Analyzing the function of TRAP1 in models of Parkinson’s disease

Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der RWTH Aachen University zur Erlangung des akademischen Grades einer Doktorin der Naturwissenschaften genehmigte Dissertation

vorgelegt von Li Zhang aus Changchun, Jilin (China)

Berichter: Universitätsprofessor Dr. med. Jörg B. Schulz Universitätsprofessor Dr. rer. nat. Marc Spehr

Tag der mündlichen Prüfung: 29.01.2016

Diese Dissertation ist auf den Internetseiten der Universitätsbibliothek online verfügbar.

Eidesstattliche Versicherung

______Zhang, Li ______Name, Vorname Matrikelnummer (freiwillige Angabe)

Ich versichere hiermit an Eides Statt, dass ich die vorliegende Arbeit/Bachelorarbeit/ Masterarbeit* mit dem Titel

______Analyzing the function of TRAP1 in models of Parkinson’s disease ______Uebersetzung: Analyse der TRAP1-Funktion in Modellen fuer Morbus Parkinson ______selbständig und ohne unzulässige fremde Hilfe erbracht habe. Ich habe keine anderen als die angegebenen Quellen und Hilfsmittel benutzt. Für den Fall, dass die Arbeit zusätzlich auf einem Datenträger eingereicht wird, erkläre ich, dass die schriftliche und die elektronische Form vollständig übereinstimmen. Die Arbeit hat in gleicher oder ähnlicher Form noch keiner Prüfungsbehörde vorgelegen.

______Aachen, 17.12.2015 ______

Ort, Datum Unterschrift

*Nichtzutreffendes bitte streichen

Belehrung:

§ 156 StGB: Falsche Versicherung an Eides Statt Wer vor einer zur Abnahme einer Versicherung an Eides Statt zuständigen Behörde eine solche Versicherung falsch abgibt oder unter Berufung auf eine solche Versicherung falsch aussagt, wird mit Freiheitsstrafe bis zu drei Jahren oder mit Geldstrafe bestraft.

§ 161 StGB: Fahrlässiger Falscheid; fahrlässige falsche Versicherung an Eides Statt (1) Wenn eine der in den §§ 154 bis 156 bezeichneten Handlungen aus Fahrlässigkeit begangen worden ist, so tritt Freiheitsstrafe bis zu einem Jahr oder Geldstrafe ein. (2) Straflosigkeit tritt ein, wenn der Täter die falsche Angabe rechtzeitig berichtigt. Die Vorschriften des § 158 Abs. 2 und 3 gelten entsprechend.

Die vorstehende Belehrung habe ich zur Kenntnis genommen:

______Aachen, 17.12.2015 ______Ort, Datum Unterschrift Members of the Thesis Committee

Supervisor Univ.-Prof. Dr. med. Jörg B. Schulz Department of Neurology University Medical Center, RWTH Aachen University Pauwelsstrasse 30 52074 Aachen

Second member of the Thesis Committee Univ.-Prof. Dr. rer. nat. Marc Spehr Institut für Biologie II, Zoologie Department of Biology, RWTH Aachen University Worringerweg 3 52074 Aachen

Third member of the Thesis Committee Univ.-Prof. Dr. rer. nat. Bernhard Lüscher Institut für Biochemie und Molekularbiologie, Medizinische Fakultät University Medical Center, RWTH Aachen University Pauwelsstrasse 30 52074 Aachen

Date of Disputation:

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List of Publications

Parts of this work have already been published with authorisation of Prof. Jörg B. Schulz, Head of the Department of Neurology, University Medical Centre of the RWTH Aachen University, on behalf of the thesis committee.

Oral presentation. Trap1, a new player in Parkinson’s disease. Regional Drosophila Meeting 2014 Heidelberg, Germany (05. 2014)

Zhang L, Karsten P, Hamm S, Pogson JH, Müller-Rischart AK, Exner N, Haass C, Whitworth AJ, Winklhofer KF, Schulz JB, Voigt A. TRAP1 rescues PINK1 loss-of-function phenotypes. Hum Mol Genet. 2013 Jul 15;22(14):2829-2841. DOI: 10.1093/hmg/ddt132

Poster Presentation. Trap1 rescues Pink1 loss-of-function phenotypes. ISN-ASN Meeting 2013 Cancun, Mexico (04. 2013)

Oral presentation. A TRAP1 rescues Pink1 loss-of-function phenotypes and mitochondrial dysfunction in vivo. Regional Drosophila Meeting 2012 Osnabrück, Germany (10. 2012)

Poster Presentation. Trap1 mitigates α-Synuclein-induced toxicity and rescues Pink1 loss- of-function phenotypes in vivo. 8th FENS Forum of Neuroscience Barcelona, Spain (07. 2012)

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Acknowledgements

Hereby I would like to express my sincere and deepest gratitude, To Prof. Jörg B. Schulz, for offering me the position at the first place, for giving me great opportunities in scientific domain and broadening my mind as an international researcher, for always providing critical suggestions for the direction of the projects, and last but not least, being an excellent role model himself. To Prof. Marc Spehr and Prof. Bernhard Lüscher, for being members in my doctoral thesis committee, for the splendid suggestions from their professional point of views which expands the horizons of my projects, for their patience and precious time. To Dr. Aaron Voigt, for being a great advisor as well as a very supportive friend, for being so generous and unreserved to spread his knowledge and exchange his valuable ideas, for his kindness to care about not only the scientific projects but also my personal career blueprint, for inspiring me for the scientific questions and offering me a friendly working and studying atmosphere. To Prof. Björn Falkenburg, Dr. Peter Karsten, Dr. Hannes Voßfeldt, Dr. Malte Butzlaff, Dr. Katja Pr in , Jane Patricia Tögel, Kavita Kaur, Dr. Theodora Saridaki and Dr. Barbara Stopschinski for showing me the experiments patiently, for sharing their scientific opinions. To Sabine Hamm and Anne Lankes, for technique supporting and their kindness. To Natalie Alexandra Burdiek-Reinhard, for always being so nice, for her considerable thoughts, and her help. To Antje Hofmeister, Xia Pan, Sarah Lenz, Stefan Esser, Daniel Komnig, Anna Hilverling, Athanasios Tarampanis, Maria Ingenerf, Simon Stilling, Larissa Kaltenhäuser, Elisabeth Dinter, Markus Nippold, Julie Schmidt-Tiedemann, and other students, for being such wonderful companion, for being so amazing and caring friends, and for the lovely times spending together. To Alexander J. Withworth and Konstanze F. Winklhofer for the cooperation, and Bundesministerium für Bildung und Forschung (Nationales Genomforschungsnetz (NGFN+) and the Kompetenznetz Degenerative Demenzen (KNDD) for financial support. Finally, to the ones who love me, only with their unconditional love, I live.

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Table of contents

LIST OF PUBLICATIONS ...... II ACKNOWLEDGEMENTS ...... III LIST OF FIGURES ...... VI LIST OF TABLES ...... VII LIST OF ABBREVIATIONS ...... VIII ABSTRACT ...... X 1 INTRODUCTION ...... 1

1.1 OVERVIEW OF PARKINSON S DISEASE ...... 1 1.2 PATHOLOGICAL HALLMARKS ...... 1 1.3 GENETICS OF PD ...... ’ 3 1.3.1 Autosomal dominant PD-causal , SNCA ...... 4 1.3.2 Autosomal recessive PD-causal gene, parkin ...... 5 1.3.3 Autosomal recessive PD-causal gene, Pink1 ...... 5 1.4 MITOCHONDRIAL DYSFUNCTION THEORY ...... 8 1.5 A MODIFIER OF -SYN-INDUCED TOXICITY, TRAP1 ...... 9 1.6 AIM OF STUDY ...... 10 Α 2 MATERIAL AND METHODS ...... 12

2.1 ORGANISM ...... 12 2.1.1 Fly stocks...... 12 2.1.2 UAS-Gal4 System ...... 12 2.1.3 siRNA in fly ...... 13 2.1.4 Transgenic flies ...... 14 2.2 CHEMICALS, , AND CONSUMABLE MATERIAL ...... 14 2.3 BUFFERS AND SOLUTIONS ...... 16 2.4 KITS ...... 17 2.5 EQUIPMENTS ...... 17 2.6 FLY BEHAVIORS/ PHENOTYPE ASSAYS ...... 17 2.6.1 Wing posture & Thorax indentation ...... 17 2.6.2 Negative geotaxis ...... 18 2.6.3 Longevity ...... 18 2.7 MITOCHONDRIAL ANALYSIS ...... 18 2.7.1 ATP content ...... 18 2.7.2 Mitochondrial complex I activity analysis ...... 20 2.7.3 Mitochondrial DNA level analysis ...... 21 2.7.4 Mitochondrial morphological analysis ...... 22 2.8 OTHER ASSAYS ...... 22 2.8.1 Analysis of DNA ...... 22 DNA Extraction ...... 22 2.8.2 Analysis of RNA ...... 22 RNA Extraction ...... 23 Real-time PCR...... 23 Oligo nucleotides (primers)...... 23 2.8.3 Analysis of protein...... 24 Preparation of protein lysate for western blot ...... 24 SDS Polyacrylamide Gel Electrophoresis and Western Blot ...... 24 Antibodies ...... 25 2.9 STATISTICAL ANALYSIS ...... 25 3 RESULTS ...... 26

