Neurofibromatosis-1 Regulates Mtor-Mediated Astrocyte Growth

Neuroﬁbromatosis-1 regulates mTOR-mediated astrocyte growth and glioma formation in a TSC/Rheb-independent manner

Sutapa Banerjee, Nikkilina R. Crouse, Ryan J. Emnett, Scott M. Gianino, and David H. Gutmann1

Department of Neurology, Washington University School of Medicine, St. Louis, MO 63110

Edited* by Webster K. Cavenee, Ludwig Institute, University of California at San Diego, La Jolla, CA, and approved August 15, 2011 (received for review December 17, 2010)

Converging evidence from the analysis of human brain tumors seizures and death, whereas Nf1 loss in these same neuroglial and genetically engineered mice has revealed that the mamma- progenitors has no effect on mouse viability or epileptogenesis (16, lian target of rapamycin (mTOR) pathway is a central regulator of 17). Importantly, although Pten-, hamartin-, and neurofibromin- glial and glioma cell growth. In this regard, mutational inactiva- deficient glial phenotypes have different biological effects, each tion of neurofibromatosis-1 (NF1), tuberous sclerosis complex is completely inhibited by rapamycin in vitro and in vivo (15, 18– (TSC), and PTEN genes is associated with glioma formation, such 21). Collectively, these observations support a model in which fi that pharmacologic inhibition of mTOR signaling results in atten- neuro bromin-regulated mTOR-dependent growth regulation uated tumor growth. This shared dependence on mTOR suggests is distinct from TSC/Rheb-mediated mTOR-dependent growth that PTEN and NF1 (neurofibromin) glial growth regulation regulation in the brain. To critically examine the requirement for the TSC/Rheb axis requires TSC/Rheb (Ras homolog enriched in brain) control of fi mTOR function. In this report, we use a combination of genetic in neuro bromin mTOR-dependent growth regulation in the silencing in vitro and conditional mouse transgenesis approaches brain, we used a combination of pharmacologic and genetic silencing approaches in vitro coupled with conditional knockout in vivo to demonstrate that neurofibromin regulates astrocyte and overexpression transgenesis strategies in vivo. We show that cell growth and glioma formation in a TSC/Rheb-independent fi Nf1 Pten neuro bromin regulation of glial cell growth and glioma for- fashion. First, we show that or inactivation, but not mation is TSC/Rheb-independent, and that the functional con- Tsc1 loss or Rheb overexpression, increases astrocyte cell growth Nf1 fi sequences of TSC/Rheb mTOR regulation in astroglial cells are in vitro. Second, -de cient increased mTOR signaling and as- distinct from those conferred by Nf1 gene inactivation. trocyte hyperproliferation is unaffected by Rheb shRNA silencing. Third, conditional Tsc1 inactivation or Rheb overexpression in Results Nf1+/− glial progenitors of mice does not lead to glioma forma- mTOR Activation Differentially Regulates Cell Growth in Astrocytes tion. Collectively, these findings establish TSC/Rheb-independent in Vitro. Previous studies have demonstrated that Nf1, Pten, and mechanisms for mTOR-dependent glial cell growth control and Tsc1 gene inactivation each results in increased mTOR activa- gliomagenesis relevant to the design of therapies for individuals tion in astrocytes in vitro (15, 19, 20). In addition, Rheb over- with glioma. expression to mimic Tsc1/Tsc2 gene inactivation similarly increases mTOR activation (15). In each case, this mTOR acti- astrocytoma | optic glioma | glia vation is completely inhibited by rapamycin treatment (Fig. 1), suggesting that mTOR signaling is a critical regulator of cell utations in several tumor suppressor genes and receptor growth in astrocytes. However, mTOR activation as a conse- Mtyrosine kinases (RTKs) deregulate cell growth and survival quence of Nf1 and Pten loss results in increased astrocyte pro- by increasing mammalian target of rapamycin (mTOR) signaling. liferation in vitro, whereas increased mTOR signaling following This convergence on mTOR is well-illustrated in brain tumors Tsc1 loss, Tsc2 loss, or Rheb overexpression (>300-fold) has no (gliomas) in which mutations lead to mTOR-dependent growth such effect. This differential effect does not reflect the magni- deregulation. In this regard, copy-number increases or gain-of- tude of mTOR activation as measured by S6 phosphorylation on function mutations in key RTK molecules (PDGFR, EGFR, and Ser residues 240/244 (Fig. 1 and Fig. S1). Moreover, the differ- ERBB2) enhance glioma growth by activating mTOR (1). Simi- ence between TSC/Rheb and neurofibromin/PTEN mTOR reg- PTEN ulation of astrocyte proliferation is not due to the activation of larly, loss-of-function mutations in the , tuberous sclerosis − − − − complex (TSC), and neurofibromatosis-1 (NF1) tumor suppres- other signaling pathways, as both Nf1 / and Pten / astrocyte sor genes result in glioma formation, which is reduced by phar- hyperproliferation is abolished following mTOR inhibition. To- macologic mTOR inhibition. The shared activation of mTOR by gether, these findings suggest that mTOR regulation by neuro- PTEN, TSC (tuberin and hamartin), and NF1 (neurofibromin) fibromin/PTEN is distinct from TSC/Rheb (Fig. 2A). loss has suggested a model in which protein kinase B (PKB)/AKT hyperactivation following PTEN or neurofibromin loss leads to Neurofibromin Growth Regulation of Astrocyte Proliferation Does tuberin phosphorylation (2–5), loss of tuberin/hamartin complex Not Depend on Rheb in Vitro. Prior analyses of neurofibromin inhibition of Ras homolog enriched in brain (Rheb) activity (6– mTOR growth control suggested that the Akt hyperactivation 10), and Rheb-mediated mTOR activation (11–14) relevant to resulting from Nf1 inactivation (Fig. S2A) leads to tuberin glioma growth and targeted therapy. However, several experimental findings call this unified TSC/ Rheb model of mTOR regulation into question. First, although Author contributions: S.B. and N.R.C. designed research; S.B., N.R.C., R.J.E., and S.M.G. Nf1 and Pten inactivation cause increased astroglial cell growth performed research; S.B. and N.R.C. analyzed data; and S.B., N.R.C., and D.H.G. wrote the in vitro, no such increase in proliferation is observed following paper. Tsc1 loss or Rheb overexpression (15). Second, Tsc1 loss in The authors declare no conflict of interest. astrocytes results in increased cell size in the brain, whereas Nf1 *This Direct Submission article had a prearranged editor. inactivation in vitro or using the same Cre driver mouse strain 1To whom correspondence should be addressed. E-mail: [email protected]. Tsc1 in vivo has no discernable effect on cell size (16, 17). Third, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. inactivation in neuroglial progenitor cells results in intractable 1073/pnas.1019012108/-/DCSupplemental.

