Recent Submissions

  • 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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  • Journal Article

    Combined Use of Unnatural Amino Acids Enables Dual Color Super-Resolution Imaging of Proteins via Click Chemistry 

    Saal, Kim-A.; Richter, Frank; Rehling, Peter; Rizzoli, Silvio O.
    ACS Nano 2018; 2018(12) p.12247-12254
    Recent advances in optical nanoscopy have brought the imaging resolution to the size of the individual macromolecules, thereby setting stringent requirements for the fluorescent labels. Such requirements are optimally fulfilled by the incorporation of unnatural amino acids (UAAs) in the proteins of interest (POI), followed by fluorophore conjugation via click chemistry. However, this approach has been limited to single POIs in mammalian cells. Here we solve this problem by incorporating different UAAs in different POIs, which are expressed in independent cell sets. The cells are then fused, thereby combining the different proteins and organelles, and are easily imaged by dual-color super-resolution microscopy. This procedure, which we termed Fuse2Click, is simple, requires only the well-established Amber codon, and allows the use of all previously optimized UAAs and tRNA/RS pairs. This should render it a tool of choice for multi-color click-based imaging.
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  • Journal Article

    Investigating the feasibility of channelrhodopsin variants for nanoscale optogenetics 

    Stahlberg, Markus A.; Ramakrishnan, Charu; Willig, Katrin I.; Boyden, Edward S.; Deisseroth, Karl; Dean, Camin
    Neurophotonics 2019; 6(1): Art. 015007
    Optogenetics has revolutionized the study of circuit function in the brain, by allowing activation of specific ensembles of neurons by light. However, this technique has not yet been exploited extensively at the subcellular level. Here, we test the feasibility of a focal stimulation approach using stimulated emission depletion/reversible saturable optical fluorescence transitions-like illumination, whereby switchable light-gated channels are focally activated by a laser beam of one wavelength and deactivated by an overlapping donut-shaped beam of a different wavelength, confining activation to a center focal region. This method requires that activated channelrhodopsins are inactivated by overlapping illumination of a distinct wavelength and that photocurrents are large enough to be detected at the nanoscale. In tests of current optogenetic tools, we found that ChR2 C128A/H134R/T159C and CoChR C108S and C108S/D136A-activated with 405-nm light and inactivated by coillumination with 594-nm light-and C1V1 E122T/C167S-activated by 561-nm light and inactivated by 405-nm light-were most promising in terms of highest photocurrents and efficient inactivation with coillumination. Although further engineering of step-function channelrhodopsin variants with higher photoconductances will be required to employ this approach at the nanoscale, our findings provide a framework to guide future development of this technique.
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  • Journal Article

    Precisely measured protein lifetimes in the mouse brain reveal differences across tissues and subcellular fractions. 

    Fornasiero, Eugenio F.; Mandad, Sunit; Wildhagen, Hanna; Alevra, Mihai; Rammner, Burkhard; Keihani, Sarva; Opazo, Felipe; Urban, Inga; Ischebeck, Till; Sakib, M. Sadman; et al.
    Fard, Maryam K.Kirli, KorayCenteno, Tonatiuh PenaVidal, Ramon O.Rahman, Raza-UrBenito, EvaFischer, AndréDennerlein, SvenRehling, PeterFeussner, IvoBonn, StefanSimons, MikaelUrlaub, HenningRizzoli, Silvio O.
    Nature Communications 2018; 9(1): Art. 4230
    The turnover of brain proteins is critical for organism survival, and its perturbations are linked to pathology. Nevertheless, protein lifetimes have been difficult to obtain in vivo. They are readily measured in vitro by feeding cells with isotopically labeled amino acids, followed by mass spectrometry analyses. In vivo proteins are generated from at least two sources: labeled amino acids from the diet, and non-labeled amino acids from the degradation of pre-existing proteins. This renders measurements difficult. Here we solved this problem rigorously with a workflow that combines mouse in vivo isotopic labeling, mass spectrometry, and mathematical modeling. We also established several independent approaches to test and validate the results. This enabled us to measure the accurate lifetimes of ~3500 brain proteins. The high precision of our data provided a large set of biologically significant observations, including pathway-, organelle-, organ-, or cell-specific effects, along with a comprehensive catalog of extremely long-lived proteins (ELLPs).
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  • Journal Article

    WDR1 is a novel EYA3 substrate and its dephosphorylation induces modifications of the cellular actin cytoskeleton 

