Role of autophagic protein ULK1 in axonal degeneration and regeneration in rat cortical neurons in vitro

In many traumatic and neurodegenerative disorders of the central nervous system, axonal degeneration is a central and early pathophysiological feature. Since the regenerative response of injured neurons is severely hampered by an intrinsically reduced growth capacity and external growth-inhibiting factors such as CSPG, axonal degeneration often results in progressive clinical disability. An improved understanding of the molecular mechanisms is hoped to unravel potential therapeutic targets, as there are currently no therapeutic options available. During axonal degeneration, macroautophagy (here: autophagy), a cellular homeostatic process responsible for the degradation of long-lived organelles and proteins, was previously shown to be activated, but its detailed role in this context, harmful or beneficial, is incompletely understood. Particularly, the autophagy-initiating kinase ULK1 accumulated in degenerating axons, leading to the speculation that ULK1-dependent autophagy might represent a deleterious executing process during axonal degeneration. To assess this hypothesis in the present study, primary cortical neurons were transduced with an adeno-associated viral vector expressing kinase-dead dominant-negative ULK1, leading to the inhibition of ULK1 function. After selective axonal lesion to transduced neurons cultured in microfluidic chambers, dominant-negative ULK1 attenuated acute axonal degeneration for up to six hours and fostered axonal regeneration up to 96 hours post-injury, as compared with control. Correspondingly, increased neurite outgrowth was observed after transduction with dominant-negative ULK1 in neurons cultured on both the permissive substrate laminin and the growth-inhibiting matrix CSPG. Mechanistically, dominant-negative ULK1 reduced autophagy activation, indicating that a decrease in autophagy is one mechanism underlying its axon-protective effects. The additional proteomic analysis of neurons transduced with dominant-negative ULK1 surprisingly outlined a strong regulation of splicing and translation-associated proteins. This finding was corroborated by a transcriptomic analysis uncovering dominant-negative ULK1-dependent differential splicing of axonal degeneration and regeneration-associated genes such as Kif1b and Ddit3. Dominant-negative ULK1 might therefore additionally elicit axon-protective and pro-regenerative properties via a modulation of Kif1b-dependent axonal transport and Ddit3-mediated endoplasmic reticulum stress. Furthermore, the beneficial effects of dominant-negative ULK1 on axonal degeneration and regeneration were connected to an activation of mTOR-S6-mediated translation, elevated expression of the growth-promoting molecule p-ERK1, and reduced levels of the degeneration-mediator and growth-inhibitor ROCK2. Together, the data obtained in this thesis reveal a key involvement of ULK1 in axonal degeneration and regeneration in vitro, and demonstrate a more complex role of ULK1 in neuronal biology than previously known, including a novel function in splicing. ULK1 inhibition attenuates the degeneration of axons and promotes their regeneration in vitro by a proposed switch from the catabolic autophagy process to the activation of axon-protective and axon growth-promoting processes. ULK1 inhibition thus represents a putative therapeutic approach in traumatic and neurodegenerative disorders of the central nervous system.2021-11-1

Disentangling drivers of colonization success in laboratory and natural systems

2017 Summer.Includes bibliographical references.Understanding why colonizing populations successfully establish is important for predicting dynamics of invasive species. Propagule pressure, or the number of individuals in a founding group, is considered the most consistent predictor of establishment success, however, there remains considerable variance around predictions that demography alone cannot explain. The identity of individuals within a founding group (e.g. level of pre-adaptation to the recipient environment, diversity) as well as how individuals are introduced (e.g. frequency and timing of discrete introduction events) can influence establishment. The relative importance of these factors is unclear, and could vary across species and environmental contexts. To address these inconsistencies, we conducted two experiments: one with Tribolium castaneum (red flour beetle) populations maintained in controlled laboratory conditions, and one with Bromus tectorum (cheatgrass) founding populations introduced to a natural environment. For the Tribolium experiment, we varied the level of prior adaptation, diversity, and introduction frequency and timing for groups of eggs colonizing in a novel environment across three levels of propagule pressure (n = 15, 30, 60). Founding groups that were larger and more adapted to the novel environment survived the founding event better than smaller and less adapted groups. Further, we found that a high frequency of smaller introductions reduced initial survival. After a generation of mating, establishment success was driven predominantly by adaptation to the novel environment and diversity of founders. In the second experiment, we introduced groups of B. tectorum seeds at a constant propagule pressure (n = 32) to a common garden in Colorado, varying in source diversity (1, 2, 4, 8, or 16 source populations) and source region (Colorado = pre-adapted or Nevada = unadapted). We evaluated establishment success by deriving the number of seeds produced by each founding group after one generation of growth and reproduction using a hierarchical Bayesian model. We found that increasing source diversity increased the number of seeds produced per founding group, but source region did not influence establishment success. Results from these experiments particularly speak to the context-dependency of the importance of pre-adaptation and diversity in predicting establishment success. This suggests that propagule pressure alone is not enough to explain why founding populations establish

