Role of cell adhesion molecule neurofascin in synapse formation and stabilization in vitro and in vivo

Affektive Störungen, wie Depressionen, Angsterkrankungen oder das Posttraumatische Stresssyndrom nehmen in der heutige Zeit einen immer höheren Stellenwert ein. Molekulare Mechanismen, die diesen Erkrankungen zugrunde liegen, sind nur unzureichend bekannt. Stress wurde bereits mehrfach als mögliche Ursache molekularer Veränderungen und Netzwerkstörungen beschrieben. Ebenso wurde Zelladhäsionsmolekülen eine große Bedeutung bei affektiven Störungen zugeschrieben. Neurofascin (NF) gehört der Ig-Superfamilie der Zelladhäsionsmoleküle an. Seine Lokalisation ist hauptsächlich auf das Axoninitialsegment (AIS) neuronaler Zellen beschränkt. In dieser Arbeit wurde die Bedeutung der neuronalen Neurofascin-Isoformen (NF166/NF180/NF186) auf inhibitorische Synapsenbildung und -stabilisierung sowohl in vitro, als auch in vivo untersucht. Dabei konnten modulatorische Effekte auf das Gerüstprotein Gephyrin in primären neuronalen Zellen am AIS beobachtet werden. Außerdem zeigte die shRNA-vermittelte Reduktion der Neurofascinexpression im Gyrus dentatus juveniler Ratten eine Abnahme prä- und postsynaptischer Strukturen am AIS. Des Weiteren konnte juveniler Stress im ventralen Gyrus dentaus von Ratten einen Anstieg der NF180-Expression bewirken, wodurch ein möglicherweise jugendlicherer Status erreicht wird. Durch Überexpression der embryonalen NF-Isoform in den Gyrus dentatus sollten mögliche Effekte einer Hochregulierung eruiert werden. Tatsächlich zeigten sich prä- und postsynaptisch Volumenzunahmen der inhibitorischen Strukturen nach Überexpression. Dies zeigt, dass Neurofascin einen Einfluss auf prä-und postsynaptische Strukturen am AIS hat. Eine weitere Analyse, welchen regulatorischen Faktoren diese Neurofascinexpression unterliegt, muss durchgeführt werden. In dieser Arbeit konnte gezeigt werden, dass eine spezifische Activinrezeptor-Inhibition die Neurofascin-Expression in Neuronen reduziert. Welche Mechanismen dieser Regulation zugrunde liegen, muss weiter untersucht werden.Nowadays, affective disorders like depression, anxiety or posttraumatic stress disorder get more and more important. Molecular mechanisms that underlie those diseases are still not well known. Stress has already been mentioned so often as cause for molecular changes and network disturbances. Due to affective disorders cell adhesion molecules appear to be important as well. Neurofascin (NF) belongs to the immunoglobulin superfamily of cell adhesion molecules. It is especially localized at the axon initial segment (AIS) of neurons. In this work we investigate the role of neuronal neurofascin isoforms (NF166/NF180/NF186) in inhibitory synapse formation and stabilization, in vitro as well as in vivo. In primary hippocampal neurons we could identify modulating effects on scaffolding protein gephyrin at the AIS. In addition we observed a reduction of pre- and postsynaptic structures at the AIS in vivo after shRNA based knockdown of neurofascin in dentate gyrus of juvenile rats. Furthermore juvenile stress appeared to increase the expression of juvenile NF180 in the ventral part of dentate gyrus of rats. This could lead to a more juvenile state of rats. By overexpressing juvenile NF we wanted to investigate possible effects of such an up regulation of juvenile neurofascin, which occurred after stress. In fact we could detect an increase in inhibitory pre- and postsynaptic structure volumes at the AIS. These results showed that neurofascin influences pre- and postsynaptic structures at the axon initial segment of neurons. Mechanisms which underlie such a neurofascin regulation have to be investigated. In this work we could already show that an inhibition of activin receptor reduces neurofascin expression in primary hippocampal neurons. However further investigations are necessary to understand the role and regulation of neurofascin expression

Expansion Microscopy for Cell Biology Analysis in Fungi

Super-resolution microscopy has evolved as a powerful method for subdiffraction-resolution fluorescence imaging of cells and cellular organelles, but requires sophisticated and expensive installations. Expansion microscopy (ExM), which is based on the physical expansion of the cellular structure of interest, provides a cheap alternative to bypass the diffraction limit and enable super-resolution imaging on a conventional fluorescence microscope. While ExM has shown impressive results for the magnified visualization of proteins and RNAs in cells and tissues, it has not yet been applied in fungi, mainly due to their complex cell wall. Here we developed a method that enables reliable isotropic expansion of ascomycetes and basidiomycetes upon treatment with cell wall degrading enzymes. Confocal laser scanning microscopy (CLSM) and structured illumination microscopy (SIM) images of 4.5-fold expanded sporidia of Ustilago maydis expressing fluorescent fungal rhodopsins and hyphae of Fusarium oxysporum or Aspergillus fumigatus expressing either histone H1-mCherry together with Lifeact-sGFP or mRFP targeted to mitochondria, revealed details of subcellular structures with an estimated spatial resolution of around 30 nm. ExM is thus well suited for cell biology studies in fungi on conventional fluorescence microscopes

The Cell Adhesion Molecule Neurofascin Stabilizes Axo-axonic GABAergic Terminals at the Axon Initial Segment*

Cell adhesion molecules regulate synapse formation and maintenance via transsynaptic contact stabilization involving both extracellular interactions and intracellular postsynaptic scaffold assembly. The cell adhesion molecule neurofascin is localized at the axon initial segment of granular cells in rat dentate gyrus, which is mainly targeted by chandelier cells. Lentiviral shRNA-mediated knockdown of neurofascin in adult rat brain indicates that neurofascin regulates the number and size of postsynaptic gephyrin scaffolds, the number of GABAA receptor clusters as well as presynaptic glutamate decarboxylase-positive terminals at the axon initial segment. By contrast, overexpression of neurofascin in hippocampal neurons increases gephyrin cluster size presumably via stimulation of fibroblast growth factor receptor 1 signaling pathways