Notch1 signaling promotes survival of glioblastoma cells via EGFR-mediated induction of anti-apoptotic Mcl-1.

The Notch1-mediated signaling pathway has a central role in the maintenance of neural stem cells and contributes to growth and progression of glioblastomas, the most frequent malignant brain tumors in adults. Here, we demonstrate that the Notch1 receptor promotes survival of glioblastoma cells by regulation of the anti-apoptotic Mcl-1 protein. Notch1-dependent regulation of Mcl-1 occurs cell type dependent at a transcriptional or post-translational level and is mediated by the induction of epidermal growth factor receptor (EGFR). Inhibition of the Notch1 pathway overcomes apoptosis resistance and sensitizes glioblastoma cells to apoptosis induced by ionizing radiation, the death ligand TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) or the Bcl-2/Bcl-XL inhibitor ABT-737. In conclusion, targeting Notch1 might represent a promising novel strategy in the treatment of glioblastomas.

Neural and cancer stem cells in tumor suppressor mouse models of malignant astrocytoma.

Malignant astrocytomas are highly invasive brain tumors that portend poor prognosis and dismal survival. Mouse models that genetically resemble the human malignancy provide insight into the nature and pathogenesis of these cancers. We previously reported tumor suppressor mouse models based on conditional inactivation of human astrocytoma-relevant genes p53, Nf1, and Pten. These mice develop, with full penetrance, varying grades of astrocytic malignancy that recapitulate the human condition histologically and molecularly. Our studies indicate a central role for neural stem cells and stem-cell-like cancer cells in tumor initiation and progression. These mouse models thus represent powerful tools for investigating various aspects of tumor development that otherwise cannot be explored in humans. Further studies will provide a better understanding of the biology of these tumors and will hopefully pave the way for more effective therapeutic approaches for these devastating diseases.

Tumor microenvironment and neurofibromatosis type I: connecting the GAPs.

The human disease von Recklinghausen's neurofibromatosis (Nf1) is one of the most common genetic disorders. It is caused by mutations in the NF1 tumor suppressor gene, which encodes a GTPase activating protein (GAP) that negatively regulates p21-RAS signaling. Dermal and plexiform neurofibromas as well as malignant peripheral nerve sheath tumors and other malignant tumors, are significant complications in Nf1. Neurofibromas are complex tumors and composed mainly of abnormal local cells including Schwann cells, endothelial cells, fibroblasts and additionally a large number of infiltrating inflammatory mast cells. Recent work has indicated a role for the microenvironment in plexiform neurofibroma genesis. The emerging evidence points to mast cells as crucial contributors to neurofibroma tumorigenesis. Therefore, further understanding of the molecular interactions between Schwann cells and their environment will provide tools to develop new therapies aimed at delaying or preventing tumor formation in Nf1 patients.

Transcriptional regulation of Trk family neurotrophin receptors.

The Trk family of neurotrophin receptors plays essential roles in cell fate specification, survival, growth, and differentiation. Their expression patterns are complex and dynamically regulated under many physiological and pathological conditions. However, the molecular mechanisms that control their tissue-specific expression are largely unknown. In this report, we review current knowledge about the transcriptional regulation of Trk receptors.

Modeling neurofibromatosis type 1 tumors in the mouse for therapeutic intervention.

Von Recklinghausen's neurofibromatosis is a dominantly inherited cancer syndrome. Its gene encodes neurofibromin, a protein with ras GTPase-activating function (rasGAP) and, therefore, all NF1-associated pathology is thought to originate from selective deregulation of the ras pathway. We have constructed a variety of mouse models for NF1 that permit recapitulation of the most common tumors seen in patients. In addition, these mouse models offer insights into tumor origin and into paracrine interactions. Given the molecular and pathological fidelity of the mouse tumors to the human counterparts, it is hoped that these mouse strains will serve as effective tools for therapeutic discovery.

Potential role of glial cell line-derived neurotrophic factor receptors in Müller glial cells during light-induced retinal degeneration.

