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- Institut für Medizinische Strahlenkunde und Zellforschung (86) (remove)
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Parkinson's disease (PD) provokes bradykinesia, resting tremor, rigidity and postural instability, and also non-motor symptoms such as depression, anxiety, sleep and cognitive impairments. Similar phenotypes can be induced in Drosophila melanogaster through modification of PD-relevant genes or the administration of PD inducing toxins. Recent studies correlated deregulation of human p21-activated kinase 4 (PAK4) with PD, leaving open the question of a causative relationship of mutations in this gene for manifestation of PD symptoms. To determine whether flies lacking the PAK4 homolog Mushroom bodies tiny (Mbt) show PD-like phenotypes, we tested for a variety of PD criteria. Here, we demonstrate that mbt mutant flies show PD-like phenotypes including age-dependent movement deficits, reduced life expectancy and fragmented sleep. They also react to a stressful situation with higher immobility, indicating an influence of Mbt on emotional behavior. Loss of Mbt function has a negative effect on the number of dopaminergic protocerebral anterior medial (PAM) neurons, most likely caused by a proliferation defect of neural progenitors. The age-dependent movement deficits are not accompanied by a corresponding further loss of PAM neurons. Previous studies highlighted the importance of a small PAM subgroup for age-dependent PD motor impairments. We show that impaired motor skills are caused by a lack of Mbt in this PAM subgroup. In addition, a broader re-expression of Mbt in PAM neurons improves life expectancy. Conversely, selective Mbt knockout in the same cells shortens lifespan. We conclude that mutations in Mbt/PAK4 can play a causative role in the development of PD phenotypes.
Purpose
Integrating moderate hypofractionation to the macroscopic tumor with elective nodal irradiation while sparing the organs at risk (OAR) in chemoradiotherapy of locally advanced non-small-cell lung cancer.
Methods
From 2010-2018, treatment, patient and tumor characteristics of 138 patients from two radiation therapy centers were assessed. Chemoradiotherapy by intensity-modulated radiation therapy (IMRT) with a simultaneous integrated boost (SIB) to the primary tumor and macroscopic lymph node metastases was used.
Results
A total of 124 (90%) patients received concurrent chemotherapy. 106 (76%) patients had UICC (Union for International Cancer Control) stage ≥IIIB and 21 (15%) patients had an oligometastatic disease (UICC stage IV). Median SIB and elective total dose was 61.6 and 50.4 Gy in 28 fractions, respectively. Furthermore, 64 patients (46%) had an additional sequential boost to the primary tumor after the SIB-IMRT main series: median 6.6 Gy in median 3 fractions. The median cumulative mean lung dose was 15.6 Gy (range 6.2-29.5 Gy). Median follow-up and radiological follow-up for all patients was 18.0 months (range 0.6-86.9) and 16.0 months (range 0.2-86.9), respectively. Actuarial local control rates at 1, 2 and 3 years were 80.4, 68.4 and 57.8%. Median overall survival and progression-free survival was 30.0 months (95% confidence interval [CI] 23.5-36.4) and 12.1 months (95% CI 8.2-16.0), respectively. Treatment-related toxicity was moderate. Radiation-induced pneumonitis grade 2 and grade 3 occurred in 13 (9.8%) and 3 (2.3%) patients.
Conclusions
Chemoradiotherapy using SIB-IMRT showed promising local tumor control rates and acceptable toxicity in patients with locally advanced and in part oligometastatic lung cancer. The SIB concept, resulting in a relatively low mean lung dose, was associated with low numbers of clinically relevant pneumonitis. The overall survival appears promising in the presence of a majority of patients with UICC stage ≥IIIB disease.
Purpose
A signal dependence on dose rate was reported for the ArcCHECK array due to recombination processes within the diodes. The purpose of our work was to quantify the necessary correction and apply them to quality assurance measurements.
Methods
Static 10 × 10 cm\(^2\) 6-MV fields delivered by a linear accelerator were applied to the detector array while decreasing the average dose rate, that is, the pulse frequency, from 500 to 30 MU/min. An ion chamber was placed inside the ArcCHECK cavity as a reference. Furthermore, the instantaneous dose rate dependence (DRD) was studied. The position of the detector was adjusted to change the dose-per-pulse, varying the distance between the focus and the diode closest to the focus between 69.6 and 359.6 cm. Reference measurements were performed with an ion chamber placed inside a PMMA slab phantom at the same source-to-detector distances (SDDs). Exponential saturation functions were fitted to the data, with different parameters to account for two generations of ArcCHECK detectors (types 2 and 3) and both DRDs. Corrections were applied to 12 volumetric modulated arc therapy plans.