15996–16001 | PNAS | September 20, 2011 | vol. 108 | no. 38 www.pnas.org/cgi/doi/10.1073/pnas.1019012108 Downloaded by guest on September 30, 2021 Fig. 1. Neurofibromin and Pten loss in astrocytes results in increased mTOR- dependent proliferation. Whereas − − − − Nf1 / (A)andPten / (B) astrocytes exhibit increased mTOR activation (phospho-S6 Ser-240/244) and proliferation sensitive to 10 nM rapamycin inhibition (C and D), Tsc1-deficient (E) and Rheb-expressing (F)astrocytesex- hibit robust mTOR activation but no increase in proliferation in vitro. Total S6 and α-tubulin were included as internal loading controls. Asterisks denote statistically significant differences − − (P ≤ 0.0001) between WT and Nf1 / or − − Pten / astrocytes. Bars indicate SEM.

− − phosphorylation and inactivation (21). First, we observed no Rheb in Nf1 / astrocytes (Fig. 2B and Fig. S2D). In contrast, change in tuberin phosphorylation (residues Thr-1462 and Ser- S6 phosphorylation is attenuated by Rheb silencing in Tsc1- − − 969) in Nf1 / astrocytes relative to wild-type astrocytes (Fig. deficient astrocytes. Third, following Rheb knockdown in Nf1- S2B). Similarly, we observed no change in tuberin Ser-969 fi −/− de cient or WT astrocytes, we observed no reproducible changes phosphorylation in Pten astrocytes compared with wild-type in proliferation (Fig. 2C and Fig. S2 E and F). Collectively, these astrocytes (Fig. S2C). Second, we found no change in S6 phos- observations demonstrate that neurofibromin regulation of as- phorylation following small hairpin RNA (shRNA) silencing of trocyte mTOR signaling and proliferation is not dependent on TSC/Rheb function.