3.1 TRAP1 FUNCTIONS DOWNSTREAM OF PINK1 ...... 26 3.1.1 Trap1 recues phenotypes caused by Pink1 loss-of-function in flies ...... 27

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3.1.2 Expression of Trap1 mitigates mitochondrial morphology and function in Pink1 loss-of-function flies ...... 33 Mitochondrial dysmorphology...... 33 ATP levels ...... 34 Mitochondrial DNA ...... 35 Mitochondrial Complex I ...... 36 Male fertility ...... 37 3.1.3 Knocking down Trap1 in Pink1 loss-of-function flies cause semi-lethality ...... 39 3.2 TRAP1 FUNCTIONS INDEPENDENTLY OF PINK1/PARKIN PATHWAY ...... 40 3.2.1 Trap1 does not influence phenotypes in parkin mutant flies...... 41 3.2.2 Expression of Trap1 does not mitigate mitochondrial function/morphology in parkin mutant flies ...... 42 3.3 TRAP1 MITIGATES MITOCHONDRIAL MORPHOLOGY/FUNCTION IN PINK1 KNOCK-OUT SH-SY5Y CELLS ...... 43 3.4 TRAP1 AND MITOCHONDRIAL COMPLEX I RESCUE EACH OTHER ...... 44 3.4.1 Trap1 rescues mitochondrial complex I subunits loss-of-function ...... 44 3.4.2 Ndi1p (mitochondrial complex I) rescues Trap1 loss-of-function ...... 46 4 DISCUSSION ...... 49

4.1 TRAP1 FUNCTIONS DOWNSTREAM OF PINK1 ...... 49 4.2 THE RESCUING EFFECT OF TRAP1 ON PINK1 LOSS-OF-FUNCTION REQUIRES MITOCHONDRIAL LOCATION OF TRAP1 AND ITS ATPASE ACTIVITY ...... 51 4.3 TRAP1 DOES NOT FUNCTION IN PINK1/PARKIN PATHWAY ...... 52 4.4 -SYNUCLEIN, PINK1, TRAP1 AND THE MITOCHONDRIAL COMPLEX I ...... 54

5 REFERENCESΑ ...... 59

List of Figures

FIGURE 1. PINK1/PARKIN PATHWAYS...... 8 FIGURE 2. THE STRUCTURE OF TRAP1...... 10 FIGURE 3. AN OVERVIEW OF THE UAS/GAL4 EXPRESSION SYSTEM...... 13 FIGURE 4. TRANSGENIC RNAI IN DROSOPHILA...... 14 FIGURE 5. THE PRINCIPLE OF ATP MEASUREMENT...... 19 FIGURE 6. THE ATP STANDARD LINE FOR FLY ATP ASSAY...... 19 FIGURE 7. DCIP ACCEPTS ELECTRONS AND BECOMES REDUCED-DCIP...... 20 FIGURE 8. EXPRESSION LEVELS OF TRAP1 IN HTRAP1 EXPRESSING FLIES...... 27 FIGURE 9. PINK1B9 FLIES WITH TRAP1 EXPRESSION REGAINED NORMAL WING POSTURE...... 28 FIGURE 10. TRAP1 RESCUED THE ABNORMAL WING POSTURE CAUSED BY NEURONAL LOSS OF PINK1...... 29 FIGURE 11. EXPRESSION OF TRAP1 MITIGATES PINK1 LOSS-OF-FUNCTION PHENOTYPES...... 31 FIGURE 12. EXPRESSION OF TRAP1 RESCUED THE DEGENERATION OF INDIRECT FLIGHT MUSCLES (LONGITUDINAL MUSCLES) IN PINK1B9 FLIES...... 32 FIGURE 13. EXPRESSION OF TRAP1 IMPROVED MITOCHONDRIAL MORPHOLOGY IN INDIRECT FLIGHT MUSCLES OF B9 PINK1 FLIES...... 33 FIGURE 14. EXPRESSION OF TRAP1 RESTORED ATP LEVELS IN THORAXES OF PINK1B9 FLIES...... 35 FIGURE 15. EXPRESSION OF HTRAP1 RESTORED MTDNA LEVELS IN PINK1B9 FLIES...... 36 FIGURE 16. HTRAP1 ELEVATED MITOCHONDRIAL COMPLEX I ACTIVITY AND THE LEVELS OF NDUFS3 IN PINK1B9 FLIES. .. 37 FIGURE 17. TRAP1 RESCUES THE STERILITY OF PINK1 MUTANT MALE FLIES...... 38 FIGURE 18. GENETIC INTERACTION OF PINK1 AND TRAP1 LOSS-OF-FUNCTION ALLELES...... 40 25 FIGURE 19. EXPRESSION OF TRAP1 DID NOT INFLUENCE PHENOTYPES IN PARK FLIES...... 42 FIGURE 20. EXPRESSION OF TRAP1 DID NOT MITIGATE MITOCHONDRIAL FUNCTION IN PARK25 FLIES...... 43 FIGURE 21. ATP LEVELS AND MITOCHONDRIAL FRAGMENTATION IN PINK1-SIRNA & PARKIN-SIRNA SH-SY5Y CELLS. ... 44 FIGURE 22. TRAP1 RESTORED ATP LEVELS IN L3 LARVE OF NDUFB1-KNOCK-DOWN FLIES...... 46 FIGURE 23. PHENOTYPES INDUCED BY TRAP1 LOSS-OF-FUNCTION IN TRAP14 FLIES...... 47 FIGURE 24. NDI1P RESCUED TRAP1 LOSS-OF-FUNCTION...... 48 FIGURE 25 TRAP1 PROTECTS THE FUNCTION/INTEGRITY OF MITOCHONDRIAL COMPLEX I...... 56 FIGURE 26. PUTATIVE ROLE(S) OF TRAP1 IN PD...... 58

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List of Tables

TABLE 1 LIST OF PD CAUSAL (SELECTION OF GENES) ...... 4 TABLE 2 LIST OF USED FLY STRAINS ...... 12 TABLE 3 INDEX OF CHEMICALS, ENZYMES, AND CONSUMABLE MATERIAL...... 14 TABLE 4 INDEX OF BUFFER AND SOLUTIONS ...... 16 TABLE 5 INDEX OF USED KITS ...... 17 TABLE 6 INDEX OF USED EQUIPMENTS ...... 17 TABLE 7 INDEX OF OLIGO NUCLEOTIDES ...... 23 TABLE 8 INDEX OF USED ANTIBODIES ...... 25 WT TABLE 9. HTRAP1 RESCUES SILENCING OF MITOCHONDRIAL COMPLEX SUBUNITS ...... 45 TABLE 10. A GENETIC SCREEN ON MITOCHONDRIALLY FUNCTIONAL BY LETHALITY ...... 70

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List of Abbreviations

Abbreviation Denotation

Acetyl CoA Acetyl coenzyme A ATP Adenosine triphosphate CG Computed Gene CoQ Coenzyme Q DA Dopamine DCIP Sodium 2,6-dichloroindophenolate hydrate d.p.e. days post eclosion ddc Dopa decarboxylase-Gal4 driver, DEPC Diethylpyrocarbonat dNTPs Deoxynuctleoside triphophates DRP1 Dynamin-related protein 1 DTNB 5,5’-dithio-bis(2-nitrobenzoic acid; 3-carboxy-4-nitrophenyl disulfide EDTA Ethylene diamine tetraacetic acid, disodium salt eIF2α Eukaryotic translation initiation factor 2α ER Endoplasmic reticulum EtBr Ethidium bromide ETC EtOH Ethanol HPLC High performance liquid chromatography hTrap1 Human Trap1

K2HPO4 di-Potassium hydrogen phosphate KAc Potassium acetate kDa Kilodalton

KH2PO4 Potassium dihydrogen phosphate LBs Lewy bodies LiCl Lithium chloride LNs Lewy neurites LRRK2 Leucine-rich repeat kinase 2 MDVs Mitochondria-derived vesicles MIM Mitochondrial inner membrane min Minutes MOM Mitochondrial outer membrane MPP Mitochondrial processing peptidase MPTP 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine mtDNA Mitochondrial DNA

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MTS Mitochondrial targeting sequence NaCl Sodium chloride NADH Nicotinamide adenine dinucleotide NDUFS NADH dehydrogenase (ubiquinone) Fe-S protein PAGE Polyacrylamide gel electrophoresis PARL Presenilinassociated rhomboid-like protease PCR Polymerase chain reaction PD Parkinson’s disease PFA Paraformaldehyde PINK1 PTEN-induced kinase 1 qPCR Quantitative polymerase chain reaction RIPA Radio immunoprecipitation assay RNAi RNA interference ROS Reactive oxygen species SDS Sodium dodecyl sulphate shRNA Short hairpin RNA siRNA Small interfering RNA SNCA Synuclein, alpha (non A4 component of amyloid precursor) SNpc Substantia nigra pars compacta SQSTM1 Sequestosome 1 TBS Tris buffered saline TCA cycle Tricarboxylic acid cycle TEMED N,N,N’,N’‐Tetramethylethylendiamide TIM23 Translocase of mitochondrial inner membrane 23 TOM70 Translocase of mitochondrial outer membrane 70 TRAP1 TNF receptor-associated protein 1 Tris-Base Tris (hydroxymethyl) aminomethane UAS Upstream-activating sequence UCHL1 carboxyl-terminal esterase L1 UPS Ubiquitin-proteasomal system VDAC1 Voltage-dependent anion channel 1 VDRC Vienna Drosophila RNAi Center w White WT Wild type y Yellow yr Years old

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Abstract

Loss-of-function mutations in the genes Pink1 and parkin cause recessive, early-onset Parkinson’s disease (PD). In Pink1/parkin-linked PD patients, mitochondrial function is impaired. Recent findings imply that PINK1 and Parkin facilitate mitochondrial quality control and target damaged or depolarized mitochondria for degradation via mitophagy. Thus, impaired mitophagy is considered to contribute to PD etiology in Pink1/parkin-linked PD.

