Recent Submissions

  • Journal Article

    Dose-dependent effect of cannabinoid WIN-55,212-2 on myelin repair following a demyelinating insult 

    Tomas-Roig, J.; Agbemenyah, H. Y.; Celarain, N.; Quintana, E.; Ramió-Torrentà, Ll.; Havemann-Reinecke, U.
    Scientific Reports 2020; 10(1): Art. 590
    Dysfunctions in the endocannabinoid system have been associated with experimental animal models and multiple sclerosis patients. Interestingly, the endocannabinoid system has been reported to confer neuroprotection against demyelination. The present study aims to assess the effects of the cannabinoid agonist WIN-55,212-2 in cuprizone fed animals on myelin repair capacity. Animals exposed to cuprizone were simultaneously treated withWIN-55,212-2, behaviorally tested and finally the corpus callosum was exhaustively studied by Western blotting, qRT-PCR and a myelin staining procedure. We report that the long-term administration of WIN-55,212-2 reduced the global amount of CB1 protein. Histological analysis revealed clear demyelination after being fed cuprizone for three weeks. However, cuprizone-fed mice subjected to 0.5 mg/Kg of WIN-55,212-2 displayed no differences when compared to controls during demyelination, although there was a robust increase in the myelinated axons during the remyelination phase. These animals displayed better performance on contextual fear conditioning which was in turn non-attributable to an antinociceptive effect. In contrast, a 1 mg/Kg dosage caused a remarkable demyelination accompanied by limited potential for myelin repair. Upon drug administration while mice ongoing demyeliniation, the expression of Aif1 (microglia) and Gfap (astrocytes) followed a dose-dependent manner whereas the expression of both markers was apparently attenuated during remyelination. Treatment with vehicle or 0.5 mg/Kg of the drug during demyelination increased the expression of Pdgfra (oligodendrocyte precursor cells) but this did not occur when 1 mg/Kg was administered. In conclusion, the drug at 0.5 mg/Kg did not alter myelin architecture while 1 mg/Kg had a deleterious effect in this model.
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  • Journal Article

    GABA-Glycine Cotransmitting Neurons in the Ventrolateral Medulla: Development and Functional Relevance for Breathing 

    Hirrlinger, Johannes; Marx, Grit; Besser, Stefanie; Sicker, Marit; Köhler, Susanne; Hirrlinger, Petra G.; Wojcik, Sonja M.; Eulenburg, Volker; Winkler, Ulrike; Hülsmann, Swen
    Frontiers in Cellular Neuroscience 2019; 13: Art. 517
    Inhibitory neurons crucially contribute to shaping the breathing rhythm in the brain stem. These neurons use GABA or glycine as neurotransmitter; or co-release GABA and glycine. However, the developmental relationship between GABAergic, glycinergic and cotransmitting neurons, and the functional relevance of cotransmitting neurons has remained enigmatic. Transgenic mice expressing fluorescent markers or the split-Cre system in inhibitory neurons were developed to track the three different interneuron phenotypes. During late embryonic development, the majority of inhibitory neurons in the ventrolateral medulla are cotransmitting cells, most of which differentiate into GABAergic and glycinergic neurons around birth and around postnatal day 4, respectively. Functional inactivation of cotransmitting neurons revealed an increase of the number of respiratory pauses, the cycle-by-cycle variability, and the overall variability of breathing. In summary, the majority of cotransmitting neurons differentiate into GABAergic or glycinergic neurons within the first 2 weeks after birth and these neurons contribute to fine-tuning of the breathing pattern.
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  • Journal Article

    The small molecule inhibitor anle145c thermodynamically traps human islet amyloid peptide in the form of non-cytotoxic oligomers 

