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    Collapsin response mediator protein-2 plays a major protective role in acute axonal degeneration. 

    Zhang, Jian-Nan; Koch, Jan C
    Neural regeneration research 2017-05; 12(5) p.692-695
    Axonal degeneration is a key pathological feature in many neurological diseases. It often leads to persistent deficits due to the inability of axons to regenerate in the central nervous system. Therefore therapeutic approaches should optimally both attenuate axonal degeneration and foster axonal regeneration. Compelling evidence suggests that collapsin response mediator protein-2 (CRMP2) might be a molecular target fulfilling these requirements. In this mini-review, we give a compact overview of the known functions of CRMP2 and its molecular interactors in neurite outgrowth and in neurodegenerative conditions. Moreover, we discuss in detail our recent findings on the role of CRMP2 in acute axonal degeneration in the optic nerve. We found that the calcium influx induced by the lesion activates the protease calpain which cleaves CRMP2, leading to impairment of axonal transport. Both calpain inhibition and CRMP2 overexpression effectively protected the proximal axons against acute axonal degeneration. Taken together, CRMP2 is further characterized as a central molecular player in acute axonal degeneration and thus evolves as a promising therapeutic target to both counteract axonal degeneration and foster axonal regeneration in neurodegenerative and neurotraumatic diseases.
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    Three-dimensional mouse brain cytoarchitecture revealed by laboratory-based x-ray phase-contrast tomography. 

    Töpperwien, Mareike; Krenkel, Martin; Vincenz, Daniel; Stöber, Franziska; Oelschlegel, Anja M.; Goldschmidt, Jürgen; Salditt, Tim
    Scientific reports 2017-02-27; 7: Art. 42847
    Studies of brain cytoarchitecture in mammals are routinely performed by serial sectioning of the specimen and staining of the sections. The procedure is labor-intensive and the 3D architecture can only be determined after aligning individual 2D sections, leading to a reconstructed volume with non-isotropic resolution. Propagation-based x-ray phase-contrast tomography offers a unique potential for high-resolution 3D imaging of intact biological specimen due to the high penetration depth and potential resolution. We here show that even compact laboratory CT at an optimized liquid-metal jet microfocus source combined with suitable phase-retrieval algorithms and a novel tissue preparation can provide cellular and subcellular resolution in millimeter sized samples of mouse brain. We removed water and lipids from entire mouse brains and measured the remaining dry tissue matrix in air, lowering absorption but increasing phase contrast. We present single-cell resolution images of mouse brain cytoarchitecture and show that axons can be revealed in myelinated fiber bundles. In contrast to optical 3D techniques our approach does neither require staining of cells nor tissue clearing, procedures that are increasingly difficult to apply with increasing sample and brain sizes. The approach thus opens a novel route for high-resolution high-throughput studies of brain architecture in mammals.
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    RIM-Binding Protein 2 Promotes a Large Number of CaV1.3 Ca2+-Channels and Contributes to Fast Synaptic Vesicle Replenishment at Hair Cell Active Zones. 

    Krinner, Stefanie; Butola, Tanvi; Jung, SangYong; Wichmann, Carolin; Moser, Tobias
    Frontiers in cellular neuroscience 2017; 11: Art. 334
    Ribbon synapses of inner hair cells (IHCs) mediate high rates of synchronous exocytosis to indefatigably track the stimulating sound with sub-millisecond precision. The sophisticated molecular machinery of the inner hair cell active zone realizes this impressive performance by enabling a large number of synaptic voltage-gated CaV1.3 Ca2+-channels, their tight coupling to synaptic vesicles (SVs) and fast replenishment of fusion competent SVs. Here we studied the role of RIM-binding protein 2 (RIM-BP2)-a multidomain cytomatrix protein known to directly interact with Rab3 interacting molecules (RIMs), bassoon and CaV1.3-that is present at the inner hair cell active zones. We combined confocal and stimulated emission depletion (STED) immunofluorescence microscopy, electron tomography, patch-clamp and confocal Ca2+-imaging, as well as auditory systems physiology to explore the morphological and functional effects of genetic RIM-BP2 disruption in constitutive RIM-BP2 knockout mice. We found that RIM-BP2 (1) positively regulates the number of synaptic CaV1.3 channels and thereby facilitates synaptic vesicle release and (2) supports fast synaptic vesicle recruitment after readily releasable pool (RRP) depletion. However, Ca2+-influx-exocytosis coupling seemed unaltered for readily releasable SVs. Recordings of auditory brainstem responses (ABR) and of single auditory nerve fiber firing showed that RIM-BP2 disruption results in a mild deficit of synaptic sound encoding.
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    Posttranslational modifications of blood-derived alpha-synuclein as biochemical markers for Parkinson's disease. 