Pink1 coded PTEN-induced Kinase 1 (PINK1) is a mitochondrial serine/threonine kinase. In SH-SY5Y cells, overexpression of PINK1 protects from oxidative stress by suppressing mitochondrial Cytochrome C release, thereby preventing cell death. Interestingly, the protective effects of PINK1 depend on phosphorylation of the downstream factor TNF receptor-associated protein 1 (TRAP1). In the absence of TRAP1, the protective effects of PINK1 overexpression are abolished. Furthermore, TRAP1 has been shown to miti ate α- Synuclein-induced toxicity. These data suggest that TRAP1 might be an important factor in PD acting downstream of PINK1.

To gain more insights in TRAP1 function, I asked whether overexpression of TRAP1 rescues Pink1 and/or parkin deficiency. My data suggest that TRAP1 mediates protective effects on mitochondrial function in pathways that are affected in PD. TRAP1 rescues dysfunction induced by Pink1 deficiency in vivo and in vitro. Especially, I show that overexpression of human Trap1 is able to mitigate Pink1 but not parkin loss-of-function phenotypes in Drosophila. Moreover, TRAP1 was able to rescue mitochondrial fragmentation and dysfunction upon siRNA silencing of Pink1 but not parkin in human neuronal SH-SY5Y cells. In addition, detrimental effects observed after RNAi-mediated silencing of mitochondrial complex I subunits were rescued by TRAP1 in Drosophila. Expression of Ndi1p, the only protein in yeast that determines complex I activity, rescued Trap1 loss-of- function induced phenotypes in flies. Thus the data suggest a functional role of TRAP1 in maintaining mitochondrial function downstream of Pink1 and mitochondrial complex I deficits but parallel or upstream of Parkin and independent of mitophagy via the PINK1/Parkin pathway. It has been reported that α-Synuclein interacts with complex I and impairs the function of complex I, and that expression of some complex I subunits also rescues Pink1 loss-of-function situations in flies. Therefore, I hypothesize that TRAP1 acts to maintain complex I function and in this way is protective against either overexpression of α- Synuclein or Pink1 loss-of-function induced toxicity. This offers a new pathway to slow or even stop the progression of neuronal degeneration in PD.

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Chapter 1 - Introduction 1

1 Introduction

1.1 Overview of Parkinson’s disease Parkinson’s disease (PD) is the second most common neurode enerative disease, after Alzheimer’s disease (AD). The prevalence of PD rises from 1% at 65 years old (yr) to 5% at 85 years (Shulman et al. 2011). Thus, age has been considered as a prominent risk factor of PD.

PD is characterized by bradykinesia, resting tremor, rigidity and postural instability (Shulman et al. 2011). Apart from motor abnormalities, PD’s clinical manifestations include depression (Aarsland et al. 2012), dementia (Aarsland et al. 2005), pain (Wasner & Deuschl 2012), olfactory dysfunction (Doty 2012), visual hallucination (Barnes & David 2001) and disturbed sleep (Peeraully et al. 2012).

These symptoms are, at least partially, believed to be caused by a loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) in the midbrain (Pakkenberg et al. 1991, Damier et al. 1999). The lesion in the SNpc in PD patients causes a severe depletion of striatal dopamine (DA), which is considered to cause the symptoms observed in PD patients (Obeso et al. 2000). Accordingly, most current therapies for PD are based on exogenous replacement of DA within the striatum, for example, L-Dopa administration. Although, such treatments improve most symptoms, they do not impede the progression of neurodegeneration. This is thought to be the main cause why these therapies lose efficacy with time (Marsden & Parkes 1977). This dissatisfaction of disease modifying PD treatment primarily results from a lack of knowledge about the cellular/molecular mechanism in PD etiology. With better mechanistic understanding of the PD, it might be possible to stop or even prevent the degeneration of dopaminergic neurons, and thereby the progression of PD.

1.2 Pathological hallmarks

Till now, two key pathological hallmarks are known in brains of PD patients: Lewy bodies (LBs)/Lewy neurites (LNs) and impairment of mitochondrial function, particularly of mitochondrial complex I.

Chapter 1 - Introduction 2

LBs and LNs are accumulation of fibrous protein deposits in neuronal cytoplasm (LBs) and nerve fibres (LNs) in the brain (Gibb & Lees 1988). α-Synuclein (α-Syn) has been identified as the primary component of LBs/LNs (Spillantini et al. 1997, Baba et al. 1998). Although α- Syn has been reported to be involved in synaptic traffic of vesicles, the specific function(s) of α-Syn is(are) still elusive (Maroteaux et al. 1988).

LBs and LNs mainly consist of accumulated α-Syn and are present in both familiar and idiopathic variants of PD (Polymeropoulos et al. 1997, Spillantini et al. 1997). Accordingly, LB and LN are considered as cardinal pathological features of PD (Hughes et al. 2001). α•Syn inclusions emerge in a predictable order in different regions of the brain with the progression of the disease, starting from olfactory bulb and/or the dorsal motor nucleus of the glossopharyngeal and vagal nerves (loss of smell), later reaching the medulla oblongata and the pontine tegmentum, followed by reaching the amygdala and the substantia nigra (onset of motor symptoms) and the temporal cortex, finally spreading the neocortex (showing cognitive problems) (Braak et al. 2003, Braak & Del Tredici 2009). In addition, recent investigations have shown a neuron-to-neuron “spread” of α-Syn in rodent brains (Kordower et al. 2008, Freundt et al. 2012, Rey et al. 2013, Braak et al. 2004, Eisbach et al. 2013). These findings susbstantiate the pathological finding that synucleopathies might spread through connected brain regions.

Impairment of mitochondrial complex I is another biochemical hallmark of PD pathology. In 1982, drug addicts were found to develop acute, severe, permanent Parkinsonism after inadvertent injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which was an unintended side-product of a heroin analog (Langston et al. 1983, Calne & Langston 1983, Langston et al. 1984). MPTP is a ‘protoxin’ that easily enters the brain where it is metabolized by glia cells to the active toxin, 1-methyl-4-phenylpyridinium ion (MPP+). As a substrate for the dopamine transporter, MPP+ is selectively transported into dopaminergic neurons. Once inside a neuron, MPP+ accumulates in mitochondria and inhibits respiration at the level of mitochondrial complex I (Langston et al. 1984). Thus, a mitochondrial complex I toxin selectively acting in dopaminergic neurons is able to produce a Parkinsonian syndrome in humans. Moreover, the MPP+-induced symptoms were astonishingly similar to symptoms observed in typical ‘idiopathic’ PD.

Chapter 1 - Introduction 3

This finding prompted researchers to wonder whether there was a defect of mitochondrial complex I in PD patients. A specific defect of mitochondrial complex I (other complexes were unaffected) has been firstly reported in necropsy specimens of substantia nigra from patients with PD already in 1990 (Schapira et al. 1990, Schapira et al. 1989). The mitochondrial complex I defects in PD appear to be systemic, also affecting tissues outside the brain. Numerous studies have reported reduced mitochondrial complex I activity in platelets and skeletal muscle samples from live PD patients (Bindoff et al. 1989, Parker et al. 1989, Haas et al. 1995, Krige et al. 1992, Yoshino et al. 1992). Therefore, a loss of mitochondrial complex I activity seems to be typically associated with PD.

In line with these findings, rats infused with rotenone, a classical, high affinity inhibitor of mitochondrial complex I, showed selective degeneration of the substantia nigra dopaminergic neurons (Betarbet et al. 2000). Many of those dying neurons presented large cytoplasmic inclusions containing α•Syn, which resembled LBs. Behaviorally, rotenone-infused rats developed symptoms of Parkinsonism, such as bradykinesia and rigidity. Accordingly, rotenone infusion is capable to recapitulate the anatomical, biochemical, pathological, and behavioral features of PD. These findings further support the assumption that defects in mitochondrial complex I can cause Parkinsonism. Although rotenone is administered systemically, dopaminergic neurons of the substantia nigra are the first cells to react as these cells are especially sensitive to mitochondrial complex I defects (Greenamyre et al. 2001).

1.3 Genetics of PD

Although LBs/LNs (α•Syn inclusions) and impairment of mitochondrial complex I were observed in dying dopaminergic neurons in the substantia nigra, the cause(s) of neuronal death in PD remain(s) unknown. Environmental factors, such as exposure to toxins (MPTP, rotenone) or ageing, were thought to be the main factor contributing to PD. However, accumulating evidences strongly suggest that genetic factors contribute to PD, as well.

