Abnormal expression of REST/NRSF and Myc in neural stem/progenitor cells causes cerebellar tumors by blocking neuronal differentiation.

Medulloblastoma, one of the most malignant brain tumors in children, is thought to arise from undifferentiated neural stem/progenitor cells (NSCs) present in the external granule layer of the cerebellum. However, the mechanism of tumorigenesis remains unknown for the majority of medulloblastomas. In this study, we found that many human medulloblastomas express significantly elevated levels of both myc oncogenes, regulators of neural progenitor proliferation, and REST/NRSF, a transcriptional repressor of neuronal differentiation genes. Previous studies have shown that neither c-Myc nor REST/NRSF alone could cause tumor formation. To determine whether c-Myc and REST/NRSF act together to cause medulloblastomas, we used a previously established cell line derived from external granule layer stem cells transduced with activated c-myc (NSC-M). These immortalized NSCs were able to differentiate into neurons in vitro. In contrast, when the cells were engineered to express a doxycycline-regulated REST/NRSF transgene (NSC-M-R), they no longer underwent terminal neuronal differentiation in vitro. When injected into intracranial locations in mice, the NSC-M cells did not form tumors either in the cerebellum or in the cerebral cortex. In contrast, the NSC-M-R cells did produce tumors in the cerebellum, the site of human medulloblastoma formation, but not when injected into the cerebral cortex. Furthermore, the NSC-M-R tumors were blocked from terminal neuronal differentiation. In addition, countering REST/NRSF function blocked the tumorigenic potential of NSC-M-R cells. To our knowledge, this is the first study in which abnormal expression of a sequence-specific DNA-binding transcriptional repressor has been shown to contribute directly to brain tumor formation. Our findings indicate that abnormal expression of REST/NRSF and Myc in NSCs causes cerebellum-specific tumors by blocking neuronal differentiation and thus maintaining the "stemness" of these cells. Furthermore, these results suggest that such a mechanism plays a role in the formation of human medulloblastoma.

REST maintains self-renewal and pluripotency of embryonic stem cells.

The neuronal repressor REST (RE1-silencing transcription factor; also called NRSF) is expressed at high levels in mouse embryonic stem (ES) cells, but its role in these cells is unclear. Here we show that REST maintains self-renewal and pluripotency in mouse ES cells through suppression of the microRNA miR-21. We found that, as with known self-renewal markers, the level of REST expression is much higher in self-renewing mouse ES cells than in differentiating mouse ES (embryoid body, EB) cells. Heterozygous deletion of Rest (Rest+/-) and its short-interfering-RNA-mediated knockdown in mouse ES cells cause a loss of self-renewal-even when these cells are grown under self-renewal conditions-and lead to the expression of markers specific for multiple lineages. Conversely, exogenously added REST maintains self-renewal in mouse EB cells. Furthermore, Rest+/- mouse ES cells cultured under self-renewal conditions express substantially reduced levels of several self-renewal regulators, including Oct4 (also called Pou5f1), Nanog, Sox2 and c-Myc, and exogenously added REST in mouse EB cells maintains the self-renewal phenotypes and expression of these self-renewal regulators. We also show that in mouse ES cells, REST is bound to the gene chromatin of a set of miRNAs that potentially target self-renewal genes. Whereas mouse ES cells and mouse EB cells containing exogenously added REST express lower levels of these miRNAs, EB cells, Rest+/- ES cells and ES cells treated with short interfering RNA targeting Rest express higher levels of these miRNAs. At least one of these REST-regulated miRNAs, miR-21, specifically suppresses the self-renewal of mouse ES cells, corresponding to the decreased expression of Oct4, Nanog, Sox2 and c-Myc. Thus, REST is a newly discovered element of the interconnected regulatory network that maintains the self-renewal and pluripotency of mouse ES cells.

