id,collection,dc.contributor.author,dc.contributor.contact,dc.contributor.firstReferee,dc.contributor.furtherReferee,dc.contributor.gender,dc.date.accepted,dc.date.accessioned,dc.date.available,dc.date.issued,dc.description,dc.description.abstract[de],dc.format.extent,dc.identifier.uri,dc.identifier.urn,dc.language,dc.rights.uri,dc.subject,dc.subject.ddc,dc.title,dc.title.translated[de],dc.type,dcterms.accessRights.dnb,dcterms.accessRights.openaire,dcterms.format[de],refubium.affiliation[de],refubium.mycore.derivateId,refubium.mycore.fudocsId "e7381ffa-815a-4d07-bbe4-31e952ad477a","fub188/14","Auer, Sebastian","auer.sebastian@mdc-berlin.de","Prof. Dr. Fritz G. Rathjen","Prof. Dr. Constance Scharff","m","2010-05-04","2018-06-07T19:12:08Z","2010-05-14T11:13:51.857Z","2010","1\. INTRODUCTION 7 1.1 Venom peptide toxins in research and therapy 7 1.2 Conotoxins: Peptide toxins derived from cone snails 8 1.2.1 α-conotoxin GID 9 1.2.2 ω-conotoxin MVIIA 9 1.2.3 ω-conotoxin MVIIC 10 1.3 Agatoxins: natural spider toxins 10 1.3.1 ω-Agatoxin IIIA 10 1.3.2 ω-Agatoxin IVA 11 1.4 Lentiviral vectors for gene delivery 12 1.5 Ion channels used for selective inhibition by t-toxins 14 1.5.1 Nicotinic acetylcholine receptors 14 1.5.2 Voltage-gated calcium channels 16 1.6 The nigro-striatal pathway 19 1.7 Origin of membrane-tethered toxins 21 1.8 Aims of this work 23 1.8.1 Aim 1: Construct optimization of t-toxins 23 1.8.2 Aim 2: Establishment of lentiviral delivery and stereotaxic injection of t-toxins 24 1.8.3 Aim 3: Generation of new t-toxins and application in vitro and in vivo 24 2\. MATERIAL AND METHODS 27 2.1 Preface 27 2.1.1 Chemicals 27 2.1.2 Composition of prepared buffers and solutions 28 2.1.3 Solutions for electrophysiology 29 2.1.4 Bacteria strains 30 2.1.5 Cell lines 30 2.1.6 Culture media 30 2.1.7 Plasmids 31 2.1.8 Primers 31 2.1.9 Oligonucleotides for toxin sequence generation 31 2.1.10 Antibodies and markers 32 2.1.11 Enzymes 32 2.1.12 Kits 33 2.1.13 Equipment and software 33 2.1.14 Statistical analyses 36 2.1.15 Animals 36 2.2 Molecular biology 36 2.2.1 Vector construction 36 2.2.2 Primer design 37 2.2.3 Amplification of DNA fragments by PCR 38 2.2.4 Agarose gel electrophoresis 39 2.2.5 Gel purification of DNA 39 2.2.6 TOPO TA cloning 39 2.2.7 Restriction digest and subcloning 40 2.2.8 Preparation of CaCl2 competent E. Coli cells 40 2.2.9 Transformation 41 2.2.10 Glycerol stock preparation 41 2.2.11 Plasmid DNA extraction 41 2.2.12 Sequencing 41 2.2.13 In vitro transcription 42 2.3 Cell culture 42 2.3.1 Cell culture of HEK293T and HeLa cells 42 2.3.2 Preparation of freezing stocks of HEK293T and HeLa cells 43 2.3.3 Thawing of HEK293T and HeLa cell freezing stocks 43 2.3.4 Lentivirus production 43 2.3.5 Lentivirus concentration 44 2.3.6 Lentivirus titration 44 2.3.7 FACS analysis 45 2.3.8 Protein extraction of transfected HEK293T cells 45 2.3.9 SDS-PAGE and Western blotting 46 2.3.10 Primary neuronal cultures 47 2.3.10.1 Rat hippocampus culture 47 2.3.10.2 Mouse cortex culture 47 2.3.11 Immunostaining of cultured cells 48 2.4 Electrophysiology 49 2.4.1 Electrophysiological recordings of nAChRs in X. laevis oocytes 49 2.4.2 Recordings of evoked calcium currents in HEK293-Cav2.2 cells 49 2.4.3 Paired-pulse recordings in rat hippocampal culture 50 2.5 In vivo analyses 51 2.5.1 Stereotaxic injections 51 2.5.2 Behavioural analysis 52 2.5.3 Perfusion of mice 52 2.5.4 Cryosections of perfused mouse brains 53 2.5.5 Immunostaining of brain sections 53 2.5.6 Quantification of immunostained brain sections 54 3\. RESULTS 55 3.1 Targeting nicotinic acetylcholine receptors with t-toxins 55 3.1.1 Toxin selection 55 3.1.2 Composition of GID t-toxin variants 55 3.1.3 Expression analyses of t-GID 56 3.1.3.1 Immunocytochemical analyses 56 3.1.3.2 Western Blot analysis 57 3.1.4 Functional analysis in Xenopus oocytes 58 3.2 Silencing neurotransmission