Although the vast majority of PD cases are not directly inherited, susceptibility to PD is increased in first-degree relatives (parents, siblings, and offspring) of both sporadic and familial cases (Marder et al. 1996). With the advantage of modern sequence technologies and studies on families in which many members have developed PD, PD causal genes were found. Genetic abnormalities in a PD causal gene alone, without the influence of other genes or environmental factors, will cause PD. A list of PD causal genes is shown Table

Chapter 1 - Introduction 4

1. Mutations in the genes SNCA (Spillantini et al. 1995), Lrrk2 (Zimprich et al. 2004) and UCHL1 (Maraganore et al. 2004) cause autosomal dominant forms of late-onset Parkinsonism (affecting patients over the age of 50). In contrast, mutations in parkin (Kitada et al. 1998), Dj-1 (Bonifati et al. 2003b), Pink1 (Valente et al. 2004), and ATP13A2 (Ramirez et al. 2006) cause autosomal recessive early-onset Parkinsonism (affecting patients before the age 50). In the present study, I focused on SNCA, parkin and Pink1. Therefore, these genes and the function of their gene products are presented in more detail.

Table 1 List of PD causal genes (Selection of genes) Gene Name of Protein function Age of onset Clinical phenotpye Protein (years old) Dominant PARK1/4 SNCA α-Syn Synaptic protein Duplications: Typical PD, sometimes (4q21-23) 38-65 associated with cognitive Triplications: decline, autonomic 24-48 dysfunction and dementia Point mutations PARK5 UCHL1 UCHL1 Hydrolyze small C- 55-58 Only one family reported so (4p14) terminal adducts of (Ile93Met) far ubiquitin PARK8 LRRK2 LRRK2 Multiple functions 50-70 Typical PD (12p12- (range, 32-79) q13.1) Recessive PARK2 parkin Parkin Ubiquitin protein 30 on average Mutations account for 50% (6q25.2- ligase (range, 16-72) familial juvenile and early- q27) onset parkinsonism, sporadic. With sleep benefit. PARK6 Pink1 PINK1 Mitochondrial 20-40 Mutations account for 1-2% (1p35- kinase familial juvenile and early- p36) onset parkinsonism, sporadic. With sleep benefit. PARK7 Dj-1 DJ-1 Oxidative stress 20-40 Mutations account for <1% (1p36) protection early-onset parkinsonism PARK9 ATP13A2 ATPase Lysosomal protein <20 Juvenile and early-onset (1p36) type 13A2 parkinsonism with pyramidal degeneration and dementia

1.3.1 Autosomal dominant PD-causal gene, SNCA The SNCA gene was firstly identified as a dominant PD causal gene (Spillantini et al. 1995). Interestingly, the SNCA gene encodes α-Syn, which has been identified as the primary component of LBs/LNs (Spillantini et al. 1997, Baba et al. 1998). Mis-sense mutations in α- Syn like A30P, E46K and A53T have been linked to familial Parkinsonism (Polymeropoulos et al. 1997, Kruger et al. 1998). SNCA duplication has been found in PD families who showed late-onset slow-progressing Parkinsonism without cognitive decline. In contrast, families with SNCA triplication developed early-onset Parkinsonism with dementia (Chartier-

Chapter 1 - Introduction 5

Harlin et al. 2004, Singleton et al. 2003). This indicates a dosage-dependent effect of SNCA- encoded α-Syn in the disease progression of PD.

1.3.2 Autosomal recessive PD-causal gene, parkin Mutations in parkin have been identified in 50% of patients with autosomal recessive juvenile-onset PD (AR-JP) (affecting patients before the age 20) and 77% of apparently sporadic cases with disease onset before the age of 20 (Kitada et al. 1998, Lucking et al. 2000, Fitzgerald & Plun-Favreau 2008). PD patients with mutations in parkin suffer a slow progression of PD frequently associated with early-onset dystonia (Schapira 2008), but most of the patients lack LBs (Hayashi et al. 2000). If at all, LBs appear in some later onset cases (Farrer et al. 2001, Pramstaller et al. 2005). Parkin encodes an E3 , an essential component of the ubiquitin-proteasomal system (UPS). Parkin localizes predominantly to the cytosol and cellular vesicles, but has been found to also associate with the mitochondrial outer membrane (MOM) (Darios et al. 2003, Shimura et al. 2000, Kubo et al. 2001).

1.3.3 Autosomal recessive PD-causal gene, Pink1 Mutations in Pink1 are the second most-common cause of autosomal recessive PD after parkin (Fitzgerald & Plun-Favreau 2008). Initially, three pedigrees were described with mutations in the Pink1 gene: an substitution G309D in one family and a truncation mutation (W437X) in two additional families (Pridgeon et al. 2007, Zhou et al. 2008, Becker et al. 2012, Valente et al. 2004). Subsequently, several studies have described other pathogenic mutations in Pink1 (Tan & Skipper 2007). Patients with Pink1 mutations respond well to L-Dopa treatment but do not suffer from dystonia at disease onset (Healy et al. 2004).

PINK1, encoded by the Pink1 gene, is a serine/threonine kinase (Pridgeon et al. 2007, Zhou et al. 2008, Becker et al. 2012, Valente et al. 2004). PINK1 is located in the mitochondrial membranes in human brain tissue, as well as a cytoplasmic pool (Gandhi et al. 2006, Weihofen et al. 2008, Haque et al. 2008).

PINK1 has been reported to be involved in numerous pathways. Among those pathways, the so-called PINK1/Parkin pathway is the most well known. In 2006, two independent groups generated Pink1 mutant fly lines (Clark et al. 2006, Park et al. 2006). Pink1 mutant flies

Chapter 1 - Introduction 6

displayed many phenotypes, closely resembling the phenotypes observed in parkin mutant flies (Clark et al. 2006, Park et al. 2006, Yang et al. 2006). This suggested that the two proteins might act in one molecular pathway. Interestingly, overexpression of parkin rescued abnormal phenotypes in Pink1 loss-of-function flies. In contrast, overexpression of Pink1 did not mitigate phenotypes caused by parkin loss-of-function. This implies that PINK1 and Parkin indeed function in a common pathway and that Pink1 acts upstream of parkin. Of note here is that PD patients with Pink1 or parkin mutations also exhibit overlapping clinical symptoms (Ibanez et al. 2006).

Very recently, the mechanism of the PINK1/Parkin pathway became unveiled. PINK1 and Parkin function together to monitor the quality of the mitochondrial population. Under healthy conditions, full-length PINK1 is synthesized in cytoplasm. According to the N- terminal mitochondrial localization sequence, PINK1 is imported through MOM via translocase of MOM 70 (TOM70) complex and then through mitochondrial inner membrane (MIM) via the translocase of MIM 23 (TIM23) (Lazarou et al. 2012, Kato et al. 2013). In the mitochondrial matrix PINK1 is cleaved by mitochondrial processing peptidase (MPP) and presenilinassociated rhomboid-like protease (PARL), and released to the cytosol for proteasomal degradation (Becker et al. 2012, Greene et al. 2012, Jin et al. 2010, Yamano & Youle 2013). The import of PINK1 to the inner mitochondrial membrane requires polarized mitochondria. Thus, in the presence of mitochondria with an intact membrane potential, PINK1 is rapidly degraded (Figure 1A).

In contrast, damaged mitochondria trigger the accumulation of full-length PINK1 at the MOM in case of, either by a depletion of mitochondrial membrane potential, accumulation of mitochondrial misfolded protein in the mitochondrial matrix or oxidative damage (Vives- Bauza et al. 2010b, Jin & Youle 2013). Full-length PINK1 on the MOM recruits Parkin to these damaged mitochondria (Geisler et al. 2010, Kawajiri et al. 2010, Michiorri et al. 2010, Narendra et al. 2010b, Narendra et al. 2010a, Vives-Bauza et al. 2010a) and also phosphorylates ubiquitin for activation (Kane et al. 2014, Koyano et al. 2014, Kazlauskaite et al. 2014). As a consequence, Parkin initiates the ubiquitination on numerous mitochondrial targets. In this way, abundant downstream pathways are switched on or off, such as mitochondrial transport (off, to halt the damaged mitochondria), mitochondrial fusion (off, to prevent damaged mitochondria to fuse and thereby alter other healthy mitochondria) and mitophagy or mitochondria-derived vesicles (MDVs) (Gegg et al. 2010, Tanaka et al. 2010,

Chapter 1 - Introduction 7

Poole et al. 2010, Glauser et al. 2011, Chen & Dorn 2013, Klionsky 2010, Wang et al. 2011, Geisler et al. 2010, McLelland et al. 2014) (Figure 1B).

Strong stimulation of the PINK1/Parkin pathway in response to low mitochondrial membrane potential or accumulation of misfolded proteins causes the targeting of damaged mitochondria to mitophagy (Klionsky 2010, Wang et al. 2011, Geisler et al. 2010). In contrast, only mild stimulation, such as mild oxidative damage, the PINK1/Parkin pathway mediates MDVs to sort out just the damaged parts for degradation by lysosomes (McLelland et al. 2014).

Intriguingly, in addition to Parkin, PINK1 is also known to interact with several other proteins encoded by PD-linked genes. DJ-1 forms a complex with PINK1 and Parkin to degrade Parkin substrates (Xiong et al. 2009). In addition, α-Syn-induced mitochondrial fragmentation is rescued by PINK1, but not the PD-associated mutation PINK1G309D (Kamp et al. 2010). Therefore, PINK1 seems to play a central role in the etiology of PD. Thus, understanding the endogenous functions of the cellular mechanisms regulated by PINK1 will help in the future design of PD therapies.

