Center Nanoscale Microscopy and Molecular Physiology of the Brain

Göttingen, Germany

Center Nanoscale Microscopy and Molecular Physiology of the Brain

Göttingen, Germany
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Wagener R.J.,University of Gottingen | Witte M.,University of Gottingen | Guy J.,University of Gottingen | Mingo-Moreno N.,University of Gottingen | And 5 more authors.
Cerebral Cortex | Year: 2016

Neuronal wiring is key to proper neural information processing. Tactile information from the rodent's whiskers reaches the cortex via distinct anatomical pathways. The lemniscal pathway relays whisking and touch information from the ventral posteromedial thalamic nucleus to layer IV of the primary somatosensory "barrel" cortex. The disorganized neocortex of the reeler mouse is a model system that should severely compromise the ingrowth of thalamocortical axons (TCAs) into the cortex. Moreover, it could disrupt intracortical wiring. We found that neuronal intermingling within the reeler barrel cortex substantially exceeded previous descriptions, leading to the loss of layers. However, viral tracing revealed that TCAs still specifically targeted transgenically labeled spiny layer IV neurons. Slice electrophysiology and optogenetics proved that these connections represent functional synapses. In addition, we assessed intracortical activation via immediate-early-gene expression resulting from a behavioral exploration task. The cellular composition of activated neuronal ensembles suggests extensive similarities in intracolumnar information processing in the wild-type and reeler brains. We conclude that extensive ectopic positioning of neuronal partners can be compensated for by cell-autonomous mechanisms that allow for the establishment of proper connectivity. Thus, genetic neuronal fate seems to be of greater importance for correct cortical wiring than radial neuronal position. © 2015 The Author 2015.


Hua Y.,Max Planck Institute for Biophysical Chemistry | Hua Y.,University of Munster | Woehler A.,Max Planck Institute for Biophysical Chemistry | Woehler A.,Center Nanoscale Microscopy and Molecular Physiology of the Brain | And 5 more authors.
Neuron | Year: 2013

It is commonly thought that clathrin-mediated endocytosis is the rate-limiting step of synaptic transmission in small CNS boutons with limited capacity for synaptic vesicles, causing short-term depression during high rates of synaptic transmission. Here, we show by analyzing synaptopHluorin fluorescence that 200 action potentials evoke the same cumulative amount of vesicle fusion, irrespective of the frequency of stimulation (5-40Hz), implying the absence of vesicle reuse, since the method used (alkaline-trapping) measures only first-round exocytosis. After blocking all slow or specifically clathrin-mediated endocytosis, however, the same stimulation patterns cause a rapid stimulation-frequency-dependent release depression. This form of depression does not reflect insufficient vesicle supply, but appears to be the result of slow clearance of vesicular components from the release site. Our findings uncover an important yet overlooked role of endocytic proteins for release site clearance in addition to their well-characterized role in endocytosis itself


Saab A.S.,Max Planck Institute for Experimental Medicine | Saab A.S.,Saarland University | Saab A.S.,University of Zürich | Tzvetavona I.D.,Max Planck Institute for Experimental Medicine | And 29 more authors.
Neuron | Year: 2016

Oligodendrocytes make myelin and support axons metabolically with lactate. However, it is unknown how glucose utilization and glycolysis are adapted to the different axonal energy demands. Spiking axons release glutamate and oligodendrocytes express NMDA receptors of unknown function. Here we show that the stimulation of oligodendroglial NMDA receptors mobilizes glucose transporter GLUT1, leading to its incorporation into the myelin compartment in vivo. When myelinated optic nerves from conditional NMDA receptor mutants are challenged with transient oxygen-glucose deprivation, they show a reduced functional recovery when returned to oxygen-glucose but are indistinguishable from wild-type when provided with oxygen-lactate. Moreover, the functional integrity of isolated optic nerves, which are electrically silent, is extended by preincubation with NMDA, mimicking axonal activity, and shortened by NMDA receptor blockers. This reveals a novel aspect of neuronal energy metabolism in which activity-dependent glutamate release enhances oligodendroglial glucose uptake and glycolytic support of fast spiking axons. © 2016 Elsevier Inc.


Busse D.,Ruhr University Bochum | Kudella P.,Ruhr University Bochum | Gruning N.-M.,Ruhr University Bochum | Gruning N.-M.,University of Cambridge | And 11 more authors.
Journal of Investigative Dermatology | Year: 2014