    Vicente Miranda, Hugo; Cássio, Rafaela; Correia-Guedes, Leonor; Gomes, Marcos António; Chegão, Ana; Miranda, Elisa; Soares, Tiago; Coelho, Miguel; Rosa, Mário Miguel; Ferreira, Joaquim J; et al.
    Outeiro, Tiago Fleming
    Scientific reports 2017-10-20; 7(1): Art. 13713
    Parkinson's disease (PD) is a progressive neurodegenerative disorder known for the typical motor features associated. Pathologically, it is characterized by the intracellular accumulation of alpha-synuclein (aSyn) in Lewy bodies and Lewy neurites. Currently, there are no established biochemical markers for diagnosing or for following disease progression, a major limitation for the clinical practice. Posttranslational modifications (PTMs) in aSyn have been identified and implicated on its pathobiology. Since aSyn is abundant in blood erythrocytes, we aimed to evaluate whether PTMs of aSyn in the blood might hold value as a biomarker for PD. We examined 58 patients with PD and 30 healthy age-matched individuals. We found that the levels of Y125 phosphorylated, Y39 nitrated, and glycated aSyn were increased in PD, while those of SUMO were reduced. A combinatory analysis of the levels of these PTMs resulted in an increased sensitivity, with an area under curve (AUC) of 0.843 for PD versus healthy controls, and correlated with disease severity and duration. We conclude that the levels of these selected PTMs hold strong potential as biochemical markers for PD. Ultimately, our findings might facilitate the monitoring of disease progression in clinical trials, opening the possibility for developing more effective therapies against PD.
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    The Insect Ortholog of the Human Orphan Cytokine Receptor CRLF3 Is a Neuroprotective Erythropoietin Receptor. 

    Hahn, Nina; Knorr, Debbra Y.; Liebig, Johannes; Wüstefeld, Liane; Peters, Karsten; Büscher, Marita; Bucher, Gregor; Ehrenreich, Hannelore; Heinrich, Ralf
    Frontiers in molecular neuroscience 2017; 10: Art. 223
    The cytokine erythropoietin (Epo) mediates various cell homeostatic responses to environmental challenges and pathological insults. While stimulation of vertebrate erythrocyte production is mediated by homodimeric "classical" Epo receptors, alternative receptors are involved in neuroprotection. However, their identity remains enigmatic due to complex cytokine ligand and receptor interactions and conflicting experimental results. Besides the classical Epo receptor, the family of type I cytokine receptors also includes the poorly characterized orphan cytokine receptor-like factor 3 (CRLF3) present in vertebrates including human and various insect species. By making use of the more simple genetic makeup of insect model systems, we studied whether CRLF3 is a neuroprotective Epo receptor in animals. We identified a single ortholog of CRLF3 in the beetle Tribolium castaneum, and established protocols for primary neuronal cell cultures from Tribolium brains and efficient in vitro RNA interference. Recombinant human Epo as well as the non-erythropoietic Epo splice variant EV-3 increased the survival of serum-deprived brain neurons, confirming the previously described neuroprotective effect of Epo in insects. Moreover, Epo completely prevented hypoxia-induced apoptotic cell death of primary neuronal cultures. Knockdown of CRLF3 expression by RNA interference with two different double stranded RNA (dsRNA) fragments abolished the neuroprotective effect of Epo, indicating that CRLF3 is a crucial component of the insect Epo-responsive receptor. This suggests that a common urbilaterian ancestor of the orphan human and insect cytokine receptor CRLF3 served as a neuroprotective receptor for an Epo-like cytokine. Our work also suggests that vertebrate CRLF3, like its insect ortholog, might represent a tissue protection-mediating receptor.
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    Imaging of neuronal tissues by x-ray diffraction and x-ray fluorescence microscopy: evaluation of contrast and biomarkers for neurodegenerative diseases. 