    Saravanan, Manikam S.; Ryazanov, Sergey; Leonov, Andrei; Nicolai, Janine; Praest, Patrique; Giese, Armin; Winter, Roland; Khemtemourian, Lucie; Griesinger, Christian; Killian, J. Antoinette
    Scientific Reports 2019; 9(1): Art. 19023
    Type 2 diabetes (T2DM) is associated with aggregation of the human islet amyloid polypeptide (hIAPP) into cytotoxic amyloid species. Here we tested the effect of a diphenylpyrazole (DPP)-derived small molecule inhibitor, anle145c, on cytotoxicity and on aggregation properties of hIAPP. We demonstrate that incubation of hIAPP with the inhibitor yields ~10 nm-sized non-toxic oligomers, independent of the initial aggregation state of hIAPP. This suggests that anle145c has a special mode of action in which anle145c-stabilized oligomers act as a thermodynamic sink for the preferred aggregation state of hIAPP and anle145c. We also demonstrate that the inhibitor acts in a very efficient manner, with sub-stoichiometric concentrations of anle145c being sufficient to (i) inhibit hIAPP-induced death of INS-1E cells, (ii) prevent hIAPP fibril formation in solution, and (iii) convert preformed hIAPP fibrils into non-toxic oligomers. Together, these results indicate that anle145c is a promising candidate for inhibition of amyloid formation in T2DM.
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  • Journal Article

    Blood-brain barrier hyperpermeability precedes demyelination in the cuprizone model 

    Berghoff, Stefan A.; Düking, Tim; Spieth, Lena; Winchenbach, Jan; Stumpf, Sina K.; Gerndt, Nina; Kusch, Kathrin; Ruhwedel, Torben; Möbius, Wiebke; Saher, Gesine
    Acta Neuropathologica Communications 2017; 5(1): Art. 94
    In neuroinflammatory disorders such as multiple sclerosis, the physiological function of the blood-brain barrier (BBB) is perturbed, particularly in demyelinating lesions and supposedly secondary to acute demyelinating pathology. Using the toxic non-inflammatory cuprizone model of demyelination, we demonstrate, however, that the onset of persistent BBB impairment precedes demyelination. In addition to a direct effect of cuprizone on endothelial cells, a plethora of inflammatory mediators, which are mainly of astroglial origin during the initial disease phase, likely contribute to the destabilization of endothelial barrier function in vivo. Our study reveals that, at different time points of pathology and in different CNS regions, the level of gliosis correlates with the extent of BBB hyperpermeability and edema. Furthermore, in mutant mice with abolished type 3 CXC chemokine receptor (CXCR3) signaling, inflammatory responses are dampened and BBB dysfunction ameliorated. Together, these data have implications for understanding the role of BBB permeability in the pathogenesis of demyelinating disease.
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  • Journal Article

    Incremental value of biomarker combinations to predict progression of mild cognitive impairment to Alzheimer’s dementia 

    Frölich, Lutz; Peters, Oliver; Lewczuk, Piotr; Gruber, Oliver; Teipel, Stefan J.; Gertz, Hermann J.; Jahn, Holger; Jessen, Frank; Kurz, Alexander; Luckhaus, Christian; et al.
    Hüll, MichaelPantel, JohannesReischies, Friedel M.Schröder, JohannesWagner, MichaelRienhoff, OttoWolf, StefanieBauer, ChrisSchuchhardt, JohannesHeuser, IsabellaRüther, EckartHenn, FritzMaier, WolfgangWiltfang, JensKornhuber, Johannes
    Alzheimer's Research & Therapy 2017; 9(1): Art. 97
    Background: Type II diabetes is an important health problem with a complex connection to obesity, leading to a broad range of cardiovascular complications. Insulin therapy often results in weight gain and does not always ensure adequate glycemic control. However, previous studies reported that insulin detemir is an efficient long-acting insulin with a weight sparing effect. The aim of this study was to determine the association of catechol O-methyltransferase (COMT) Val108/158Met and dopamine-beta-hydroxylase (DBH) 1021C/T polymorphisms with the effectiveness of insulin detemir in achieving glucose control and body weight control. Participants and methods: This 52-week observational study included 185 patients with inadequate glycemic control treated with premix insulin analogues, which were replaced with insulin aspart and insulin detemir, and 156 healthy controls. After DNA isolation from blood samples, genotyping of DBH-1021C/T polymorphism (rs1611115) and COMT Val108/158Met polymorphism (rs4680) was performed. Results: Our results confirmed that insulin detemir did not lead to weight gain. The most significant finding was that A carriers (the combined AG and AA genotype) of the COMT Val108/158Met achieved significantly better hemoglobin A1c (HbA1c) values compared to patients carrying GG genotype. No association between DBH-1021C/T genotypes and weight and/or glucose control was detected in diabetes patients or in healthy control subjects. Conclusions: This study showed that the presence of one or two A allele of the COMT Val108/158Met was associated with improved glycemic response, and with a better response to insulin detemir therapy in patients with type II diabetes, separating them as best candidates for detemir therapy.
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  • Journal Article