Results
The sensitivity decreased by up to 2.8% with a decrease in average dose rate and by 9% with a decrease in instantaneous dose rate. Correcting the average DRD, the mean gamma pass rates (2%/2-mm criterion) of the treatment plans were improved by 5 percentage points (PP) for diode type 3 and 0.4 PP for type 2. Correcting the instantaneous DRD, the improvement was 8.4 PP for type 3 and 0.9 PP for type 2.
Conclusions
The instantaneous DRD was identified as the prevailing effect on the diode sensitivity. We developed and validated a method to correct this behavior. The number of falsely not passed treatment plans could be considerably reduced.
Ischemia-like stress conditions stimulate trophic activities of adipose-derived stromal/stem cells
(2020)
Adipose-derived stromal/stem cells (ASCs) have been shown to exert regenerative functions, which are mainly attributed to the secretion of trophic factors. Upon transplantation, ASCs are facing an ischemic environment characterized by oxygen and nutrient deprivation. However, current knowledge on the secretion capacity of ASCs under such conditions is limited. Thus, the present study focused on the secretory function of ASCs under glucose and oxygen deprivation as major components of ischemia. After exposure to glucose/oxygen deprivation, ASCs maintained distinct viability, but the metabolic activity was greatly reduced by glucose limitation. ASCs were able to secrete a broad panel of factors under glucose/oxygen deprivation as revealed by a cytokine antibody array. Quantification of selected factors by ELISA demonstrated that glucose deprivation in combination with hypoxia led to markedly higher secretion levels of the angiogenic and anti-apoptotic factors IL-6, VEGF, and stanniocalcin-1 as compared to the hypoxic condition alone. A conditioned medium of glucose/oxygen-deprived ASCs promoted the viability and tube formation of endothelial cells, and the proliferation and migration of fibroblasts. These findings indicate that ASCs are stimulated by ischemia-like stress conditions to secrete trophic factors and would be able to exert their beneficial function in an ischemic environment.
In einem sich entwickelnden multizellulären Organismus ist die räumlich-zeitliche Regulation der Genexpression von entscheidender Bedeutung für die Bildung, Identität und Funktion von Zellen. Der REST (repressor element silencing transcription factor) Komplex spielt bei der neuronalen Differenzierung und bei der Aufrechterhaltung des neuronalen Status eine essentielle Rolle, indem er in nicht neuronalen Zellen und neuralen Vorläufern die Expression neuronaler Gene unterdrückt, in deren Promotorregion eine RE1 (repressor element 1) Erkennungssequenz vorhanden ist. Während der neuronalen Differenzierung wird der REST-Komplex schrittweise inaktiviert, was zur Einleitung eines neuronalen Genexpression-Programms führt. Es wird daher angenommen, dass die Inhibierung des REST-Komplexes ein essentieller Vorgang der Neurogenese ist. Wichtige Bestandteile für die transkriptionell repressive Funktion des REST-Komplexes sind kleine Phosphatasen (CTDSP = C-terminal domain small phosphatases), welche die Polymerase-II-Aktivität an Zielgenen inhibieren. Im Zebrafisch wurde gezeigt, dass ctdsp2 durch die miR-26b negativ reguliert wird. Alle miR-26 Familienmitglieder sind in Vertebraten evolutionär konserviert und in Introns von Ctdsp Genen kodiert. Sie sind in der Lage, die Expression ihres eigenen Wirtsgens mittels einer autoregulatorischen Rückkopplungsschleife zu regulieren.