Simulation of the Directional Dark Matter Detector (D3) and Directional Neutron Observer (DiNO)

Preliminary simulation and optimization studies of the Directional Dark Matter Detector and the Directional Neutron Observer are presented. These studies show that the neutron interaction with the gas-target in these detectors is treated correctly by GEANT4 and that by lowering the pressure, the sensitivity to low-mass WIMP candidates is increased. The use of negative ion drift might allow us to search the WIMP mass region suggested by the results of the non-directional experiments DAMA/LIBRA, CoGeNT and CRESST-II.Comment: Proceedings of the 3rd International conference on Directional Detection of Dark Matter (CYGNUS 2011), Aussois, France, 8-10 June 201

Application of Time Projection Chambers with GEMs and Pixels to WIMP Searches and Fast Neutron Detection

We present work on the detection of neutral particles via nuclear recoils in gas-filled Time Projection Chambers (TPCs). We employ Gas Electron Multipliers (GEMs) to amplify the signal and silicon pixel electronics to detect the avalanche charge. These technologies allow ionization in the target gas to be detected with low noise, improved position and time resolution, and high efficiency. We review experimental results obtained in previous years, and report on ongoing simulation studies and construction of the first prototype at the University of Hawaii. We also present prospects of using such detectors to perform direction-sensitive searches for WIMP dark matter and fast neutron from fissionable material.Comment: Proceedings of 2nd International Conference on Technology and Instrumentation in Particle Physics (TIPP 2011), to be published in Physics Procedia, 8 pages, 9 figure

Escuelas Universitarias de Trabajo Social en Alemania.

Sin resume

Charge-Focusing Readout of Time Projection Chambers

Time projection chambers (TPCs) have found a wide range of applications in particle physics, nuclear physics, and homeland security. For TPCs with high-resolution readout, the readout electronics often dominate the price of the final detector. We have developed a novel method which could be used to build large-scale detectors while limiting the necessary readout area. By focusing the drift charge with static electric fields, we would allow a small area of electronics to be sensitive to particle detection for a much larger detector volume. The resulting cost reduction could be important in areas of research which demand large-scale detectors, including dark matter searches and detection of special nuclear material. We present simulations made using the software package Garfield of a focusing structure to be used with a prototype TPC with pixel readout. This design should enable significant focusing while retaining directional sensitivity to incoming particles. We also present first experimental results and compare them with simulation.Comment: 5 pages, 17 figures, Presented at IEEE Nuclear Science Symposium 201

Die Rolle der 12/15-Lipoxygenase in der Pathogenese der Insulinresistenz : Untersuchungen an der Alox15-Knockout-Maus