with t-toxins by targeting calcium channels 61 3.2.1 Toxin selection 61 3.2.2 Expression analyses of calcium channel t-toxins in mammalian cells 61 3.2.3 Expression analyses of calcium channel t-toxins in neurons 62 3.2.4 Functional in vitro analyses 64 3.2.4.1 Electrophysiological recordings in HEK293-Cav2.2 cells 64 3.2.4.2 Electrophysiological recordings in rat hippocampal neurons 66 3.2.5 Influence of t-toxins on neuronal survival and cellular properties 70 3.2.6 Analyses of inducible t-toxin constructs 73 3.2.7 Analyses of Cre-dependent t-toxin constructs 77 3.2.8 Functional in vivo analyses 80 3.2.8.1 Stereotaxic injection of t-toxin lentivirus in mice 80 3.2.8.2 Behavioral analysis 81 3.2.8.3 Immunohistochemistry 82 4\. DISCUSSION 83 4.1 Influence of linker length on t-toxin activity 83 4.2 Comparison of soluble GID and t-toxin GID activity 84 4.3 The paired-pulse ratio as a measure of neurotransmission 84 4.4 Lack of functionality in multi-target t-toxins 85 4.5 Inducible and Cre-recombinase dependent expression 86 4.6 Inhibition of dopamine release in the nigro-striatal pathway by t-toxins 88 4.7 Advantages of t-toxins over other approaches 89 4.8 Possible applications of t-toxins 91 4.8.1 T-toxins in research 91 4.8.2 Therapeutic potential of t-toxins 91 4.8.3 Application of t-toxins for drug discovery 93 4.9 Possible further optimizations of t-toxins 94 5\. CONCLUSIONS 95 6\. APPENDIX 97 6.1 Abbreviations 97 6.2 Plasmid maps 100 6.3 Index of figures 107 6.4 Index of tables 108 6.5 Publication and presentation list 110 6.6 Lebenslauf 111 7\. REFERENCES 113","Based on the structural homology of the endogenous prototoxin lynx1 with the snake α-bungarotoxin, our lab has recently developed the tethered toxin (t-toxin) strategy for recombinant expression of functionally active, membrane-bound toxins, by using the biological scaffold of lynx1 (secretory signal and GPI signal). The work presented here expands the t-toxin approach and establishes for the first time the utility of t-toxins to specifically inhibit calcium currents in-vivo in mice. This has been accomplished by the integration of new modules and peptide toxins to generate novel t-toxins, and by using lentiviral vectors for gene delivery to targeted cells. The optimized constructs were generated by incorporation of several well-characterized peptide toxins, to achieve cell-specific and autonomous blockade of voltage gated calcium channels (Cav2.1 and Cav2.2; toxins: AgaIIIA, AgaIVA, MVIIA and MVIIC), as well as of nicotinic acetylcholine receptors (nAChRs; toxin: GID). In addition, fluorescent reporter proteins (EGFP, Venus and mCherry) were integrated to enable constant monitoring of the expression and subcellular localization. Furthermore, to achieve efficient insertion of t-toxins into the plasma membrane, the PDGF-receptor transmembrane domain or a glycophosphatidylinositol (GPI) anchor were attached. We show here that expression of calcium channel specific t-toxins by constitutive, inducible, as well as Cre-recombinase dependent lentiviral constructs can be efficiently used to inhibit Cav2.1 and Cav2.2 ionic currents in vitro in rat hippocampal neurons. Moreover, complete silencing of neurotransmission was achieved by simultaneous blockade of Cav2.1 and Cav2.2 by t-toxin co-transduction in these neurons. And importantly, the in vivo efficacy of this approach to block neurotransmission could be demonstrated by inhibition of dopaminergic signaling in the nigro-striatal pathway in lentivirus injected mice. In addition to calcium-channel t-toxins, the functionality of nAChR specific constructs was demonstrated in this work by inhibition of α7 and α3β4 nAChRs in Xenopus laevis oocytes. In conclusion, the optimized t-toxins generated in this work provide a straightforward new method to inhibit Cav2.1 and Cav2.2 voltage-gated calcium channels and nicotinic acetylcholine receptors (nAChRs) on long-term scale by recombinant and cell-autonomous expression in targeted cells. Given the extreme diversity of natural peptide venoms, membrane- tethered toxins are promising new tools for long-term modulation of neurotransmission by inhibition of specific ionic currents, and for characterization of the contribution of very diverse channels and receptors to physiological functions in a wide variety of species.