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Converging evidence from the analysis of human brain tumors and genetically engineered mice has revealed that the mammalian target of rapamycin (mTOR) pathway is a central regulator of glial and glioma cell growth. In this regard, mutational inactivation of neurofibromatosis-1 (NF1), tuberous sclerosis complex (TSC), and PTEN genes is associated with glioma formation, such that pharmacologic inhibition of mTOR signaling results in attenuated tumor growth. This shared dependence on mTOR suggests that PTEN and NF1 (neurofibromin) glial growth regulation requires TSC/Rheb (Ras homolog enriched in brain) control of mTOR function. In this report, we use a combination of genetic silencing in vitro and conditional mouse transgenesis approaches in vivo to demonstrate that neurofibromin regulates astrocyte cell growth and glioma formation in a TSC/Rheb-independent fashion. First, we show that Nf1 or Pten inactivation, but not Tsc1 loss or Rheb overexpression, increases astrocyte cell growth in vitro. Second, Nf1-deficient increased mTOR signaling and astrocyte hyperproliferation is unaffected by Rheb shRNA silencing. Third, conditional Tsc1 inactivation or Rheb overexpression in glial progenitors of Nf1(+/-) mice does not lead to glioma formation. Collectively, these findings establish TSC/Rheb-independent mechanisms for mTOR-dependent glial cell growth control and gliomagenesis relevant to the design of therapies for individuals with glioma.

The neurofibromatosis type 1 (NF1) tumor predisposition syndrome provides an illustrative example of brain tumor formation and growth in which a permissive microenvironment (stroma) is required for the expansion and maintenance of the neoplastic cells. In this chapter, we review the experimental evidence that supports the emerging concept that brain tumors are dynamic ecosystems where interactions between non-neoplastic and neoplastic cell types dictate where and when gliomas (astrocytomas) form and grow. The notion that brain tumors require a confluence of supportive stromal cell types and signals, susceptible preneoplastic/neoplastic cells, and genomic influences allows researchers and clinicians to develop strategies that effectively disrupt these critical relationships in a targeted and developmentally appropriate fashion., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Amyloid-beta (Abeta) is a naturally occurring 40- or 42-residue peptide fragment with a primary role in Alzheimer's disease (AD). Aggregated Abeta accumulates as both dense core plaques and diffuse deposits in the brains of AD patients. Abeta plaques are surrounded by activated microglia, some of which are believed to be derived from peripheral blood monocytes that have infiltrated the central nervous system and differentiated into phagocytes in response to Abeta. We have modeled this process using THP-1 human monocytes and found Abeta(1-42) to be as effective as phorbol myristate acetate at differentiating THP-1 monocytes based on cell adhesion, fibronectin binding, CD11b cell-surface expression, and morphological changes. Cell adhesion studies and atomic force microscopy imaging revealed an inverse correlation between Abeta(1-42)-induced monocyte maturation and aggregation progression. Freshly reconstituted Abeta(1-42) solutions were the most effective, yet continued aggregation reduced, and eventually abolished, the ability to induce monocyte adhesion. Abeta(1-40), lower aggregation concentrations of Abeta(1-42), and an aggregation-restricted Abeta(1-42) L34P mutant had little effect on monocyte adhesion under the same conditions as Abeta(1-42). These findings implicated an oligomeric, but not monomeric or fibrillar, Abeta(1-42) aggregation species in the monocyte maturation process. The rapidly-formed Abeta(1-42) oligomers were distinct from Abeta-derived diffusible ligands which did not elicit significant THP-1 monocyte adhesion. These data demonstrate that a specific oligomeric Abeta(1-42) aggregation species can potently initiate the THP-1 monocyte maturation process.

The primary molecules for mediating the innate immune response are the Toll-like family of receptors (TLRs). Recent work has established that amyloid-beta (Abeta) fibrils, the primary components of senile plaques in Alzheimer's disease (AD), can interact with the TLR2/4 accessory protein CD14. Using antibody neutralization assays and tumor necrosis factor alpha release in the human monocytic THP-1 cell line, we determined that both TLR2 and TLR4 mediated an inflammatory response to aggregated Abeta(1-42). This was in contrast to exclusive TLR ligands lipopolysaccharide (LPS) (TLR4) and tripalmitoyl cysteinyl seryl tetralysine (Pam(3)CSK(4)) (TLR2). Atomic force microscopy imaging showed a fibrillar morphology for the proinflammatory Abeta(1-42) species. Pre-treatment of the cells with 10 microg/mL of a TLR2-specific antibody blocked approximately 50% of the cell response to fibrillar Abeta(1-42), completely blocked the Pam(3)CSK(4) response, and had no effect on the LPS-induced response. A TLR4-specific antibody (10 microg/mL) blocked approximately 35% of the cell response to fibrillar Abeta(1-42), completely blocked the LPS response, and had no effect on the Pam(3)CSK(4) response. Polymyxin B abolished the LPS response with no effect on Abeta(1-42) ruling out bacterial contamination of the Abeta samples. Combination antibody pre-treatments indicated that neutralization of TLR2, TLR4, and CD14 together was much more effective at blocking the Abeta(1-42) response than the antibodies used alone. These data demonstrate that fibrillar Abeta(1-42) can trigger the innate immune response and that both TLR2 and TLR4 mediate Abeta-induced tumor necrosis factor alpha production in a human monocytic cell line.