PINK1 has also been shown to affect mitochondrial complex I activity. Pink1 deficiency or clinical mutations in the gene cause reduced mitochondrial complex I activity in rat and Drosophila (Morais et al. 2009, Park et al. 2006). Phenotypes in Pink1 deficient flies can be rescued by the expression of Saccharomyces cerevisiae Ndi1p (an that bypasses mammalian ETC complex I), but not by sea squirt Ciona intestinalis AOX (an enzyme bypassing mammalian ETC Complex III and IV). In contrast, expression of Ndi1p failed to rescue any of the parkin mutant phenotypes in flies, and flies deficient for parkin did not show reduced activity of complex I. In summary, these data suggest that mitochondrial complex I acts downstream of Pink1, but upstream or independent of Parkin (Vilain et al. 2012). This assumption is further supported by the finding that flies deficient for certain mitochondrial complex I subunits display phenotypes similar to Pink1 deficient flies, which suggest strong genetic link between Pink1 and mitochondrial complex I (Vilain et al. 2012).

Chapter 1 - Introduction 8

Figure 1. PINK1/Parkin pathways. (A) In healthy mitochondria, after being synthesized in cytoplasm, PINK1 translocates to mitochondria and stops at MOM. As a result of mitochondrial membrane potential (ΔΨm), PINK1 is imported through the MOM via TOM70, and docks at TIM23 complex on the MIM. In case of depolarized mitochondria, PINK1 is cleaved by the matrix proteases MPP and PARL. Cleaved PINK1 is released to the cytosol for proteasomal degradation. In damaged mitochondria, newly synthesized PINK1 accumulates on the MOM. (B) Accumulation of PINK1 is required to recruit Parkin to damaged mitochondria. This recruitment probably involves increasing amounts of phosphorylated ubiquitin, another direct target of PINK1. Recruited to the mitochondria, Parkin ubiquitinates several targets to prevent the damaged mitochondria from fusing with other healthy mitochondria. Finally, Parkin initiates either mitophagy to clear the damaged mitochondria, or MDVs to remove only damaged parts of mitochondria.

1.4 Mitochondrial dysfunction theory

As mentioned, deficiency of ETC in mitochondria has been reported in PD (Schapira et al. 1990, Schapira et al. 1989, Parker et al. 2008, Parker et al. 1989, Haas et al. 1995, Krige et al. 1992, Yoshino et al. 1992, Bindoff et al. 1989), and inhibitors of the ETC, such as Rotenone or MPTP cause Parkinsonian symptoms (Langston et al. 1983, Calne & Langston 1983, Langston et al. 1984, Betarbet et al. 2000, Greenamyre et al. 2001). Therefore, it is generally accepted that mitochondrial dysfunction at least contributes to PD pathology.

Chapter 1 - Introduction 9

In addition, accumulating evidence strengthened the idea that mitochondrial dysfunction is a critical event in PD pathology. Mitochondria are key regulators of cell survival and have a central role in ageing, which is the highest risk factor for PD. Mitochondria are thought to contribute to ageing through the accumulation of mitochondrial DNA (mtDNA) mutations and production of reactive oxygen species (ROS). Oxidative stress and mtDNA damage have been commonly detected, especially in substantia nigra neurons, and are thought to induce the degeneration of dopaminergic neurons of PD patients (Gu et al. 1998, Ikebe et al. 1990, Jenner et al. 1992, Bender et al. 2006).

Moreover, proteins encoded by several PD-genes have indispensable roles for mitochondrial function. As mentioned, PINK1 and Parkin monitor the quality of mitochondrial population. DJ-1 is protective against oxidative stress. Consequently, this anti-oxidative stress function keeps mitochondria healthy and prevents cell death (Taira et al. 2004, Menzies et al. 2005). α-Syn induces mitochondrial fragmentation (Kamp et al. 2010). Moreover, α-Syn has been reported to associate with, and thereby inhibit mitochondrial complex I activity. This in turn causes increased production of ROS, causing additional damage to the ETC (Devi et al. 2008).

1.5 A modifier of α-Syn-induced toxicity, Trap1 Despite the knowledge of the hallmarks of PD pathology and the theory of mitochondrial dysfunction, the cause(s) of PD is(are) still indistinct. To gain insights in the role of α-Syn in PD etiology, an unbiased, genome-wide screen for genetic modifiers of α-Syn-induced toxicity was conducted (Butler et al. 2012). This screen utilized a Drosophila (fruit fly) model.

The fruit fly is a reliable model to study α-Syn-induced toxicity. Albeit flies do not possess a SNCA ortholog, human SNCA transgenic flies carrying a disease causing mutation were reported to present an impairment of locomotion as seen in humans (Feany & Bender 2000). LBs-like inclusions in some neurons were also observed (Feany & Bender 2000). Moreover, these SNCA transgenic flies showed age-dependent loss of dopaminergic neurons, while other neurons appeared to be fairly unaffected (Feany & Bender 2000, Auluck et al. 2001). Thus, the SNCA transgenic fly model closely recapitulated many of the characteristic features of PD.

Chapter 1 - Introduction 10

Expression of the α-SynA53T (a PD causing mutant variant of α-Syn) in flies also recapitulated the key features of PD. Flies with α-SynA53T expression in aminergic neurons (including dopaminergic neurons as a subgroup) displayed shortened life span, reduced age-dependent locomotion defects and reduced level of dopamine (Butler et al. 2012). Using the dopamine level of brain as an indicator for the dysfunction of dopaminergic neurons, modifier(s) of α- SynA53T-induced toxicity were screened for. Among several other candidates, TNF receptor- associated protein 1 (TRAP1) was found to modulating α-SynA53T-induced toxicity. A reduction of endogenous Trap1 (by 50%) strongly enhanced α-SynA53T-induced toxicity, whereas overexpression of human Trap1 in flies suppressed the toxicity (Butler et al. 2012).

TRAP1 is a mitochondrial molecular chaperone with similarity to 90 () (also called Hsp75). The domain structure of TRAP1 is very similar to Hsp90 (Figure 2). Although the proteins share a similar domain structure, they seem to function slightly different. So far, there has no co-chaperone partner for TRAP1 identified (as it is for the Hsp90s) and TRAP1 fails to execute the classical Hsp90 activity assays (Matassa et al. 2012).

Figure 2. The structure of TRAP1. TRAP1, as a mitochondrial chaperone, contains a 59 amino acids N-terminal Mitochondria-Targeting Sequence (MTS), an ATPase domain with four ATP-binding sites (ATP binding sites are located at amino acid positions 119, 158, 171 and 205) and a C-terminal Hsp90-like domain (Matassa et al. 2012).

Interestingly, TRAP1 was also discovered as a substrate of PINK1 (Pridgeon et al. 2007, Zhou et al. 2008, Becker et al. 2012, Valente et al. 2004). PINK1 binds and co-localizes with TRAP1 in the mitochondria and PINK1 was shown to phosphorylate TRAP1 both in vitro and in vivo (Pridgeon et al. 2007). Moreover, in the same study, they also found that PINK1 protects against oxidative-stress-induced cell death by suppressing cytochrome c release from mitochondria. This protection depended on the phosphorylation of TRAP1 by PINK1. Furthermore, the ability of PINK1 to promote TRAP1 phosphorylation and cell survival was impaired by PD-linked PINK1 G309D, L347P, and W437X mutations.

1.6 Aim of study

Chapter 1 - Introduction 11

On the one hand, TRAP1 rescues the toxicity caused by α-SynA53T, a PD-linked mutant variant of α-Syn. On the other hand, TRAP1 is a substrate of a serine/threonine kinase PINK1, encoded by an autosomal recessive PD gene Pink1. Therefore, TRAP1 seems a crucial molecular factor of PD as it connects autosomal dominant PD-causal gene SCNA to autosomal recessive PD-causal gene Pink1.

In this study, I examined whether and how TRAP1 involved in the pathways/components relating to PINK1 or α-Syn. In addition, I analyzed Trap1 to address its molecular mechanism. Better understanding of TRAP1 is required to gain a more complete view of the mechanism of PD. Finally, I hope that my research will unravel new pathways to slow or even stop the progression of neuronal degeneration in PD.

Chapter 2 - Material and Methods 12

2 Material and Methods 2.1 Organism 2.1.1 Fly stocks The fly stocks in this study are from Bloomington Drosophila Stock Center (BL), Vienna Drosophila RNAi Center (VDRC) and National Institute of Genetics (Japan) (NIG-fly). Unless otherwise noted, flies were raised on standard cornmeal medium at 25º C . The fly stocks used in this study are listed in Table 2.