    Carboni, Eleonora; Nicolas, Jan-David; Töpperwien, Mareike; Stadelmann-Nessler, Christine; Lingor, Paul; Salditt, Tim
    Biomedical optics express 2017-10-01; 8(10) p.4331-4347
    We have used scanning X-ray diffraction (XRD) and X-ray fluorescence (XRF) with micro-focused synchrotron radiation to study histological sections from human substantia nigra (SN). Both XRF and XRD mappings visualize tissue properties, which are inaccessible by conventional microscopy and histology. We propose to use these advanced tools to characterize neuronal tissue in neurodegeneration, in particular in Parkinson's disease (PD). To this end, we take advantage of the recent experimental progress in x-ray focusing, detection, and use automated data analysis scripts to enable quantitative analysis of large field of views. XRD signals are recorded and analyzed both in the regime of small-angle (SAXS) and wide-angle x-ray scattering (WAXS). The SAXS signal was analyzed in view of the local myelin structure, while WAXS was used to identify crystalline deposits. PD tissue scans exhibited increased amounts of crystallized cholesterol. The XRF analysis showed increased amounts of iron and decreased amounts of copper in the PD tissue compared to the control.
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    The Serotonin Receptor Subtype 5b Specifically Interacts with Serotonin Receptor Subtype 1A. 

    Niebert, Sabine; van Belle, Gijsbert J.; Vogelgesang, Steffen; Manzke, Till; Niebert, Marcus
    Frontiers in molecular neuroscience 2017; 10: Art. 299
    Previously, we described the dysregulation of serotonin (5-HT) receptor subtype 5b (5-ht5b) in a mouse model of Rett syndrome (RTT). 5-ht5b has not been extensively studied, so we set out to characterize it in more detail. Unlike common cell surface receptors, 5-ht5b displays no membrane expression, while receptor clusters are located in endosomes. This unusual subcellular localization is at least in part controlled by glycosylation of the N-terminus, with 5-ht5b possessing fewer glycosylation sites than related receptors. We analyzed whether the localization to endosomes has any functional relevance and found that 5-ht5b receptors can specifically interact with 5-HT1A receptors and retain them in endosomal compartments. This interaction reduces 5-HT1A surface expression and is mediated by interactions between the fourth and fifth trans-membrane domain (TMD). This possibly represents a mechanism by which 5-ht5b receptors regulate the activity of other 5-HT receptor.
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    Activating de novo mutations in NFE2L2 encoding NRF2 cause a multisystem disorder. 

    Huppke, Peter; Weissbach, Susann; Church, Joseph A.; Schnur, Rhonda; Krusen, Martina; Dreha-Kulaczewski, Steffi; Kühn-Velten, W. Nikolaus; Wolf, Annika; Huppke, Brenda; Millan, Francisca; et al.
    Begtrup, AmberAlmusafri, FatimaThiele, HolgerAltmüller, JanineNürnberg, PeterMüller, MichaelGärtner, Jutta
    Nature communications 2017-10-10; 8(1): Art. 818
    Transcription factor NRF2, encoded by NFE2L2, is the master regulator of defense against stress in mammalian cells. Somatic mutations of NFE2L2 leading to NRF2 accumulation promote cell survival and drug resistance in cancer cells. Here we show that the same mutations as inborn de novo mutations cause an early onset multisystem disorder with failure to thrive, immunodeficiency and neurological symptoms. NRF2 accumulation leads to widespread misregulation of gene expression and an imbalance in cytosolic redox balance. The unique combination of white matter lesions, hypohomocysteinaemia and increased G-6-P-dehydrogenase activity will facilitate early diagnosis and therapeutic intervention of this novel disorder.The NRF2 transcription factor regulates the response to stress in mammalian cells. Here, the authors show that activating mutations in NRF2, commonly found in cancer cells, are found in four patients with a multisystem disorder characterized by immunodeficiency and neurological symptoms.
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    Sexual dimorphism of AMBRA1-related autistic features in human and mouse. 