    The influence of dopamine-beta-hydroxylase and catechol O-methyltransferase gene polymorphism on the efficacy of insulin detemir therapy in patients with type 2 diabetes mellitus 

    Bozek, Tomislav; Blazekovic, Antonela; Perkovic, Matea Nikolac; Jercic, Kristina Gotovac; Sustar, Aleksandra; Smircic-Duvnjak, Lea; Outeiro, Tiago F.; Pivac, Nela; Borovecki, Fran
    Diabetology & Metabolic Syndrome 2017; 9(1): Art. 97
    Background: Type II diabetes is an important health problem with a complex connection to obesity, leading to a broad range of cardiovascular complications. Insulin therapy often results in weight gain and does not always ensure adequate glycemic control. However, previous studies reported that insulin detemir is an efficient long-acting insulin with a weight sparing effect. The aim of this study was to determine the association of catechol O-methyltransferase (COMT) Val108/158Met and dopamine-beta-hydroxylase (DBH) 1021C/T polymorphisms with the effectiveness of insulin detemir in achieving glucose control and body weight control. Participants and methods: This 52-week observational study included 185 patients with inadequate glycemic control treated with premix insulin analogues, which were replaced with insulin aspart and insulin detemir, and 156 healthy controls. After DNA isolation from blood samples, genotyping of DBH-1021C/T polymorphism (rs1611115) and COMT Val108/158Met polymorphism (rs4680) was performed. Results: Our results confirmed that insulin detemir did not lead to weight gain. The most significant finding was that A carriers (the combined AG and AA genotype) of the COMT Val108/158Met achieved significantly better hemoglobin A1c (HbA1c) values compared to patients carrying GG genotype. No association between DBH-1021C/T genotypes and weight and/or glucose control was detected in diabetes patients or in healthy control subjects. Conclusions: This study showed that the presence of one or two A allele of the COMT Val108/158Met was associated with improved glycemic response, and with a better response to insulin detemir therapy in patients with type II diabetes, separating them as best candidates for detemir therapy.
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  • Journal Article

    Editorial: Molecular Chaperones and Neurodegeneration 

    Roodveldt, Cintia; Outeiro, Tiago F.; Braun, Janice E. A.
    Frontiers in Neuroscience 2017; 11: Art. 565
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  • Journal Article

    Homogenous generation of dopaminergic neurons from multiple hiPSC lines by transient expression of transcription factors 

    Mahajani, Sameehan; Raina, Anupam; Fokken, Claudia; Kügler, Sebastian; Bähr, Mathias
    Cell Death & Disease 2019; 10(12): Art. 898
    A major hallmark of Parkinson's disease is loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). The pathophysiological mechanisms causing this relatively selective neurodegeneration are poorly understood, and thus experimental systems allowing to study dopaminergic neuron dysfunction are needed. Induced pluripotent stem cells (iPSCs) differentiated toward a dopaminergic neuronal phenotype offer a valuable source to generate human dopaminergic neurons. However, currently available protocols result in a highly variable yield of dopaminergic neurons depending on the source of hiPSCs. We have now developed a protocol based on HBA promoter-driven transient expression of transcription factors by means of adeno-associated viral (AAV) vectors, that allowed to generate very consistent numbers of dopaminergic neurons from four different human iPSC lines. We also demonstrate that AAV vectors expressing reporter genes from a neuron-specific hSyn1 promoter can serve as surrogate markers for maturation of hiPSC-derived dopaminergic neurons. Dopaminergic neurons differentiated by transcription factor expression showed aggravated neurodegeneration through α-synuclein overexpression, but were not sensitive to γ-synuclein overexpression, suggesting that these neurons are well suited to study neurodegeneration in the context of Parkinson's disease.
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  • Journal Article

