Supplemental Data Tuberous sclerosis complex-associated CNS abnormalities depend on hyperactivation of mTORC1 and Akt Paola Zordan1, Manuela Cominelli2, Federica Cascino1, Elisa Tratta2, Pietro Luigi Poliani2 and Rossella Galli1* 1 Neural Stem Cell Biology Unit, Division of Regenerative Medicine, Stem Cells and Gene Therapy, San Raffaele Scientific Institute, Via Olgettina 58, Milan, Italy, 20132; 2 Department of Pathology, University of Brescia, Spedali Civili of Brescia, Brescia, Italy, 25124. 1 Supplemental Figure 1 – Efficient early post-natal targeted inactivation of Tsc1 and Pten in Nestin- expressing SVZ pNSCs results in macroscopic phenotype alterations and very short mouse lifespan. (A) Macroscopic appearance of Tsc1c/cPtenc/c/Nestin-CreERT2+(TPN) and Tsc1c/cPtenc/c mice control mice activated at P10 and analyzed at 38 days of life. Brains of mutant mice were significantly enlarged as compared to controls; in particular the cerebellum appears to be expanded (arrow). (B) Kaplan-Meier survival curve for TPN mutant mice and controls (n=15). 2 (C) PCR analysis of genomic DNA from the tail of mutant mice showing the presence of Cre recombinase and tdTomato reporter gene. Endogenous tdT protein fluorescence highlights efficient Cre-mediated recombination in the cells lining the lateral ventricles in tdTc/c/Tsc1c/cPtenc/c/Nestin-CreERT2+(tdT-TPN) mice 48 hours after TMX administration (magnification 40x). (D) tdT positive cells (red) that were also immunoreactive (-IR) for nestin (green, left panels) and S100 (green, right panels) were detected 48 hours after Tamoxifen administration in the SVZ of TPN-tdT mice (merge, yellow; magnification 600x). (E) Some of the tdT-IR cells (red) also expressed GFAP (green; 600x). 3 Supplemental Figure 2 – Lesions developing in TPN mice activated at P10 and P10-11 display features of SENs. (A) Endogenous tdT protein fluorescence (red) highlights efficient Cre-mediated recombination in the lesions verging into the lateral ventricles in tdTc/c/Tsc1c/cPtenc/c/Nestin-CreERT2+(tdT-TPN) mice 27 days after TMX administration (mouse 1; 40x, inset 400x). The same pattern is observed when the endogenous tdT expression was detected by IHC with an antibody raised against the red fluorescent protein (RFP) (brown; mouse 2; 40x, 4 inset 400x). White solid arrow: upper SVZ expansion. *: SEN-like nodular lesions. **: bulb-like nodular abnormalities found in the proximity of the interventricular foramen (IF). (B) Most of the tdT-positive cells within the nodules were pS6-IR (green; 600x). (C) Many tdT-positive cells were also GFAP-IR (arrows, green; 600x). (D) pERK hyperactivation was found in cells within the SVZ, whereas it was absent in cells making up the nodular lesions (green; control, 400x; mutant, 600x ). (E) A significant enlargement of the SVZ was evident 19 day after TMX administration in P10 TPN mice as compared to controls (H&E; 200x). 5 Supplemental Figure 3 - Efficient targeted inactivation of Tsc1 and Pten in Nestin-expressing SVZ pNSCs at P15-17 results in macroscopic phenotypic alterations and reduced mouse lifespan, whereas no differences in the same features were detected when activation was performed at very late time points. 6 (A) Macroscopic appearance of Tsc1c/cPtenc/c/Nestin-CreERT2+(TPN) and Tsc1c/cPtenc/c mice control mice activated at P15-17 and analyzed at 63 days of life. Brains of mutant mice are significantly enlarged as compared to controls; in particular the cerebellum appears to be expanded (arrow). (B) Kaplan-Meier survival curve for TPN mutant mice and controls activated at P15-17 (n=10) and P24-26 (n=7). (C) Endogenous tdT protein fluorescence highlights efficient Cre-mediated recombination in the cells lining the lateral ventricles in tdTc/c/Tsc1c/cPtenc/c/Nestin-CreERT2+(tdT-TPN) mice 48 hours after TMX administration at P15-17 and P24-26 (magnification 40x). (D) Many tdT positive cells (red) that were immunoreactive (-IR) for nestin (green, upper panels), S100 (green, middle panels) and GFAP were detected 48 hours after Tamoxifen administration in the SVZ of in TPN-tdT mice activated at P15-17 (merge, yellow; magnification 400x). (E) Few tdT positive cells (red) that were immunoreactive (-IR) for nestin (green, upper panels), S100 (green, middle panels) and GFAP were detected 48 hours after Tamoxifen administration in the SVZ of in TPN-tdT mice activated at 24-26 (merge, yellow; magnification 400x). 7 Supplemental Figure 4 - Late/very late post-natal targeted inactivation of Tsc1 and Pten in SVZ pNSCs promotes the development of pNSC-derived SEGA-like lesions. 