Neuropeptide Y receptor Y5 as an inducible pro-survival factor in neuroblastoma: implications for tumor chemoresistance.

Neuroblastoma (NB) is a pediatric tumor of neural crest origin with heterogeneous phenotypes. Although low-stage tumors carry a favorable prognosis, >50% of high-risk NB relapses after treatment with a fatal outcome. Thus developing therapies targeting refractory NB remains an unsolved clinical problem. Brain-derived neurotrophic factor (BDNF) and its TrkB receptor are known to protect NB cells from chemotherapy-induced cell death, while neuropeptide Y (NPY), acting via its Y2 receptor (Y2R), is an autocrine proliferative and angiogenic factor crucial for maintaining NB tumor growth. Here we show that in NB cells, BDNF stimulates the synthesis of NPY and induces expression of another one of its receptors, Y5R. In human NB tissues, the expression of NPY and Y5R positively correlated with the expression of BDNF and TrkB. Functionally, BDNF triggered Y5R internalization in NB cells, whereas Y5R antagonist inhibited BDNF-induced p44/42 mitogen-activated protein kinase activation and its pro-survival activity. These observations suggested TrkB-Y5R transactivation that resulted in cross-talk between their signaling pathways. Additionally, NPY and Y5R were upregulated in a BDNF-independent manner in NB cells under pro-apoptotic conditions, such as serum deprivation and chemotherapy, as well as in cell lines and tissues derived from posttreatment NB tumors. Blocking Y5R in chemoresistant NB cells rich in this receptor sensitized them to chemotherapy-induced apoptosis and inhibited their growth in vivo by augmenting cell death. In summary, the NPY/Y5R axis is an inducible survival pathway activated in NB by BDNF or cellular stress. Upon such activation, Y5R augments the pro-survival effect of BDNF via its interactions with TrkB receptor and exerts an additional BDNF-independent anti-apoptotic effect, both of which contribute to NB chemoresistance. Therefore, the NPY/Y5R pathway may become a novel therapeutic target for patients with refractory NB, thus far an incurable form of this disease.

Hyperactivity, startle reactivity and cell-proliferation deficits are resistant to chronic lithium treatment in adult Nr2e1(frc/frc) mice.

The NR2E1 region on Chromosome 6q21-22 has been repeatedly linked to bipolar disorder (BP) and NR2E1 has been associated with BP, and more specifically bipolar I disorder (BPI). In addition, patient sequencing has shown an enrichment of rare candidate-regulatory variants. Interestingly, mice carrying either spontaneous (Nr2e1(frc) ) or targeted (Tlx(-) ) deletions of Nr2e1 (here collectively known as Nr2e1-null) show similar neurological and behavioral anomalies, including hypoplasia of the cerebrum, reduced neural stem cell proliferation, extreme aggression and deficits in fear conditioning; these are the traits that have been observed in some patients with BP. Thus, NR2E1 is a positional and functional candidate for a role in BP. However, no Nr2e1-null mice have been fully evaluated for behaviors used to model BP in rodents or pharmacological responses to drugs effective in treating BP symptoms. In this study we examine Nr2e1(frc/frc) mice, homozygous for the spontaneous deletion, for abnormalities in activity, learning and information processing, and cell proliferation; these are the phenotypes that are either affected in patients with BP or commonly assessed in rodent models of BP. The effect of lithium, a drug used to treat BP, was also evaluated for its ability to attenuate Nr2e1(frc/frc) behavioral and neural stem cell-proliferation phenotypes. We show for the first time that Nr2e1-null mice exhibit extreme hyperactivity in the open field as early as postnatal day 18 and in the home cage, deficits in open-field habituation and passive avoidance, and surprisingly, an absence of acoustic startle. We observed a reduction in neural stem/progenitor cell proliferation in Nr2e1(frc/frc) mice, similar to that seen in other Nr2e1-null strains. These behavioral and cell-proliferation phenotypes were resistant to chronic-adult-lithium treatment. Thus, Nr2e1(frc/frc) mice exhibit behavioral traits used to model BP in rodents, but our results do not support Nr2e1(frc/frc) mice as pharmacological models for BP.

DAPK2 is a novel E2F1/KLF6 target gene involved in their proapoptotic function.

