Recent Submissions

  • 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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  • Journal Article

    Cytosolic Trapping of a Mitochondrial Heat Shock Protein Is an Early Pathological Event in Synucleinopathies 

    Szegő, Éva M.; Dominguez-Meijide, Antonio; Gerhardt, Ellen; König, Annekatrin; Koss, David J.; Li, Wen; Pinho, Raquel; Fahlbusch, Christiane; Johnson, Mary; Santos, Patricia; et al.
    Villar-Piqué, AnnaThom, TobiasRizzoli, SilvioSchmitz, MatthiasLi, JiayiZerr, IngaAttems, JohannesJahn, OlafOuteiro, Tiago F.
    Cell Reports 2019; 28(1) p.65-77
    Alpha-synuclein (aSyn) accumulates in intracellular inclusions in synucleinopathies, but the molecular mechanisms leading to disease are unclear. We identify the 10 kDa heat shock protein (HSP10) as a mediator of aSyn-induced mitochondrial impairments in striatal synaptosomes. We find an age-associated increase in the cytosolic levels of HSP10, and a concomitant decrease in the mitochondrial levels, in aSyn transgenic mice. The levels of superoxide dismutase 2, a client of the HSP10/HSP60 folding complex, and synaptosomal spare respiratory capacity are also reduced. Overexpression of HSP10 ameliorates aSyn-associated mitochondrial dysfunction and delays aSyn pathology in vitro and in vivo. Altogether, our data indicate that increased levels of aSyn induce mitochondrial deficits, at least partially, by sequestering HSP10 in the cytosol and preventing it from acting in mitochondria. Importantly, these alterations manifest first at presynaptic terminals. Our study not only provides mechanistic insight into synucleinopathies but opens new avenues for targeting underlying cellular pathologies.
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  • Journal Article

    Thalamus exhibits less sensory variability quenching than cortex 

    Poland, E.; Donner, T. H.; Müller, K. -M.; Leopold, D. A.; Wilke, M.
    Scientific Reports 2019; 9(1): Art. 7590
    Spiking activity exhibits a large degree of variability across identical trials, which has been shown to be significantly reduced by stimulus onset in a wide range of cortical areas. Whether similar dynamics apply to the thalamus and in particular to the pulvinar is largely unknown. Here, we examined electrophysiological recordings from two adult rhesus macaques performing a perceptual task and comparatively investigated trial-to-trial variability in higher-order thalamus (ventral and dorsal pulvinar), the lateral geniculate nucleus (LGN) and visual cortex (area V4) prior to and following the presentation of a visual stimulus. We found spiking variability during stable fixation prior to stimulus onset to be considerably lower in both pulvinar and the LGN as compared to area V4. In contrast to the prominent variability reduction in V4 upon stimulus onset, variability in the thalamic nuclei was largely unaffected by visual stimulation. There was a small but significant variability decrease in the dorsal pulvinar, but not in the ventral portion of the pulvinar, which is closely connected to visual cortices and would thus have been expected to reflect cortical response properties. This dissociation did not stem from differences in response strength or mean firing rates and indicates fundamental differences in variability quenching between thalamus and cortex.
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  • xmlui.Mirage2.DIM-type-Erratum

    Publisher Correction: NRG1 type I dependent autoparacrine stimulation of Schwann cells in onion bulbs of peripheral neuropathies 

    Fledrich, Robert; Akkermann, Dagmar; Schütza, Vlad; Abdelaal, Tamer A.; Hermes, Doris; Schäffner, Erik; Soto-Bernardini, M. Clara; Götze, Tilmann; Klink, Axel; Kusch, Kathrin; et al.
    Krueger, MartinKungl, TheresaFrydrychowicz, ClaraMöbius, WiebkeBrück, WolfgangMueller, Wolf C.Bechmann, IngoSereda, Michael W.Schwab, Markus H.Nave, Klaus-ArminStassart, Ruth M.
    Nature Communications 2019; 10(1): Art. 1840
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  • Journal Article

    Spreading of α-Synuclein and Tau: A Systematic Comparison of the Mechanisms Involved 

    Vasili, Eftychia; Dominguez-Meijide, Antonio; Outeiro, Tiago Fleming
    Frontiers in Molecular Neuroscience 2019; 12: Art. 107
    Alzheimer's disease (AD) and Parkinson's disease (PD) are age-associated neurodegenerative disorders characterized by the misfolding and aggregation of alpha-synuclein (aSyn) and tau, respectively. The coexistence of aSyn and tau aggregates suggests a strong overlap between tauopathies and synucleinopathies. Interestingly, misfolded forms of aSyn and tau can propagate from cell to cell, and throughout the brain, thereby templating the misfolding of native forms of the proteins. The exact mechanisms involved in the propagation of the two proteins show similarities, and are reminiscent of the spreading characteristic of prion diseases. Recently, several models were developed to study the spreading of aSyn and tau. Here, we discuss the mechanisms involved, the similarities and differences between the spreading of the two proteins and that of the prion protein, and the different cell and animal models used for studying these processes. Ultimately, a deeper understanding of the molecular mechanisms involved may lead to the identification of novel targets for therapeutic intervention in a variety of devastating neurodegenerative diseases.
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  • Journal Article

    Neuronal Redox-Imbalance in Rett Syndrome Affects Mitochondria as Well as Cytosol, and Is Accompanied by Intensified Mitochondrial O2 Consumption and ROS Release 

