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Analysis of ALS and FTLD-U Linked Protein TDP-43 in Drosophila Melanogaster

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Analysis of ALS and FTLD-U Linked Protein TDP-43 in Drosophila Melanogaster Analysis of ALS and FTLD-U linked protein TDP-43 in Drosophila melanogaster Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der RWTH Aachen University zur Erlangung des akademische Grades einer Doktorin der Naturwissenschaften genehmigte Dissertation vorgelegt von Kavita Kaur MSc. aus Amritsar, Indien. Berichter: Prof. Dr. med. J.B. Schulz Prof. Dr. rer. nat. habil Hermann Wagner Tag der Mündlichen Prüfung: 6 Juli 2015 Diese Dissertation ist auf den Internetseiten der Universitätsbibliothek online verfügbar. Thesis Committee Thesis Committee Members of the thesis committee Supervisor Prof. Dr. med. J.B. Schulz Head, Department of neurology University medical center, RWTH Aachen University Pauwelsstrasse 30 52074 Aachen Prof. Dr. rer. nat. habil Hermann Wagner Institute leader, Chair and institute of biology II (Zoology) Sammelbau 2 Biologie Worringerweg 3 52074 Aachen Prof. Marc Spehr Abteilung Chemosensorik Institute of biology II (Zoology) Sammelbau 2 Biologie Worringerweg 3 52074 Aachen List of publications List of Publications and Author’s contributions Part of this thesis has already been published with authorisation of Prof J.B. Schulz, Head of the Department of Neurology, University Medical Centre RWTH Aachen. Original article:” TDP-43-mediated neuron loss in vivo requires RNA-binding activity Aaron Voigt, David Herholz, Fabienne Fiesel, Kavita Kaur, Daniel Mueller, Peter Karsten, Stephanie S. Weber, Phillipp J. Kahle, Till Marquardt, J.B.Schulz PLoS ONE August 2010 5(8): e 12247 doi10.1317/journal.pone.0012247 Conceived and designed the experiments: Aaron Voigt, David Herholz, Till Marquardt Performed the experiments: Aaron Voigt, David Herholz, Fabienne Fiesel, Kavita Kaur (fly experiments), Daniel Mueller, Peter Karsten, Stephanie S. Weber. Contributed reagents/ materials/ analysis tools: David Herholz, Fabienne Fiesel, Phillipp J. Kahle, J.B. Schulz Poster: “A fly model of TDP-43 proteinopathy” Kavita Kaur, J.B.Schulz, Aaron Voigt Regional Drosophila meeting 2009, Muenster (28 August 2009). Poster Abstract: “Genome-wide screen for modifiers of TDP-43 induced neurodegeneration” Kavita kaur, Peter Karsten, Sabine Hamm, J.B.Schulz and Aaron Voigt. International Society for Neurochemistry 2011, J Neurochem. 118 (Suppl. 1) Poster WE04-14, 165-244 Summary Summary TAR-DNA binding protein (TDP-43) is a multifunctional ribonucleoprotein, which is involved in transcription, splicing and mRNA stablilisation. TDP-43 is predominantly localised in the nucleus but shuttles between nucleus and the cytoplasm. In a pathological situation it is depleted from the nucleus and found mislocalised in the cytoplasm where it is abnormally cleaved, phosphorylated, ubiquitinated and aggregated. TDP-43 has been implicated in wide range of neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration-U (FTLD-U). The relative impact of the endogenous protein function, alteration and mutations on disease progression or pathology remains unclear. In this study I addressed many questions pertaining to TDP-43 biology with both random and strict comparable expression levels. This study focused on developing and characterising Drosophila models of TDP-43 proteinopathy. In addition, this study investigated the role of ALS/FTLD linked mutations and alterations on neural integrity utilising Drosophila as model system. A direct role of TDP-43 in neurodegeneration is highlighted by the fact that neuronal expression of human TDP-43 in Drosophila causes age- and dose-dependent locomotion deficits and early lethality. Further, targeted expression of TDP-43 to the developing fly eye results in a rough eye phenotype (REP). In this study TDP-43 RNA-binding was found to be absolutely required for toxicity. ALS/FTLD linked mutations and other reported pathological events like mislocalisation or truncation were relatively less toxic when compared to TDP-43 wild-type. Further I performed a large-scale toxicity modifier screen for TDP-43 induced toxicity using REP as readout. I identified novel modifiers of TDP-43-induced toxicity in this genetic screen. The most prominent candidates were RNA/DNA related genes, ubiquitin proteasome pathway related genes and neuron related genes. In addition, I found a novel protein interactor DBNDD2/CK1BP of TDP-43 in a yeast-2- hybrid screen. In the end, I present a very potent model of TDP-43 proteinopathy, which recapitulates a wide range of neuropathological, biochemical and functional features of human TDP-43 proteinopathy. Further working with this model I could uncover many endogenous functions and toxicity modifiers of TDP-43 induced toxicity. This study enhances our knowledge on TDP-43 biology and potentially widens future research strategies. Table of contents Table of contents 1 Introduction ........................................................................................................................... 