Glial cell line-derived neurotrophic factor (GDNF), neurturin (NTN) and their receptors (GFRalpha1, GFRalpha2 and Ret) play an important role in the survival of neurons in the central and peripheral nervous system. For example, GDNF as well as other trophic factors promotes photoreceptor survival during retinal degeneration. Recent studies have proposed that part of neurotophic rescue of photoreceptors may be indirect, mediated by interaction of the neurotrophic factors with other cell types, that in turn release secondary factors that act directly on photoreceptors. In the present study, we examined the GDNF receptor expression in control and light-damaged retina, and found that GFRalpha2 protein is upregulated in retina-specific Müller glial cells during photoreceptor degeneration. We also examined the effect of GDNF or NTN on cultured Müller cells. Exogenous GDNF increased brain-derived neurotrophic factor, basic fibroblast growth factor and GDNF, but not NTN mRNA production. On the other hand, NTN increased NTN, but not GDNF mRNA production in cultured Müller cells. These observations suggest that GDNF, NTN and their receptors are involved in the regulation of trophic factor production in retinal glial cells, and that functional glia-neuron network may utilize GDNF family for the protection of neural cells during retinal degeneration.

Heterozygosity for the neurofibromatosis 1 (NF1) tumor suppressor results in abnormalities in cell attachment, spreading and motility in astrocytes.

Individuals with the neurofibromatosis 1 (NF1) tumor predisposition syndrome develop low-grade pilocytic astrocytomas at an increased frequency. Previously, we demonstrated that astrocytes from mice heterozygous for a targeted mutation in the Nf1 gene (Nf1+/- astrocytes) exhibit a cell autonomous growth advantage associated with increased RAS pathway activation. In this report, we extend our initial characterization of the effect of reduced Nf1 gene expression on astrocyte function by demonstrating that Nf1+/- astrocytes exhibit decreased cell attachment, actin cytoskeletal abnormalities during the initial phases of cell spreading, and increased cell motility. Whereas these cytoskeletal abnormalities were also observed in Nf1-/- astrocytes, astrocytes expressing a constitutively active RAS molecule showed increased cell motility and abnormal actin cytoskeleton organization during cell spreading, but exhibited normal cell attachment. Based on ongoing gene expression profiling experiments on human astrocytoma tumors, we demonstrate increased expression of two proteins implicated in cell attachment, spreading and motility (GAP43 and T-cadherin) in Nf1+/- and Nf1-/- astrocytes. These results support the emerging notion that tumor suppressor gene heterozygosity results in abnormalities in cell function that may contribute to the pathogenesis of non-tumor phenotypes in NF1.

Embryonic neuronal death due to neurotrophin and neurotransmitter deprivation occurs independent of Apaf-1.

Apoptotic protease-activating factor-1 (Apaf-1), dATP, and procaspase-9 form a multimeric complex that triggers programmed cell death through the activation of caspases upon release of cytochrome c from the mitochondria into the cytosol. Although cell death pathways exist that can bypass the requirement for cytochrome c release and caspase activation, several gene knockout studies have shown that the cytochrome c-mediated apoptotic pathway is critical for neural development. Specifically, the number of neuronal progenitor cells is abnormally increased in Apaf-1-, caspase-9-, caspase-3-deficient mice. However, the role of the cytochrome c cell death pathway for apoptosis of postmitotic, differentiated neurons in the developing brain has not been investigated in vivo. In this study we investigated embryonic neuronal cell death caused by trophic factor deprivation or lack of neurotransmitter release by analyzing Apaf-1/tyrosine kinase receptor A (TrkA) and Apaf-1/Munc-18 double mutant mice. Histological analysis of the double mutants' brains (including cell counting and terminal (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) staining) reveals that neuronal cell death caused by these stimuli can proceed independent of Apaf-1. We propose that a switch between apoptotic programs (and their respective proteins) characterizes the transition of a neuronal precursor cell from the progenitor pool to the postmitotic population of differentiated neurons.

The Ras-ERK pathway is required for the induction of neuronal nitric oxide synthase in differentiating PC12 cells.