    Can, Karolina; Menzfeld, Christiane; Rinne, Lena; Rehling, Peter; Kügler, Sebastian; Golubiani, Gocha; Dudek, Jan; Müller, Michael
    Frontiers in Physiology 2019; 10: Art. 479
    Rett syndrome (RTT), an X chromosome-linked neurodevelopmental disorder affecting almost exclusively females, is associated with various mitochondrial alterations. Mitochondria are swollen, show altered respiratory rates, and their inner membrane is leaking protons. To advance the understanding of these disturbances and clarify their link to redox impairment and oxidative stress, we assessed mitochondrial respiration in defined brain regions and cardiac tissue of male wildtype (WT) and MeCP2-deficient (Mecp2-/y) mice. Also, we quantified for the first time neuronal redox-balance with subcellular resolution in cytosol and mitochondrial matrix. Quantitative roGFP1 redox imaging revealed more oxidized conditions in the cytosol of Mecp2-/y hippocampal neurons than in WT neurons. Furthermore, cytosol and mitochondria of Mecp2-/y neurons showed exaggerated redox-responses to hypoxia and cell-endogenous reactive oxygen species (ROS) formation. Biochemical analyzes exclude disease-related increases in mitochondrial mass in Mecp2-/y hippocampus and cortex. Protein levels of complex I core constituents were slightly lower in Mecp2-/y hippocampus and cortex than in WT; those of complex V were lower in Mecp2-/y cortex. Respiratory supercomplex-formation did not differ among genotypes. Yet, supplied with the complex II substrate succinate, mitochondria of Mecp2-/y cortex and hippocampus consumed more O2 than WT. Furthermore, mitochondria from Mecp2-/y hippocampus and cortex mediated an enhanced oxidative burden. In conclusion, we further advanced the molecular understanding of mitochondrial dysfunction in RTT. Intensified mitochondrial O2 consumption, increased mitochondrial ROS generation and disturbed redox balance in mitochondria and cytosol may represent a causal chain, which provokes dysregulated proteins, oxidative tissue damage, and contributes to neuronal network dysfunction in RTT.
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  • Journal Article

    Acute Complexin Knockout Abates Spontaneous and Evoked Transmitter Release 

    López-Murcia, Francisco José; Reim, Kerstin; Jahn, Olaf; Taschenberger, Holger; Brose, Nils
    Cell Reports 2019; 26(10): Art. 2530.e5
    SNARE-mediated synaptic vesicle (SV) fusion is controlled by multiple regulatory proteins that determine neurotransmitter release efficiency. Complexins are essential SNARE regulators whose mode of action is unclear, as available evidence indicates positive SV fusion facilitation and negative "fusion clamp"-like activities, with the latter occurring only in certain contexts. Because these contradictory findings likely originate in part from different experimental perturbation strategies, we attempted to resolve them by examining a conditional complexin-knockout mouse line as the most stringent genetic perturbation model available. We found that acute complexin loss after synaptogenesis in autaptic and mass-cultured hippocampal neurons reduces SV fusion probability and thus abates the rates of spontaneous, synchronous, asynchronous, and delayed transmitter release but does not affect SV priming or cause "unclamping" of spontaneous SV fusion. Thus, complexins act as facilitators of SV fusion but are dispensable for "fusion clamping" in mammalian forebrain neurons.
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  • Journal Article

    Anillin facilitates septin assembly to prevent pathological outfoldings of central nervous system myelin 

    Erwig, Michelle S.; Patzig, Julia; Steyer, Anna M.; Dibaj, Payam; Heilmann, Mareike; Heilmann, Ingo; Jung, Ramona B.; Kusch, Kathrin; Möbius, Wiebke; Jahn, Olaf; et al.
    eLife 2019; 8: Art. e43888
    Myelin serves as an axonal insulator that facilitates rapid nerve conduction along axons. By transmission electron microscopy, a healthy myelin sheath comprises compacted membrane layers spiraling around the cross-sectioned axon. Previously we identified the assembly of septin filaments in the innermost non-compacted myelin layer as one of the latest steps of myelin maturation in the central nervous system (CNS) (Patzig et al., 2016). Here we show that loss of the cytoskeletal adaptor protein anillin (ANLN) from oligodendrocytes disrupts myelin septin assembly, thereby causing the emergence of pathological myelin outfoldings. Since myelin outfoldings are a poorly understood hallmark of myelin disease and brain aging we assessed axon/myelin-units in Anln-mutant mice by focused ion beam-scanning electron microscopy (FIB-SEM); myelin outfoldings were three-dimensionally reconstructed as large sheets of multiple compact membrane layers. We suggest that anillin-dependent assembly of septin filaments scaffolds mature myelin sheaths, facilitating rapid nerve conduction in the healthy CNS.
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  • Journal Article

    X10 expansion microscopy enables 25-nm resolution on conventional microscopes 

    Truckenbrodt, Sven; Maidorn, Manuel; Crzan, Dagmar; Wildhagen, Hanna; Kabatas, Selda; Rizzoli, Silvio O.
    EMBO Reports 2018; 19(9): Art. e45836
    Expansion microscopy is a recently introduced imaging technique that achieves super-resolution through physically expanding the specimen by ~4×, after embedding into a swellable gel. The resolution attained is, correspondingly, approximately fourfold better than the diffraction limit, or ~70 nm. This is a major improvement over conventional microscopy, but still lags behind modern STED or STORM setups, whose resolution can reach 20-30 nm. We addressed this issue here by introducing an improved gel recipe that enables an expansion factor of ~10× in each dimension, which corresponds to an expansion of the sample volume by more than 1,000-fold. Our protocol, which we termed X10 microscopy, achieves a resolution of 25-30 nm on conventional epifluorescence microscopes. X10 provides multi-color images similar or even superior to those produced with more challenging methods, such as STED, STORM, and iterative expansion microscopy (iExM). X10 is therefore the cheapest and easiest option for high-quality super-resolution imaging currently available. X10 should be usable in any laboratory, irrespective of the machinery owned or of the technical knowledge.
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