Conditional Rheb Activation in Mice Results in Increased Astrocyte mTOR Signaling and Proliferation in Vivo. To extend the above in vitro studies, we next generated conditional Rheb-expressing mice (Fig. 3A). The conditional Rheb transgene was engineered into the Rosa locus by homologous recombination, resulting in two independent strains of LSL-Rheb mice (lines 13431 and 13433). In addition, the transgene contained a reverse tetracycline control element, allowing doxycycline regulation of Rheb expression as well as a downstream enhanced green fluorescent protein (eGFP) driven from an internal ribosomal entry site (IRES). We verified the inducible and regulatable expression of Rheb in primary forebrain astrocytes from these mice. Following infection with Ad5-Cre virus, robust GFP and Rheb expression was observed, which was abrogated following doxycycline treatment (line 13431; Fig. 3B). No GFP or Rheb expression was observed in identical astrocytes infected with Ad5-LacZ. Similar results were also obtained using line 13433 (Fig. S3). Moreover, as seen following viral Rheb expression (Fig. 1F), transgenic expression of Rheb resulted in mTOR activation (S6 Ser-240/244 and Ser-235/236 phosphorylation; Fig. 3C), but no increase in astrocyte proliferation (Fig. 3D). Next, we sought to determine whether Rheb overexpression in GFAP+ cells had similar effects as Tsc1 or Nf1 inactivation in vivo. For these experiments, both LSL-Rheb lines were intercrossed with GFAP-Cre mice. In contrast to our previous Fig. 2. Nf1-deficient astrocyte hyperproliferation is not dependent on Rheb Tsc1GFAP fi results in CKO mice, we found no increase in body or expression. (A) Convergence on mTOR pathway by neuro bromin, PTEN, A Tsc1GFAP TSC, and Rheb. (B) Rheb shRNA silencing reduces mTOR activation in Tsc1- brain weight (Fig. 4 ). However, similar to CKO mice − − deficient astrocytes (B; Right), but has no effect on mTOR activity in Nf1 / (17), Rheb overexpression resulted in increased mTOR signaling astrocytes (B; Left). Representative blots are shown. (C) Rheb shRNA (number of S6 phospho-Ser-240/244-immunoreactive cells; Fig. knockdown in Nf1-deficient astrocytes does not reduce cell proliferation. 4B), proliferation (Fig. 4C), and astrocytes (GFAP-immunore- Bars indicate SEM of six replicate wells per condition for the representative active cells; Fig. 4D) without any change in apoptosis (Fig. S4). experiment shown in B (Left). Similar results were obtained with both LSL-Rheb strains. These MEDICAL SCIENCES

Banerjee et al. PNAS | September 20, 2011 | vol. 108 | no. 38 | 15997 Downloaded by guest on September 30, 2021 Fig. 3. Conditional expression of Rheb in astrocytes does not increase cell growth in vitro. (A) Conditional targeting strategy for the generation of LSL-Rheb mice (line 13431). (B) Ad5 virus-mediated Cre delivery results in eGFP (Left; immunoﬂuorescence microscopy) and Rheb (Right; Western blot) expression compared with LacZ delivery. Both eGFP and Rheb expression in Cre-transduced astrocytes is elim- inated with doxycycline. (C) Cre-mediated Rheb expression results in increased mTOR signaling (phospho-S6 Ser-240/244 and Ser-235/236). (D) No change in astrocyte proliferation was observed following Cre-mediated Rheb expression as assessed by [3H]thymidine incorporation. Bars indicate SEM.

