Erik J. Uhlmann

Astrocyte-specific TSC1 conditional knockout mice exhibit abnormal neuronal organization and seizures.

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 p27Kip1 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.

Pten Loss Causes Hypertrophy and Increased Proliferation of Astrocytes In vivo

Somatic mutations of PTEN are found in many types of cancers including glioblastoma, the most malignant astrocytic tumor. PTEN mutation occurs in 25 to 40% of glioblastomas but is rarely observed in low-grade glial neoplasms. To determine the role of Pten in astrocytes and glial tumor formation, we inactivated Pten by a Cre-loxP approach with a GFAP-cre transgenic mouse that induced Cre-mediated recombination in astrocytes. Pten conditional knockout mice showed a striking progressive enlargement of the entire brain. Increased nuclear and soma size was observed in both astrocytes and neurons, which contributed in part to the increase in brain size. Pten-deficient astrocytes showed accelerated proliferation in vitro and aberrant ongoing proliferation in adult brains in vivo. In contrast, neurons lacking Pten did not show alterations in proliferation. This study shows cell-type dependent effects of Pten loss in the adult brain, including increased astrocyte proliferation that may render astroglial cells susceptible to neoplastic transformation or malignant progression.

Impaired glial glutamate transport in a mouse tuberous sclerosis epilepsy model

Excessive astrocytosis in cortical tubers in tuberous sclerosis complex (TSC) suggests that astrocytes may be important for epileptogenesis in TSC. We previously demonstrated that astrocyte‐specific Tsc1 gene inactivation in mice (Tsc1 cKO mice) results in progressive epilepsy. Here, we report that glutamate transporter expression and function is impaired in Tsc1 cKO astrocytes. Tsc1 cKO mice exhibit decreased GLT‐1 and GLAST protein expression. Electrophysiological assays demonstrate a functional decrease in glutamate transport currents of Tsc1 cKO astrocytes in hippocampal slices and astrocyte cultures. These findings suggest that Tsc1 inactivation in astrocytes causes dysfunctional glutamate homeostasis, leading to seizure development in TSC. Ann Neurol 2003

Epileptogenesis and Reduced Inward Rectifier Potassium Current in Tuberous Sclerosis Complex-1-Deficient Astrocytes

Summary:  Purpose: Individuals with tuberous sclerosis complex (TSC) frequently have intractable epilepsy. To gain insights into mechanisms of epileptogenesis in TSC, we previously developed a mouse model of TSC with conditional inactivation of the Tsc1 gene in glia (Tsc1GFAPCKO mice). These mice develop progressive seizures, suggesting that glial dysfunction may be involved in epileptogenesis in TSC. Here, we investigated the hypothesis that impairment of potassium uptake through astrocyte inward rectifier potassium (Kir) channels may contribute to epileptogenesis in Tsc1GFAPCKO mice.

Identification of Dominant Negative Mutants of Rheb GTPase and Their Use to Implicate the Involvement of Human Rheb in the Activation of p70S6K

Rheb GTPases represent a unique family of the Ras superfamily of G-proteins. Studies on Rheb in Schizosaccharomyces pombe and Drosophila have shown that this small GTPase is essential and is involved in cell growth and cell cycle progression. The Drosophila studies also raised the possibility that Rheb is involved in the TOR/S6K signaling pathway. In this paper, we first report identification of dominant negative mutants of S. pombe Rheb (SpRheb). Screens of a randomly mutagenized SpRheb library yielded a mutant, SpRhebD60V, whose expression in S. pombe results in growth inhibition, G1 arrest, and induction of fnx1+, a gene whose expression is induced by the disruption of Rheb. Alteration of the Asp-60 residue to all possible amino acids by site-directed mutagenesis led to the identification of two particularly strong dominant negative mutants, D60I and D60K. Characterization of these dominant negative mutant proteins revealed that D60V and D60I exhibit preferential binding of GDP, while D60K lost the ability to bind both GTP and GDP. A possible use of the dominant negative mutants in the study of mammalian Rheb was explored by introducing dominant negative mutations into human Rheb. We show that transient expression of the wild type Rheb1 or Rheb2 causes activation of p70S6K, while expression of Rheb1D60K mutant results in inhibition of basal level activity of p70S6K. In addition, Rheb1D60K and Rheb1D60V mutants blocked nutrient- or serum-induced activation of p70S6K. This provides critical evidence that Rheb plays a role in the mTOR/S6K pathway in mammalian cells.

Brain metastases in patients with EGFR-mutated or ALK-rearranged non-small-cell lung cancers

INTRODUCTION Brain metastases (BM) are common in non-small-cell lung cancer (NSCLC). However, the baseline incidence and evolution of BM over time in oncogene-driven NSCLCs are seldom reported. In this study, we evaluated the frequency of BM in patients with epidermal growth factor receptor (EGFR)-mutated or anaplastic lymphoma kinase (ALK)-rearranged NSCLC. METHODS The presence of BM, clinicopathologic data, and tumor genotype were retrospectively compiled and analyzed from a cohort of 381 patients. RESULTS We identified 86 EGFR-mutated (90.7% with metastatic disease; 85.9% received an EGFR inhibitor) and 23 ALK-rearranged (91.3% with metastatic disease; 85.7% received an ALK inhibitor) NSCLCs. BM were present in 24.4% of EGFR-mutated and 23.8% of ALK-rearranged NSCLCs at the time of diagnosis of advanced disease. This study did not demonstrate a difference in the cumulative incidence of BM over time between the two cohorts (EGFR/ALK cohort competing risk regression [CRR] coefficient of 0.78 [95% CI 0.44-1.39], p=0.41). In still living patients with advanced EGFR-mutated NSCLC, 34.2% had BM at 1 year, 38.4% at 2 years, 46.7% at 3 years, 48.7% at 4 years, and 52.9% at 5 years. In still living patients with advanced ALK-rearranged NSCLC, 23.8% had BM at 1 year, 45.5% at 2 years, and 58.4% at 3 years. CONCLUSIONS BM are frequent in advanced EGFR-mutated or ALK-rearranged NSCLCs, with an estimated >45% of patients with CNS involvement by three years of survival with the use of targeted therapies. These data point toward the CNS as an important unmet clinical need in the evolving schema for personalized care in NSCLC.