Death-associated protein kinase 2 (DAPK2) belongs to a family of proapoptotic Ca(2+)/calmodulin-regulated serine/threonine kinases. We recently identified DAPK2 as an enhancing factor during granulocytic differentiation. To identify transcriptional DAPK2 regulators, we cloned 2.7 kb of the 5'-flanking region of the DAPK2 gene. We found that E2F1 and Krüppel-like factor 6 (KLF6) strongly activate the DAPK2 promoter. We mapped the E2F1 and KLF6 responsive elements to a GC-rich region 5' of exon 1 containing several binding sites for KLF6 and Sp1 but not for E2F. Moreover, we showed that transcriptional activation of DAPK2 by E2F1 and KLF6 is dependent on Sp1 using Sp1/KLF6-deficient insect cells, mithramycin A treatment to block Sp1-binding or Sp1 knockdown cells. Chromatin immunoprecipitation revealed recruitment of Sp1 and to lesser extent that of E2F1 and KLF6 to the DAPK2 promoter. Activation of E2F1 in osteosarcoma cells led to an increase of endogenous DAPK2 paralleled by cell death. Inhibition of DAPK2 expression resulted in significantly reduced cell death upon E2F1 activation. Similarly, KLF6 expression in H1299 cells increased DAPK2 levels accompanied by cell death that is markedly decreased upon DAPK2 knockdown. Moreover, E2F1 and KLF6 show cooperation in activating the DAPK2 promoter. In summary, our findings establish DAPK2 as a novel Sp1-dependent target gene for E2F1 and KLF6 in cell death response.

Physical sciences research plans for the International Space Station.

The restructuring of the research capabilities of the International Space Station has forced a reassessment of the Physical Sciences research plans and a re-targeting of the major scientific thrusts. The combination of already selected peer-reviewed flight investigations with the initiation of new research and technology programs will allow the maximization of the ISS scientific and technological potential. Fundamental and applied research will use a combination of ISS-based facilities, ground-based activities, and other experimental platforms to address issues impacting fundamental knowledge, industrial and medical applications on Earth, and the technology required for human space exploration. The current flight investigation research plan shows a large number of principal investigators selected to use the remaining planned research facilities.

Research priorities and plans for the International Space Station-results of the 'REMAP' Task Force.

Recent events in the International Space Station (ISS) Program have resulted in the necessity to re-examine the research priorities and research plans for future years. Due to both technical and fiscal resource constraints expected on the International Space Station, it is imperative that research priorities be carefully reviewed and clearly articulated. In consultation with OSTP and the Office of Management and budget (OMB), NASA's Office of Biological and Physical Research (OBPR) assembled an ad-hoc external advisory committee, the Biological and Physical Research Maximization and Prioritization (REMAP) Task Force. This paper describes the outcome of the Task Force and how it is being used to define a roadmap for near and long-term Biological and Physical Research objectives that supports NASA's Vision and Mission. Additionally, the paper discusses further prioritizations that were necessitated by budget and ISS resource constraints in order to maximize utilization of the International Space Station. Finally, a process has been developed to integrate the requirements for this prioritized research with other agency requirements to develop an integrated ISS assembly and utilization plan that maximizes scientific output.

Regulation of the retinoblastoma-dependent Mdm2 and E2F-1 signaling pathways during neuronal apoptosis.

We have previously demonstrated that the apoptotic signaling pathway in K(+)-deprived cerebellar granule neurons involves a caspase-dependent cleavage of the retinoblastoma protein (Rb). Here, we have further investigated the functional consequences of this cleavage on two Rb-binding partners: the oncoprotein Mdm2 and the transcription factor E2F-1. A K(+) deprivation time course leads to a caspase inhibitor-sensitive degradation of Mdm2. Experimental blockade of Mdm2 expression with antisense oligodeoxynucleotides (ODN) results in neuronal death, suggesting an active role of Mdm2 in neuroprotection. By contrast, the E2F-1 protein accumulates in a caspase-independent manner following K(+) withdrawal, a consequence of increased gene transcription. This is likely to result from the rapid cyclin-dependent kinase 4 activation observed in LK, that correlates with a transient Rb phosphorylation. Counteracting E2F-1 upregulation with antisense ODNs prevents neuronal loss. Taken together, these data demonstrate that Rb is a central player in regulating both caspase-dependent and -independent events leading to apoptosis.

Compressibility effects on steady streaming from a noncompact rigid sphere.

