Stochastic model of Tsc1 lesions in mouse brain

Abstract

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder due to mutations in either TSC1 or TSC2 that affects many organs with hamartomas and tumors. TSC-associated brain lesions include subependymal nodules, subependymal giant cell astrocytomas and tubers. Neurologic manifestations in TSC comprise a high frequency of mental retardation and developmental disorders including autism, as well as epilepsy. Here, we describe a new mouse model of TSC brain lesions in which complete loss of Tsc1 is achieved in multiple brain cell types in a stochastic pattern. Injection of an adeno-associated virus vector encoding Cre recombinase into the cerebral ventricles of mice homozygous for a Tsc1 conditional allele on the day of birth led to reduced survival, and pathologic findings of enlarged neurons, cortical heterotopias, subependymal nodules, and hydrocephalus. The severity of clinical and pathologic findings as well as survival was shown to be dependent upon the dose and serotype of Cre virus injected. Although several other models of TSC brain disease exist, this model is unique in that the pathology reflects a variety of TSC-associated lesions involving different numbers and types of cells. This model provides a valuable and unique addition for therapeutic assessment.

Figure 1. Survival of Tsc1^c/c mice injected ICV with AAVrh8-CBA-GFP or AAVrh8-CBA-GFP at P0.

Survival is shown for Tsc1^c/c pups injected with the AAV-Cre (N = 15) or the AAV-GFP (N = 13) vectors as a control, both at 2×10¹⁰ g.c. per 2 µl into each ventricle. Median survival of the Cre injected mice was 38 days; controls survived >90 days. Difference between groups p<0.0001.

Figure 2. Cre-mediated recombination in Tsc1^c/c mice after injection of AAVrh8-CBA-Cre at birth.

In Tsc1^c/c mice, Cre-mediated recombination knocks-out the floxed Tsc1 allele and knocks-on a transgenic lacZgene. These mice were injected at P0 with AAV8-CBA-Cre vector (2×10¹⁰g.c. in each ventricle), and lacZ expression throughout the brain was analyzed at ∼P28 as a marker of recombination efficiency. (A) AAV-treated Tsc1^c/cROSA mice displayed intense lacZ expression throughout the brain, (B) while uninjected controls were devoid of any lacZ activity. Ten µm sections were stained with X-gal solution and counterstained with Nuclear Fast Red. Scale bars: 1 mm. (C & D) Staining for NeuN shows that neurons above the lateral ventricles in AAVrh8-CBA-Cre injected mice were considerably larger than in control uninjected mice Scale bars: 200 µm. (E) The periventricular neurons of the brains of AAV-Cre injected and non-injected mice (P28) were immunostained for the neuronal marker, NeuN and the widest diameter of stained cell bodies was measured in >180 randomly selected cells from several fields with 3 animals per group. The AAV-Cre injected cortical neurons had an almost 2-fold increase in diameter, shown as mean±SEM (*p<0.001).

Figure 3. LacZ and GFAP expression in AAVrh8-CBACre injected Tsc1^c/cROSA mice.

Tsc1^c/cROSA mice were injected at P0 with AAVrh8-CBA-Cre vector (2×10⁹g.c. in each ventricle), and lacZ expression in the brain was analyzed at 6 months (N = 2). (A & B) LacZ+ cells were observed scattered as single cells or in patches throughout the cortex. (C & D) Immunocytochemical staining for GFAP with DAPI dye staining of nuclei revealed numerous foci of GFAP+ (red) cells in the cortex (C) while uninjected controls were devoid of any GFAP+ cells in the cortex (D). Scale bars: A–D: 200 µm.

Figure 4. GFAP, NeuN and pS6 staining in brains of AAVrh8-CBA-Cre injected and non-injected Tsc1^c/cROSA mice at 1 month.