    Temporal stability of fMRI in medetomidine-anesthetized rats 

    Sirmpilatze, Nikoloz; Baudewig, Jürgen; Boretius, Susann
    Scientific Reports 2019; 9(1)
    Medetomidine has become a popular choice for anesthetizing rats during long-lasting sessions of blood-oxygen-level dependent (BOLD) functional magnetic resonance imaging (fMRI). Despite this, it has not yet been thoroughly established how commonly reported fMRI readouts evolve over several hours of medetomidine anesthesia and how they are affected by the precise timing, dose, and route of administration. We used four different protocols of medetomidine administration to anesthetize rats for up to six hours and repeatedly evaluated somatosensory stimulus-evoked BOLD responses and resting state functional connectivity. We found that the temporal evolution of fMRI readouts strongly depended on the method of administration. Intravenous administration of a medetomidine bolus (0.05 mg/kg), combined with a subsequent continuous infusion (0.1 mg/kg/h), led to temporally stable measures of stimulus-evoked activity and functional connectivity throughout the anesthesia. Deviating from the above protocol-by omitting the bolus, lowering the medetomidine dose, or using the subcutaneous route-compromised the stability of these measures in the initial two-hour period. We conclude that both an appropriate protocol of medetomidine administration and a suitable timing of fMRI experiments are crucial for obtaining consistent results. These factors should be considered for the design and interpretation of future rat fMRI studies.
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  • Journal Article

    Chronic exposure to cannabinoids during adolescence causes long-lasting behavioral deficits in adult mice 

    Tomas-Roig, J; Benito, E; Agis-Balboa, RC; Piscitelli, F; Hoyer-Fender, S; Di Marzo, V; Havemann-Reinecke, U
    Addiction Biology 2016; 22(6) p.1778-1789: Art.
    Regular use of marijuana during adolescence enhances the risk of long-lasting neurobiological changes in adulthood. The present study was aimed at assessing the effect of long-term administration of the synthetic cannabinoid WIN55212.2 during adolescence in young adult mice. Adolescent mice aged 5 weeks were subjected daily to the pharmacological action of WIN55212.2 for 3 weeks and were then left undisturbed in their home cage for a 5-week period and finally evaluated by behavioral testing. Mice that received the drug during adolescence showed memory impairment in the Morris water maze, as well as a dose-dependent memory impairment in fear conditioning. In addition, the administration of 3 mg/kg WIN55212.2 in adolescence increased adult hippocampal AEA levels and promoted DNA hypermethylation at the intragenic region of the intracellular signaling modulator Rgs7, which was accompanied by a lower rate of mRNA transcription of this gene, suggesting a potential causal relation. Although the concrete mechanisms underlying the behavioral observations remain to be elucidated, we demonstrate that long-term administration of 3 mg/kg of WIN during adolescence leads to increased endocannabinoid levels and altered Rgs7 expression in adulthood and establish a potential link to epigenetic change
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  • Journal Article