STUDY OF REST AS A NEGATIVE REGULATOR OF P16INK4A

STUDY OF REST AS A NEGATIVE REGULATOR OF P16INK4A Monica Gireud, B.S. Thesis Advisor: Vidya Gopalakrishnan, Ph.D. The RE1 Silencing Transcription Factor (REST) is a negative regulator of neuronal differentiation. It is expressed ubiquitously in early embryos, but downregulated in neural progenitors concomitant with onset of neuronal differentiation in these cells. REST has been widely studied as a negative regulator of neuronal differentiation genes. Our recent work identified a novel role for REST in control of cell proliferation. However, the underlying molecular mechanism(s) are not known and is a focus of the current thesis project. Here, we provide evidence that REST signaling controls the expression of the cyclin-dependent kinase inhibitor, p16Ink4a, a negative regulator of the cell cycle and passage through G1. We determined that REST expression in the proliferating granule progenitors of the cerebellum and its lack of expression in the differentiated neurons is reciprocally correlated with that of p16Ink4a. Decline in REST levels in differentiating primary and neural stem cells immortalized with v-myc (NSC-M) granule progenitors in vitro was also associated with upregulation of p16Ink4a expression. Conversely, constitutive human REST transgene expression in NSC-M cells (NSC-MRs) blocked p16Ink4 upregulation, even under neuronal differentiation conditions. However, the lack of a consensus REST DNA binding RE1 element in the regulatory regions of p16Ink4a locus suggested an indirect regulation of p16Ink4a by REST. Based on work from other groups that showed repression of p16Ink4a transcription by the polycomb protein Bmi-1, and its negative regulation by microRNA-203 (miR-203) and our identification of a RE1 element in the downstream regulatory region of miR-203, we asked if the p16Ink4a expression was controlled by REST through a series of negative regulatory events involving miR-203 and Bmi-1. We observed that Bmi1 -expression mirrored that of REST and inversely correlated with that of miR-203 in the postnatal cerebellum and in vitro differentiated granule and NSC-M progenitors. In contrast, forced REST transgene expression in NSC-MR cells abrogated the decrease in Bmi-1 levels and elevation in miR-203 expression. Significant REST binding to the miR-203 RE1 element was also observed in NSC-M cells, indicating that REST had the potential to directly regulate miR-203 expression. In conclusion, our studies suggest a role for REST in control of cell cycle transit in neural progenitors through negative regulation of p16Ink4a. Further validation of these results in REST knockout mice is needed, and is ongoing.

The role of the bed nucleus of the stria terminalis in stress: A behavioral, neurophysiological and neuropharmacological study

During the fifty-five years since the origin of the modern concept of stress, a variety of neurochemical, physiological, behavioral and pathological data have been collected in order to define stress and catalogue the components of the stress response. Over the last twenty-five years, as interest in the neural mechanisms underlying the stress response grew, most of the studies have focused on the hypothalamus and major limbic structures such as the amygdala or on nuclei involved in neurochemical changes observed during stress. There are other CNS sites, such as the bed nucleus of the stria terminalis (BNST), that neuroanatomical and neurochemical studies suggest may be involved in stress, but these sites have rarely been studied. Four experiments were performed for this dissertation, the goal of which was to examine the BNST to determine its role in the regulation of the stress response. The first experiment demonstrated that electrical stimulation of BNST was sufficient to produce stress-like behaviors. The second experiment demonstrated that single BNST neurons altered their firing rate in response to both a noxious somatosensory stimulus such as tail pinch and electrical stimulation of the amygdala (AmygS). The third experiment showed that the opioid, cholinergic, and noradrenergic systems, three neurotransmitter systems implicated in the control of the stress response, were effective in altering the firing rate of BNST neurons. The fourth experiment demonstrated that the cholinergic effects were mediated via muscarinic receptors and showed that the effects of AmygS were not mediated via cholinergic pathways. Collectively, these findings provide a possible explanation for the nonspecificity in causation of stress and the invariability of the stress response and suggest a neurochemical basis for its pharmacological control. ^

REST-based sensor networks with OData

RESTful services gained a lot of attention recently, even in the enterprise world, which is traditionally more web-service centric. Data centric RESfFul services, as previously mainly known in web environments, established themselves as a second paradigm complementing functional WSDL-based SOA. In the Internet of Things, and in particular when talking about sensor motes, the Constraint Application Protocol (CoAP) is currently in the focus of both research and industry. In the enterprise world a protocol called OData (Open Data Protocol) is becoming the future RESTful data access standard. To integrate sensor motes seamlessly into enterprise networks, an embedded OData implementation on top of CoAP is desirable, not requiring an intermediary gateway device. In this paper we introduce and evaluate an embedded OData implementation. We evaluate the OData protocol in terms of performance and energy consumption, considering different data encodings, and compare it to a pure CoAP implementation. We were able to demonstrate that the additional resources needed for an OData/JSON implementation are reasonable when aiming for enterprise interoperability, where OData is suggested to solve both the semantic and technical interoperability problems we have today when connecting systems

Correction to Variations of Ice Bed Coupling Beneath and Beyond Ice Streams: The Force Balance

A geometrical force balance that links stresses to ice bed coupling along a flow band of an ice sheet was developed in 1988 for longitudinal tension in ice streams and published 4 years later. It remains a work in progress. Now gravitational forces balanced by forces producing tensile, compressive, basal shear, and side shear stresses are all linked to ice bed coupling by the floating fraction phi of ice that produces the concave surface of ice streams. These lead inexorably to a simple formula showing how phi varies along these flow bands where surface and bed topography are known: phi = h(O)/h(I) with h(O) being ice thickness h(I) at x = 0 for x horizontal and positive upslope from grounded ice margins. This captures the basic fact in glaciology: the height of ice depends on how strongly ice couples to the bed. It shows how far a high convex ice sheet (phi = 0) has gone in collapsing into a low flat ice shelf (phi = 1). Here phi captures ice bed coupling under an ice stream and h(O) captures ice bed coupling beyond ice streams.