− findings demonstrate that Rheb overexpression, similar to Tsc1 (n = 5 mice analyzed). In this regard, optic nerves in Nf1+/ ; inactivation, is sufficient to increase astrocyte numbers in vivo. RhebGFAP mice were indistinguishable from their wild-type counterparts on gross examination and had optic nerve volumes − Rheb Activation Is Insufficient for Gliomagenesis in Vivo. Previous similar to control mice (Fig. 5B). As observed in the brain, Nf1+/ ; studies from our laboratory have shown that Nf1 loss in astro- RhebGFAP mouse optic nerves had more proliferating cells (Fig. cytes alone is not sufficient for glioma formation unless coupled 5C) but no optic glioma, as judged by focal collections of GFAP- − − with reduced Nf1 expression in the brain (Nf1+/ mice) (16, 22). immunoreactive cells (Fig. 5D). In contrast, Nf1+/ GFAPCKO − The requirement for both an Nf1+/ brain environment and Nf1 mice developed optic gliomas with kinking of the prechiasmatic loss in astrocytes provides a unique in vivo platform to assess the optic nerves and chiasmal enlargement (Fig. 5B), increased impact of neurofibromin downstream signaling pathway activity numbers of proliferating cells (average number of Ki67+ cells = on gliomagenesis. Consistent with the known role of neuro- 6 per nerve; Fig. 5C), and more GFAP immunostaining (Fig. − fibromin as a negative regulator of KRas in astrocytes, Nf1+/ 5D). These observations demonstrate that Tsc1 loss or Rheb mice with astrocyte expression of an activated KRas allele de- overexpression is not equivalent to neurofibromin loss in vivo, − velop optic glioma similar to Nf1+/ GFAPCKO mice. The ability and establishes TSC/Rheb-independent mechanisms of mTOR of activated KRas to replace neurofibromin loss and result in regulation in the brain relevant to glial cell growth and tumor − optic glioma formation in Nf1+/ mice was exploited to evaluate formation. the impact of other mTOR-activating events on gliomagenesis. First, we used an LSL-myr-Akt strain (23) to determine whether Discussion constitutive Akt activation is sufficient for optic glioma forma- Numerous studies have highlighted the central role of Akt axis − tion in Nf1+/ mice. Unfortunately, due to tissue mosaicism, the signaling in astrocyte differentiation and growth. Astrocyte dif- LSL-Akt transgene was not expressed in the optic nerve. This ferentiation from neural stem cells is controlled by Akt signaling limitation precluded our ability to directly examine the impact of through STAT3, N-CoR, or p27 deregulation (24–26), whereas Akt hyperactivation on optic nerve gliomagenesis. Akt/mTOR hyperactivation is observed in reactive astrocytes Because mice with GFAP conditional Pten inactivation die in responding to spinal cord injury (27). Similarly, Akt drives as- the early postnatal period (18), we focused on experiments using trocyte survival and proliferation initiated by RTK activation Tsc1 conditional knockout mice. For these studies, we inter- (28, 29). The importance of Akt/mTOR signaling to astrocyte fl fl − crossed Tsc1 ox/ ox mice with Nf1+/ and GFAP-Cre mice to proliferation is also underscored by glioma-associated tumor − develop Nf1+/ ; Tsc1GFAPCKO mice. Similar to our previous mutations, including the PTEN, TSC, and NF1 tumor suppressor studies using Tsc1GFAPCKO mice, Tsc1 inactivation resulted in genes. Investigations using genetically engineered mutant mice seizures and death by 6–10 wk of age. Whereas the vast majority and derivative astrocytes demonstrate that growth control is − of Nf1+/ ; Tsc1GFAPCKO mice died before 3 mo of age (n = 12), dependent on mTOR activation, such that Nf1-deficient and − − a time when Nf1+/ GFAPCKO mice and Nf1+/ ; KRasGFAP Pten-deficient astrocytes exhibit increased astrocyte proliferation transgenic mice develop optic glioma, three mice survived to in vitro and in vivo, which is inhibited by pharmacologic in- 12 wk of age. None of these mice (11.5, 12, and 13 wk of age) hibition of mTOR pathway function (18, 20, 30). were found to have optic gliomas on gross histologic analysis In contrast to PTEN and neurofibromin loss, Tsc1 loss and despite loss of Tsc1 expression. Rheb overexpression do not increase astrocyte growth in vitro. Because RhebGFAP mice appear healthy and do not develop Moreover, Rheb silencing did not reduce Nf1-deficient astrocyte − seizures, we intercrossed LSL-Rheb mice with Nf1+/ and proliferation or mTOR activation (pS6) in vitro, whereas Rheb − GFAP-Cre mice to generate Nf1+/ ; RhebGFAP mice. Unlike silencing attenuated mTOR activity in Tsc1-deficient astrocytes. LSL-Akt mice, there was robust Rheb transgene expression in Following Rheb overexpression or Tsc1 loss in astrocytes in vivo, the optic nerve (Fig. 5A). However, optic gliomas were not ob- there was a modest increase in cell proliferation and astrocyte − served in Nf1+/ ; RhebGFAP mice from either line at 3 mo of age numbers. This increase in astrocyte number prompted us to