    Membrane protein megahertz crystallography at the European XFEL 

    Gisriel, Chris; Coe, Jesse; Letrun, Romain; Yefanov, Oleksandr M.; Luna-Chavez, Cesar; Stander, Natasha E.; Lisova, Stella; Mariani, Valerio; Kuhn, Manuela; Aplin, Steve; et al.
    Grant, Thomas D.Dörner, KaterinaSato, TokushiEchelmeier, AustinCruz Villarreal, JorvaniHunter, Mark S.Wiedorn, Max O.Knoska, JurajMazalova, VictoriaRoy-Chowdhury, ShatabdiYang, Jay-HowJones, AlexBean, RichardBielecki, JohanKim, YoonheeMills, GrantWeinhausen, BrittaMeza, Jose D.Al-Qudami, NasserBajt, SašaBrehm, GerritBotha, SabineBoukhelef, DjelloulBrockhauser, SandorBruce, Barry D.Coleman, Matthew A.Danilevski, CyrilDiscianno, ErinDobson, ZacharyFangohr, HansMartin-Garcia, Jose M.Gevorkov, YaroslavHauf, SteffenHosseinizadeh, AhmadJanuschek, FriederikeKetawala, Gihan K.Kupitz, ChristopherMaia, LuisManetti, MaurizioMesserschmidt, MarcMichelat, ThomasMondal, JyotirmoyOurmazd, AbbasPrevitali, GianpietroSarrou, IosifinaSchön, SilvanSchwander, PeterShelby, Megan L.Silenzi, AlessandroSztuk-Dambietz, JolantaSzuba, JanuszTurcato, MonicaWhite, Thomas A.Wrona, KrzysztofXu, ChenAbdellatif, Mohamed H.Zook, James D.Spence, John C. H.Chapman, Henry N.Barty, AntonKirian, Richard A.Frank, MatthiasRos, AlexandraSchmidt, MariusFromme, RaimundMancuso, Adrian P.Fromme, PetraZatsepin, Nadia A.
    Nature Communications 2019; 10(1)
    The world's first superconducting megahertz repetition rate hard X-ray free-electron laser (XFEL), the European XFEL, began operation in 2017, featuring a unique pulse train structure with 886 ns between pulses. With its rapid pulse rate, the European XFEL may alleviate some of the increasing demand for XFEL beamtime, particularly for membrane protein serial femtosecond crystallography (SFX), leveraging orders-of-magnitude faster data collection. Here, we report the first membrane protein megahertz SFX experiment, where we determined a 2.9 Å-resolution SFX structure of the large membrane protein complex, Photosystem I, a > 1 MDa complex containing 36 protein subunits and 381 cofactors. We address challenges to megahertz SFX for membrane protein complexes, including growth of large quantities of crystals and the large molecular and unit cell size that influence data collection and analysis. The results imply that megahertz crystallography could have an important impact on structure determination of large protein complexes with XFELs.
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  • Journal Article

    α‐Synuclein toxicity in yeast and human cells is caused by cell cycle re‐entry and autophagy degradation of ribonucleotide reductase 1 

    Sampaio‐Marques, Belém; Guedes, Ana; Vasilevskiy, Igor; Gonçalves, Susana; Outeiro, Tiago F.; Winderickx, Joris; Burhans, William C.; Ludovico, Paula
    Aging Cell 2019; 18(4)
    ƒ¿ ]Synuclein (aSyn) toxicity is associated with cell cycle alterations, activation of DNA damage responses (DDR), and deregulation of autophagy. However, the relationships between these phenomena remain largely unknown. Here, we demonstrate that in a yeast model of aSyn toxicity and aging, aSyn expression induces Ras2 ]dependent growth signaling, cell cycle re ]entry, DDR activation, autophagy, and autophagic degradation of ribonucleotide reductase 1 (Rnr1), a protein required for the activity of ribonucleotide reductase and dNTP synthesis. These events lead to cell death and aging, which are abrogated by deleting RAS2, inhibiting DDR or autophagy, or overexpressing RNR1. aSyn expression in human H4 neuroglioma cells also induces cell cycle re ]entry and S ]phase arrest, autophagy, and degradation of RRM1, the human homologue of RNR1, and inhibiting autophagic degradation of RRM1 rescues cells from cell death. Our findings represent a model for aSyn toxicity that has important implications for understanding synucleinopathies and other age ]related neurodegenerative diseases.
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  • Journal Article

    Two adhesive systems cooperatively regulate axon ensheathment and myelin growth in the CNS 