Im Rahmen dieser Dissertation wurde als Modellsystem für die Neurogenese ein neurales Differenzierungssystem, welches auf murinen, embryonalen Stammzellen (ESCs) aufbaut, eingesetzt. Zur funktionellen Analyse der miR-26 Familie wurden mit Hilfe der CRISPR/Cas9-Methode verschiedene miR-26 Knockout (KO) ESC-Linien hergestellt. Hierbei wurden die Sequenzen der einzelnen Familienmitglieder und der gesamten miR-26 Familie im Genom von Wildtyp (Wt) ESCs deletiert. Diese miR-26-defizienten ESCLinien behielten ihre Pluripotenz und zeigten keinen Phänotyp hinsichtlich Proliferation, Morphologie und Identität der Zellen während der Differenzierung bis zum neuralen Vorläuferzellstadium (NPCs, engl.: neural progenitor cells). Jedoch führte die Deletion sowohl der gesamten miR-26 Familie als auch einzelner Mitglieder bei der terminalen Differenzierung zu einem spezifischen Entwicklungsstillstand im NPC Stadium und infolgedessen zu einer starken Reduktion der Anzahl von Neuronen und Astroglia. Die Transkriptom-Analyse der differenzierten miR-26-KO ESCs mittels RNA-Seq zeigte, dass die Expression von Genen die mit der Neurogenese und der neuronalen Differenzierung, aber auch der Gliogenese assoziert sind, herunterreguliert war. Die Abwesenheit der miR-26 Familie führte außerdem zu einer selektiven Reduzierung bestimmter miRNAs (REST-miRs), die einerseits die Expression von REST-Komplex Komponenten unterdrücken können, und andererseits selbst unter dessen transkriptioneller Kontrolle stehen. Zu diesem REST-miR Netzwerk gehören einige miRNAs (miR-9, miR-124, miR-132 und miR-218), die wichtige Funktionen bei verschiedenen Prozessen der neuronalen Entwicklung haben. Weiterhin führte der miR-26-KO zu einer Derepression der Proteinlevel von REST und CTDSP2 während der terminalen Differenzierung. Funktionelle Analysen mit miRNA mimics zeigten, dass erhöhte miR-26 Level zu einer Hochregulation von REST-miRs führen. Weitere Experimente, die darauf zielten, die Hierarchie des REST-miR Netwerks aufzuklären zeigten, dass die miR-26 Familie stromaufwärts die REST-miR Expression reguliert.
Zusammengefasst weisen die in dieser Arbeit gezeigten Daten darauf hin, dass die miR-26 Familie als Initiator der schrittweisen Inaktivierung des REST-Komplexes eine zentrale Rolle bei der Differenzierung von neuralen Vorläuferzellen zu postmitotischen Neuronen spielt.
Mbt/PAK4 together with SRC modulates N-Cadherin adherens junctions in the developing Drosophila eye
(2019)
Tissue morphogenesis is accompanied by changes of adherens junctions (AJ). During Drosophila eye development, AJ reorganization includes the formation of isolated N-Cadherin AJ between photoreceptors R3/R4. Little is known about how these N-Cadherin AJ are established and maintained. This study focuses on the kinases Mbt/PAK4 and SRC, both known to alter E-Cadherin AJ across phyla. Drosophila p21-activated kinase Mbt and the non-receptor tyrosine kinases Src64 and Src42 regulate proper N-Cadherin AJ. N-Cadherin AJ elongation depends on SRC kinase activity. Cell culture experiments demonstrate binding of both Drosophila SRC isoforms to N-Cadherin and its subsequent tyrosine phosphorylation. In contrast, Mbt stabilizes but does not bind N-Cadherin in vitro. Mbt is required in R3/R4 for zipping the N-Cadherin AJ between these cells, independent of its kinase activity and Cdc42-binding. The mbt phenotype can be reverted by mutations in Src64 and Src42. Because Mbt neither directly binds to SRC proteins nor has a reproducible influence on their kinase activity, the conclusion is that Mbt and SRC signaling converge on N-Cadherin. N-Cadherin AJ formation during eye development requires a proper balance between the promoting effects of Mbt and the inhibiting influences of SRC kinases.
Endogenous molecular circadian clocks drive daily rhythmic changes at the cellular, physiological, and behavioral level for adaptation to and anticipation of environmental signals. The core molecular system consists of autoregulatory feedback loops, where clock proteins inhibit their own transcription. A complex and not fully understood interplay of regulatory proteins influences activity, localization and stability of clock proteins to set the pace of the clock. This study focuses on the molecular function of Ribosomal S6 Kinase (RSK) in the Drosophila melanogaster circadian clock. Mutations in the human rsk2 gene cause Coffin–Lowry syndrome, which is associated with severe mental disabilities. Knock-out studies with Drosophila ortholog rsk uncovered functions in synaptic processes, axonal transport and adult behavior including associative learning and circadian activity. However, the molecular targets of RSK remain elusive. Our experiments provide evidence that RSK acts in the key pace maker neurons as a negative regulator of Shaggy (SGG) kinase activity, which in turn determines timely nuclear entry of the clock proteins Period and Timeless to close the negative feedback loop. Phosphorylation of serine 9 in SGG is mediated by the C-terminal kinase domain of RSK, which is in agreement with previous genetic studies of RSK in the circadian clock but argues against the prevailing view that only the N-terminal kinase domain of RSK proteins carries the effector function. Our data provide a mechanistic explanation how RSK influences the molecular clock and imply SGG S9 phosphorylation by RSK and other kinases as a convergence point for diverse cellular and external stimuli.