We have studied the role of MAP kinase pathways in neuronal nitric oxide synthase (nNOS) induction during the differentiation of PC12 cells. In nerve growth factor (NGF)-treated PC12 cells, we find nNOS induced at RNA and protein levels, resulting in increased NOS activity. We note that neither nNOS mRNA, nNOS protein nor NOS activity is induced by NGF treatment in cells that have been infected with a dominant negative Ras adenovirus. We have also used drugs that block MAP kinase pathways and assessed their ability to inhibit nNOS induction. Even though U0126 and PD98059 are both MEK inhibitors, we find that U0126, but not PD98059, blocks induction of nNOS protein and NOS activity in NGF-treated PC12 cells. Also, the p38 kinase inhibitor, SB203580, does not block nNOS induction in our clone of PC12 cells. Since the JNK pathway is not activated in NGF-treated PC12 cells, we conclude that the Ras-ERK pathway and not the p38 or JNK pathway is required for nNOS induction in NGF-treated PC12 cells. We find that U0126 is much more effective than PD98059 in blocking the Ras-ERK pathway, thereby explaining the discrepancy in nNOS inhibition. We conclude that the Ras-ERK pathway is required for nNOS induction.

Retinoic acid combined with neurotrophin-3 enhances the survival and neurite outgrowth of embryonic sympathetic neurons.

Both nerve growth factor (NGF) and neurotrophin-3 (NT-3) are necessary for the survival of embryonic sympathetic neurons in vivo. All-trans retinoic acid (atRA) has been shown to promote neurite outgrowth and long-term survival of chick embryonic sympathetic neurons cultured in the presence of NGF. The present study shows that atRA can also potentiate the survival and neurite outgrowth-promoting activities of NT-3. This was accomplished by enhancing the survival of existing neurons, as cell proliferation was unaffected by exposure to atRA. atRA also enhanced neurite outgrowth of the NT-3-treated cells; however, the neurites appeared thicker and less branched than cells treated with atRA in combination with NGF. Using a quantitative PCR assay, trkA and p75(NTR) mRNAs, but not trkC mRNA, were increased ( approximately 1.5- to 2-fold) after 72 and 48 hr of exposure of the cultures to atRA, respectively. The atRA-induced increase in trkA mRNA may play a role in the enhanced survival of neurons cultured in the presence of either NGF or NT-3, as both neurotrophins have been shown to signal through this receptor. The time course of these mRNA changes would indicate that atRA does not regulate the neurotrophin receptor mRNA directly, rather, intervening gene transcription is required. Thus, during development, atRA may play a role in fine-tuning embryonic responsiveness to both NT-3 and NGF.

Ablation of NF1 function in neurons induces abnormal development of cerebral cortex and reactive gliosis in the brain.

Neurofibromatosis type 1 (NF1) is a prevalent genetic disorder that affects growth properties of neural-crest-derived cell populations. In addition, approximately one-half of NF1 patients exhibit learning disabilities. To characterize NF1 function both in vitro and in vivo, we circumvent the embryonic lethality of NF1 null mouse embryos by generating a conditional mutation in the NF1 gene using Cre/loxP technology. Introduction of a Synapsin I promoter driven Cre transgenic mouse strain into the conditional NF1 background has ablated NF1 function in most differentiated neuronal populations. These mice have abnormal development of the cerebral cortex, which suggests that NF1 has an indispensable role in this aspect of CNS development. Furthermore, although they are tumor free, these mice display extensive astrogliosis in the absence of conspicuous neurodegeneration or microgliosis. These results indicate that NF1-deficient neurons are capable of inducing reactive astrogliosis via a non-cell autonomous mechanism.

mKlf7, a potential transcriptional regulator of TrkA nerve growth factor receptor expression in sensory and sympathetic neurons.

Development of the nervous system relies on stringent regulation of genes that are crucial to this process. TrkA, the receptor for nerve growth factor (NGF), is tightly regulated during embryonic development and is essential for the survival and differentiation of neural crest-derived sensory and sympathetic neurons. We have previously identified a mouse TrkA enhancer and have characterized several cis regulatory elements that are important for appropriate TrkA expression in vivo. We now report the cloning of a novel gene encoding a Kruppel-like factor from a mouse dorsal root ganglion expression library. This Kruppel-like factor, named mKlf7, binds specifically to an Ikaros core binding element that is crucial for in vivo TrkA enhancer function. Using in situ hybridization, we demonstrate that mKlf7 is coexpressed with TrkA in sensory and sympathetic neurons during embryogenesis and in adulthood. These data are consistent with the idea that mKlf7 may directly regulate TrkA gene expression in the peripheral nervous system.