1 1.1 TDP-43 proteinopathies ..................................................................................................... 1 1.2 Amyotrophic lateral sclerosis (ALS) ................................................................................. 3 1.3 Frontotemporal lobar dementia (FTLD-U) ......................................................................... 5 1.4 TAR-DNA binding protein 43 (TDP-43) .............................................................................. 6 1.4.1 Physiological functions of TDP-43 ...................................................................................... 7 1.4.2 Pathological characteristics of TDP-43 ............................................................................... 8 1.5 Modelling general neurodegeneration in flies ................................................................ 11 1.5.1 The UAS/Gal4 expression system .................................................................................... 11 1.5.2 The rough eye phenotype ................................................................................................ 13 2 Aim of the study .................................................................................................................. 14 3 Material and Methods .......................................................................................................... 15 3.1 Chemicals, buffers and equipment ................................................................................. 15 3.2 Transgenic Drosophila strains and fly keeping conditions ........................................... 18 3.2.1 The UAS/Gal4 expression system and Mating Procedure ................................................ 21 3.2.2 Longevity assays .............................................................................................................. 22 3.2.3 Climbing analyses ............................................................................................................ 23 3.2.4 Screening procedure and rough eye phenotype modification ........................................... 23 3.2.5 Drug administration/pharmacological treatment................................................................ 24 3.3 Protein biochemistry ........................................................................................................ 24 3.3.1 Head lysates preparation ................................................................................................. 24 3.3.2 SDS-polyacrylamide-gel-electrophoresis and Western blot .............................................. 25 3.4 Quantitative PCR analysis. .............................................................................................. 26 3.5 Statistical analysis ........................................................................................................... 27 3.6 Yeast-2-hybrid screen ...................................................................................................... 27 4 Results ................................................................................................................................. 28 4.1 Characterisation of TDP-43 proteinopathy in Drosophila .............................................. 29 4.1.1 GFP-tagged TDP-43 transgenic lines exhibiting Variable expression ............................... 29 4.1.2 Subcellular localisation of TDP-43:GFP tagged variants .................................................. 31 4.1.3 Expressing TDP-43 in eyes results in rough eye phenotype ............................................. 32 4.1.4 Pan-neural TDP-43 expression causes neurodegeneration. ............................................ 34 4.1.5 Expressing TDP-43 in muscles leads to muscle loss ........................................................ 35 4.1.6 TDP-43 toxicity is dose-dependent ................................................................................... 36 4.2 Site directed TDP-43 variants with comparable level of expression ............................. 38 Table of contents 4.2.1 Subcellular localisation of untagged TDP-43 variants in Drosophila ................................. 39 4.2.2 Pan-neural expression of TDP-43 reduces longevity in Drosophila. ................................. 43 4.2.3 Motor neuron specific expression of TDP-43 impaired locomotion ................................... 44 4.2.4 TDP-43 expression leads to loss of neuromuscular function ............................................ 46 4.2.5 Adult- onset model of TDP-43 proteinopathy. ..................................................................
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