15998 | www.pnas.org/cgi/doi/10.1073/pnas.1019012108 Banerjee et al. Downloaded by guest on September 30, 2021 Fig. 4. Conditional Rheb expression in astroglial cells results in increased glial numbers and proliferation in vivo. (A) No effect of Rheb expression on body size or brain weight was observed in RhebGFAP mice. (B) GFAP-Cre-mediated Rheb expression results in increased numbers of pS6 (Ser-240/244) cells, (C) Ki67+ proliferating cells, and (D) GFAP+ cells in vivo. Magniﬁcation = 10×.(Insets) Representative pS6+ (B) and Ki67+ (C) cells. (Scale bars, 100 μm.) Bars indicate SEM.

determine whether hamartin loss or Rheb overexpression could neurofibromin astrocyte growth control and gliomagenesis is substitute for neurofibromin loss in the genesis of optic glioma. TSC/Rheb-independent. Our findings are consistent with pre- − In contrast to Nf1 loss, gliomas were not observed in Nf1+/ ; vious studies in Drosophila demonstrating that tuberin is not − RhebGFAP or Nf1+/ ; Tsc1GFAPCKO mice. These findings dem- a critical substrate of Akt relevant to fly development (31). In this onstrate that whereas TSC/Rheb signaling is important for mTOR regard, we have also shown that the adhesion molecule on glia activation and glial cell proliferation, it is not equivalent to neu- regulates Akt/mTOR/S6K activation independently of Rheb (32). rofibromin loss, and establish two independent pathways to mTOR- The observation that neurofibromin regulates glial cell pro- regulated cell growth with different functional consequences. liferation in a TSC/Rheb-independent manner raises several Importantly, our results do not support previous conclusions issues. First, there are likely differences in signaling pathways that neurofibromin growth control mediated by mTOR is TSC/ activated following Nf1 loss in distinct cell types. We have re- Rheb-dependent (21). In this report, the TSC dependence was cently shown that neural stem cells from different brain regions largely based on increased tuberin phosphorylation on residues have varying responses to Nf1 inactivation, attributable to Thr-1462 and Ser-939 in Nf1-deficient mouse embryonic fibro- changes in mTOR complex protein (rictor versus raptor) levels blasts and tumor cells as well as the use of a phospho-Thr-1462/ (25). Similarly, PDK1-dependent Akt activation is differentially Ser-939 double mutant tuberin molecule. In contrast, we used regulated in neurons versus astrocytes as a result of cell type- a combination of Rheb silencing in vitro, Tsc1 inactivation specific activation of regulatory feedback pathways (33). In ad- in vivo, and conditional Rheb overexpression in vivo to show that dition, there are differences in mTOR pathway activation and MEDICAL SCIENCES

Banerjee et al. PNAS | September 20, 2011 | vol. 108 | no. 38 | 15999 Downloaded by guest on September 30, 2021 Fig. 5. Conditional Rheb expression in Nf1+/− mice is insufficient for glioma formation. (A) Robust eGFP expression is seen in the optic nerves of RhebGFAP mice. Magnification, 10×. (Scale bar, 100 − μm.) (B) Nf1+/ ; RhebGFAP mice have normal-appearing optic nerves and no increase in optic nerve volume, whereas Nf1+/−GFAPCKO mice develop prechiasmatic optic nerve and chiasmal fl fl gliomas. FF, Nf1 ox/ ox(WT). Magnifica- tion, 10×. (Scale bar, 100 μm.) (C and D) − Nf1+/ ; RhebGFAP mice have increased numbers of Ki67+ proliferating cells in the optic nerve but no focal areas of GFAP+ cells indicative of optic glioma, − whereas Nf1+/ GFAPCKO mice demonstrated both increased numbers of Ki67+ cells and GFAP immunostaining. Magni- fication, 20×. (Scale bars, 100 μm.) The arrows denote Ki67+ cells. Bars indicate SEM.