||Basierend auf der strukturellen Ähnlichkeit des endogenen Prototoxins lynx1 mit dem Schlangengift α-Bungarotoxin hat unsere Arbeitsgruppe die sogenannte „tethered-toxin“ (T-Toxin) Methode entwickelt, bei welcher die Grundsequenz von lynx1, bestehend aus einem Sekretionssignal und einem Glycophosphatidylinositol (GPI)-Membrananker genutzt wird, um funktionale Peptidtoxine als Fusionsproteine membranständig zu exprimieren. In der vorliegenden Arbeit wurde diese Methode weiterentwickelt und die Funktionalität von T-Toxinen zur Inhibierung von Kalzium-Kanälen konnte zum ersten Mal in-vivo im Mausmodell demonstriert werden. Dies wurde zum einen durch die Integration neuer Module und Peptidtoxine, als auch durch die Verwendung lentiviraler Vektoren zum Gentransport in die Zielzellen ermöglicht. Der Einbau von genau charakterisierten Neurotoxinen ermöglicht die zell-spezifische und zell-autonome Inhibierung von Kalzium-Kanälen (Cav2.1 und Cav2.2; Toxine: AgaIIIA, AgaIVA, MVIIA und MVIIC), als auch von nikotinischen Azetylcholinrezeptoren (nAChR; toxin: GID). Zusätzlich wurden erstmals Fluoreszenzmarker integriert (EGFP, Venus und mCherry), welche eine ständige Expressions- und Lokalisationsanalyse der Fusionsproteine erlauben. Um die Membranständigkeit der Kostrukte zu gewährleisten wurde die Transmembrandomäne des PDGF-Rezeptors oder ein GPI- Membrananker verwendet. Wir konnten zeigen, dass die Inhibierung der Kalzium-Kanäle Cav2.1 und Cav2.2 duch spezifische T-Toxine von konstitutiven, induzierbaren und Cre-rekombinase abhängigen lentiviralen Vektoren zu einer Beeinflussung der Neurotransmission in kultivierten Hippocampus Neuronen führt. Zudem wurde eine vollständige Blockierung der Neurotransmission durch die gleichzeitige Inhibierung beider Kanäle nach Ko-Transduktion der T-Toxine erzielt. Bedeutenderweise konnte die Funktionalität dieser Konstrukte auch in vivo, durch Blockierung der Dopaminausschüttung im nigro-striatalen Signalübertragungsweg in Lentivirus- injizierten Mäusen gezeigt werden. Desweiteren wurde die Funktionalität von nAChR-spezifischen T-Toxinen durch Inhibierung von α7 und α3β4 nACh-Rezeptoren in Xenopus laevis Oocyten nachgewiesen. Die aus dieser Arbeit hervorgegangenen T-Toxine stellen eine Erweiterung der vorhandenen rekombinanten und pharmakologischen Methoden zur Untersuchung der physiologischen Funktionen von Ionenkanälen und Rezeptoren dar. Sie erlauben deren zell-spezifische und zell- autonome Langzeit-Inhibierung und können damit für die genauere Charakterisierung dieser Ionenkanäle und Rezeptoren in verschiedensten Tiermodellen verwendet werden.","120 S.","https://refubium.fu-berlin.de/handle/fub188/5819||http://dx.doi.org/10.17169/refubium-10018","urn:nbn:de:kobv:188-fudissthesis000000017398-2","eng","http://www.fu-berlin.de/sites/refubium/rechtliches/Nutzungsbedingungen","toxins||neuron||neurotransmission||voltage-gated calcium channels||Cav||inhibition||silencing||tethered toxins","500 Naturwissenschaften und Mathematik::570 Biowissenschaften; Biologie::570 Biowissenschaften; Biologie","Development of new membrane-tethered toxins as genetic tools for in vitro and in vivo silencing of ion channels","Entwicklung von neuen membrangebundenen Toxinen zur Inhibierung von Ionenkanälen in vitro und in vivo","Dissertation","free","open access","Text||Bild","Biologie, Chemie, Pharmazie","FUDISS_derivate_000000007566","FUDISS_thesis_000000017398"