Regulation of Trk receptors following contusion of the rat spinal cord.

Neurotrophins function through high-affinity tyrosine kinase (Trk) receptors to promote growth and survival of cells in the injured nervous system. To investigate the role of Trk receptors in the adult nervous system, we examined TrkA, TrkB, and TrkC mRNA expression in spinal cord and brain after spinal contusion. At 1 day postinjury, all Trk receptor transcripts were down regulated at and around the site of injury, a situation that persisted through the first week. By 42 days, Trk expression was absent only within the cavity. In addition, truncated TrkB expression was substantially increased in ependymal cells and astrocytes surrounding the lesion cavity of chronically injured spinal cords. Rostral and caudal to the injury site, TrkA, TrkB, and TrkC mRNA expression did not differ from that of uninjured control spinal cords. Furthermore, no changes were observed in TrkB or TrkC expression in the axotomized corticospinal and rubrospinal neurons. These studies suggest that loss of Trk receptors at the injury site may contribute to the early progressive cellular loss in injured spinal cords, while increased presence of truncated TrkB receptors in the chronic injured spinal cord may sequester and restrict BDNF availability to support axonal regeneration and neuronal survival. The persistence of Trk receptors on supraspinal neurons suggests that neurotrophin application can support growth and survival in the acute and chronic injury states.

Hormones in male sexual development.

Classical embryology has provided a clear view of the timing and hormonal cues that govern sexual differentiation. Molecular biology has added important details to this picture. The cloning of SRY, MIS, and INSL3 provide insight into the molecular signals that provide important cues at the cellular level. Continued understanding of these pathways may provide the necessary information to one day reverse defects of sexual differentiation.

A molecular basis for estrogen-induced cryptorchidism.

Male sexual differentiation relies upon testicular secretion of the hormones testosterone, Mullerian inhibiting substance, and insulin-3 (Insl3). Insl3 is responsible for testicular descent through virilization and outgrowth of the embryonic gubernaculum. In mouse, prenatal exposure to 17beta-estradiol and the nonsteroidal synthetic estrogen diethylstilbestrol (DES) disturbs the endocrine balance, causing demasculinizing and feminizing effects in the male embryo, including impaired testicular descent (cryptorchidism). In the current study, we show that maternal exposure to estrogens, including 17alpha- and beta-estradiol, as well as DES, specifically down regulates Insl3 expression in embryonic Leydig cells, thereby providing a mechanism for cryptorchidism. These experiments may have implications for the widespread use of estrogenic substances in agriculture and the environment.

Molecular characterization of the TrkA/NGF receptor minimal enhancer reveals regulation by multiple cis elements to drive embryonic neuron expression.

Neural development relies on stringent regulation of key genes that mediate specialized function. TrkA is primarily expressed in neural crest-derived sensory and sympathetic neurons where it transmits critical survival information. We have identified a 457 base pair sequence upstream of the murine first TrkA coding exon that is conserved in human and in chick, and is sufficient for expression in the correct cells with appropriate timing. Mutation analysis of consensus transcription factor binding domains within the minimal enhancer reveals a complex positive regulation that includes sites required for global expression and sites that are specifically required for DRG, trigeminal or sympathetic expression. These results provide a foundation for identification of the transcriptional machinery that specifies neurotrophin receptor expression.

Role of N-myc in the developing mouse kidney.

N-myc is a transcription factor expressed in the developing metanephric kidney and other organs. In mice, complete disruption of the N-myc gene results in fetal death on the first day of renal organogenesis. In addition to the null N-myc allele, others have generated a hypomorphic N-myc allele. In this study, combinations of these mutant genes were used to demonstrate that reduction in N-myc protein levels correlate with fewer developing glomeruli and collecting ducts in embryonic kidney explants. Histological sections revealed that the mutant kidneys were hypoplastic with normal developing structures. The data indicate that the hypoplasia is due to a reduction in proliferation rather than an increase in apoptosis. Thus, N-myc loss causes a decrease in numbers of ureteric bud tips and developing glomeruli in explants and hypoplastic kidneys in vivo, in a dose-dependent manner.