Table 2 List of used fly strains Symbol Genotype Detail Origin/Donor

Pink1B9 w*,Pink1B9/FM7i, P{w+mC.ActGFP} Pink1 loss-of-function BL34749 Park25 (y)w;;park25/TM3,Ser,Sb parkin loss-of-function Alex Whitworth Trap14 w;Trap14 / (CyO) Trap1 loss-of-function Miguel Martins y1w[67c23];P{y+mDint2wBR.E.BR= SUPor- Trap1KG Trap1 loss-of-function BL14032 P}Trap1KG06242 The Yeast mitochondrial Ndi1p w; P{w+mC=UAS-Ndi1p} Patrik Verstreken complex I Equivalent elav P{ w+mC.hs.GawB}elavC155 Pan-neuronal driver BL458 Da w*;;P{w+mW.hs.Gal4-da.G32} Ubiquitous driver BL5460 white-RNAi w[*];;P{GD14981}v30033 Invert repeats of white VDRC30033 w*;P{w+mC=UAS-Trap1.B}4M/TM3, Trap1-RNAi Invert repeats of Trap1 BL58766 Sb1 Pink1-RNAi w1118; P{GD11336}v21860/CyO Invert repeats of Pink1 VDRC21860 y[*],w[*];P(acman){w[+]=UAS- Bestgene, strains: hTRAP1/(CyO), hTrap1WT UAS-hTrap1WT 9723 (28E7) y[*],w[*];;P(acman){w[+]=UAS- 9732 (76A2) hTRAP1[WT]/(TM3, Sb) y[*],w[*];P(acman){w[+]=UAS- Bestgene, strains: hTRAP1/(CyO), hTrap1D158N UAS-hTrap1D158N 9723 (28E7) y[*],w[*];;P(acman){w[+]=UAS- 9732 (76A2) hTRAP1[D158N]/(TM3, Sb)

2.1.2 UAS-Gal4 System The UAS-Gal4 system is a widely used tool to express target gene(s) in specific tissues. Gal4 is a yeast transcriptional activator. A plethora of different Gal4-expressing lines (so called drivers) are available in public stock centers. Using this well characterized set of drivers, Gal4 expression can be restricted to virtually every cell type in a spaiotemporal manner. Upstream-activating sequence (UAS) are the the target of Gal4. A given sequence under

Chapter 2 - Material and Methods 13

control of UAS will only be expressed in presence of Gal4. Thus, by crosssing a specific Gal4 driver to transgenes carrying a UAS-transgene, expression of the UAS-controlled sequence mimicks Gal4 expression in the F1-generation. By choosing a suitable dirver, spatiotemporal expression of UAS-controlled sequences is facilitated (Figure 3). The Gal4 drivers used in this study are listed in Table 2.

Figure 3. An overview of the UAS/Gal4 expression system. Target gene follows UAS sequence and only express when Gal4 binds on UAS. Gal4 with a tissue specific expression enhancer expresses only in certain tissue. Thus, only in Gal4-driven tissue, Gal4 is expressed and binds to UAS, thus target gene is translated.

2.1.3 siRNA in fly The UAS/Gal4 system can also be used to knock down a specific gene in certain tissue by inducing transgenic RNAi. Instead of a target gene under UAS control, a short gene fragments (300-400bp) as inverted repeats (IR) in the antisense-sense orientation is inserted in a modified pUAST vector pMF3 and then transfected into fly. In Gal4 driven tissue, the IR is transcripted into hairpin RNAs (hpRNAs), which later is cleaved into siRNA by a dicer (Figure 4). Next, the siRNAs target at the endogenous mRNA of the target gene, and in this way the target mRNA is degraded and the expression of target gene is halted.

Chapter 2 - Material and Methods 14

Figure 4. Transgenic RNAi in Drosophila. The UAS/Gal4 system is used to drive the expression of a hairpin RNA (hpRNAs), which is coded by inverted repeat sequence. Dicer processes these double-stranded RNAs into siRNAs, which direct sequence-specific degradation of the target mRNA.

2.1.4 Transgenic flies Transgenic flies carrying UAS-hTRAP1 (wild-type and D158N) on second and third were generated by BestGene, Inc. using sitedirected integration (on second location 28E7, strain 9723;on third location 76A2, strain 9732) (Butler et al. 2012).

2.2 Chemicals, Enzymes, and Consumable Material

Table 3 Index of chemicals, enzymes, and consumable material Name Source & Number

Acetyl CoA(acetyl coenzyme A) lithium salt, C23H38N7O17P3S Li SIGMA, A2056-5MG

Acetic acid, CH3COOH MERCK, 1.00063.1000 LE Agarose PeQlab, 35-1020 Antimycin-A Fluka, 10792-5MG APS (ammonium peroxodisulfate) ROTH, 9592.2

ATP (Adenosine- 5'-triphosphate), C10H14N5O13P3Na2 SIGMA, A-7699-1G BSA (Bovine serum albumin) SIGMA, A9418-10G

Chloroform, CHCl3 ROTH, 3313.1 Complete™ Protease Inhibitors Roche, 11873580

DCIP (2,6-dichloroindophenolate hydrate), C12H7Cl2NO2•xH2O SIGMA, 119814-5G

Decyl-Ubiquinone, C19H30O4 SIGMA, D7911-10MG DEPC (Diethylpyrocarbonat) ROTH, K028.1

Chapter 2 - Material and Methods 15

DMSO, C2H6OS ROTH, A994.2 DNA ladder (100bp, 1kb), Fermentas, GeneRulerTM DNA loading Buffer 6 × Thermo, R0611 dNTPs (Deoxynuctleoside triphophates Mix 10 mM) Fermentas, R0193 DTNB -dithio-bis(2-nitrobenzoic acid; 3-carboxy-4- (5,5’ SIGMA, D-8130-5g nitrophenyl disulfide, Ellman’s rea ent), C14H8N2O8S2 EDTA (ethylene diamine tetraacetic acid), disodium salt, dehydrate, MERCK, 1.00944.1000 C10H14N2O8Na2•2H2O

EtOH (Ethanol), C2H5OH, ≥ 99.8% p.a. ROTH, 9065.4 EtBr (Ethidium bromide) ROTH, Art.2218.1 Fluoromount Southern Biotech, 0100-01

Glycine, H2NCH2COOH, ≥ 99% p.a. ROTH, 3908.2 Lithium chloride (LiCl) MERCK, Art. 5675

KAc (Potassium acetate), CH3COOK MERCK 1.04820.1000

KH2PO4 (Potassium dihydrogen phosphate) MERCK, 1.04873.1000

K2HPO4 (di-Potassium hydrogen phosphate) MERCK, 1.05099.1000

Isopropanol, (CH3)2CHOH ROTH, T910.1

ß-mercaptoethanol, C2H6OS ROTH, 4227.3

Methanol, CH3OH ROTH, 717.1

Methyl benzoate, C8H8O2 MERCK, 822330.1000 NaCl (Sodium chloride), ≥ 95.5% p.a. ROTH, P029.2

NADH (Nicotinamide adenine dinucleotide), C21H27N7O14P2Na2 Roche, 10128015001

Oxalacetic acid (oxobutanedioic acid), C4H4O5 SIGMA, O4126-1G PFA (Paraformaldehyde) ROTH, 0335.2 Proteinase K ROTH, 7588.1 RNase Inhibitor Fermentas, EO0381

Rotenone, C23H22O6 SIGMA, R8875-5G SDS (Sodium dodecyl sulphate, AccuGene), 10% Cambrex, 51213 Silk milk ROTH, T145.2

Succinate, C4H4O4Na2• 6H2O SIGMA, S-5047 1008 Sucrose ROTH, 4621.1 Sodium citrate MERCK, 1.06448.0500 Taq polymerase Genecraft, GC-002-1000

TEMED, C6H16N2, N,N,N’,N’‐Tetramethylethylendiamide Applichem, A1148,0100

Triethanolamine (2,2’,2’’-nitrilotriethanol), C6H15NO3 ROTH, 6300.1 Tris-Base, NH C(CH OH) , 2 2 3 (Tris(hydroxymethyl)aminomethane), ≥ ROTH, AE15.2 99.3%

Triton X-100, C34H62O11 ROTH, 3051.3

Chapter 2 - Material and Methods 16

Tween 20, C58H114O26 ROTH, 9127.1 TRIZOL-Reagent pepGOLD TriFast TM Ambion, 15596026

2.3 Buffers and Solutions

Table 4 Index of buffer and solutions Description Constitution Application

10mM Tris-HCl pH7.6 12.1 g/l Tris-HCl pH 7.6 H2O Mitochondria isolation mitochondrial complex I 0.25M pH 7.4 Potassium phosphate Buffer mitochondrial complex Incubation Buffer I activity measurement Bovine serum albumin (BSA) 3,5 g/l, DCIP 60 µM, Decyl- Ubichinon in DMSO, 70 µM, Antimycin-A in DMSO 1 µM,

0.1M, pH 7.4, 3.8 ml, 1M KH2PO4, 16.2 ml, 1M K HPO , 180 ml H O Potassium phosphate 2 4 2 Buffer (200 ml), DNA Extraction buffer 10 mM Tris-HCl pH 8.2, 1 mM EDTA, 25 Genomic DNA mM NaCl, 0.2 µg/µl Proteinase K isolation Drosophila Ringer 182 mM KCl, 46 mM NaCl, 3 mM CaCl2 x 2 H2O, 10 mM Tris pH 7.2 LiCl/KAc Solution 5M KAc: 6M LiCl, Volume 1 : 2.5 TE Buffer (1L) 10 ml 1 M Tris pH 8.0, 200 µl 0.5 M Na2EDTA pH 8.0, add H2O to 1 L

TAE Buffer (1L) 242 g Tris Base, 100 ml 0.5 M Na2EDTA, DNA, cDNA analysis pH 8.0, 57.1 ml glacial acetic acid, add H2O to 1 L Western Blot Running 0.1 M Tris, 1 M Glycine and 0.5% SDS Western blot Buffer Western Blot Semi-Dry 25 mM Tris, 192 mM Glycine, 20% Buffer Methanol SDS PAGE running Buffer 0.4% SDS, 1.5 M Tris, pH 8.8 SDS PAGE stacking Buffer 4% SDS, 0.25 M Tris, pH 6.8 Tris Buffered Saline (TBS) 25 mM Tris, 140 mM NaCl, pH 7.5 Buffer TBST Buffer 25 mM Tris, 140 mM NaCl, pH 7.5, 0.05% Tween Blocking Buffer 5% silk milk in TBST Laemmli buffer (SDS 1.25% Bromphenol Blue, 50% Glycerol, PAGE sample buffer) EDTA 10 mM, 10% SDS, 250 mM Tris, pH 6.8, ß- Mercaptoethanol 5%,