Loss of function mutations in the rsk2 gene cause Coffin-Lowry syndrome (CLS), which is associated with multiple symptoms including severe mental disabilities. Despite the characterization of ribosomal S6 kinase 2 (RSK2) as a protein kinase acting as a downstream effector of the well characterized ERK MAP-kinase signaling pathway, it turns out to be a challenging task to link RSK2 to specific neuronal processes dysregulated in case of mutation. Animal models such as mouse and Drosophila combine advanced genetic manipulation tools with in vivo imaging techniques, high-resolution connectome analysis and a variety of behavioral assays, thereby allowing for an in-depth analysis for gene functions in the nervous system. Although modeling mental disability in animal systems has limitations because of the complexity of phenotypes, the influence of genetic variation and species-specific characteristics at the neural circuit and behavioral level, some common aspects of RSK2 function in the nervous system have emerged, which will be presented. Only with this knowledge our understanding of the pathophysiology of CLS can be improved, which might open the door for development of potential intervention strategies.
In der vorliegenden Arbeit wurden die Verteilungsmuster von PG-Donorzellen in Gehirnen von Mäusechimären, die nach dem Aggregations- und dem ESC-Verfahren generiert wurden, im Embryonalstadium E14.5 untersucht und miteinander verglichen. Während in Aggregations-Chimären eine Präferenz von PG-Donorzellen für eine Besiedelung des Cortex und des Striatum zu beobachten ist, zeigen Gehirnbereiche in ESC-Chimären eine gleichmäßige Verteilung der PG-Donorzellen.
Um die unterschiedliche Besiedelung von PG-Stammzellen in den Chimären erklären zu können, wurden neuronale und gliale Zellfrequenzanalysen durchgeführt. Sowohl bei der relativen Neuronen- als auch bei der relativen Astrozytenhäufigkeit ist kein signifikanter Unterschied zwischen den Aggregations- und den ESC-Chimären festzustellen. Beide Chimärtypen unterscheiden sich nicht in der Zahl der aus PG-Donorzellen differenzierten Nerven- und Stützzellen.
Das Potenzial von PG-Stammzellen, funktionsfähige dopaminerge Neuronen zu bilden, wurde in den beiden Chimärtypen vergleichend analysiert. In beiden Chimärtypen wurden von PG-Donorzellen abstammende dopaminerge Neuronen nachgewiesen. Sowie für die Neuronen- und die Astrozytenzahl konnte auch für die Anzahl dopaminerger Neuronen kein signifikanter Unterschied zwischen Aggregations- und ESC-Chimären beobachtet werden.
Mushroom body defect is required in parallel to Netrin for midline axon guidance in Drosophila
(2016)
The outgrowth of many neurons within the central nervous system is initially directed towards or away from the cells lying at the midline. Recent genetic evidence suggests that a simple model of differential sensitivity to the conserved Netrin attractants and Slit repellents is insufficient to explain the guidance of all axons at the midline. In the Drosophila embryonic ventral nerve cord, many axons still cross the midline in the absence of the Netrin genes (NetA and NetB) or their receptor frazzled. Here we show that mutation of mushroom body defect (mud) dramatically enhances the phenotype of Netrin or frazzled mutants, resulting in many more axons failing to cross the midline, although mutations in mud alone have little effect. This suggests that mud, which encodes a microtubule-binding coiled-coil protein homologous to NuMA and LIN-5, is an essential component of a Netrin-independent pathway that acts in parallel to promote midline crossing. We demonstrate that this novel role of Mud in axon guidance is independent of its previously described role in neural precursor development. These studies identify a parallel pathway controlling midline guidance in Drosophila and highlight a novel role for Mud potentially acting downstream of Frizzled to aid axon guidance.
