Von der Form zur Richtung

Growth cones, the terminal structures of elongating neurites are important for development and regeneration. The second messenger cAMP regulates both processes. cAMP is also known to induce growth cone turning. By using time-lapse microscopy and release of cAMP from biological inactive caged compounds the directional response of neuronal growth cones was investigated. Intracellular gradients were induced by asymmetric release of cAMP on one side of the growth cone. Using a new method, which released the same amount cAMP independent of the stimulation pattern it was possible to show that pulsed release in contrast to continuous release was more effective in inducing growth cone turning. From experiments with several pharmacological agents it can be concluded that the protein kinase A plays a major role for the cellular responses and that the gradient steepness causes the different reactions. In cooperation with other scientists it was ruled out that the different responses were caused by non-linearities of the caged compound. Furthermore cAMP gradients were visualized directly in HeLa cells expressing cAMP sensitive ion channels as a reporter system. With this cell line it was also possible to show that the stimulation pattern, meaning pulsed and continuous release respectively, influences the gradient shape. This finding was in accordance with published theories and numerical calculations. Here it was shown for the first time that the spatio-temporal properties of the intracellular gradient are crucial for growth cone turning.Sowohl bei der Entwicklung als auch der Regeneration des Nervensystems spielen die wachsenden Enden von Nerven, sogenannte neuronale Wachstumskegel, eine wichtige Rolle. Es ist bekannt, dass beide Prozesse durch den Zweitbotenstoff cAMP reguliert werden und dass dieser das Richtungswachstum beeinflussen kann. In der vorliegenden Arbeit wurde mit Hilfe von Zeitraffer-Mikroskopie und der Freisetzung von cAMP aus biologisch inaktiven Käfigsubstanzen das Richtungswachstum von neuronalen Wachstumskegeln untersucht. Durch asymmetrische Freisetzung des cAMP auf einer Seite des Wachstumskegels wurden intrazelluläre cAMP Gradienten erzeugt. Mit einer neu entwickelten Methode, bei der mit verschiedenen Freisetzungsmustern immer die gleiche Menge cAMP freigesetzt wurde, konnte gezeigt werden, dass eine gepulste Freisetzung im Gegensatz zur kontinuierlichen das Richtungswachstum beeinflusst. Experimente mit verschiedenen Pharmaka legen den Schluss nahe, dass die Protein Kinase A für die Antworten der Zellen und die Gradientensteilheit für die unterschiedliche Wirksamkeit von gepulster und kontinuierlicher Erzeugung des cAMP verantwortlich sind. In Zusammenarbeit mit anderen Wissenschaftlern konnte des Weiteren ausgeschlossen werden, dass die unterschiedlichen Ergebnisse bei kontinuierlicher und gepulster Freisetzung durch einen Artefakt oder nichtlineare Effekte bei der Freisetzung der Käfigsubstanz zustandekamen. Zudem konnte mit Hilfe einer Zellinie, die cAMP sensitive Kanälen stabil exprimiert, gezeigt werden, dass es möglich ist, intrazelluläre cAMP Gradienten zu erzeugen und dass unterschiedliche Freisetzungsmuster, das heißt kontinuierliche und gepulste Freisetzung, zu unterschiedlichen Gradienten führen. Dieser Befund konnte des Weiteren mit Hilfe einer numerischen Simulation mit den gängigen Theorien abgeglichen werden. In der vorliegenden Arbeit wurde erstmals gezeigt, dass die raum-zeitliche Form von intrazellulären Gradienten für die Orientierung von neuronalen Fortsätzen ausschlaggebend ist