As the outermost barrier of the body, the skin is exposed to multiple environmental factors, including temperature, humidity, mechanical stress, and chemical stimuli such as odorants that are often used in cosmetic articles. Keratinocytes, the major cell type of the epidermal layer, express a variety of different sensory receptors that enable them to react to various environmental stimuli and process information in the skin. Here we report the identification of a novel type of chemoreceptors in human keratinocytes, the olfactory receptors (ORs). We cloned and functionally expressed the cutaneous OR, OR2AT4, and identified Sandalore, a synthetic sandalwood odorant, as an agonist of this receptor. Sandalore induces strong Ca 2+ signals in cultured human keratinocytes, which are mediated by OR2AT4, as demonstrated by receptor knockdown experiments using RNA interference. The activation of OR2AT4 induces a cAMP-dependent pathway and phosphorylation of extracellular signal-regulated kinases (Erk1/2) and p38 mitogen-activated protein kinases (p38 MAPK). Moreover, the long-term stimulation of keratinocytes with Sandalore positively affected cell proliferation and migration, and regeneration of keratinocyte monolayers in an in vitro wound scratch assay. These findings combined with our studies on human skin organ cultures strongly indicate that the OR 2AT4 is involved in human keratinocyte re-epithelialization during wound-healing processes. © 2014 The Society for Investigative Dermatology.


Massberg D.,Ruhr University Bochum | Simon A.,Ruhr University Bochum | Haussinger D.,Heinrich Heine University Düsseldorf | Keitel V.,Heinrich Heine University Düsseldorf | And 4 more authors.
Archives of Biochemistry and Biophysics | Year: 2015

Terpenes are the major constituents of essential oils in plants. In recent years, terpenes have become of clinical relevance due to their ability to suppress cancer development. Their effect on cellular proliferation has made them promising agents in the prevention or treatment of many types of cancer. In the present study, a subset of different monoterpenes was investigated for their molecular effects on the hepatocellular carcinoma cell line Huh7. Using fluorometric calcium imaging, acyclic monoterpene (-)-citronellal was found to induce transient Ca2+ signals in Huh7 cells by activating a cAMP-dependent signaling pathway. Moreover, we detected the (-)-citronellal-activated human olfactory receptor OR1A2 at the mRNA and protein levels and demonstrated its potential involvement in (-)-citronellal-induced calcium signaling in Huh7 cells. Furthermore, activation of OR1A2 results in phosphorylation of p38 MAPK and reduced cell proliferation, indicating an effect on hepatocellular carcinoma progression. Here, we provide for the first time data on the molecular mechanism evoked by (-)-citronellal in human hepatocellular carcinoma cells. The identified olfactory receptor could serve as a potential therapeutic target for cancer diagnosis and treatment. © 2014 Elsevier Inc. All rights reserved.


Kehrein K.,University of Stockholm | Schilling R.,University of Stockholm | Moller-Hergt B.,University of Stockholm | Wurm C.,Max Planck Institute for Biophysical Chemistry | And 7 more authors.
Cell Reports | Year: 2015

Mitochondria contain their own genetic system that provides subunits of the complexes driving oxidativephosphorylation. A quarter of the mitochondrial proteome participates in gene expression, but how all these factors are orchestrated and spatially organized is currently unknown. Here, we established amethod to purify and analyze native and intact complexes of mitochondrial ribosomes. Quantitative mass spectrometry revealed extensive interactions of ribosomes with factors involved in all the steps of posttranscriptional gene expression. These interactions result in large expressosome-like assemblies that we termed mitochondrial organization of gene expression (MIOREX) complexes. Superresolution microscopy revealed that most MIOREX complexes are evenly distributed throughout the mitochondrial network, whereas a subset is present as nucleoid-MIOREX complexes that unite the whole spectrum of organellar gene expression. Our work therefore provides a conceptual framework for the spatial organization of mitochondrial protein synthesis that likely developed to facilitate gene expression in the organelle. © 2015 The Authors.


Saal K.-A.,University of Gottingen | Koch J.C.,University of Gottingen | Tatenhorst L.,University of Gottingen | Szego T.M.,University of Gottingen | And 8 more authors.
Neurobiology of Disease | Year: 2015

Parkinson's disease (PD) is a neurodegenerative disorder with prominent neuronal cell death in the substantia nigra (SN) and other parts of the brain. Previous studies in models of traumatic and neurodegenerative CNS disease showed that pharmacological inhibition of Rho-associated kinase (ROCK), a molecule involved in inhibitory signaling in the CNS, by small-molecule inhibitors improves neuronal survival and increases regeneration. Most small-molecule inhibitors, however, offer only limited target specificity and also inhibit other kinases, including both ROCK isoforms. To establish the role of the predominantly brain-expressed ROCK2 isoform in models of regeneration and PD, we used adeno-associated viral vectors (AAV) to specifically knockdown ROCK2 in neurons.Rat primary midbrain neurons (PMN) were transduced with AAV expressing short-hairpin-RNA (shRNA) against ROCK2 and LIM-domain kinase 1 (LIMK1), one of the downstream targets of ROCK2. While knock-down of ROCK2 and LIMK1 both enhanced neurite regeneration in a traumatic scratch lesion model, only ROCK2-shRNA protected PMN against 1-methyl-4-phenylpyridinium (MPP+) toxicity. Moreover, AAV.ROCK2-shRNA increased levels of the pro-survival markers Bcl-2 and phospho-Erk1.In vivo, AAV.ROCK2-shRNA vectors were injected into the ipsilateral SN and a unilateral 6-OHDA striatal lesion was performed. After four weeks, behavioral, immunohistochemical and biochemical alterations were investigated. Downregulation of ROCK2 protected dopaminergic neurons in the SN from 6-OHDA-induced degeneration and resulted in significantly increased TH-positive neuron numbers. This effect, however, was confined to nigral neuronal somata as striatal terminal density, dopamine and metabolite levels were not significantly preserved. Interestingly, motor behavior was improved in the ROCK2-shRNA treated animals compared to control after four weeks.Our studies thus confirm ROCK2 as a promising therapeutic target in models of PD and demonstrate that neuron-specific inhibition of ROCK2 promotes survival of lesioned dopaminergic neurons. © 2014 Elsevier Inc.