    Mitjans, M.; Begemann, M.; Ju, A.; Dere, E.; Wüstefeld, L.; Hofer, S.; Hassouna, I.; Balkenhol, J.; Oliveira, B.; van der Auwera, S.; et al.
    Tammer, R.Hammerschmidt, K.Völzke, H.Homuth, G.Cecconi, F.Chowdhury, K.Grabe, H.Frahm, J.Boretius, S.Dandekar, T.Ehrenreich, H.
    Translational psychiatry 2017-10-10; 7(10): Art. e1247
    Ambra1 is linked to autophagy and neurodevelopment. Heterozygous Ambra1 deficiency induces autism-like behavior in a sexually dimorphic manner. Extraordinarily, autistic features are seen in female mice only, combined with stronger Ambra1 protein reduction in brain compared to males. However, significance of AMBRA1 for autistic phenotypes in humans and, apart from behavior, for other autism-typical features, namely early brain enlargement or increased seizure propensity, has remained unexplored. Here we show in two independent human samples that a single normal AMBRA1 genotype, the intronic SNP rs3802890-AA, is associated with autistic features in women, who also display lower AMBRA1 mRNA expression in peripheral blood mononuclear cells relative to female GG carriers. Located within a non-coding RNA, likely relevant for mRNA and protein interaction, rs3802890 (A versus G allele) may affect its stability through modification of folding, as predicted by in silico analysis. Searching for further autism-relevant characteristics in Ambra1(+/-) mice, we observe reduced interest of female but not male mutants regarding pheromone signals of the respective other gender in the social intellicage set-up. Moreover, altered pentylentetrazol-induced seizure propensity, an in vivo readout of neuronal excitation-inhibition dysbalance, becomes obvious exclusively in female mutants. Magnetic resonance imaging reveals mild prepubertal brain enlargement in both genders, uncoupling enhanced brain dimensions from the primarily female expression of all other autistic phenotypes investigated here. These data support a role of AMBRA1/Ambra1 partial loss-of-function genotypes for female autistic traits. Moreover, they suggest Ambra1 heterozygous mice as a novel multifaceted and construct-valid genetic mouse model for female autism.
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    In vivo mouse and live cell STED microscopy of neuronal actin plasticity using far-red emitting fluorescent proteins. 

    Wegner, Waja; Ilgen, Peter; Gregor, Carola; van Dort, Joris; Mott, Alexander C.; Steffens, Heinz; Willig, Katrin I.
    Scientific reports 2017-09-18; 7(1) p.1-10: Art. 11781
    The study of proteins in dendritic processes within the living brain is mainly hampered by the diffraction limit of light. STED microscopy is so far the only far-field light microscopy technique to overcome the diffraction limit and resolve dendritic spine plasticity at superresolution (nanoscopy) in the living mouse. After having tested several far-red fluorescent proteins in cell culture we report here STED microscopy of the far-red fluorescent protein mNeptune2, which showed best results for our application to superresolve actin filaments at a resolution of ~80 nm, and to observe morphological changes of actin in the cortex of a living mouse. We illustrate in vivo far-red neuronal actin imaging in the living mouse brain with superresolution for time periods of up to one hour. Actin was visualized by fusing mNeptune2 to the actin labels Lifeact or Actin-Chromobody. We evaluated the concentration dependent influence of both actin labels on the appearance of dendritic spines; spine number was significantly reduced at high expression levels whereas spine morphology was normal at low expression.
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    EV-3, an endogenous human erythropoietin isoform with distinct functional relevance 