HDAC6 is a bruchpilot deacetylase that facilitates neurotransmitter release

Presynaptic densities are specialized structures involved in synaptic vesicle tethering and neurotransmission; however, the mechanisms regulating their function remain understudied. In Drosophila, Bruchpilot is a major constituent of the presynaptic density that tethers vesicles. Here, we show that HDAC6 is necessary and sufficient for deacetylation of Bruchpilot. HDAC6 expression is also controlled by TDP-43, an RNA-binding protein deregulated in amyotrophic lateral sclerosis (ALS). Animals expressing TDP-43 harboring pathogenic mutations show increased HDAC6 expression, decreased Bruchpilot acetylation, larger vesicle-tethering sites, and increased neurotransmission, defects similar to those seen upon expression of HDAC6 and opposite to hdac6 null mutants. Consequently, reduced levels of HDAC6 or increased levels of ELP3, a Bruchpilot acetyltransferase, rescue the presynaptic density defects in TDP-43-expressing flies as well as the decreased adult locomotion. Our work identifies HDAC6 as a Bruchpilot deacetylase and indicates that regulating acetylation of a presynaptic release-site protein is critical for maintaining normal neurotransmission

Collaborating by courier, imaging by mail

Core facilities offer visiting scientists access to equipment and expertise to generate and analyze data. For some projects, it might however be more efficient to collaborate remotely by sending in samples

Constitutive hippocampal cholesterol loss underlies poor cognition in old rodents

Cognitive decline is one of the many characteristics of aging. Reduced long-term potentiation (LTP) and long-term depression (LTD) are thought to be responsible for this decline, although the precise mechanisms underlying LTP and LTD dampening in the old remain unclear. We previously showed that aging is accompanied by the loss of cholesterol from the hippocampus, which leads to PI3K/Akt phosphorylation. Given that Akt de-phosphorylation is required for glutamate receptor internalization and LTD, we hypothesized that the decrease in cholesterol in neuronal membranes may contribute to the deficits in LTD typical of aging. Here, we show that cholesterol loss triggers p-Akt accumulation, which in turn perturbs the normal cellular and molecular responses induced by LTD, such as impaired AMPA receptor internalization and its reduced lateral diffusion. Electrophysiology recordings in brain slices of old mice and in anesthetized elderly rats demonstrate that the reduced hippocampal LTD associated with age can be rescued by cholesterol perfusion. Accordingly, cholesterol replenishment in aging animals improves hippocampal-dependent learning and memory in the water maze test.publishedVersionFil: Martín, Mauricio Gerardo. Consejo Superior de Investigaciones Científicas. Centro de Biología Molecular Severo Ochoa; España.Fil: Martín, Mauricio Gerardo. Universidad Autónoma de Madrid. Centro de Biología Molecular Severo Ochoa; España.Fil: Martín, Mauricio Gerardo. Katholieke Universiteit Leuven. Center for Human Genetics. VIB Center for the Biology of Disease; Bélgica.Fil: Ahmed, Tariq. Katholieke Universiteit Leuven. Faculty of Psychology and Educational Sciences. Laboratory of Biological Psychology; Bélgica.Fil: Korovaichuk, Alejandra. Consejo Superior de Investigaciones Científicas. Instituto Cajal. Departamento de Neurobiología Funcional y de Sistemas; España.Fil: Venero, César. Universidad Nacional de Educación a Distancia. Facultad de Psicología. Departamento de Psicobiología; España.Fil: Menchón, Silvia Adriana. Katholieke Universiteit Leuven. Center for Human Genetics. VIB Center for the Biology of Disease; Bélgica.Fil: Menchón, Silvia Adriana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Física Enrique Gaviola; Argentina.Fil: Menchón, Silvia Adriana. Universidad Nacional de Córdoba. Facultad de Matemática, Astronomía y Física; Argentina.Fil: Salas, Isabel. Consejo Superior de Investigaciones Científicas. Centro de Biología Molecular Severo Ochoa; España.Fil: Salas, Isabel. Universidad Autónoma de Madrid. Centro de Biología Molecular Severo Ochoa; España.Fil: Salas, Isabel. Consejo Superior de Investigaciones Científicas. Centro de Biología Molecular Severo Ochoa; España.Fil: Munck, Sebastian. Katholieke Universiteit Leuven. Center for Human Genetics. VIB Center for the Biology of Disease; Bélgica.Fil: Herreras, Oscar. Consejo Superior de Investigaciones Científicas. Instituto Cajal. Departamento de Neurobiología Funcional y de Sistemas; España.Fil: Balschun, Detlef. Katholieke Universiteit Leuven. Faculty of Psychology and Educational Sciences. Laboratory of Biological Psychology; Bélgica.Fil: Dotti, Carlos Gerardo. Consejo Superior de Investigaciones Científicas. Centro de Biología Molecular Severo Ochoa; España.Fil: Dotti, Carlos Gerardo. Universidad Autónoma de Madrid. Centro de Biología Molecular Severo Ochoa; España.Fil: Dotti, Carlos Gerardo. Katholieke Universiteit Leuven. Center for Human Genetics. VIB Center for the Biology of Disease; Bélgica.Biofísic