Background
Fabry disease is characterized by a progressive deposition of sphingolipids in different organ systems, whereby cardiac involvement leads to death. We hypothesize that lysosomal storage of sphingolipids in the heart as occurring in Fabry disease does not reflect in higher cardiac lipid concentrations detectable by \(^1\)H magnetic resonance spectroscopy (MRS) at 3 Tesla.
Methods
Myocardial lipid content was quantified in vivo by \(^1\)H-MRS in 30 patients (12 male, 18 female; 18 patients treated with enzyme replacement therapy) with genetically proven Fabry disease and in 30 healthy controls. The study protocol combined \(^1\)H-MRS with cardiac cine imaging and LGE MRI in a single examination.
Results
Myocardial lipid content was not significantly elevated in Fabry disease (p = 0.225). Left ventricular (LV) mass was significantly higher in patients suffering from Fabry disease compared to controls (p = 0.019). Comparison of patients without signs of myocardial fibrosis in MRI (LGE negative; n = 12) to patients with signs of fibrosis (LGE positive; n = 18) revealed similar myocardial lipid content in both groups (p > 0.05), while the latter showed a trend towards elevated LV mass (p = 0.076).
Conclusions
This study demonstrates the potential of lipid metabolic investigation embedded in a comprehensive examination of cardiac morphology and function in Fabry disease. There was no evidence that lysosomal storage of sphingolipids influences cardiac lipid content as measured by \(^1\)H-MRS. Finally, the authors share the opinion that a comprehensive cardiac examination including three subsections (LGE; \(^1\)H-MRS; T\(_1\) mapping), could hold the highest potential for the final assessment of early and late myocardial changes in Fabry disease.
Pluripotente Zellen sind sowohl in der Stammzellforschung als auch für regenerative Therapieansätze von großer Bedeutung. Erste Stammzelltherapien sind bereits erfolgreich am Menschen durchgeführt worden. Besonders wichtig ist die Sicherheit der Therapie, um Risiken, wie die „Entartung“ von Stammzellen zu Tumorzellen, zu minimieren. Als Ansatzpunkt für einheitliche Therapie-Standards, sind z.B. genaue Angaben zur Anzahl injizierter Zellen, dem Injektionsort und Biomarker (wie Pluripotenz- und Differenzierungs-Marker) zur Kategorisierung der Stammzellen zu nennen. Während der Embryonalentwicklung spielen die Polycomb-Proteinkomplexe PCR1 und PCR2 eine maßgebliche Rolle beim Aufrechterhalten der Pluripotenz, weil sie Chromatin-Modifikationen, wie z.B. Histonmethylierungen vermitteln und so die Genexpression kontrollieren können. Lange Zeit wurde angenommen, dass Histon-Methylierungen irreversibel sind, doch mit Entdeckung der Lysin-spezifischen Demethylase 1 (LSD1) wurde diese Sichtweise revidiert. Ein Mitglied der derzeit bekannten 32 Histon-Demethylasen ist Kdm6a (UTX), die die Histon-Demethylierung des Lysins an der Aminosäure-Position 27 von Histon H3 (H3K27me2/3) katalysiert. Kdm6a spielt eine wichtige Rolle bei der Embryogenese und wurde in der hier vorgestellten Arbeit am Teratommodell, einem benignen Keimzelltumor, untersucht.
In dieser Arbeit wurden Teratome von Mäusen untersucht, die aus embryonalen Stammzellen (ESC) mit Wildtyp- und shRNA vermittelter reduzierter Expression oder durch genetisch kontrollierten Knockdown sowie Knockout entstand sind. Diese wurden anschließend nach histologischen (H&E-Färbungen), histochemischen (PCNA-, SSEA-1- und TUNEL-Färbungen) sowie Analyse der Genexpressionsmuster aller drei Keimblätter mittels RT-PCR untersucht und ausgewertet.
Sowohl Wildtyp als auch Kdm6a-Knockdown und Knockout-Teratome bildeten Gewebe der drei Keimblätter aus. In Teratomen mit supprimierter Kdm6a-Expression gab es jedoch Unterschiede in der Bildung mesodermaler und endodermaler Gewebe mit einer signifikanten Abnahme von Knorpel- und Muskelgewebe. Da sich Kdm6a-defiziente Teratome zu wesentlich größeren Tumoren als Wildtyp-Teratome entwickelten, wurde deren Proliferations-, Pluripotenz- und Apoptose-Verhalten mittels PCNA und SSEA-1 und TUNEL histochemischen Färbungen untersucht. Wir beobachteten in Knockout-Teratomen eine höhere Anzahl von PCNA- und SSEA-1-positiven Zellen. Daraus folgt, dass Kdm6a-defiziente ESCs - im Gegensatz zu Wildtyp ESCs - zur Bildung von Teratomen mit einer höheren Anzahl von proliferierenden und pluripotenten Zellen neigen. In der Fraktion apoptotischer Zellen (TUNEL positiver Zellen) der Kdm6a-defizienten Teratome gab es keinen signifikanten Unterschied zu Teratomen, die aus Wildtyp-ESCs entstanden.