Loss of tuberous sclerosis complex 1 (Tsc1) expression results in increased Rheb/S6K pathway signaling important for astrocyte cell size regulation.

Individuals with tuberous sclerosis complex (TSC) develop central nervous system abnormalities that may reflect astrocyte dysfunction. In an effort to model astrocyte dysfunction in TSC, we generated mice lacking Tsc1 (hamartin) expression in astrocytes and demonstrated that Tsc1‐null astrocytes exhibit abnormalities in contact inhibition growth arrest. In this study, we demonstrate that hamartin‐deficient astrocytes are also defective in cell size regulation. We show that the increase in Tsc1‐null astrocyte size is associated with increased activation of the S6‐kinase pathway. In keeping with recent reports that the hamartin/tuberin complex may regulate Rheb and downstream S6K activation, we demonstrate that expression of either Rheb or S6K in primary astrocytes results in increased S6 pathway activation, and that inhibition of Rheb activity in Tsc1‐deficient astrocytes using either pharmacologic or genetic strategies markedly reduces S6 activation. Collectively, these observations suggest that TSC inactivation in astrocytes results in defective cell size regulation associated with dysregulated Rheb/mTOR/S6K pathway activity.

Prestorage universal WBC reduction of RBC units does not affect the incidence of transfusion reactions

BACKGROUND: Febrile nonhemolytic transfusion reaction (FNHTR) has been identified as a pivotal reason for prestorage universal WBC reduction. A regional blood center implemented universal prestorage WBC reduction for RBCs on January 1, 2000. Whether prestorage universal WBC reduction of RBC units will affect FNHTR is not known.

Heterozygosity for the tuberous sclerosis complex (TSC) gene products results in increased astrocyte numbers and decreased p27-Kip1 expression in TSC2+/- cells.

Tuberous sclerosis complex (TSC) is an autosomal dominant tumor predisposition syndrome characterized by benign proliferations (hamartomas). In the brain, individuals with TSC develop autism, mental retardation and seizures associated with focal cortical dysplasias, subependymal nodules, and subependymal giant cell astrocytomas (SEGAs). We hypothesize that dysregulated astrocyte function due to mutations in the tumor suppressor genes, TSC1 and TSC2, may contribute to the pathogenesis of these brain abnormalities. In this report, we demonstrate that mice heterozygous for a targeted defect in either the Tsc1 or Tsc2 genes(Tsc1+/− and Tsc2+/− mice) exhibit a 1.5-fold increase in the number of astrocytes in vivo. Whereas increased astrocyte numbers in vivo were suggestive of a proliferative advantage, Tsc2+/− primary astrocyte cultures did not show a cell-autonomous growth advantage, anchorage-independent growth, increased saturation density, or increased fluid-phase endocytosis compared to wild type astrocytes. Tsc2 null mouse embryonic fibroblasts (MEFs) however, did exhibit increased saturation density compared to Tsc2 wild type controls. In both Tsc2+/− astrocytes and Tsc2 null mouse embryonic fibroblasts, p27-Kip1 expression was decreased compared to wild type cells, and was reversed by tuberin re-expression in Tsc2−/− MEFs. In contrast, no change in endocytosis was observed upon tuberin re-expression in Tsc2−/− MEFs. Collectively, these results suggest Tsc heterozygosity may provide a non-cell-autonomous growth advantage for astrocytes that may involve p27-Kip1 expression.

A Potent Cell Death Activity Associated with Transient High Level Expression of BCL-2

The BCL-2 proto-oncogene contains unusually long untranslated 5′ and 3′ sequences. Deletion of the sequences flanking the BCL-2 open reading frame dramatically increases the level of protein expression. Transient high level BCL-2 protein expression mediated by plasmid transfection or by infection with recombinant adenovirus results in potent apoptosis of several cell lines. Detailed mutational (deletion and add-back) analysis reveals that both 5′- and 3′-flanking sequences contribute to the negative modulation of protein expression from the BCL-2 open reading frame. It appears that these sequences exert the negative regulatory effect in an orientation-dependent manner. Analysis of BCL-2RNA levels indicate that elevated levels of mRNA may be the primary cause of elevated levels of protein expression. Apoptosis induced by adenovirus vectors expressing elevated levels of BCL-2 can be readily inhibited by the caspase inhibitor z-VAD-fmk, suggesting that high levels of BCL-2 expression induce apoptosis via the caspase cascade. Mutational analysis of BCL-2 indicates that its pro-apoptotic activity is separable from its anti-apoptosis activity. Our results raise the possibility that oncogenic conversion of BCL-2 may require somatic mutations in the pro-apoptotic activity, in addition to other activating mutations that result in enhanced expression. Consistent with this hypothesis, a somatic mutation of BCL-2 observed in multiple human tumors results in reduced apoptosis activity.