The problem of steady streaming around a rigid isolated sphere in a plane standing acoustic field is considered. Existing results in the literature have been generalized to allow for noncompactness of the sphere, and the influence of fluid compressibility on the streaming behavior has been included. It is found that in the high-frequency limit of interest for which the streaming is strongest, the effective steady slip velocity at the edge of the inner boundary layer region that is responsible for driving the steady streaming in the bulk of the fluid in the outer region, has a complex variation over the surface of the sphere that depends on (i) the sphere position (with respect to the node/antinode of the acoustic field), (ii) the extent of sphere compactness, and (iii) on a well-defined function (representing compressibility effects) of the fluid Prandtl number and its ratio of specific heats. Not surprisingly, the contribution from this function is negligible when the host fluid is a liquid. The steady streaming behavior around the sphere is demonstrated with the help of flow streamlines for various cases in the diffusive limit of weak outer flow for low streaming Reynolds numbers.

Caspase-dependent cleavage of the retinoblastoma protein is an early step in neuronal apoptosis.

Rb-deficient embryos (Rb-/-) show abnormal degeneration of neurons and die at mid-gestation, suggesting that RB may protect against apoptosis. Having previously shown that cyclin D1 accumulates during K+-induced apoptosis of granule neurons, we chose to investigate the role of RB under these conditions. We show that RB is cleaved in its C-terminus during the onset of neuronal apoptosis. Caspase 3-like activity increases following K+ deprivation and the time course correlates with RB cleavage and apoptosis. Although the use of a specific caspase 3-like inhibitor (z-DEBD.fmk) delays RB cleavage and reduces DNA fragmentation, data implicate other caspases in these processes. However, K+ deprivation induces a gradual production of the active p20 subunit of caspase 3 (CPP32) that coincides with RB disappearance at the cellular level. Nuclear detection of a transfected HA-tagged caspase cleavage-resistant RB mutant (DEAG/D to DEAA/D) revealed a significant decrease in apoptosis of neurons expressing the RB mutant (less than 5%) relative to the wild type form of RB (40%) during K+ deprivation. Taken together, these data show that caspase-dependent cleavage of RB is an early permissive step of the apoptosis-inducing signaling pathway in neurons. They indicate a major role of RB in neuronal protection.

Brain-derived neurotrophic factor exerts opposing effects on beta2-adrenergic receptor according to depolarization status of cerebellar neurons.

To investigate the molecular mechanisms underlying brain-derived neurotrophic factor (BDNF)-controlled synaptic plasticity, we studied beta2-adrenergic receptor (beta2-AR) expression in cultured cerebellar granule cells. We show that, depending on the state of depolarization, BDNF exerts opposite effects on beta2-AR expression. In neurons maintained in low K+ medium (5 mM K+) that will enter apoptosis, BDNF increases beta2-AR and beta2-AR transcripts. In contrast, in depolarized neurons (high K+ medium, 25 mM K+) BDNF represses beta2-AR expression. The use of reporter genes (driven by the beta2-AR promoter or restricted regulatory elements) revealed that BDNF exerts its opposite effects at the transcriptional level by recruiting a cyclic AMP response element (CRE) and the trans-acting factor CRE binding protein. These results provide the first evidence that a neurotrophin, e.g., BDNF, may exert an opposite effect on receptor expression and function (beta2-AR) according to the depolarization status of the neuron. Based on this finding, we propose that BDNF not only mediates neuronal survival, but is also involved in the modulation of the general sensitivity of the neuron to external signals, thus maintaining its optimal functional integration within the neuronal network.

Internal circulation in a drop in an acoustic field.

An investigation of the internal flow field for a drop at the antinode of a standing wave has been carried out. The main difference from the solid sphere case is the inclusion of the shear stress and velocity continuity conditions at the liquid-gas interface. To the leading order of calculation, the internal flow field was found to be quite weak. Also, this order being fully time dependent has a zero mean flow. At the next higher order, steady internal flows are predicted and, as in the case of a solid sphere, there is a recirculating layer consisting of closed streamlines near the surface. In the case of a liquid drop, however, the behavior of this recirculating Stokes layer is quite interesting. It is predicted that the layer ceases to have recirculation when [formula: see text], where [symbol: see text] is the liquid viscosity, mu is the exterior gas-phase viscosity, and M is the dimensionless frequency parameter for the gas phase, defined by M = i omega a2 rho/mu, with a being the drop radius. Thorough experimental confirmation of this interesting new development needs to be conducted. Although it seems to agree with many experiments with levitated drops where no recirculating layer has been clearly observed, a new set of experiments for specifically testing this interesting development need to be carried out.