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.

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Tuberous sclerosis complex (TSC) is an autosomal dominant syndrome in which affected individuals develop nervous system abnormalities that might reflect astrocyte dysfunction. The TSC2 gene product, tuberin, encodes a GTPase-activating protein (GAP) domain, which regulates the activity of Rap1 in vitro. To determine whether dysregulated Rap1, resulting from TSC2 inactivation, leads to increased astrocyte proliferation in vivo, we generated transgenic mice expressing activated Rap1 G12V specifically in astrocytes. We observed no statistically significant difference in the number of astrocytes between wild-type and GFAP-Rap1 G12V littermates in vivo; however, during log-phase growth, we observed a 25% increase in GFAP-Rap1 G12V astrocyte doubling times compared to wild-type controls. This decreased proliferation was associated with delayed MAP kinase, but not AKT, activation. Lastly, to determine whether constitutive Rap1 activation could reverse the increased astrocyte proliferation observed in transgenic mice expressing oncogenic Ras G12V , we generated transgenic mice expressing both Ras G12V and Rap1 G12V in astrocytes. These double transgenic mice showed a striking reversion of the Ras G12V astrocyte growth phenotype. Collectively, these results argue that the tumor suppressor properties of tuberin are unlikely to be related to Rap1 inactivation and that Rap1 inhibits mitogenic Ras pathway signaling in astrocytes.

The development of malignant gliomas (astrocytomas) involves the accumulation of multiple genetic changes, including mutations in the p53 and retinoblastoma (Rb) cell cycle regulatory pathways. One Rb pathway alteration seen in high-grade astrocytomas is ampli®ca-tion of cyclin dependent kinase-4 (CDK4). To de®ne the function of CDK4 ampli®cation/overexpression in astrocytoma pathogenesis, we generated three transgenic mouse lines that overexpress human CDK4 (hCDK4) in astrocytes using the human glial ®brillary acidic protein (GFAP) promoter. GFAP-hCDK4 mice do not develop brain tumors, but exhibit a small increase in astrocyte number. Cultured astrocytes from these mice do not demonstrate a cell-autonomous growth advantage in vitro and lack properties of transformed cells. To determine whether cdk4 overexpression provides a cooperative growth advantage in vitro, CDK4-overexpressing C6 glioma cell lines were generated and found to exhibit increased cell growth. In addition, GFAP-hCDK4; p53+/ 7 as well as p53+/7; Rb+/7 mice exhibited increased numbers of astrocytes compared to GFAP-hCDK4, p53+/7, or Rb+/7 mice in vivo. No cooperative e ect was observed with GFAP-hCDK4; Rb+/7 mice. These results support the hypothesis that cdk4 overexpression alone is not su cient for astrocytoma formation, but can provide a cooperative growth advantage in concert with genetic alterations in the p53 pathway.