8 (A) Endogenous tdT protein fluorescence (red) highlights efficient Cre-mediated recombination in the lesions verging into the lateral ventricles in tdTc/c/Tsc1c/cPtenc/c/Nestin-CreERT2+(tdT-TPN) mice 86 days after TMX administration at P15-17 (40x). (B) Most of the tdT-positive cells within the nodules were pS6-IR (green; 400x). (C) Some tdT-positive cells were also GFAP-IR (arrows, green; 400x). (D) Significant pERK hyperactivation was retrieved in the tdT cells making up the nodular lesions (green; 400x). (E) Endogenous tdT protein fluorescence (red) highlights efficient Cre-mediated recombination in focal lesions verging into the lateral ventricles in tdTc/c/Tsc1c/cPtenc/c/Nestin-CreERT2+(tdT-TPN) mice 120 days after TMX administration at P24-26 (40x). (F) H&E and IHC for pS6, pErk and pNrdg1 confirm the enhanced SEGA nature of the focal abnormalities developing in P24-26 TPN mice, such as the presence of spindle-shaped cells (inset in H&E) and pNdrg1- activating giant cells (inset in IHC). (G) Some of the tdT-positive cells within the lesions were pS6-IR (green; 600x) and GFAP-IR (green; 600x). Strong pERK hyperactivation was found in most cells within the P24-26 TPN lesions (green; control, 400x; mutant, 600x). 9 Supplemental Figure 5 - Postnatal NSCs isolated from SENs and SEGAs developing in TPN mice are reminiscent of the molecular and cellular characteristics of TSC brain abnormalities. (A) PCR analysis of genomic DNA extracted from pNSCs showed Cre activation and complete deletion of Tsc1 and Pten in mutant pNSC lines with respect to control pNSC lines. (B) Some cells in P15-17 TPN pNSC-derived differentiated progeny were IR for both neuronal and glial markers, suggesting that the differentiation of mutant pNSCs was impaired (Tuj1, red; GFAP, green; merge, yellow). (C) Treatment of P10 and P15-17 TPN mutant pNSCs with 100 nM rapamycin did not rescue the impaired differentiation into neuronal cells (Tuj1, red), whereas it partially restored mutant pNSC differentiation into morphologically normal GFAP- and GalC-IR cells (green) in both undifferentiated and differentiated pNSC cultures (magnification 400x). 10 Supplemental Figure 6 - SEGA pNSCs give rise to tumors that express the same markers found in the corresponding human lesions. 11 Tumors generated by the subcutaneous implantation of distinct pNSC lines (i.e. L90, L1 and L3) reproduced the heterogeneity in marker expression observed in human SEGA samples. (A) Tumors generated by pNSC line L90 were characterized by the hyperactivation of pS6 and pAkt in most cells as well as by the expression of GFAP, DCX, S100 ox2 and Pax6 in subset of cells (IHC, magnification 600x; Sox2, 200x). They also hyperactivated pErk and pNdrg1 in focal clusters of cells (100x and 600x). (B) Tumors generated by pNSC line L1 strongly activated pS6 and pAkt in the majority of cells. GFAP, DCX, S100 ox2 and Pax6 were also retrieved in the majority of cells (Magnification 600x; Sox2, 200x). They hyperactivated pErk and pNdrg1 in many cells (100x and 600x). Multinucleated cells were also detected (arrow in GFAP IHC). (C) Tumors generated by pNSC line L3 expressed GFAP and DCX in most cells (100x and 600x). S100 ox2 and Pax6 were retrieved less frequently (600x). 12 Supplemental Figure 7 – Genes expressed in mouse SEGAs are also upregulated in the corresponding human lesion. Heatmap showing 15 selected human SEGA-specific genes that were also increased/decreased in control pNSCs vs. mouse SEGAs, ranked based on their p value (p<0.001) and colored according to the scale blue=low and red=high (n=3 independent biological replicate for each condition). 13 Supplemental Table 1 – Demographic information of SEGA-bearing TSC patients. # Internal reference Age Gender 1 15-B-02346 10 y M 2 12-B-03845 4 m F 3 11-B-01049 13 y M 4 11-B-00081 1 y F 5 10-B-03157 11 y M 6 08-B-00287 13 y M 7 06-B-03242 19 y F 8 11395-1997 6 y F 9 13568-2007 9 y F 10 15579-2001 8 y F 11 9225-2002 9 y M 12 I-04-1727 5 y M 13 I-13-3839 3 y M 14 I-04-1268 16 y M 15 I-04-1614 8 y F Gender: 8 males (53%) and 7 females (47%) Median age at surgery: 8.8 years (range 4 months-19 years) 14 Supplemental Table 2 – Frequency of cells activating pS6, pAKT, pNDRG1, and pERK in TPN P10 and P15-17 pNSC-derived differentiated cultures. TPN P10 TPN P15-17 % IR pNSC-derived pNSC-derived cells end cells end cells control mutant control mutant pS6 16.5 8.0 98.7 2.1**** a 0 99.1 1.1**** a pAkt 0 99.0 0.4**** a 0 95.9 3.8**** a a a/ b pNDRG1 0 8.5 4.2** 0 22.5 8.4*** ** pERK 0 41.3 15.3*** a 0 78.6 19.5**** a/*** b Results for continuous variables were expressed as mean ± standard deviation. Two-group comparisons were performed with the independent samples’ t test. P values <0.05 were considered statistically significant. Statistical significance of the data is indicated as follows: *: p< 0.05; **: p< 0.01; ***: p< 0.001; ****: p< 0.0001.