    Bonnas, Christel; Wüstefeld, Liane; Winkler, Daniela; Kronstein-Wiedemann, Romy; Dere, Ekrem; Specht, Katja; Boxberg, Melanie; Tonn, Torsten; Ehrenreich, Hannelore; Stadler, Herbert; et al.
    Sillaber, Inge
    Scientific Reports 2017; 7(1): Art. 3684
    Generation of multiple mRNAs by alternative splicing is well known in the group of cytokines and has recently been reported for the human erythropoietin (EPO) gene. Here, we focus on the alternatively spliced EPO transcript characterized by deletion of exon 3 (hEPOΔ3). We show co-regulation of EPO and hEPOΔ3 in human diseased tissue. The expression of hEPOΔ3 in various human samples was low under normal conditions, and distinctly increased in pathological states. Concomitant up-regulation of hEPOΔ3 and EPO in response to hypoxic conditions was also observed in HepG2 cell cultures. Using LC-ESI-MS/MS, we provide first evidence for the existence of hEPOΔ3 derived protein EV-3 in human serum from healthy donors. Contrary to EPO, recombinant EV-3 did not promote early erythroid progenitors in cultures of human CD34+ haematopoietic stem cells. Repeated intraperitoneal administration of EV-3 in mice did not affect the haematocrit. Similar to EPO, EV-3 acted anti-apoptotic in rat hippocampal neurons exposed to oxygen-glucose deprivation. Employing the touch-screen paradigm of long-term visual discrimination learning, we obtained first in vivo evidence of beneficial effects of EV-3 on cognition. This is the first report on the presence of a naturally occurring EPO protein isoform in human serum sharing non-erythropoietic functions with EPO.
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    Increased Mitochondrial Mass and Cytosolic Redox Imbalance in Hippocampal Astrocytes of a Mouse Model of Rett Syndrome: Subcellular Changes Revealed by Ratiometric Imaging of JC-1 and roGFP1 Fluorescence 

    Bebensee, Dörthe F.; Can, Karolina; Müller, Michael
    Oxidative Medicine and Cellular Longevity 2017; 2017 p.1-15
    Rett syndrome (RTT) is a neurodevelopmental disorder with mutations in the MECP2 gene. Mostly girls are affected, and an apparently normal development is followed by cognitive impairment, motor dysfunction, epilepsy, and irregular breathing. Various indications suggest mitochondrial dysfunction. In Rett mice, brain ATP levels are reduced, mitochondria are leaking protons, and respiratory complexes are dysregulated. Furthermore, we found in MeCP2-deficient mouse (Mecp2−/y) hippocampus an intensified mitochondrial metabolism and ROS generation. We now used emission ratiometric 2-photon imaging to assess mitochondrial morphology, mass, and membrane potential (ΔΨm) in Mecp2−/y hippocampal astrocytes. Cultured astrocytes were labeled with the ΔΨm marker JC-1, and semiautomated analyses yielded the number of mitochondria per cell, their morphology, and ΔΨm. Mecp2−/y astrocytes contained more mitochondria than wild-type (WT) cells and were more oxidized. Mitochondrial size, ΔΨm, and vulnerability to pharmacological challenge did not differ. The antioxidant Trolox opposed the oxidative burden and decreased the mitochondrial mass, thereby dampening the differences among WT and Mecp2−/y astrocytes; mitochondrial size and ΔΨm were not markedly affected. In conclusion, mitochondrial alterations and redox imbalance in RTT also involve astrocytes. Mitochondria are more numerous in Mecp2−/y than in WT astrocytes. As this genotypic difference is abolished by Trolox, it seems linked to the oxidative stress in RTT.
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    Loss of Neuroligin3 specifically downregulates retinal GABAAα2 receptors without abolishing direction selectivity. 