Pielecka-Fortuna J.,University of Gottingen | Wagener R.J.,University of Gottingen | Martens A.-K.,University of Gottingen | Goetze B.,University of Gottingen | And 4 more authors.
Brain Structure and Function | Year: 2015

A hallmark of neocortical circuits is the segregation of processing streams into six distinct layers. The importance of this layered organization for cortical processing and plasticity is little understood. We investigated the structure, function and plasticity of primary visual cortex (V1) of adult mice deficient for the glycoprotein reelin and their wild-type littermates. In V1 of rl−/− mice, cells with different laminar fates are present at all cortical depths. Surprisingly, the (vertically) disorganized cortex maintains a precise retinotopic (horizontal) organization. Rl−/− mice have normal basic visual capabilities, but are compromised in more challenging perceptual tasks, such as orientation discrimination. Additionally, rl−/− animals learn and memorize a visual task as well as their wild-type littermates. Interestingly, reelin deficiency enhances visual cortical plasticity: juvenile-like ocular dominance plasticity is preserved into late adulthood. The present data offer an important insight into the capabilities of a disorganized cortical system to maintain basic functional properties. © 2014, The Author(s).


Tatenhorst L.,University of Gottingen | Tonges L.,University of Gottingen | Saal K.-A.,University of Gottingen | Koch J.C.,University of Gottingen | And 5 more authors.
Journal of Neuropathology and Experimental Neurology | Year: 2014

Chronic degeneration of nigrostriatal projections, followed by nigral dopaminergic cell death, is a key feature of Parkinson disease (PD). This study examines the neuroprotective potential of the rho kinase inhibitor fasudil in the 6-hydroxydopamine (6-OHDA) mouse model of PD in vivo. C57Bl/6 mice were lesioned by striatal stereotactic injections with 4 μg of 6-OHDA and treated with fasudil 30 or 100 mg/kg body weight via drinking water. Motor behavior was tested biweekly; histologic and biochemical analyses were performed at 4 and 12 weeks after lesion. Motor behavior was severely impaired after 6-OHDA lesion and was not improved by fasudil treatment. Fasudil 100 mg/kg did not significantly increase the number of dopaminergic cells in the substantia nigra after 12 weeks versus lesion controls. Interestingly, however, high-performance liquid chromatography analysis of dopamine metabolites revealed that striatal levels of 3,4-dihydroxyphenylacetic acid were significantly increased after 12 weeks, suggesting a regenerative response. In contrast to recent findings in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridin model, fasudil effects seem limited in this severe 6-OHDA model of PD. Nevertheless, high therapeutic concentrations of fasudil are suggestive of a proregenerative potential for dopaminergic neurons, making further evaluations of rho kinase inhibition as a proregenerative therapeutic strategy in PD promising.Copyright © 2014 by the American Association of Neuropathologists, Inc.


Eckermann K.,University Hospital Freiburg | Eckermann K.,Center Nanoscale Microscopy and Molecular Physiology of the Brain
NeuroMolecular Medicine | Year: 2013

Parkinson's disease (PD) is one of the most common degenerative disorders of the central nervous system that produces motor and non-motor symptoms. The majority of cases are idiopathic and characterized by the presence of Lewy bodies containing fibrillar α-synuclein. Small ubiquitin-related modifier (SUMO) immunoreactivity was observed among others in cases with PD. Key disease-associated proteins are SUMO-modified, linking this posttranslational modification to neurodegeneration. SUMOylation and SUMO-mediated mechanisms have been intensively studied in recent years, revealing nuclear and extranuclear functions for SUMO in a variety of cellular processes, including the regulation of transcriptional activity, modulation of signal transduction pathways, and response to cellular stress. This points to a role for SUMO more than just an antagonist to ubiquitin and proteasomal degradation. The identification of risk and age-at-onset gene loci was a breakthrough in PD and promoted the understanding of molecular mechanisms in the pathology. PD has been increasingly linked with mitochondrial dysfunction and impaired mitochondrial quality control. Interestingly, SUMO is involved in many of these processes and up-regulated in response to cellular stress, further emphasizing the importance of SUMOylation in physiology and disease. © 2013 Springer Science+Business Media New York.

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