Nach Analyse der Genexpressionsmuster fanden wir in Zellen, in denen Kdm6a reprimiert bzw. deaktiviert wurde, einen Verlust der Pluripotenz und folglich eine starke Reduzierung der Pluripotenzmarker Oct4, Sox2 und Nanog. Die Analyse des Genexpressionsmusters läßt vermuten, dass der Verlust bzw. die Abnahme der Kdm6a-Aktivität in direkten Zusammenhang mit einer Abnahme der Pluripotenz durch Methylierung von H3K27 steht. Weitere Analysen, z.B. durch ChIP (Chromatin Immun-Präzipitations-) Assays mit H3K27me2/3 spezifischen Antikörpern, sind nötig, um dies endgültig zu beweisen. Unsere Arbeiten zeigten, dass die Kdm6-Demethylase-Aktivität essentiell für den Erhalt der Pluripotenz von embryonalen Stammzellen ist.
Cord blood hematopoietic stem cells (CB-HSCs) are an outstanding source for transplantation approaches. However, the amount of cells per donor is limited and culture expansion of CB-HSCs is accompanied by a loss of engraftment potential. In order to analyze the molecular mechanisms leading to this impaired potential we profiled global and local epigenotypes during the expansion of human CB hematopoietic stem and progenitor cells (HPSCs). Human CB-derived CD34+ cells were cultured in serum-free medium together with SCF, TPO, FGF, with or without Igfbp2 and Angptl5 (STF/STFIA cocktails). As compared to the STF cocktail, the STFIA cocktail maintains in vivo repopulation capacity of cultured CD34+ cells. Upon expansion, CD34+ cells genome-wide remodel their epigenotype and depending on the cytokine cocktail, cells show different HK4me3 and H3K27me3 levels. Expanding cells without Igfbp2 and Angptl5 leads to higher global H3K27me3 levels. ChIPseq analyses reveal a cytokine cocktail-dependent redistribution of H3K27me3 profiles. Inhibition of the PRC2 component EZH2 counteracts the culture-associated loss of NOD scid gamma (NSG) engraftment potential. Collectively, our data reveal chromatin dynamics that underlie the culture-associated loss of engraftment potential. We identify PRC2 component EZH2 as being involved in the loss of engraftment potential during the in vitro expansion of HPSCs.
Plastic changes in synaptic properties are considered as fundamental for adaptive behaviors. Extracellular-signal-regulated kinase (ERK)-mediated signaling has been implicated in regulation of synaptic plasticity. Ribosomal S6 kinase 2 (RSK2) acts as a regulator and downstream effector of ERK. In the brain, RSK2 is predominantly expressed in regions required for learning and memory. Loss-of-function mutations in human RSK2 cause Coffin-Lowry syndrome, which is characterized by severe mental retardation and low IQ scores in affected males. Knockout of RSK2 in mice or the RSK ortholog in Drosophila results in a variety of learning and memory defects. However, overall brain structure in these animals is not affected, leaving open the question of the pathophysiological consequences. Using the fly neuromuscular system as a model for excitatory glutamatergic synapses, we show that removal of RSK function causes distinct defects in motoneurons and at the neuromuscular junction. Based on histochemical and electrophysiological analyses, we conclude that RSK is required for normal synaptic morphology and function. Furthermore, loss of RSK function interferes with ERK signaling at different levels. Elevated ERK activity was evident in the somata of motoneurons, whereas decreased ERK activity was observed in axons and the presynapse. In addition, we uncovered a novel function of RSK in anterograde axonal transport. Our results emphasize the importance of fine-tuning ERK activity in neuronal processes underlying higher brain functions. In this context, RSK acts as a modulator of ERK signaling.
Background
Myc proteins are essential regulators of animal growth during normal development, and their deregulation is one of the main driving factors of human malignancies. They function as transcription factors that (in vertebrates) control many growth- and proliferation-associated genes, and in some contexts contribute to global gene regulation.