−/− proliferation between astrocytes and fibroblasts in response mechanism underlying mTOR activation in Nf1 glial cells. to Nf1, Tsc1, and Pten inactivation (7). For example, 4EBP1 The identification of Akt signaling intermediates responsible for phosphorylation is unaltered following Nf1 and Pten inactivation neurofibromin mTOR regulation will require further studies −/− in astrocytes, whereas Tsc1 astrocytes exhibit increased 4EBP1 aimed at discovering new potential regulators of mTOR or phosphorylation relative to their wild-type counterparts (Fig. S5). mTOR complex molecules in astrocytes. Second, TSC and Rheb may have additional functions that In summary, our results establish Akt-mediated TSC/Rheb- influence Akt/mTOR signaling. For example, loss of TSC ex- dependent and -independent mechanisms for mTOR growth pression disrupts Akt signaling through down-regulation of the regulation in the brain and demonstrate that neurofibromin platelet-derived growth factor receptor (PDGFR) (34). In ad- controls astrocyte growth in an Akt-dependent, but TSC/Rheb- dition, phosphodiesterase-4B (PDE4B) can bind to and inhibit independent, fashion relevant to gliomagenesis. As we move into Rheb function in a cAMP-dependent manner (35). Previous an era of targeted therapeutics, these differences in mTOR studies from our laboratory have revealed reduced cAMP levels growth regulation will need to be considered to develop the most in Nf1-deficient astrocytes (36). These lower cAMP levels would effective therapies for cancers with deregulated TOR signaling. be predicted to reduce mTOR signaling as a result of enhanced PDE4B–Rheb binding; however, we observed no changes in Methods mTOR activity following pharmacologic treatments that raise Mice. Nf1flox/flox, Tsc1flox/flox, and Ptenflox/flox mice were generated as pre- − − cAMP levels in Nf1-deficient astrocytes (Fig. S6A). Whereas viously described (16–18), whereas Nf1+/ ; LSL-Rheb; GFAP-Cre and Nf1+/ ; fl fl tuberin/Rheb has been reported to regulate MAPK activity via Tsc1 ox/ ox; GFAP-Cre mice were generated by successive intercrossing (16, B-RAF (37), we observed no change in MAPK activation in ei- 22, 41) with age-matched mice as controls. GFAP-Cre and LSL-Rheb litter- ther Tsc1-deficient or Rheb-expressing astrocytes (Fig. S6B). The mates were indistinguishable from C57BL/6 mice. All mice were maintained increase in MAPK activity in Nf1-deficient astrocytes results on a C57BL/6 background and used in accordance with active animal studies from Ras hyperactivation and is not attenuated by PI3K in- protocols at Washington University School of Medicine. hibition, as might be predicted if Akt activation silenced TSC function by tuberin phosphorylation to increase Rheb/BRAF- Generation of Conditional Rheb-Expressing Mice. LSL-Rheb mice were gener- mediated MAPK signaling. ated by a knock-in mouse strategy using a backbone knock-in ROSA-26- Third, although neurofibromin astrocyte growth regulation tetracycline-regulated transcriptional activator (tTA) vector provided by does not require TSC/Rheb signaling, the precise mechanism S. Miyazaki (Osaka University Graduate School of Medicine, Osaka, Japan). − − fl underlying Nf1 / astrocyte mTOR activation is unclear. Al- This construct included a splice acceptor sequence, a loxP- anked neomycin though neurofibromin activation of mTOR is Akt-dependent phosphotransferase gene with a polyA sequence, the pUHD15-1 tTA gene, (Fig. S6C), it does not involve direct mTOR phosphorylation a separate polyA sequence, an insulator sequence, the CMV*1 promoter D A responsive to tTA, the rabbit β-globin second intron, the rat Rheb2 gene (Fig. S6 ), cAMP signaling (Fig. S6 ), or changes in either (HA-Rheb generated by PCR), an IRES, the eGFP gene, and another polyA DEPTOR expression (38) (Fig. S6E) or phospholipase D activity fi F sequence (42) (Fig. 3A). Three ES cell lines were chosen, veri ed by kar- (39) (Fig. S6 ). Interestingly, we found that PRAS40 phos- yotyping, and used to generate LSL-Rheb knock-in mice by blastocyst in- Nf1−/− Tsc1−/− phorylation was increased in , but not , astrocytes jection. Two chimeric founder mice with germ-line transmission (lines 13431 (Fig. S7 A and B), suggesting that neurofibromin loss might lead and 13433) were identified. to Akt-mediated PRAS40 phosphorylation (inactivation) and reduced mTOR inhibition (40). However, shRNA-mediated Primary Astrocytes. PN2 forebrain astrocyte cell cultures from LSL-Rheb, PRAS40 silencing in WT astrocytes, to mimic PRAS40 in- Nf1flox/flox, Tsc1flox/flox,andPtenflox/flox mice were generated as previously activation in Nf1-deficient astrocytes, decreased S6 activation described (16, 17, 20). Tsc2-deficient and control astrocytes were provided by (Fig. S7C) and reduced astrocyte proliferation (Fig. S7D), argu- Michael Wong (Washington University). Adenovirus type 5 containing β-ga- ing against differential PRAS40 phosphorylation as the likely lactosidase (Ad5-LacZ) and Cre recombinase (Ad5-Cre) (University of Iowa