Die Aufrechterhaltung der Glukosehomöostase wird im erster Linie von dem Polypeptidhormon Insulin gesteuert. Durch Bindung von Insulin an den Insulinrezeptor wird die Insulinsignalkaskade aktiviert und somit die Translokation des Glukosetransporters GLUT4 zur Plasmamembran reguliert. Dort vermittelt GLUT4 die Aufnahme von Glukose in die Zelle. Diese Translokation geschieht über ein Aktinfilament-Netzwerk, das sich nach Insulinstimulus aus Aktinfilamenten umbildet. Es gilt als erwiesen, dass Metaboliten der 12/15-Lipoxygenase an der Regulation dieses Netzwerkes beteiligt sind und somit Einfluss auf den Transport von GLUT4 zur Plasmamembran und damit auf die Glukoseaufnahme haben. In Untersuchungen konnte eine vollständige Hemmung der Glukoseaufnahme in Kardiomyozyten, die mit einem Lipoxygenase-Inhibitor behandelt wurden, festgestellt werden. Aufgrund dieser Daten sollte die Funktion der 12/15-Lipoxygenase in einem Knockoutmodell näher charakterisiert werden. In der vorliegenden Arbeit konnte keine veränderte GLUT4-Expression festgestellt werden. Die Akt-Expression war lediglich im Skelettmuskel der männlichenAlox15-Knockout-Tiere signifikant vermindert Die Phosphorylierung der Akt nach Insulinstimulus war in Herz und Skelettmuskel der Knockout-Tiere bei beiden Geschlechtern unverändert. In beiden Geweben der Alox15-Knockout-Tiere konnte keine veränderte GSK3alpha- oder -beta-Expression festgestellt werden. Die Phosphorylierung der beiden Isoformen nach Insulinstimulus war ebenfalls unverändert. Aufgrund der vorliegenden Ergebnisse hat der Verlust der 12/15-Lipoxygenase keinen Einfluss auf den GLUT4-Gehalt. Die Insulinsignalkaskade wird ebenfalls nicht beeinflusst. Eine Aussage über eine veränderte GLUT4-Translokation kann anhand dieser Daten nicht gemacht werden. Um eine Störung durch den Verlust des Enzyms genauer feststellen zu können, müsste das Aktinfilament-Netzwerk und die GLUT4-Translokation in den Knockout-Tieren näher untersucht werden. Die Insulinresistenz ist die erste pathologische Störung, die bei der Entwicklung eines Diabetes mellitus Typ 2 auftritt. Durch eine verminderte Wirkung des Insulins in den insulinsensitiven Geweben (Skelettmuskel, Leber, Fettgewebe, Herz), kommt es zu einer verminderten Aufnahme von Glukose in die Zelle und damit zu einem erhöhten Blutglukosespiegel. Umgekehrt kann aber auch eine Störung der GLUT4-Translokation eine Insulinresistenz auslösen. In der vorliegenden Arbeit sollten deshalb die Knockout-Tiere auch in Hinblick auf die Entwicklung einer Insulinresistenz untersucht werden. Es konnten keine veränderten Plasmaglukosespiegel bei den Alox15-Knockout-Tieren nachgewiesen werden. Auch nach Insulinstimulus kam es bei der Knockout-Maus zu einem physiologischen Abfall des Plasmaglukosespiegels. Die GLUT4-Expression sowie die Expression und Phosphorylierung der Signalelemente Akt und GSK3 waren unverändert (s.o.). Lediglich die Akt-Expression im Skelettmuskel der männlichen Tiere war signifikant vermindert. Aufgrund dieser Daten haben die Knockout-Tiere keine Insulinresistenz entwickelt.Die verminderte Akt-Expression gibt ebenfalls keinen Hinweis auf eine Insulinresistenz. Um diese Daten abzusichern, sollten weitere Untersuchungen vorgenommen werden. Anhand der vorliegenden Ergebnisse ist davon auszugehen, dass in den Alox15-Knockout-Tieren spezifische Kompensationsmechanismen aktiviert wurden, die die Entwicklung einer Insulinresistenz verhindert haben. Weitere Untersuchungen dieser Mechanismen müssten durchgeführt werden.The maintenance of glucose homeostasis is primarily regulated by the polypeptidehormone insulin. Insulin rapidly stimulates glucose transport by inducing the translocation of vesicles containing the glucose transporter isoform 4 (GLUT4) from intracellular pools to the plasma membrane. When inserted in the plasma membrane, GLUT4 catalyses the glucose uptake into the cell. Actin cytoskeletal elements are involved in mediating the insulin-induced GLUT4 translocation. Metabolites of the 12/15-Lipoxygenase have recently been shown to contribute to the regulation of actin cytoskeleton rearrangement and therefore to the GLUT4 translocation and glucose uptake. Inhibition of 12/15-lipoxygenase activity completely abrogated insulin induced glucose uptake and GLUT4 translocation in cardiomyocytes. Based on these data the implication of 12/15-lipoxygenase was investigated in knockout mouse model. The results obtained show no effect on the total expression of GLUT4. Akt expression was only decreased in skeletal muscle of the male knockout mice. The phosphorylation of Akt after stimulation with insulin was not altered in heart and skeletal muscle of male and female knockout mice. In both tissues the expression and phosphorylation of GSK3alphabeta were also unchanged. These data suggest that the loss of 12/15-lipoxygenase has no effect on GLUT4 level and the insulin signal transduction. Studies on actin cytoskeleton structure and GLUT4 translocation will be required to potentially demonstrate the functional consequences of 12/15-lipoxygenase knockout. Insulin resistance is the first pathological disorder in the development of diabetes mellitus type 2. The decreased effect of insulin in insulin sensitive tissues (skeletal muscle, liver, adipose tissue, heart) results in a decreased glucose uptake into the cell and therefore in increased blood glucose level. Oppositely a disorder in GLUT4 translocation can also produce an insulin resistance.Therefore in this work the development of insulin resistance should be examinded in the Alox15 knockout mouse model. These results show that plasma glucose levels in Alox15-knockout animals are unchanged. Also, after stimulation with insulin the decrease of blood glucose levels was physiological. These data suggest that the knockout animals develop no insulin resistance. Also the decreased Akt expression gives no evidence for an insulin resistance. To confirm these data, further investigations should be done. It seems that in the Alox15 knockout mouse a specific mechanism of compensation has been activated, which prevents the development of insulin resistance. Further investigations of these mechanisms should be done