Variations of Ice Bed Coupling Beneath and Beyond Ice Streams: The Force Balance

A geometrical force balance that links stresses to ice bed coupling along a flow band of an ice sheet was developed in 1988 for longitudinal tension in ice streams and published 4 years later. It remains a work in progress. Now gravitational forces balanced by forces producing tensile, compressive, basal shear, and side shear stresses are all linked to ice bed coupling by the floating fraction phi of ice that produces the concave surface of ice streams. These lead inexorably to a simple formula showing how phi varies along these flow bands where surface and bed topography are known: phi = h(O)/h(I) with h(O) being ice thickness h(I) at x = 0 for x horizontal and positive upslope from grounded ice margins. This captures the basic fact in glaciology: the height of ice depends on how strongly ice couples to the bed. It shows how far a high convex ice sheet (phi = 0) has gone in collapsing into a low flat ice shelf (phi = 1). Here phi captures ice bed coupling under an ice stream and h(O) captures ice bed coupling beyond ice streams.

Ice-Bed Coupling Beneath and Beyond Ice Streams: Byrd Glacier, Antarctica

Ice sheet thickness is determined mainly by the strength of ice-bed coupling that controls holistic transitions from slow sheet flow to fast streamflow to buttressing shelf flow. Byrd Glacier has the largest ice drainage system in Antarctica and is the fastest ice stream entering Ross Ice Shelf. In 2004 two large subglacial lakes at the head of Byrd Glacier suddenly drained and increased the terminal ice velocity of Byrd Glacier from 820 m yr(-1) to 900 m yr(-1). This resulted in partial ice-bed recoupling above the lakes and partial decoupling along Byrd Glacier. An attempt to quantify this behavior is made using flowband and flowline models in which the controlling variable for ice height above the bed is the floating fraction phi of ice along the flowband and flowline. Changes in phi before and after drainage are obtained from available data, but more reliable data in the map plane are required before Byrd Glacier can be modeled adequately. A holistic sliding velocity is derived that depends on phi, with contributions from ice shearing over coupled beds and ice stretching over uncoupled beds, as is done in state-of-the-art sliding theories.

Evidence for a Frozen Bed, Byrd Glacier, Antarctica

Ice thickness, computed within the fjord region of Byrd Glacier on the assumptions that Byrd Glacier is in mass-balance equilibrium and that ice velocity is entirely due to basal sliding, are on average 400 m less than measured ice thicknesses along a radio-echo profile. We consider four explanations for these differences: (1) active glacier ice is separated from a zone of stagnant ice near the base of the glacier by a shear zone at depth; (2) basal melting rates are some 8 m/yr; (3) internal shear occurs with no basal sliding in much of the region above the grounding zone; or (4) internal creep and basal sliding contribute to the flow velocity in varying proportions above the grounding zone. Large gradients of surface strain rate seem to invalidate the first explanation. Computed values of basal shear stress (140 to 200 kPa) provide insufficient frictional heat to melt the ice demanded by the second explanation. Both the third and fourth explanations were examined by making simplifying assumptions that prevented a truly quantitative evaluation of their merit. Nevertheless, there is no escaping the qualitative conclusion that internal shear contributes strongly to surface velocities measured on Byrd Glacier, as is postulated in both these explanations.

Thermal Conditions at the Bed of the Laurentide Ice Sheet in Maine During Deglaciation: Implications for Esker Formation

The University of Maine Ice Sheet Model was used to study basal conditions during retreat of the Laurentide ice sheet in Maine. Within 150 km of the margin, basal melt rates average similar to 5 mm a(-1) during retreat. They decline over the next 100km, so areas of frozen bed develop in northern Maine during retreat. By integrating the melt rate over the drainage area typically subtended by an esker, we obtained a discharge at the margin of similar to 1.2 m(3) s(-1). While such a discharge could have moved the material in the Katahdin esker, it was likely too low to build the esker in the time available. Additional water from the glacier surface was required. Temperature gradients in the basal ice increase rapidly with distance from the margin. By conducting upward into the ice all of the additional viscous heat produced by any perturbation that increases the depth of flow in a flat conduit in a distributed drainage system, these gradients inhibit the formation of sharply arched conduits in which an esker can form. This may explain why eskers commonly seem to form near the margin and are typically segmented, with later segments overlapping onto earlier ones.