Results
We combine chromatin immunoprecipitation-sequencing (ChIPseq) and RNAseq approaches in Drosophila tissue culture cells to identify a core set of less than 500 Myc target genes, whose salient function resides in the control of ribosome biogenesis. Among these genes we find the non-coding snoRNA genes as a large novel class of Myc targets. All assayed snoRNAs are affected by Myc, and many of them are subject to direct transcriptional activation by Myc, both in Drosophila and in vertebrates. The loss of snoRNAs impairs growth during normal development, whereas their overexpression increases tumor mass in a model for neuronal tumors.
Conclusions
This work shows that Myc acts as a master regulator of snoRNP biogenesis. In addition, in combination with recent observations of snoRNA involvement in human cancer, it raises the possibility that Myc’s transforming effects are partially mediated by this class of non-coding transcripts.
Parent-of-origin imprints have been implicated in the regulation of neural differentiation and brain development. Previously we have shown that, despite the lack of a paternal genome, human parthenogenetic (PG) embryonic stem cells (hESCs) can form proliferating neural stem cells (NSCs) that are capable of differentiation into physiologically functional neurons while maintaining allele-specific expression of imprinted genes. Since biparental ("normal") hESC-derived NSCs (N NSCs) are targeted by immune cells, we characterized the immunogenicity of PG NSCs. Flow cytometry and immunocytochemistry revealed that both N NSCs and PG NSCs exhibited surface expression of human leukocyte antigen (HLA) class I but not HLA-DR molecules. Functional analyses using an in vitro mixed lymphocyte reaction assay resulted in less proliferation of peripheral blood mononuclear cells (PBMC) with PG compared with N NSCs. In addition, natural killer (NK) cells cytolyzed PG less than N NSCs. At a molecular level, expression analyses of immune regulatory factors revealed higher HLA-G levels in PG compared with N NSCs. In line with this finding, MIR152, which represses HLA-G expression, is less transcribed in PG compared with N cells. Blockage of HLA-G receptors ILT2 and KIR2DL4 on natural killer cell leukemia (NKL) cells increased cytolysis of PG NSCs. Together this indicates that PG NSCs have unique immunological properties due to elevated HLA-G expression.
All organisms have to adapt to acute as well as to regularly occurring changes in the environment. To deal with these major challenges organisms evolved two fundamental mechanisms: the p38 mitogen-activated protein kinase (MAPK) pathway, a major stress pathway for signaling stressful events, and circadian clocks to prepare for the daily environmental changes. Both systems respond sensitively to light. Recent studies in vertebrates and fungi indicate that p38 is involved in light-signaling to the circadian clock providing an interesting link between stress-induced and regularly rhythmic adaptations of animals to the environment, but the molecular and cellular mechanisms remained largely unknown. Here, we demonstrate by immunocytochemical means that p38 is expressed in Drosophila melanogaster's clock neurons and that it is activated in a clock-dependent manner. Surprisingly, we found that p38 is most active under darkness and, besides its circadian activation, additionally gets inactivated by light. Moreover, locomotor activity recordings revealed that p38 is essential for a wild-type timing of evening activity and for maintaining ∼ 24 h behavioral rhythms under constant darkness: flies with reduced p38 activity in clock neurons, delayed evening activity and lengthened the period of their free-running rhythms. Furthermore, nuclear translocation of the clock protein Period was significantly delayed on the expression of a dominant-negative form of p38b in Drosophila's most important clock neurons. Western Blots revealed that p38 affects the phosphorylation degree of Period, what is likely the reason for its effects on nuclear entry of Period. In vitro kinase assays confirmed our Western Blot results and point to p38 as a potential "clock kinase" phosphorylating Period. Taken together, our findings indicate that the p38 MAP Kinase is an integral component of the core circadian clock of Drosophila in addition to playing a role in stress-input pathways.
The aim of this study was to investigate the prognostic value of 18F-fluoro-deoxyglucose positron emission tomography–computed tomography (18F-FDG-PET/CT) in 37 patients with a history of multiple myeloma (MM) and suspected or confirmed recurrence after stem cell transplantation (SCT). All patients had been heavily pre-treated. Time to progression (TTP) and overall survival (OS) were correlated to a number of different PET-derived as well as clinical parameters. Impact on patient management was assessed.