Probing neutralino dark matter in the MSSM & the NMSSM with directional detection

We investigate the capability of directional detectors to probe neutralino dark matter in the Minimal Supersymmetric Standard Model and the Next-to-Minimal Supersymmetric Standard Model with parameters defined at the weak scale. We show that directional detectors such as the future MIMAC detector will probe spin dependent dark matter scattering on nucleons that are beyond the reach of current spin independent detectors. The complementarity between indirect searches, in particular using gamma rays from dwarf spheroidal galaxies, spin dependent and spin independent direct search techniques is emphasized. We comment on the impact of the negative results on squark searches at the LHC. Finally, we investigate how the fundamental parameters of the models can be constrained in the event of a dark matter signal.Comment: 21 pages, 16 figure

Functional analysis of the apoptosis-inducing factor (AIF)

The apoptosis-inducing factor (AIF) is a phylogenetically old flavoprotein that is localised in mitochondria of healthy cells. It has been known to play an important role in the process of apoptosis, a genetically programmed cell suicide that removes supernumerous or potentially dangerous cells from a multicellular organism. Upon apoptosis induction, AIF translocates from the mitochondria via the cytosol to the nucleus, where it is involved in apoptosis-associated chromatinolysis. Here, I describe a hitherto unknown role of AIF in the functioning of the respiratory chain, the cell’s major source of energy. AIF-deficient murine embryonic stem cells were found to be highly dependent on glycolysis. This abnormal dependency was due to defective oxidative phosphorylation affecting in particular complex I and III of the respiratory chain. Similar observations were made in HeLa cells, in which the AIF expression had been downregulated by RNA interference. However, in the human cell line, reduced activity was found in complex I, but not in complex III. The respiratory chain defect was detectable not only at the functional but also at the protein level. All tested complex I subunits as well as the whole complex I were found to be less abundant in AIF-deficient cells than in their wild-type counterparts. This seemed to be a post-transcriptional phenomenon since the absence of AIF did not modify the levels of mRNA’s. Moreover, AIF does not seem to be a part of complex I or any other known respiratory chain complex, but retransfection with mitochondrially targeted AIF did cause re-expression of complex I and III subunits in AIF-KO cells. Since AIF has an oxidoreductase domain, it was speculated that AIF protected the respiratory chain complexes against ROS-induced damage, but treatment with several antioxidants could not restore the expression of complex I subunits in AIF-deficient cells. Therefore, AIF seems to play a role in the assembly or the maintenance of complex I and maybe other complexes. The existence of so-called supercomplexes, which comprise several respiratory chain complexes, could explain why the stability of more than one complex is affected. Harlequin mice, which exhibit only 20% of AIF expression due to a retroviral insertion in the Aif gene, were also found to have a complex I defect in different tissues including brain and retina but not heart and liver. That allowed for a re-interpretation of the harlequin phenotype, which is characterised by progressive neuronal and retinal degradation resembling symptoms of mitochondriopathies in humans. Harlequin mice can therefore serve as a mouse model to study complex I diseases. Moreover, it was shown that conditional knock-out mice (developed in Dr.Penninger’s laboratory) that did not express AIF in heart and muscle tissues and suffered from cardiomyopathy and muscle atrophy, had a respiratory chain defect in the affected tissues. In this animal model a complex I defect was also detected in the heart. It thus seems that AIF’s role in respiration is tissue-specific and also threshold-dependent. In a second part of this work, AIF’s role in apoptosis was further characterised by defining the interaction of AIF with nucleic acids. While the interaction of AIF with DNA had been described before, the binding of AIF to RNA was a new discovery. Recombinant AIF retained DNA fragments as well as RNA extracted from HeLa cells in a gel retention assay in a size and sequence independent manner. The direct interaction was also observed in electron microscopy experiments, where the presence of AIF induced the compaction of DNA and the formation of large aggregates of both DNA and RNA. Moreover, AIF showed a preferential binding to single-stranded over double-stranded DNA. Since different RNA molecules inhibited the retention of DNA by AIF, it was hypothesized that the nucleic acids bind to a similar or the same site of the protein. This hypothesis was further supported by the finding that two AIF mutants lacking positively charged amino acids, which had been described to mediate the AIF-DNA interaction, were less efficient in retaining both DNA and RNA in a horizontal agarose gel electrophoresis. Interestingly, the affinity for nucleic acids was enhanced by the co-factor NADP. While the role of the AIF-RNA interaction in vivo remains a conundrum, the AIF-DNA interaction plays an important role in chromatin condensation and, in certain cell death scenarios, large-scale DNA fragmentation