    Hoon, Mrinalini; Krishnamoorthy, Vidhyasankar; Gollisch, Tim; Falkenburger, Bjoern; Varoqueaux, Frederique
    PloS one 2017; 12(7): Art. e0181011
    The postsynaptic adhesion proteins Neuroligins (NLs) are essential for proper synapse function, and their alterations are associated with a variety of neurodevelopmental disorders. It is increasingly clear that each NL isoform occupies specific subsets of synapses and is able to regulate the function of discrete networks. Studies of NL2 and NL4 in the retina in particular have contributed towards uncovering their role in inhibitory synapse function. In this study we show that NL3 is also predominantly expressed at inhibitory postsynapses in the retinal inner plexiform layer (IPL), where it colocalizes with both GABAA- and glycinergic receptor clusters in a 3:2 ratio. In the NL3 deletion-mutant (knockout or KO) mouse, we uncovered a dramatic reduction of the number of GABAAα2-subunit containing GABAA receptor clusters at the IPL. Retinal activity was thereafter assessed in KO and wild-type (WT) littermates by multi-electrode-array recordings of the output cells of retina, the retinal ganglion cells (RGCs). RGCs in the NL3 KO showed reduced spontaneous activity and an altered response to white noise stimulation. Moreover, upon application of light flashes, the proportion of cells firing at light offset (OFF RGCs) was significantly lower in the NL3 KO compared to WT littermates, whereas the relative number of cells firing at light onset (ON RGCs) increased. Interestingly, although GABAAα2-bearing receptors have been related to direction-selective circuits of the retina, features of direction selective-retinal ganglion cells recorded remained unperturbed in the NL3 KO. Together our data underscore the importance of NL3 for the integrity of specific GABAAergic retinal circuits and identifies NL3 as an important regulator of retinal activity.
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    Amyloid-precursor Like Proteins APLP1 and APLP2 Are Dispensable for Normal Development of the Neonatal Respiratory Network. 

    Han, Kang; Müller, Ulrike C.; Hülsmann, Swen
    Frontiers in molecular neuroscience 2017; 10: Art. 189
    Recent studies using animal models indicated that the members of the amyloid precursor protein (APP) gene family are important for the formation, maintenance, and plasticity of synapses. Despite this, the specific role of the APP homologs APLP1 and APLP2 within the CNS and PNS is still poorly understood. In contrast to the subtle phenotypes of single mutants, double knockout mice (DKO) lacking APP/APLP2 or APLP1/APLP2 die within the first day after birth. Whereas APP/APLP2-DKO mice show severe deficits of neuromuscular morphology and transmission, the underlying cause of lethality of APLP1/APLP2-DKO mice remains unclear. Since expression of both proteins was confirmed by in situ hybridization, we aimed to test the role of APLP1/APLP2 in the formation and maintenance of synapses in the brainstem, and assessed a potential dysfunction of the most vital central neuronal network in APLP1/APLP2-DKO mice by analyzing the respiratory network of the medulla. We performed in vivo unrestrained whole body plethysmography in newborn APLP1/APLP2-DKO mice at postnatal day zero. Additionally, we directly tested the activity of the respiratory network in an acute slice preparation that includes the pre-Bötzinger complex. In both sets of experiments, no significant differences were detected regarding respiratory rate and cycle variability, strongly arguing against central respiratory problems as the primary cause of death of APLP1/APLP2-DKO mice. Thus, we conclude that APLP1 and APLP2 are dispensable for the development of the network and the generation of a normal breathing rhythm.
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    Riluzole: A potential therapeutic intervention in human brain tumor stem-like cells 