Absence of FDG-avid MM foci was a positive prognostic factor for both TTP and OS (p<0.01). Presence of >10 focal lesions correlated with both TTP (p<0.01) and OS (p<0.05). Interestingly, presence of >10 lesions in the appendicular skeleton proved to have the strongest association with disease progression. Intensity of glucose uptake and presence of extramedullary disease were associated with shorter TTP (p=0.037 and p=0.049, respectively). Manifestations in soft tissue structures turned out to be a strong negative predictor for both, TTP and OS (p<0.01, respectively). PET resulted in a change of management in 30% of patients.
Our data underline the prognostic value of 18F-FDG-PET/CT in MM patients also in the setting of post-SCT relapse. PET/CT has a significant impact on patient management.
Cell growth and cell division are two interconnected yet distinct processes. Initiation of proliferation of central brain progenitor cells (neuroblasts) after the late embryonic quiescence stage requires cell growth, and maintenance of proper cell size is an important prerequisite for continuous larval neuroblast proliferation. Beside extrinsic nutrition signals, cell growth requires constant supply with functional ribosomes to maintain protein synthesis.
Mutations in the mushroom body miniature (mbm) gene were previously identified in a screen for structural brain mutants. This study focused on the function of the Mbm protein as a new nucleolar protein, which is the site of ribosome biogenesis. The comparison of the relative expression levels of Mbm and other nucleolar proteins in different cell types showed a pronounced expression of Mbm in neuroblasts, particularly in the fibrillar component of the nucleolus, suggesting that in addition to nucleolar components generally required for ribosome biogenesis, more neuroblast specific nucleolar factors exist. Mutations in mbm cause neuroblast proliferation defects but do not interfere with cell polarity, spindle orientation or asymmetry of cell division of neuroblasts. Instead a reduction in cell size was observed, which correlates with an impairment of ribosome biogenesis. In particular, loss of Mbm leads to the retention of the small ribosomal subunit in the nucleolus resulting in decreased protein synthesis. Interestingly, the defect in ribosome biogenesis was only observed in neuroblasts. Moreover, Mbm is apparently not required for cell size and proliferation control in wing imaginal disc and S2 cells supporting the idea of a neuroblast-specific function of Mbm.
Furthermore, the transcriptional regulation of the mbm gene and the functional relevance of posttranslational modifications were analyzed. Mbm is a transcriptional target of dMyc. A common feature of dMyc target genes is the presence of a conserved E-box sequence in their promoter regions. Two E-box motifs are found in the vicinity of the transcriptional start site of mbm. Gene reporter assays verified that only one of them mediates dMyc-dependent transcription. Complementary studies in flies showed that removal of dMyc function in neuroblasts resulted in reduced Mbm expression levels.
At the posttranslational level, Mbm becomes phosphorylated by protein kinase CK2. Six serine and threonine residues located in two acidic amino acid rich clusters in the C-terminal half of the Mbm protein were identified as CK2 phosphorylation sites.
Mutational analysis of these sites verified their importance for Mbm function in vivo and indicated that Mbm localization is controlled by CK2-mediated phosphorylation.
Although the molecular function of Mbm in ribosome biogenesis remains to be determined, the results of this study emphasize the specific role of Mbm in neuroblast ribosome biogenesis to control cell growth and proliferation.
The CK2 Kinase Stabilizes CLOCK and Represses Its Activity in the Drosophila Circadian Oscillator
(2013)
Phosphorylation is a pivotal regulatory mechanism for protein stability and activity in circadian clocks regardless of their evolutionary origin. It determines the speed and strength of molecular oscillations by acting on transcriptional activators and their repressors, which form negative feedback loops. In Drosophila, the CK2 kinase phosphorylates and destabilizes the PERIOD (PER) and TIMELESS (TIM) proteins, which inhibit CLOCK (CLK) transcriptional activity. Here we show that CK2 also targets the CLK activator directly. Downregulating the activity of the catalytic alpha subunit of CK2 induces CLK degradation, even in the absence of PER and TIM. Unexpectedly, the regulatory beta subunit of the CK2 holoenzyme is not required for the regulation of CLK stability. In addition, downregulation of \(CK2\alpha\) activity decreases CLK phosphorylation and increases per and tim transcription. These results indicate that CK2 inhibits CLK degradation while reducing its activity. Since the CK1 kinase promotes CLK degradation, we suggest that CLK stability and transcriptional activity result from counteracting effects of CK1 and CK2.