    Djannatian, Minou; Timmler, Sebastian; Arends, Martina; Luckner, Manja; Weil, Marie-Theres; Alexopoulos, Ioannis; Snaidero, Nicolas; Schmid, Bettina; Misgeld, Thomas; Möbius, Wiebke; et al.
    Schifferer, MartinaPeles, EliorSimons, Mikael
    Nature Communications 2019; 10(1)
    Central nervous system myelin is a multilayered membrane produced by oligodendrocytes to increase neural processing speed and efficiency, but the molecular mechanisms underlying axonal selection and myelin wrapping are unknown. Here, using combined morphological and molecular analyses in mice and zebrafish, we show that adhesion molecules of the paranodal and the internodal segment work synergistically using overlapping functions to regulate axonal interaction and myelin wrapping. In the absence of these adhesive systems, axonal recognition by myelin is impaired with myelin growing on top of previously myelinated fibers, around neuronal cell bodies and above nodes of Ranvier. In addition, myelin wrapping is disturbed with the leading edge moving away from the axon and in between previously formed layers. These data show how two adhesive systems function together to guide axonal ensheathment and myelin wrapping, and provide a mechanistic understanding of how the spatial organization of myelin is achieved.
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  • Journal Article

    Correction to: Ketogenic diet ameliorates axonal defects and promotes myelination in Pelizaeus–Merzbacher disease 

    Stumpf, Sina K.; Berghoff, Stefan A.; Trevisiol, Andrea; Spieth, Lena; Düking, Tim; Schneider, Lennart V.; Schlaphoff, Lennart; Dreha-Kulaczewski, Steffi; Bley, Annette; Burfeind, Dinah; et al.
    Kusch, KathrinMitkovski, MisoRuhwedel, TorbenGuder, PhilippRöhse, HeikoDenecke, JonasGärtner, JuttaMöbius, WiebkeNave, Klaus-ArminSaher, Gesine
    Acta Neuropathologica 2019; 138(4) p.673-674
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  • Journal Article

    Depopulation of dense α-synuclein aggregates is associated with rescue of dopamine neuron dysfunction and death in a new Parkinson’s disease model 

    Wegrzynowicz, Michal; Bar-On, Dana; Calo’, Laura; Anichtchik, Oleg; Iovino, Mariangela; Xia, Jing; Ryazanov, Sergey; Leonov, Andrei; Giese, Armin; Dalley, Jeffrey W.; et al.
    Griesinger, ChristianAshery, UriSpillantini, Maria Grazia
    Acta Neuropathologica 2019; 138(4) p.575-595
    Parkinson's disease (PD) is characterized by the presence of α-synuclein aggregates known as Lewy bodies and Lewy neurites, whose formation is linked to disease development. The causal relation between α-synuclein aggregates and PD is not well understood. We generated a new transgenic mouse line (MI2) expressing human, aggregation-prone truncated 1-120 α-synuclein under the control of the tyrosine hydroxylase promoter. MI2 mice exhibit progressive aggregation of α-synuclein in dopaminergic neurons of the substantia nigra pars compacta and their striatal terminals. This is associated with a progressive reduction of striatal dopamine release, reduced striatal innervation and significant nigral dopaminergic nerve cell death starting from 6 and 12 months of age, respectively. In the MI2 mice, alterations in gait impairment can be detected by the DigiGait test from 9 months of age, while gross motor deficit was detected by rotarod test at 20 months of age when 50% of dopaminergic neurons in the substantia nigra pars compacta are lost. These changes were associated with an increase in the number and density of 20-500 nm α-synuclein species as shown by dSTORM. Treatment with the oligomer modulator anle138b, from 9 to 12 months of age, restored striatal dopamine release, prevented dopaminergic cell death and gait impairment. These effects were associated with a reduction of the inner density of large α-synuclein aggregates and an increase in dispersed small α-synuclein species as revealed by dSTORM. The MI2 mouse model recapitulates the progressive dopaminergic deficit observed in PD, showing that early synaptic dysfunction is associated to fine behavioral motor alterations, precedes dopaminergic axonal loss and neuronal death that become associated with a more consistent motor deficit upon reaching a certain threshold. Our data also provide new mechanistic insight for the effect of anle138b's function in vivo supporting that targeting α-synuclein aggregation is a promising therapeutic approach for PD.
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  • Journal Article