    Sperling, Swetlana; Aung, Thiha; Martin, Sabine; Rohde, Veit; Ninkovic, Milena
    Oncotarget
    A small subpopulation of tumor stem-like cells has the capacity to initiate tumors and mediate radio- and chemoresistance in diverse cancers hence also in glioblastoma (GBM). It has been reported that this capacity of tumor initiation in the brain is mainly dependent on the body’s nutrient supply. This population of so-called brain tumor initiating or brain tumor stem-like cells (BTSCs) is able to extract nutrients like glucose with a higher affinity. Riluzole, a drug approved for treating amyotrophic lateral sclerosis (ALS), was reported to possess anticancer properties, affecting the glutamate metabolism. We report that riluzole treatment inhibits the growth of brain tumor stem-like cells enriched cultures isolated from two human glioblastomas. The effects of riluzole on these cells were associated with an inhibition of a poor prognostic indicator: glucose transporter 3 (GLUT3). A decrease in GLUT3 is associated with a decrease in the p-Akt/HIF1α pathway. Further, downregulation of the DNA (Cytosine- 5-)-methyltransferase 1 (DNMT1) gene that causes hypermethylation of various tumor-suppressor genes and leads to a poor prognosis in GBM, was detected. Two hallmarks of cancer cells—proliferation and cell death—were positively influenced by riluzole treatment. Finally, we observed that riluzole reduced the tumor growth in in vivo CAM assay, suggesting it could be a possible synergistic drug for the treatment of glioblastoma.
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    Monitoring ATP dynamics in electrically active white matter tracts 

    Trevisiol, Andrea; Saab, Aiman S.; Winkler, Ulrike; Marx, Grit; Imamura, Hiromi; Möbius, Wiebke; Kusch, Kathrin; Nave, Klaus-Armin; Hirrlinger, Johannes
    eLife 2017; 6: Art. e24241
    In several neurodegenerative diseases and myelin disorders, the degeneration profiles of myelinated axons are compatible with underlying energy deficits. However, it is presently impossible to measure selectively axonal ATP levels in the electrically active nervous system. We combined transgenic expression of an ATP-sensor in neurons of mice with confocal FRET imaging and electrophysiological recordings of acutely isolated optic nerves. This allowed us to monitor dynamic changes and activity-dependent axonal ATP homeostasis at the cellular level and in real time. We find that changes in ATP levels correlate well with compound action potentials. However, this correlation is disrupted when metabolism of lactate is inhibited, suggesting that axonal glycolysis products are not sufficient to maintain mitochondrial energy metabolism of electrically active axons. The combined monitoring of cellular ATP and electrical activity is a novel tool to study neuronal and glial energy metabolism in normal physiology and in models of neurodegenerative disorders.
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    Modulation of Microglial Activity by Rho-Kinase (ROCK) Inhibition as Therapeutic Strategy in Parkinson’s Disease and Amyotrophic Lateral Sclerosis 

    Roser, Anna-Elisa; Tönges, Lars; Lingor, Paul
    Frontiers in Aging Neuroscience 2017; 9: Art. 94
    Neurodegenerative diseases are characterized by the progressive degeneration of neurons in the central and peripheral nervous system (CNS, PNS), resulting in a reduced innervation of target structures and a loss of function. A shared characteristic of many neurodegenerative diseases is the infiltration of microglial cells into affected brain regions. During early disease stages microglial cells often display a rather neuroprotective phenotype, but switch to a more pro-inflammatory neurotoxic phenotype in later stages of the disease, contributing to the neurodegeneration. Activation of the Rho kinase (ROCK) pathway appears to be instrumental for the modulation of the microglial phenotype: increased ROCK activity in microglia mediates mechanisms of the inflammatory response and is associated with improved motility, increased production of reactive oxygen species (ROS) and release of inflammatory cytokines. Recently, several studies suggested inhibition of ROCK signaling as a promising treatment option for neurodegenerative diseases. In this review article, we discuss the contribution of microglial activity and phenotype switch to the pathophysiology of Parkinson’s disease (PD) and Amyotrophic lateral sclerosis (ALS), two devastating neurodegenerative diseases without disease-modifying treatment options. Furthermore, we describe how ROCK inhibition can influence the microglial phenotype in disease models and explore ROCK inhibition as a future treatment option for PD and ALS.
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    Antagonistic Functions of MBP and CNP Establish Cytosolic Channels in CNS Myelin 

