Persons affected with tuberous sclerosis complex (TSC) develop a wide range of neurological abnormalities including aberrant neuronal migration and seizures. In an effort to model TSC-associated central nervous system abnormalities in mice, we generated two independent lines of astrocyte-specific Tsc1 conditional knockout mice by using the Cre-LoxP system. Astrocyte-specific Tsc1-null mice exhibit electroencephalographically proven seizures after the first month of age and begin to die at 3 to 4 months. Tsc1-null mice show significant increases in astrocyte numbers throughout the brain by 3 weeks of age and abnormal neuronal organization in the hippocampus between 3 and 5 weeks. Moreover, cultured Tsc1-null astrocytes behave similar to wild-type astrocytes during log phase growth but demonstrate increased saturation density associated with reduced p27(Kip1) expression. Collectively, our results demonstrate that astrocyte-specific disruption of Tsc1 in mice provides a context-dependent growth advantage for astrocytes that results in abnormalities in neuronal organization and epilepsy.

Individuals with the neurofibromatosis 1 (NF1) inherited tumor syndrome develop low-grade gliomas (astrocytomas) at an increased frequency, suggesting that the NF1 gene is a critical growth regulator for astrocytes. In an effort to determine the contribution of the NF1 gene product, neurofibromin, to astrocyte growth regulation and NF1-associated astrocytoma formation, we generated astrocyte-specific Nf1 conditional knockout mice (Nf1 GFAP CKO) by using Cre/LoxP technology. Transgenic mice were developed in which Cre recombinase was specifically expressed in astrocytes by embryonic day 14.5. Successive intercrossing with mice bearing a conditional Nf1 allele (Nf1flox) resulted in GFAP-Cre Nf1flox/flox (Nf1 GFAP CKO) animals. No astrocytoma formation or neurological impairment was observed in Nf1 GFAP CKO mice after 20 months, but increased numbers of proliferating astrocytes were observed in several brain regions. To determine the consequence of Nf1 inactivation at different developmental times, the growth properties of embryonic day 12.5 and postnatal day 2 Nf1 null astrocytes were analyzed. Nf1 null astrocytes exhibited increased proliferation but lacked tumorigenic properties in vitro and did not form tumors when injected into immunocompromised mouse brains in vivo. Collectively, our results suggest that loss of neurofibromin is not sufficient for astrocytoma formation in mice and that other genetic or environmental factors might influence NF1-associated glioma tumorigenesis.Neurofibromatosis 1 (NF1) is the most common cancer predisposition syndrome affecting the nervous system, with an incidence of 1 in 3,500 births worldwide (16). Early in life, individuals with NF1 develop café-au-lait spots, skinfold freckling, and iris hamartomas (Lisch nodules). In addition, approximately 15 to 20% of children with NF1 develop glial cell tumors (astrocytomas) involving the optic nerve, chiasm, hypothalamus, and brain stem (31). Although classified as grade I juvenile pilocytic astrocytomas, these tumors can be associated with significant morbidity as a result of visual loss or neurological compromise.Since individuals affected with NF1 develop tumors at an increased frequency, the NF1 gene is hypothesized to function as a tumor suppressor. Identification of the NF1 gene (10,42,43) and its protein product, the 220-to 250-kDa cytoplasmic neurofibromin protein, revealed that a small portion of the molecule has sequence similarity with the catalytic domain of a family of proteins termed GTPase-activating proteins (GAPs) (4,34,45). GAP molecules function as negative regulators of small mitogenic GTPase proteins, like p21 ras , and inactivate these signaling proteins by accelerating their conversion from active, GTP-bound conformations to inactive, GDP-bound forms (6). In NF1-associated tumors, loss of neurofibromin results in increased p21 ras mitogenic signaling and augments cell proliferation, leading to tumor formation (5,7,12).

Development 5578EGF receptors has been well documented in diffusely infiltrative malignant astrocytomas (Holland, 2001;Kleihues and Cavenee, 2000;Maher et al., 2001;. However, similar molecular alterations have not been observed in pilocytic astrocytomas (Gutmann et al., 2002;Li et al., 2001). It has been suggested that the NF1 gene might be involved in regulating the proliferation of mature astrocytes (Bajenaru et al., 2002). Although neurofibromin is expressed below detection levels in normal astrocytes (Daston and Ratner, 1992; Daston et al., 1992; Huynh et al., 1994), it was reported that loss of NF1 confers a growth advantage to neonatal astrocytes in vitro (Bajenaru et al., 2002). Conventional NF1 knockout mice (Nf1 -/-) are embryonic lethal (Brannan et al., 1994; Jacks et al., 1994) and although heterozygous mice (Nf1 +/-) are cancer prone, they do not develop astrocytomas (Jacks et al., 1994). Conditional mutant mice lacking NF1 specifically in neurons also fail to develop astrocytomas although increased number of non-neoplastic GFAP (glial fibrillary acidic protein) expressing reactive astrocytes was observed (Zhu et al., 2001). These results suggest that NF1 can regulate the growth of astrocytes both intrinsically and also indirectly through neurons.During embryonic development, multipotent neural stem/progenitor cells progressively lose developmental potential and become lineage-restricted neuronal progenitor cells or glial progenitor cells (Gage, 2000;Temple, 2001). Gliogenesis occurs after neurogenesis and extends into postnatal stages. In the setting of NF1, the greatest risk for development of optic glioma is the first 6 years of life (Listernick et al., 1999). This observation suggests that the NF1 gene might play a role in regulating the proliferation of progenitor cells. Furthermore, recent reports demonstrate that pilocytic astrocytomas express molecular markers reminiscent of glial progenitor cells (Gutmann et al., 2002;Li et al., 2001).To determine the role of NF1 in the development of neural cell types and understand cellular and molecular basis of NF1-associated astrocytoma formation, we used a bacteriophage Cre/loxP system to target a Nf1 mutation (Zhu et al., 2001) into multipotent neural stem/progenitor cells and their derivatives, including glia and neurons (Zhuo et al., 2001). We show that loss of NF1 promotes the proliferation of glial progenitor cells resulting in increased numbers of GFAP-expressing astrocytes in both developing and adult brains. Furthermore, NF1 also plays an indispensable role in the maintenance of the differentiation state of mature astrocytes. Finally, we describe a new mouse model for NF1-associated optic pathway glioma. Materials and methods Control and mutant miceThe control mice used in this study are the pool of phenotypically indistinguishable mice with genotypes ;hGFAP-cre+, which have similar phenotypes. The genotyping procedures for the Nf1flox allele, Nf1-null allele and Cre transgene have been described previously (Zhu et al., 2001). Size and weigh...

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High-grade gliomas are devastating brain tumors associated with a mean survival of <50 weeks. Two of the most common genetic changes observed in these tumors are overexpression/ mutation of the epidermal growth factor receptor (EGFR) vIII and loss of PTEN/MMAC1 expression. To determine whether somatically acquired EGFRvIII expression or Pten loss accelerates high-grade glioma development, we used a previously characterized RasB8 glioma-prone mouse strain, in which these specific genetic changes were focally introduced at 4 weeks of age. We show that both postnatal EGFRvIII expression and Pten inactivation in RasB8 mice potentiate high-grade glioma development. Moreover, we observe a concordant loss of Pten and EGFR overexpression in nearly all high-grade gliomas induced by either EGFRvIII introduction or Pten inactivation. This novel preclinical model of highgrade glioma will be useful in evaluating brain tumor therapies targeted to the pathways specifically dysregulated by EGFR expression or Pten loss. (Cancer Res 2006; 66(15): 7429-37)