    Mentel, Mihaela; Ionescu, Aura E; Puscalau-Girtu, Ioana; Helm, Martin S; Badea, Rodica A; Rizzoli, Silvio O; Szedlacsek, Stefan E
    Scientific Reports 2018; 8(1) p.2910-2910: Art.
    Eyes absent (EYA) proteins are unusual proteins combining in a single polypeptide chain transactivation, threonine phosphatase, and tyrosine phosphatase activities. They play pivotal roles in organogenesis and are involved in a variety of physiological and pathological processes including innate immunity, DNA damage repair or cancer metastasis. The molecular targets of EYA tyrosine phosphatase activity are still elusive. Therefore, we sought to identify novel EYA substrates and also to obtain further insight into the tyrosine-dephosphorylating role of EYA proteins in various cellular processes. We show here that Src kinase phosphorylates tyrosine residues in two human EYA family members, EYA1 and EYA3. Both can autodephosphorylate these residues and their nuclear and cytoskeletal localization seems to be controlled by Src phosphorylation. Next, using a microarray of phosphotyrosine-containing peptides, we identified a phosphopeptide derived from WD-repeat-containing protein 1 (WDR1) that is dephosphorylated by EYA3. We further demonstrated that several tyrosine residues on WDR1 are phosphorylated by Src kinase, and are efficiently dephosphorylated by EYA3, but not by EYA1. The lack of phosphorylation generates major changes to the cellular actin cytoskeleton. We, therefore, conclude that WDR1 is an EYA3-specific substrate, which implies that EYA3 is a key modulator of the cytoskeletal reorganization.
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  • Journal Article

    OTTO: a new strategy to extract mental disease-relevant combinations of GWAS hits from individuals. 

    Ehrenreich, H.; Mitjans, M.; Van der Auwera, S.; Centeno, T. P.; Begemann, M.; Grabe, H. J.; Bonn, S.; Nave, K.-A.
    Molecular Psychiatry 2018; 23(2) p.476-486
    Despite high heritability of schizophrenia, genome-wide association studies (GWAS) have not yet revealed distinct combinations of single-nucleotide polymorphisms (SNPs), relevant for mental disease-related, quantifiable behavioral phenotypes. Here we propose an individual-based model to use genome-wide significant markers for extracting first genetic signatures of such behavioral continua. 'OTTO' (old Germanic=heritage) marks an individual characterized by a prominent phenotype and a particular load of phenotype-associated risk SNPs derived from GWAS that likely contributed to the development of his personal mental illness. This load of risk SNPs is shared by a small squad of 'similars' scattered under the genetically and phenotypically extremely heterogeneous umbrella of a schizophrenia end point diagnosis and to a variable degree also by healthy subjects. In a discovery sample of >1000 deeply phenotyped schizophrenia patients and several independent replication samples, including the general population, a gradual increase in the severity of 'OTTO's phenotype' expression is observed with an increasing share of 'OTTO's risk SNPs', as exemplified here by autistic and affective phenotypes. These data suggest a model in which the genetic contribution to dimensional behavioral traits can be extracted from combinations of GWAS SNPs derived from individuals with prominent phenotypes. Even though still in the 'model phase' owing to a world-wide lack of sufficiently powered, deeply phenotyped replication samples, the OTTO approach constitutes a conceptually novel strategy to delineate biological subcategories of mental diseases starting from GWAS findings and individual subjects.
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  • Journal Article

    Synaptic Alterations in Mouse Models for Alzheimer Disease—A Special Focus on N-Truncated Abeta 4-42 

    Dietrich, Katharina; Bouter, Yvonne; Müller, Michael; Bayer, Thomas
    Molecules 2018; 23(4) p.1-14: Art. 718
    This commentary reviews the role of the Alzheimer amyloid peptide Aβ on basal synaptic transmission, synaptic short-term plasticity, as well as short- and long-term potentiation in transgenic mice, with a special focus on N-terminal truncated Aβ4-42. Aβ4-42 is highly abundant in the brain of Alzheimer’s disease (AD) patients. It demonstrates increased neurotoxicity compared to full length Aβ, suggesting an important role in the pathogenesis of AD. Transgenic Tg4-42 mice, a model for sporadic AD, express human Aβ4-42 in Cornu Ammonis (CA1) neurons, and develop age-dependent hippocampal neuron loss and neurological deficits. In contrast to other transgenic AD mouse models, the Tg4-42 model exhibits synaptic hyperexcitability, altered synaptic short-term plasticity with no alterations in short- and long-term potentiation. The outcomes of this study are discussed in comparison with controversial results from other AD mouse models.
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  • Journal Article

    ATP-Dependent Chromatin Remodeling During Cortical Neurogenesis 

    Sokpor, Godwin; Castro-Hernandez, Ricardo; Rosenbusch, Joachim; Staiger, Jochen F.; Tuoc, Tran
    Frontiers in Neuroscience 2018; 12 p.1-25: Art. 226
    The generation of individual neurons (neurogenesis) during cortical development occurs in discrete steps that are subtly regulated and orchestrated to ensure normal histogenesis and function of the cortex. Notably, various gene expression programs are known to critically drive many facets of neurogenesis with a high level of specificity during brain development. Typically, precise regulation of gene expression patterns ensures that key events like proliferation and differentiation of neural progenitors, specification of neuronal subtypes, as well as migration and maturation of neurons in the developing cortex occur properly. ATP-dependent chromatin remodeling complexes regulate gene expression through utilization of energy fromATP hydrolysis to reorganize chromatin structure. These chromatin remodeling complexes are characteristically multimeric, with some capable of adopting functionally distinct conformations via subunit reconstitution to perform specific roles in major aspects of cortical neurogenesis. In this review, we highlight the functions of such chromatin remodelers during cortical development. We also bring together various proposed mechanisms by which ATP-dependent chromatin remodelers function individually or in concert, to specifically modulate vital steps in cortical neurogenesis.
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  • Journal Article

    Sumoylation Protects Against β-Synuclein Toxicity in Yeast. 