    Snaidero, Nicolas; Velte, Caroline; Myllykoski, Matti; Raasakka, Arne; Ignatev, Alexander; Werner, Hauke B.; Erwig, Michelle S.; Möbius, Wiebke; Kursula, Petri; Nave, Klaus-Armin; et al.
    Simons, Mikael
    Cell Reports 2017; 18(2) p.314-323
    The myelin sheath is a multilamellar plasma membrane extension of highly specialized glial cells laid down in regularly spaced segments along axons. Recent studies indicate that myelin is metabolically active and capable of communicating with the underlying axon. To be functionally connected to the neuron, oligodendrocytes maintain non-compacted myelin as cytoplasmic nanochannels. Here, we used high-pressure freezing for electron microscopy to study these cytoplasmic regions within myelin close to their native state. We identified 2,030-cyclic nucleotide 30-phosphodiesterase (CNP), an oligodendrocyte- specific protein previously implicated in the maintenance of axonal integrity, as an essential factor in generating and maintaining cytoplasm within the myelin compartment. We provide evidence that CNP directly associates with and organizes the actin cytoskeleton, thereby providing an intracellular strut that counteracts membrane compaction by myelin basic protein (MBP). Our study provides amolecular and structural framework for understanding how myelin maintains its cytoplasm to function as an active axon-glial unit.
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    Analysis of the Serotonergic System in a Mouse Model of Rett Syndrome Reveals Unusual Upregulation of Serotonin Receptor 5b 

    Vogelgesang, Steffen; Niebert, Sabine; Renner, Ute; Möbius, Wiebke; Hülsmann, Swen; Manzke, Till; Niebert, Marcus
    Frontiers in Molecular Neuroscience 2017; 10: Art. 61
    Mutations in the transcription factor methyl-CpG-binding-protein 2 (MeCP2) cause a delayed-onset neurodevelopmental disorder known as Rett syndrome (RTT). Although alteration in serotonin levels have been reported in RTT patients, the molecular mechanisms underlying these defects are not well understood. Therefore, we chose to investigate the serotonergic system in hippocampus and brainstem of male Mecp2􀀀=y knock-out mice in the B6.129P2(C)-Mecp2(tm1.1Bird) mouse model of RTT. The serotonergic system in mouse is comprised of 16 genes, whose mRNA expression profile was analyzed by quantitative RT-PCR. Mecp2􀀀=y mice are an established animal model for RTT displaying most of the cognitive and physical impairments of human patients and the selected areas receive significant modulation through serotonin. Using anatomically and functional characterized areas, we found regionspecific differential expression between wild type and Mecp2􀀀=y mice at post-natal day 40. In brainstem, we found five genes to be dysregulated, while in hippocampus, two genes were dysregulated. The one gene dysregulated in both brain regions was dopamine decarboxylase, but of special interest is the serotonin receptor 5b (5-ht5b), which showed 75-fold dysregulation in brainstem of Mecp2􀀀=y mice. This dysregulation was not due to upregulation, but due to failure of down-regulation in Mecp2􀀀=y mice during development. Detailed analysis of 5-ht5b revealed a receptor that localizes to endosomes and interacts with Gai proteins.
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    Limitations of Sulforhodamine 101 for Brain Imaging 

    Hülsmann, Swen; Hagos, Liya; Heuer, Heike; Schnell, Christian
    Frontiers in Cellular Neuroscience 2017; 11: Art. 44
    Since 2004, the red fluorescent dye Sulforhodamine 101 (SR101) has been boosting the functional analysis of astrocytes in a functional environment in an unprecedented way. However, two major limitations have been challenging the usefulness of this tool for cellular imaging: (i) SR101 is not as specific for astrocytes as previously reported; and (ii) discoveries of severe excitatory side effects of SR101 are bearing the risk of unwanted alteration of the system of interest. In this article, we summarize the current knowledge about SR101-labeling protocols and discuss the problems that arise from varying of the staining protocols. Furthermore, we provide a testable hypothesis for the observed hyper-excitability that can be observed when using SR101.
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