(A) Co-immunostaining for GFAP and NeuN in AAVrh8-CBA-Cre injected Tsc1^c/cROSA mice showed clusters of GFAP+ cells in the cortex, some of which co-stained for NeuN (arrows). Scale bars: a–c-:200 µm; d–f: 50 µm. (B) Co-immunostaining for pS6 and NeuN in AAVrh8-CBA-Cre injected Tsc1^c/cROSA mice and non-injected mice (a–c) Cells were larger in AAVrh8-CBA-Cre injected mice than in controls and stained more strongly for pS6. Scale bars: a–f: 200 µm.

Figure 5. Survival data (Kaplan-Meier curve) of Tsc1^c/cROSA mice injected ICV with AAV1-CBA-GFP or AAV1-CBA-Cre at P0.

Death was determined by the point at which animals were in severe distress at which time they were euthanized. Tsc1^c/cROSApups (N = 20) injected with the AAV-CBA-Cre vector died over a range of 30–150 day postnatal period, with a mean of 66.5 survival days. The same P0 pups injected ICV with AAV-GFP as a control (dashed line) all survived >200 days, the longest time point analyzed (N = 8). p<0.0001.

Figure 6. NeuN staining for neuronal diameter measurements.

Immunostaining for NeuN (counterstained with haematoxylin) shows that neurons above the lateral ventricles in (A) AAV1-CBA-Cre injected Tsc1^c/c mice were considerably larger than those in controls in the same region: (B) AAV1-CBA-GFP injected and (C) uninjected mice. Magnification = 40X. (D) Periventricular neurons in the brains of AAV1-Cre and AAV1-GFP injected, as well as non-injected mice (P30) were stained for the neuronal marker, NeuN and the widest diameter of stained cell bodies was measured in >30 randomly selected cells from several fields with 3 animals per group. Neurons in the AAV1-CBA-Cre injected brains had an almost 2-fold increase in diameter, as compared to controls, shown as mean±SEM (*p<0.0001).

Figure 7. pS6 and GFAP double immunostaining to assess astrocyte transduction.

Tsc1^c/cROSA homozygous pups were injected ICV at P0 with either an AAV1-CBA-GFP or AAV1-CBA-Cre vector at a concentration of 2×10¹⁰g.c. One month later two of the pups injected with AAV1-CBA-Cre virus who developed distress were sacrificed and showed severe hydrocephalus by neuropathological examination. To assess astrocyte transduction, the brains were double stained for pS6 and GFAP and counter stained with haematoxylin. Intermediate or mixed cells which stain for both the markers were seen in the (A) periventricular region and (B) subependymal lining. No co-localization was seen in the cortex except for a very few cells(C). No double staining was seen in any region of the brain in AAV1-GFP injected brain (D). Arrows indicate double stained cells. Magnification = 40X.

Figure 8. Quantitative volumetric analysis of MR images of Tsc1^c/c pups at 1 month of age after P0 ICV injection.

Injection of AAV1-CBA-Cre (N = 4) or AAV1-CBA-GFP (N = 2) 2×10¹⁰ g.c. per 2 µl into each ventricle was carried out on P0 and MR images were evaluated on P30. (A) Measurement of ventricle size in voxels (each voxel = 0.0976×0.130×1.302 mm).Difference between groups is significant (p<0.044). (B) Measurement of brain parenchyma (excluding ventricle volume) in voxels. Difference between groups, p<0.23, not significant. Measurements were made by observer blinded to genotype.

Figure 9. Representative MR images of AAV1-CBA-Cre and AAV1-CBA-GFP injected mouse brains.

AAV1-CBA-Cre injected mouse brains (see Fig. 8 legend) showed abnormalities associated with the ventricular regions. (A) Multiple apparent subependymal nodules (arrowheads) were seen in the ventricles in the brains of two AAV1-CBA-Cre injected mice. First two images are from the same mouse. (B) Ventricles also appeared to have thickening of the ependymal lining (arrowheads; two left panels). (C) None of these abnormalities were observed in the control vector (AAV1-CBA-GFP)-injected brains.