Hsc70-4 Deforms Membranes to Promote Synaptic Protein Turnover by Endosomal Microautophagy

SummarySynapses are often far from their cell bodies and must largely independently cope with dysfunctional proteins resulting from synaptic activity and stress. To identify membrane-associated machines that can engulf synaptic targets destined for degradation, we performed a large-scale in vitro liposome-based screen followed by functional studies. We identified a presynaptically enriched chaperone Hsc70-4 that bends membranes based on its ability to oligomerize. This activity promotes endosomal microautophagy and the turnover of specific synaptic proteins. Loss of microautophagy slows down neurotransmission while gain of microautophagy increases neurotransmission. Interestingly, Sgt, a cochaperone of Hsc70-4, is able to switch the activity of Hsc70-4 from synaptic endosomal microautophagy toward chaperone activity. Hence, Hsc70-4 controls rejuvenation of the synaptic protein pool in a dual way: either by refolding proteins together with Sgt, or by targeting them for degradation by facilitating endosomal microautophagy based on its membrane deforming activity

Regulation of branching dynamics by axon-intrinsic asymmetries in Tyrosine Kinase Receptor signaling

Axonal branching allows a neuron to connect to several targets, increasing neuronal circuit complexity. While axonal branching is well described, the mechanisms that control it remain largely unknown. We find that in the Drosophila CNS branches develop through a process of excessive growth followed by pruning. In vivo high-resolution live imaging of developing brains as well as loss and gain of function experiments show that activation of Epidermal Growth Factor Receptor (EGFR) is necessary for branch dynamics and the final branching pattern. Live imaging also reveals that intrinsic asymmetry in EGFR localization regulates the balance between dynamic and static filopodia. Elimination of signaling asymmetry by either loss or gain of EGFR function results in reduced dynamics leading to excessive branch formation. In summary, we propose that the dynamic process of axon branch development is mediated by differential local distribution of signaling receptors

Challenges and advances in optical 3D mesoscale imaging

Optical mesoscale imaging is a rapidly developing field that allows the visualisation of larger samples than is possible with standard light microscopy, and fills a gap between cell and organism resolution. It spans from advanced fluorescence imaging of micrometric cell clusters to centimetre-size complete organisms. However, with larger volume specimens, new problems arise. Imaging deeper into tissues at high resolution poses challenges ranging from optical distortions to shadowing from opaque structures. This manuscript discusses the latest developments in mesoscale imaging and highlights limitations, namely labelling, clearing, absorption, scattering, and also sample handling. We then focus on approaches that seek to turn mesoscale imaging into a more quantitative technique, analogous to quantitative tomography in medical imaging, highlighting a future role for digital and physical phantoms as well as artificial intelligence

Adenosine induces growth-cone turning of sensory neurons

The formation of appropriate connections between neurons and their specific targets is an essential step during development and repair of the nervous system. Growth cones are located at the leading edges of the growing neurites and respond to environmental cues in order to be guided to their final targets. Directional information can be coded by concentration gradients of substrate-bound or diffusible-guidance molecules. Here we show that concentration gradients of adenosine stimulate growth cones of sensory neurons (dorsal root ganglia) from chicken embryos to turn towards the adenosine source. This response is mediated by adenosine receptors. The subsequent signal transduction process involves cAMP. It may be speculated that the in vivo function of this response is concerned with the formation or the repair and regeneration of the peripheral nervous system