    Contrast enhancement for visualizing neuronal cytoarchitecture by propagation-based x-ray phase-contrast tomography 

    Töpperwien, Mareike; Markus, Andrea; Alves, Frauke; Salditt, Tim
    NeuroImage 2019; 199 p.70-80
    Knowledge of the three-dimensional (3d) neuronal cytoarchitecture is an important factor in order to understand the connection between tissue structure and function or to visualize pathological changes in neurodegenerative diseases or tumor development. The gold standard in neuropathology is histology, a technique which provides insights into the cellular organization based on sectioning of the sample. Conventional histology, however, misses the complete 3d information as only individual two-dimensional slices through the object are available. In this work, we use propagation-based phase-contrast x-ray tomography to perform 3d virtual histology on cerebellar tissue from mice. This technique enables us to non-invasively visualize the entire 3d density distribution of the examined samples at isotropic (sub-)cellular resolution. One central challenge, however, of the technique is the fact that contrast for important structural features can be easily lost due to small electron density differences, notably between the cells and surrounding tissue. Here, we evaluate the influence of different embedding media, which are intermediate steps in sample preparation for classical histology, on contrast formation and examine the applicability of the different sample preparations both at a synchrotron-based holotomography setup as well as a laboratory source.
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  • Journal Article

    Blue and Long-Wave Ultraviolet Light Induce in vitro Neutrophil Extracellular Trap (NET) Formation 

    Neubert, Elsa; Bach, Katharina Marie; Busse, Julia; Bogeski, Ivan; Schön, Michael P.; Kruss, Sebastian; Erpenbeck, Luise
    Frontiers in Immunology 2019; 10: Art. 2428
    Neutrophil Extracellular Traps (NETs) are produced by neutrophilic granulocytes and consist of decondensed chromatin decorated with antimicrobial peptides. They defend the organism against intruders and are released upon various stimuli including pathogens, mediators of inflammation, or chemical triggers. NET formation is also involved in inflammatory, cardiovascular, malignant diseases, and autoimmune disorders like rheumatoid arthritis, psoriasis, or systemic lupus erythematosus (SLE). In many autoimmune diseases like SLE or dermatomyositis, light of the ultraviolet-visible (UV-VIS) spectrum is well-known to trigger and aggravate disease severity. However, the underlying connection between NET formation, light exposure, and disease exacerbation remains elusive. We studied the effect of UVA (375 nm), blue (470 nm) and green (565 nm) light on NETosis in human neutrophils ex vivo. Our results show a dose- and wavelength-dependent induction of NETosis. Light-induced NETosis depended on the generation of extracellular reactive oxygen species (ROS) induced by riboflavin excitation and its subsequent reaction with tryptophan. The light-induced NETosis required both neutrophil elastase (NE) as well as myeloperoxidase (MPO) activation and induced histone citrullination. These findings suggest that NET formation as a response to light could be the hitherto missing link between elevated susceptibility to NET formation in autoimmune patients and photosensitivity for example in SLE and dermatomyositis patients. This novel connection could provide a clue for a deeper understanding of light-sensitive diseases in general and for the development of new pharmacological strategies to avoid disease exacerbation upon light exposure.
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  • Journal Article