Figure 10. Cre-mediated recombination in Tsc1^c/c mice after neonatal delivery of AAV1-CBA-Cre vector (2×10¹⁰ g.c. per 2 µl into each ventricle).

(A) Three months after ICV vector injection into Tsc1^c/cROSA homozygous pups, frozen sections were stained with X-gal and counterstained with Nuclear Fast Red. Intense lacZ staining was visualized in clusters throughout the brain(coronal section, anterior to hippocampus region); note enlarged ventricles (V). Magnification = 2X. (B) The ventricular lining showed patches of lacZ staining and thickening. (C) Nodule-like structure, composed of cells in which Cre recombination has been induced as visualized by X-gal staining, were observed in the ventricles. (D) H&E staining showing nodule along ventricular lining. The regions in B, C & D are taken at a level close to the anterior amygdala. Magnification B, C & D = 10X.

Figure 11. Hydrocephalus and enlarged pS6+ cortical cells in Tsc1^c/c mice injected with AAV1-CBA-Cre vector.

Tsc1^c/cROSA homozygous pups were injected ICV at P0 with either an AAV1-CBA-GFP (N = 6) or AAV1-CBA-Cre (N = 10) vector at a concentration of 2×10¹⁰g.c per ventricle. One month later pups which had received AAV1-CBA-Cre virus developed tremor (N = 8) and were found to have hydrocephalus by pathologic analysis (N = 2). (A) Representative images of brain (upper panels) and cerebral cortex (lower panels) showing in AAV1-CBA-Cre injected animals severe hydrocephalus, large portion of enlarged cortical cells with strong pS6-positivity, and condensed cortical thickness due to swelling of ventricles. Scale bars = 1 mm, upper; 100 µm, lower. (B) Representative pS6 staining in different brain regions. Enlarged pS6+ cells were ectopically present in striatum oriens of hippocampus (arrowhead) due to migration defect of Tsc1-null cells. Some pS6+ Purkinje cells in the cerebellum as well as neurons in the caudate were notably enlarged (arrowheads). Neural cells in deep nuclei such as the brain stem showed similar distribution of pS6 positivity in Cre and GFP injected mice. Scale bars = 50 µm.

Figure 12. DCX, GFAP, GPNMB and NeuN staining in brains of AAV1-CBA-Cre and AAV1-CBA-GFP P0 injected Tsc1^c/c mice at 1 month.

Tsc1^c/cROSA homozygous pups were injected ICV at P0 with either an AAV1-CBA-GFP or AAV1-CBA-Cre vector at a concentration of 2×10¹⁰g.c. One month later pups injected with AAV1-CBA-Cre virus were sacrificed and processed for immunohistochemistry and showed severe hydrocephalus (N = 2). The pups (A) Staining for DCX revealed positive regions along the ventricles(anterior to the striatum, at the level of septal nuclei) in control animals (Fig. 12A, top left panel), but those regions were enlarged in AAV1-CBA-Cre injected animals (Fig. 12A, top right). In addition, the latter animals showed small DCX positive nodules [Fig. 12A, bottom left (level close to anterior amygdale) and right (level of septal nuclei)], which were not seen in control animals. (B) No strong GFAP staining were seen in the ventricular lining (level of septal nuclei) of control brains, but was intense in some subventricular regions (anterior to striatum) of the AAV1-CBA-Cre injected animals (Fig. 12B, top panels), including some GFAP positive nodules in the CSF space at the level of septal nuclei (Fig. 12B, bottom panel). (C) GPNMB staining was virtually null in control brains, with some positive cells near the ventricles anterior to striatum in AAV1-CBA-Cre injected animals (Fig. 12C). (D) NeuN staining of brains revealed rare heterotopias (arrow) consisting of outgrowth of glial cells into the cerebral cortex. Magnification = 20X.