Adaption of cardio-respiratory balance during day-rest compared to deep sleep—An indicator for quality of life?

Heart rate and breathing rate fluctuations represent interacting physiological oscillations. These interactions are commonly studied using respiratory sinus arrhythmia (RSA) of heart rate variability (HRV) or analyzing cardiorespiratory synchronization. Earlier work has focused on a third type of relationship, the temporal ratio of respiration rate and heart rate (HRR). Each method seems to reveal a specific aspect of cardiorespiratory interaction and may be suitable for assessing states of arousal and relaxation of the organism. We used HRR in a study with 87 healthy subjects to determine the ability to relax during 5 day-resting periods in comparison to deep sleep relaxation. The degree to which a person during waking state could relax was compared to somatic complaints, health-related quality of life, anxiety and depression. Our results show, that HRR is barely connected to balance (LF/HF) in HRV, but significantly correlates to the perception of general health and mental well-being as well as to depression. If relaxation, as expressed in HRR, during day-resting is near to deep sleep relaxation, the subjects felt healthier, indicated better mental well-being and less depressive moods.

Wake up. Back to Bed – Zugang zum nächtlichen Training?

Einleitung Ein Klartraum ist definiert als ein Traum, in dem der Träumende weiß, dass er träumt. In der Fachliteratur finden sich verschiedene Induktionstechniken, um die Klartraum-häufigkeit zu steigern (z.B. Stumbrys, Erlacher, Schädlich & Schredl, 2012). Zudem wurde in einer Pilotstudie gezeigt, dass ein Training im Klartraum zu Verbesserungen in einer Zielwurfaufgabe am nächsten Morgen führt (Erlacher & Schredl, 2010). Um ein regelmäßiges Training im Traum zu ermöglichen, besteht für die Sportpraxis das Problem, Klarträume gezielt zu induzieren. In dieser Studie wurde im Schlaflabor die so genannte Memnotische Induktion von luziden Träumen (MILT) – eine Autosugges-tionstechnik in der die Intention, einen Klartraum zu erleben, an Traumhinweise ge-koppelt wird – im Morgenschlaf überprüft. Methoden Insgesamt wurden 52 Versuchsteilnehmer (32 männlich und 20 weiblich) im Alter von 24 Jahren (± 2.2) im Schlaflabor untersucht. Die Personen waren in 4 Gruppen aufge-teilt. Alle Personen schliefen zunächst für ca. 6 Stunden, wurden dann aus einer REM-Phase geweckt und sollten einen Traum berichten. Im Anschluss blieben die Teilnehmer 30 bzw. 60 Minuten wach und praktizierten entweder MILT oder beschäf-tigten sich mit einer kognitiven oder motorischen Kontrollaufgabe. Im Anschluss durf-ten alle Teilnehmer für max. 4 weitere Stunden schlafen. Das Auftreten eines Klartraums in der morgendlichen Schlafphase diente als abhängige Variable. Ergebnisse und Diskussion Die Ergebnisse zeigen, dass MILT zu einer gesteigerten Klartraumhäufigkeit (33-70%) im Vergleich zur Kontrollbedingung (9-14%) führt. Ein Unterschied zwischen 30 Minuten (50%) zu 60 Minuten MILT (70%) ist marginal. Das Auftreten von Klarträumen kann durch MILT im Morgenschlaf signifikant gestei-gert werden. Die Erfolgsquote schwankt jedoch mit Blick auf die genaue Definition ei-nes Klartraums. Es konnten bei nicht klartraumerfahrenen Versuchsteilnehmerinnen mehr Klarträume induziert werden. Für die Sportpraxis könnten solche Induktions-techniken dem Sportler ermöglichen, im Traum zu trainieren. In weiteren Studien wäre zu untersuchen, ob Athleten ebenfalls Klarträume induziert werden können. Ebenso sollte die Auswirkung eines regelmäßigen Klartraumtrainings in der Sportpraxis wei-ter untersucht werden. Literatur Stumbrys, T., Erlacher, D., Schädlich, M. & Schredl, M. (2012). Induction of lucid dreams: a systematic review of evidence. Consciousness and Cognition, 21(3), 1456-1475. Erlacher, D. & Schredl, M. (2010). Practicing a motor task in a lucid dream enhances subsequent performance: A pilot study. The Sport Psychologist, 24(2), 157-167.