    Popova, Blagovesta; Kleinknecht, Alexandra; Arendarski, Patricia; Mischke, Jasmin; Wang, Dan; Braus, Gerhard H.
    Frontiers in molecular neuroscience 2018; 11 p.1-17: Art. 94
    Aggregation of α-synuclein (αSyn) plays a central role in the pathogenesis of Parkinson's disease (PD). The budding yeast Saccharomyces cerevisiae serves as reference cell to study the interplay between αSyn misfolding, cytotoxicity and post-translational modifications (PTMs). The synuclein family includes α, β and γ isoforms. β-synuclein (βSyn) and αSyn are found at presynaptic terminals and both proteins are presumably involved in disease pathogenesis. Similar to αSyn, expression of βSyn leads to growth deficiency and formation of intracellular aggregates in yeast. Co-expression of αSyn and βSyn exacerbates the cytotoxicity. This suggests an important role of βSyn homeostasis in PD pathology. We show here that the small ubiquitin-like modifier SUMO is an important determinant of protein stability and βSyn-induced toxicity in eukaryotic cells. Downregulation of sumoylation in a yeast strain, defective for the SUMO-encoding gene resulted in reduced yeast growth, whereas upregulation of sumoylation rescued growth of yeast cell expressing βSyn. This corroborates a protective role of the cellular sumoylation machinery against βSyn-induced toxicity. Upregulation of sumoylation significantly reduced βSyn aggregate formation. This is an indirect molecular process, which is not directly linked to βSyn sumoylation because amino acid substitutions in the lysine residues required for βSyn sumoylation decreased aggregation without changing yeast cellular toxicity. αSyn aggregates are more predominantly degraded by the autophagy/vacuole than by the 26S ubiquitin proteasome system. We demonstrate a vice versa situation for βSyn, which is mainly degraded in the 26S proteasome. Downregulation of sumoylation significantly compromised the clearance of βSyn by the 26S proteasome and increased protein stability. This effect is specific, because depletion of functional SUMO did neither affect βSyn aggregate formation nor its degradation by the autophagy/vacuolar pathway. Our data support that cellular βSyn toxicity and aggregation do not correlate in their cellular impact as for αSyn but rather represent two distinct independent molecular functions and molecular mechanisms. These insights into the relationship between βSyn-induced toxicity, aggregate formation and degradation demonstrate a significant distinction between the impact of αSyn compared to βSyn on eukaryotic cells.
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  • Journal Article

    Glyoxal as an alternative fixative to formaldehyde in immunostaining and super-resolution microscopy 

    Richter, Katharina N.; Revelo, Natalia H.; Seitz, Katharina J.; Helm, Martin S.; Sarkar, Deblina; Saleeb, Rebecca S.; D'Este, Elisa; Eberle, Jessica; Wagner, Eva; Vogl, Christian; et al.
    Lazaro, Diana F.Richter, FrankCoy-Vergara, JavierCoceano, GiovannaBoyden, Edward S.Duncan, Rory R.Hell, Stefan W.Lauterbach, Marcel A.Lehnart, Stephan E.Moser, TobiasOuteiro, Tiago F.Rehling, PeterSchwappach, BlancheTesta, IlariaZapiec, BolekRizzoli, Silvio O.
    The EMBO Journal 2018; 37(1) p.139-159
    Paraformaldehyde (PFA) is the most commonly used fixative for immunostaining of cells, but has been associated with various problems, ranging from loss of antigenicity to changes in morphology during fixation. We show here that the small dialdehyde glyoxal can successfully replace PFA Despite being less toxic than PFA, and, as most aldehydes, likely usable as a fixative, glyoxal has not yet been systematically tried in modern fluorescence microscopy. Here, we tested and optimized glyoxal fixation and surprisingly found it to be more efficient than PFA-based protocols. Glyoxal acted faster than PFA, cross-linked proteins more effectively, and improved the preservation of cellular morphology. We validated glyoxal fixation in multiple laboratories against different PFA-based protocols and confirmed that it enabled better immunostainings for a majority of the targets. Our data therefore support that glyoxal can be a valuable alternative to PFA for immunostaining.
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  • Journal Article

    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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