    SRpHi ratiometric pH biosensors for super-resolution microscopy 

    Richardson, Douglas S.; Gregor, Carola; Winter, Franziska R.; Urban, Nicolai T.; Sahl, Steffen J.; Willig, Katrin I.; Hell, Stefan W.
    Nature Communications 2017; 8(1)
    Fluorescence-based biosensors have become essential tools for modern biology, allowing real-time monitoring of biological processes within living cells. Intracellular fluorescent pH probes comprise one of the most widely used families of biosensors in microscopy. One key application of pH probes has been to monitor the acidification of vesicles during endocytosis, an essential function that aids in cargo sorting and degradation. Prior to the development of super-resolution fluorescence microscopy (nanoscopy), investigation of endosomal dynamics in live cells remained difficult as these structures lie at or below the ~250 nm diffraction limit of light microscopy. Therefore, to aid in investigations of pH dynamics during endocytosis at the nanoscale, we have specifically designed a family of ratiometric endosomal pH probes for use in live-cell STED nanoscopy.Ratiometric fluorescent pH probes are useful tools to monitor acidification of vesicles during endocytosis, but the size of vesicles is below the diffraction limit. Here the authors develop a family of ratiometric pH sensors for use in STED super-resolution microscopy, and optimize their delivery to endosomes.
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  • Journal Article

    Iron-mediated aggregation and toxicity in a novel neuronal cell culture model with inducible alpha-synuclein expression 

    Bartels, Martin; Weckbecker, Daniel; Kuhn, Peer-Hendrik; Ryazanov, Sergey; Leonov, Andrei; Griesinger, Christian; Lichtenthaler, Stefan F.; Bötzel, Kai; Giese, Armin
    Scientific Reports 2019; 9(1): Art. 9100
    Parkinson’s disease (PD) represents an increasing problem in society. The oligomerization of alphasynuclein (αSyn) is a suggested key event in its pathogenesis, yet the pathological modes of action remain to be fully elucidated. To identify potential disease-modifying therapeutics and to study αSynmediated toxic mechanisms, we established cell lines with inducible overexpression of different αSyn constructs: αSyn, αSyn coupled to the fluorescence protein Venus (αSyn-Venus), and αSyn coupled to the N-terminal or C-terminal part of Venus (V1S and SV2, respectively) for a bimolecular fluorescence complementation assay (BiFC). Inducibility was achieved by applying modified GAL4-UAS or Cre-loxP systems and addition of tebufenozide or 4-OH-tamoxifen, respectively. Expression constructs were stably integrated into the host genome of H4 neuroglioma cells by lentiviral transduction. We here demonstrate a detailed investigation of the expression characteristics of inducible H4 cells showing low background expression and high inducibility. We observed increased protein load and aggregation of αSyn upon incubation with DMSO and FeCl3 along with an increase in cytotoxicity. In summary, we present a system for the creation of inducibly αSyn-overexpressing cell lines holding high potential for the screening for modulators of αSyn aggregation and αSyn-mediated toxicity.
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  • Journal Article

    Ketogenic diet ameliorates axonal defects and promotes myelination in Pelizaeus–Merzbacher disease 

    Stumpf, Sina K.; Berghoff, Stefan A.; Trevisiol, Andrea; Spieth, Lena; Düking, Tim; Schneider, Lennart V.; Schlaphoff, Lennart; Dreha-Kulaczewski, Steffi; Bley, Annette; Burfeind, Dinah; et al.
    Kusch, KathrinMitkovski, MisoRuhwedel, TorbenGuder, PhilippRöhse, HeikoDenecke, JonasGärtner, JuttaMöbius, WiebkeNave, Klaus-ArminSaher, Gesine
    Acta Neuropathologica 2019; 138(1) p.147-161
    Pelizaeus-Merzbacher disease (PMD) is an untreatable and fatal leukodystrophy. In a model of PMD with perturbed blood-brain barrier integrity, cholesterol supplementation promotes myelin membrane growth. Here, we show that in contrast to the mouse model, dietary cholesterol in two PMD patients did not lead to a major advancement of hypomyelination, potentially because the intact blood-brain barrier precludes its entry into the CNS. We therefore turned to a PMD mouse model with preserved blood-brain barrier integrity and show that a high-fat/low-carbohydrate ketogenic diet restored oligodendrocyte integrity and increased CNS myelination. This dietary intervention also ameliorated axonal degeneration and normalized motor functions. Moreover, in a paradigm of adult remyelination, ketogenic diet facilitated repair and attenuated axon damage. We suggest that a therapy with lipids such as ketone bodies, that readily enter the brain, can circumvent the requirement of a disrupted blood-brain barrier in the treatment of myelin disease.
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