Chapter 2 - Material and Methods 17

Protein Extraction Buffer 70 mM HEPES, pH 7.5, 100 mM KCl, 10 Protein extraction RIPA mM EDTA, 70 mM ß –glycerophophate, 0.1 mM Na3NO4 pH 11, 5% glycerin, Triton X-100

2.4 Kits

Table 5 Index of used kits Description Application Origin

iQTM SYBR R Green Supermix Real-time PCR BIO-RAD, 170-8882 iScriptTM Select cDNA synthesis Kit Reverse Transcription BIO-RAD, 170-8897 Mitochondria Isolation Kit Mitochondria extraction Sigma, MITOISO1 ATP Bioluminescence Assay Kit HS II ATP content Roche, 11699709001 Qiagen RNeasy Mini Kit RNA purification Qiagen, 74106

2.5 Equipments

Table 6 Index of used equipments Description Application SZX10 with ring light S80-55 RL and camera SC30, Dissecting microscope Olympus, Germany Documentation microscope BX51, Olympus, Germany Camera for microscopy documentation DP72, Olympus, Germany UV-light source X-Cite® 120 Q, Olympus, Germany Homogenisator Speedmill P12 Analytik Jena AG, Germany PCR Cycler T Professional Basic, Biometra Germany Western blot documentation Alliance LD4.777.WL.Auto, Biometra, Germany UV documentation UV Solo TS, Biometra, Germany UV Transilluminator UVStar 20, Biometra, Germany Scanning electron microscope ESEM XL 30 FEG, FEI, Netherlands Photometer Plate reader infinite M200, Tecan, Switzerland Real-time PCR Cycler BIO-RAD, MyiQTM2 Two color Detection System

2.6 Fly behaviors/ phenotype assays 2.6.1 Wing posture & Thorax indentation Wing posture and thorax indentation were assessed by visual inspection, and the presence of abnormal wing posture and indentations was scored regardless of severity or number. Both

Chapter 2 - Material and Methods 18

assays were scored 5 days post eclosion (d.p.e.) with flies raised at 25º C and shifted to 29º C after eclosion. In case of pan-neural (elav-Gal4 driver) induction of RNAi, wing posture defects were scored 20 d.p.e. At least 100 flies of each genotype were tested.

2.6.2 Negative geotaxis Negative geotaxis (climbing) analysis was performed 5 d.p.e. with flies raised at 25ºC and shifted to 29ºC after eclosion. Groups of 10 flies per vial (2.5 cm diameter) were gently tapped to the bottom and the number of flies crossing a line at 8 cm height within a time period of 10 s was scored. Each analysis was repeated 10 times with 60 seconds resting interval. The experiments were always conducted at the same time period of a day (between 10-11 am) to avoid the influence of circadian rhythm.

2.6.3 Longevity Freshly hatched male flies of designed genotype were selected according to the relative phenotype of corresponding genotype. Flies were maintained in vials and transferred to new vials every 2 days and counted every day. 10 flies were maintained in each vial.

For stress study, 1 ml of 1% H2O2 in 5% sucrose or 5 mM Rotenone in 5% sucrose was added to 3 pieces of round filter paper which were located at the bottom of the vial as food source. Several drops of fresh 1% H2O2 in 5% sucrose or 5 mM Rotenone in 5% sucrose were added each day. Dead flies were counted every day.

2.7 Mitochondrial analysis 2.7.1 ATP content ATP Bioluminescence Assay Kit HS II from Roche company was applied. The luciferase from Photinus pyralis (American firefly) in the kit catalyzes the following reaction:

ATP + D-luciferin + O2→ oxyluciferin + PPi + AMP + CO2 + light

The quantum yield for this reaction is about 90%. The resulting green light has an emission maximum at 562 nm. The Michaelis equation has the following form:

light intensity = (Vmax × CATP)/(Km + CATP)

Chapter 2 - Material and Methods 19

At low ATP concentrations (CATPm), the formula is simplified to light intensity = Vmax ×

CATP/Km. From this equation, it becomes obvious that the light output is directly proportional to the ATP concentration (CATP), and is dependent on the amount of luciferase (Vmax) present in the assay. Therefore, for maximum sensitivity, the sample ATP must be in a minimum volume, and the luciferase reagent must not be diluted.

Figure 5. The principle of ATP measurement. ATP is catalyted by D-luciferin with the help of oxygen, to produce AMP, PPi, CO2, oyxluciferin and light with an emission maximum at 562 nm. Then the emission can be read out by a photometer reader.

Standard line was generated by four standard samples with ATP content of 10-8, 10-9, 10-10, 10-11 mole. The readout of the sample was accorded to the standard line to find out the according ATP content.

6,776036343 7 Bioluminescene log10 6,5 5,816229033 6 5,5 4,842184721 5 4,5 3,862787098 y = 0,9714x + 14,552 4 3,5 -11 -10,5 -10 -9,5 -9 -8,5 -8 log10 ATP mole Figure 6. The ATP standard line for fly ATP assay. Standard line was created by samples with ATP content of 10-8, 10-9, 10-10, 10-11 moles. Then, the readout of the sample was accorded to the standard line to caculate the according ATP content.

For flies, as described in Song Liu et al. 2010, 2 thoraxes of flies were dissected in lysis buffer on ice and immediately homogenized in 100 μl lysis buffer for 1 min 30 sed. The

Chapter 2 - Material and Methods 20

sample was boiled at 95°C for 5 min before centrifuged at 4°C for 1 min with max speed. 2.5 μl clear lysis was added to 187.5 μl dilution buffer from the kit. 10 μl luciferase was added shortly before the measurement.

2.7.2 Mitochondrial complex I activity analysis Mitochondria Isolation Kit (Sigma) was applied for mitochondrial isolation from the whole- amount fly. Intact mitochondria were isolated from 30 fresh flies according to the manufacturer’s instructions. The final mitochondrial pellet was re-suspended in 60μl 10mM Tris-HCl buffer, pH 7.6 and diluted to 1:10 in 10 mM Tris-HCl pH 7.6.

Mitochondrial complex I (NADH dehydrogenase) is an enzyme complex, locating in the MIM. Mitochondrial complex I catalyzes the transfer of electrons from NADH to coenzyme Q (CoQ).

+ + + + NADH + H + CoQ + 4H in → NAD + CoQH2 + 4H out

Mitochondrial complex I oxidizes NADH and the electrons produced reduce the artificial substrate decyl-ubiquinone that subsequently delivers the electrons to 2,6- dichloroindophenolate (DCIP), the terminal acceptor (Figure 7). The reduction of DCIP is followed spectrophotometrically at 600 nm. Since the electrons produced by other NADH- dehydrogenases are not accepted by decyl-ubiquinone, only mitochondrial complex I activity contributes the reduction of DCIP.

Figure 7. DCIP accepts electrons and becomes reduced-DCIP. The reduction of DCIP can be followed spectrophotometrically at 600 nm. The electrons accepted by DCIP come from mitochondrial complex I, but not other NADH-dehydrogenases.

Mitochondrial complex I activity in fly was analyzed using 2 μl from mitochondrial extraction, 4 μl of 10mM NADH and 194 μl of freshly made incubation buffer. This solution was incubated for 1 min and measured at 37°C every 30 s for 10 min at 600 nm wavelength. Activity of the mitochondrial complex I is expressed per milligram protein (mU/mg protein):

Chapter 2 - Material and Methods 21

mU: activity of the enzyme is expressed per mg protein (mU/mg protein) Net A: rate of absorbance change (min-1) -1 -1 εNADH: extinction coefficient of NADH at 600 nm and pH 8.1 (= 0.0191 mM •cm ) d: dilution factor of the solution (= 100)

Mitochondrial complex I activity was normalized to protein concentration of lysates (measured using DC Protein Assay, Bio-Rad). Each lysate was measured in triplicate. Results from triplicate measurements were depicted as fold change compared to control genotype. The number of independent repetitions was n ≥ 5 per enotype.

2.7.3 Mitochondrial DNA level analysis Total (nuclear and mitochondrial) DNA was extracted from 30 anesthetized flies. Quantitative polymerase chain reaction (qPCR) was performed to determine mtDNA content via iQTM SYBR Green Supermix Kit (BIO-RAD).

For each sample (15 μl): 1 μl cDNA 1 μl for primer 1 μl rev primer 12 μl SYBR Green Mix

Program: Initialization (activation of HotStar Taq): 15 min 95°C Denaturation: 95°C, 15 s Annealing: 50-60°C, 30s 40 cycles Extension: 72°C, 30s Final extension: 72°C, 10min

Description Sequence 5’→3’ Application nCOX5A-206Fw TATGAACGATCTGGTGGGCATGGA 5’ primer for enomic DNA nCOX5A-322Rv CAAATAGGGATAGAGGGTGGCCTT 3’ primer for enomic DNA mtCOI-375Fw AACTGTTTACCCACCTTTATCTGCTG 5’ primer for mtDNA mtCOI-458Rv CCCGCTAAGTGTAAAGAAAAAATAGC 3’ primer for mtDNA

Chapter 2 - Material and Methods 22

The relative quantification of the mtDNA was performed using Ct values. Ct is the number of cycles that it takes each reaction to reach an arbitrary amount of fluorescence. The mean value of Ct is calculated from the three parallel samples as for both control gene (express constant, equation [1]) and interested gene (equation [2]). Then the difference in Ct values for the gene of interested and the endogenous control is calculated as Ct. These values should remain constant for sample genotype and I have expressed my results comparing Ct values. △ △

2.7.4 Mitochondrial morphological analysis Thoraces were prepared from 5-day-old adult flies (raised at 25°C and adults shifted to 29°C and treated as previously described. Semi-thin sections were stained with Toluidine blue, whereas ultra-thin sections were examined using a transmission electron microscope (FEI tecnai G2 Spirit, 120 kV). Transmission electron microscopy, FEI tecnai G2 Spirit, 120 kV.

