dc.contributor.author
Auer, Sebastian
dc.date.accessioned
2018-06-07T19:12:08Z
dc.date.available
2010-05-14T11:13:51.857Z
dc.identifier.uri
https://refubium.fu-berlin.de/handle/fub188/5819
dc.identifier.uri
http://dx.doi.org/10.17169/refubium-10018
dc.description
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
dc.description.abstract
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.
de
dc.description.abstract
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.
de
dc.rights.uri
http://www.fu-berlin.de/sites/refubium/rechtliches/Nutzungsbedingungen
dc.subject
neurotransmission
dc.subject
voltage-gated calcium channels
dc.subject
tethered toxins
dc.subject.ddc
500 Naturwissenschaften und Mathematik::570 Biowissenschaften; Biologie::570 Biowissenschaften; Biologie
dc.title
Development of new membrane-tethered toxins as genetic tools for in vitro and
in vivo silencing of ion channels
dc.contributor.contact
auer.sebastian@mdc-berlin.de
dc.contributor.firstReferee
Prof. Dr. Fritz G. Rathjen
dc.contributor.furtherReferee
Prof. Dr. Constance Scharff
dc.date.accepted
2010-05-04
dc.identifier.urn
urn:nbn:de:kobv:188-fudissthesis000000017398-2
dc.title.translated
Entwicklung von neuen membrangebundenen Toxinen zur Inhibierung von
Ionenkanälen in vitro und in vivo
de
refubium.affiliation
Biologie, Chemie, Pharmazie
de
refubium.mycore.fudocsId
FUDISS_thesis_000000017398
refubium.mycore.derivateId
FUDISS_derivate_000000007566
dcterms.accessRights.dnb
free
dcterms.accessRights.openaire
open access
