D) Oligodendrocyte analysis using CC1 revealed a 1.25-fold increase in CC1-labeled cells in the mutant (n = 4, data not shown) compared with the control. Colocalization of pS6 and CC1 (arrowheads) showed that most oligodendrocytes in the mutant had up-regulated mTORC1 activity.
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