16000 | www.pnas.org/cgi/doi/10.1073/pnas.1019012108 Banerjee et al. Downloaded by guest on September 30, 2021 − − Gene Transfer Core, Iowa City, IA) were used to produce wild-type and Nf1 / , chemistry was performed on adjacent paraffinsectionswithGFAP − − − − Tsc1 / , and Pten / or Rheb-expressing astrocytes, respectively. Neuro- (Invitrogen), Ki67 (BD PharMingen), and pS6 (Cell Signaling) antibodies (16). fibromin, hamartin, and Pten loss or Rheb overexpression was confirmed Immunoreactivity was visualized with the Vectastain avidin–biotin complex by immunoblot analysis 5 d after infection (Fig. S1). All experiments were and 3,3′-diaminobenzidine (Vector Laboratories). TUNEL labeling was performed on passage-1 or -2 cultures at least three times with similar results. performed as previously described (44). Representative sections were photographed with a digital camera (Optronics) attached to an inverted Retroviral and Lentiviral Constructs and Viral Delivery. Mouse-specific shRNA microscope (Nikon). lentivirus against Rheb (Sigma; GenBank accession no. NM_053075; − − − − TRCN0000075607) was used to silence Rheb in Tsc1 / and Nf1 / astrocytes Western Blotting. Cells were lysed in Nonidet P-40 lysis buffer with protease following two infections (25). Empty pLKO.1 virus was used as a control. Murine stem cell virus (MSCV)-containing rat Rheb2 was generated follow- and phosphatase inhibitors. All antibodies were used at the stated dilutions ing 293T cell transfection with τ-helper DNA using Fugene HD (Roche) (20). (Table S1). Western blotting was performed as previously described (43). Forty-eight hours later, virus-containing supernatants were filtered through 0.45-μm syringe filters, and astrocytes were infected thrice and harvested Optic Glioma Measurements. The entire optic nerve with the eye and intact 72 h later. MSCV-GFP was used as a control. chiasm was microdissected and photographed with a scale bar. Prechiasmatic optic nerve volumes were calculated as previously reported (44). Pharmacologic Inhibitors. Rapamycin (LC Laboratories) was used at a concen- tration of 10 nM for treating the cells for 16–18 h unless otherwise indicated. ACKNOWLEDGMENTS. We thank Dr. Renate Lewis for generating the LSL- Rheb targeting construct, Dr. Joshua Rubin for additional Nf1flox/flox astro- −/− Cell Proliferation. Cell proliferation was performed using [3H]thymidine cytes, and Dr. Michael Wong for the Tsc2 astrocytes. We also thank Ms. Cori DeSanto for expert technical assistance. This study was partially (1 μCi/mL) as previously described (43). funded by Grant NF050176 from the Department of Defense (to D.H.G.), National Cancer Institute Grant CA127008 (to D.H.G.), and the Hope Center Immunohistochemistry. Mice were perfused transcardially with 4% para- Transgenic Vector Core Facility at the Washington University School of formaldehyde in PBS. Following vibratome sectioning, immunohisto- Medicine.

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