2.8 Other assays 2.8.1 Analysis of DNA DNA Extraction 20-30 adult flies were homogenized in 500 μl DNA extraction buffer plus 10 µl 10mg/ml Proteinase K, and then incubated for 2 h at 56°C. Afterwards, supernatant was kept and centrifuged for 10 min at 12000 rpm at 4°C. Supernatant was collected. 2.5 μl RNAase A (10mg/ml) was added. The solution was incubated for 20 min at 50°C. Next, 500 μl Phenol/Chloroform/Isoamylalcohol (25/24/1) was added and centrifuged for 5 min at 2000- 3000 rpm. The upper phase was kept and Phenol/Chloroform separation was repeated once more. Then, 1 ml NaAcetat/Isopropanol (1/19) was added to the upper phase. Afterwards, the solution was centrifuged for 10 min at 12000 rpm at 4°C. The supernatant was discarded and the pellet was kept and washed with cold 75% Ethanol. The solution was centrifuged for 10 min at 12000 rpm at 4°C. Ethanol was removed and DNA pellet was dissolved in TE buffer.

2.8.2 Analysis of RNA

Chapter 2 - Material and Methods 23

RNA Extraction 20 whole flies were homogenized in 100 µl Trizol buffer via speedmill and then 50 µl chloroform was added to the lysate. After vortexing for 15 s and incubation on ice for 5 min, the samples were centrifuged at 4°C at 17.5 g speed for 15 s and the supernatant was transfered into 100 µl 70% ethanol. Next, RNA was purified by Qiagen RNAeasy Kit. RNA sample was disovled in RNAase free water and stored at -80°C.

Real-time PCR 1 µg of extracted RNA reversely transcribes into cDNA via Iscript cDNA synthesis Kit followin the manufacturer’s instructions. RNA expression was normalized with respect to endogenous reference genes: human b-actin; Drosophila ribosomal protein 49 (rp49). Relative expression was calculated for each gene using the delta delta cycle threshold method.

For each sample (15 μl): 1 μl cDNA 1 μl for primer 1 μl rev primer 12 μl SYBR Green Mix

Program: Initialization( activation of HotStar Taq): 5 min 95°C Denaturation: 94°C, 15 s Annealing: 50-60°C, 30s 40 cycles Extension: 72°C, 30s Final extension: 72°C, 10min

Oligo nucleotides (primers) The following forward (for) and reverse (rev) primers were used to analyze mRNA abundance of respective human genes: Table 7 Index of oligo nucleotides Description Sequence 5’→3’

b-actin for TGGACTTCGAGCAAGAGA b-actin rev AGGAAGGAAGGCTGGAAGAG parkin for CGA CCC TCA ACT TGG CTA CT

Chapter 2 - Material and Methods 24

parkin rev GAC ACA CTC CTC TGC ACC ATA C Pink1 for CCA ACA GGC TCA CAG AGA AG Pink1 rev AGC GTT TCA CAC TCC AGG TT rp49 for TCG GAT CGA TAT GCT AAG CTG TCG CAC rp49 rev AGG CGA CCG TTG GGG TTG GTG AG dTrap1 for AGG CAG AGT CAC CGA TCC dTrap1 rev TGA TGC CTG CTT GGT CTC hTrap1 for TCG CTG GAA AAC TCC TTG hTrap1 rev GAG GAC ATT CCC CTG AAC CT hTrap1 for and rev were from metabion. The rest were from InvitrogenTM.

The relative quantification of the cDNA was performed using 2- Ct values. △△

2.8.3 Analysis of protein Preparation of protein lysate for western blot 5 flies of interest were homogenized in 100 μl radio immunoprecipitation assay (RIPA) buffer by Speedmill P12 Analytik Jena AG. Lysates of homogenized heads were centrifuged at 13 krpm for 20 min at 4°C. Supernatant was collected and stored at -20°C. 12 μl of lysate was incubated at 95° C for 5 min after added with 3 μl 5x Laemmli lysis buffer.

SDS Polyacrylamide Gel Electrophoresis and Western Blot SDS Polyacrylamid Gel Eletrophoresis: 10% Polyacrylamide gel electrophoresis (PAGE) running gel for complex I unit NDUFS3 was prepared. Samples were run on the PAGE gel it at 110v for approximately 1-2 h.

Blotting: Prepare transfer membrane by soaking in Semi-Dry Buffer for 5 min. Wet four pieces of filter paper in Semi-Dry Buffer, as well. Place 2 filter papers on cathode plate of blotter one by one. Then place membrane on top of filter paper stack and gel on top of it. Apply a constant current of 225 mA for one membrane for 1 h.

Blocking: After transfer is complete, transfer membrane was incubated in Blocking Solution at RT for 2 hours.

Chapter 2 - Material and Methods 25

1st/2nd Antibodies: Then the membrane was incubated overnight at 4°C in primary antibody reagent. After three washes at RT for 15 min with TBST, the membrane was incubated at RT for 3 hours in an appropriate secondary antibody reagent. The membrane was washed afterwards at RT for 15 min three times with TBST and was incubated with Immune-Startm chemiluminescent reagent (WesternCTM kit, BioRad, USA). Documentation of chemiluminescent signals was achieved using Alliance LD4 documentation system.

Data Analysis: A reference protein should always be applied. In my case, syntaxin functions as the reference protein, which is expressed constantly and remains similar expression amount among different genotypes and different groups of age.

Antibodies Table 8 Index of used antibodies Antibody Dilution Animal Weight Source Application BD Transduction Anti-TRAP1/Hsp75 1:1000 mouse 75kD laboratoriesTM Primary Anti-syntaxin 1:2000 mouse 35kD DSHB antibody for western blot anti-NDUFS3 1:1000 mouse 25kD Abcam, ab14711 Secondary Anti-mouse 1:10000 sheep - GE Healthcare antibody for western blot

2.9 Statistical analysis

Experimental data were plotted and statistically analyzed by GraphPad Prism software. Statistical test for each experiment is described in the respective results section.

Chapter 3 - Results 26

3 Results 3.1 Trap1 functions downstream of Pink1

Transgenic fly lines UAS-hTrap1WT (wild-type hTrap1 variant) and UAS-hTrap1D158N (a point mutation in one ATP-binding site) were generated (Butler et al. 2012). Flies expressing hTrap1D158N did not show any protective effect against α-SynA53T-induced-toxicity as flies expressing hTrap1WT did (Butler et al. 2012). Therefore, hTrap1D158N is believed to be an inactive TRAP1 variant and was used as a control in this study.

To exclude the possibility that quantitative inequality might account for potential differences in my assays, the expression levels of the hTRAP1 were measured in both hTrap1WT and hTrap1D158N expressing flies. The RNA expression levels of Trap1 were detected by real-time PCR. Flies with ubiquitous expression of hTrap1WT and hTrap1D158N driven by DaG-Gal4 showed similar levels of hTrap1 mRNA, while the control flies display no hTrap1 mRNA (Figure 8A). To rule out the effect of exogenous hTrap1 on endogenous Drosophila Trap1 (dTrap1), the mRNA levels of endogenous dTrap1 were also tested. The mRNA levels of dTrap1 did not vary among flies expressing hTrap1WT and hTrap1D158N and the control fly lines (Figure 8A). In addition, protein levels of hTRAP1 were detected via Western blot. Flies with ubiquitous expression (DaG-Gal4) of hTrap1WT and hTrap1D158N displayed very similar/almost identical levels of hTRAP1 protein (Figure 8B). In summary, hTrap1WT and hTrap1D158N transgenic flies, driven by Gal4 driver, express almost identical amounts of hTRAP1.

Flies with the amorphic Pink1B9 allele were chosen for rescue experiments. The Pink1 gene is localized on the X in flies. Accordingly, in this study, hemizygous Pink1B9 flies (Pink1B9/Y;;DaG-Gal4/+) were analyzed as Pink1 deficient (Pink1B9 in the text), while heterozygous Pink1B9 females (Pink1B9/+;;DaG-Gal4/+) served as control. Pink1B9 flies were originally generated and described by Jeehye Park and co-workers. These flies display a variety of well characterized phenotypes, including abnormal wing posture, collapsed thoraxes, disturbed climbing ability, lack of flight ability, dysmorphic and dysfunctional mitochondria (reduced ATP levels, low protein levels of the mitochondrial complex I subunit NDUFS3 and loss of mtDNA content), loss of dopamine content and loss of dopaminergic neurons (Park et al. 2006). To analyze whether there is a genetic interaction between Trap1 and Pink1, the two human Trap1 variants, hTrap1WT and hTrap1D158N, were expressed in a

Chapter 3 - Results 27

Pink1B9 mutant background. In the F1 generation, I asked the question whether TRAP1 was able to mitigated Pink1B9 phenotypes.

Figure 8. Expression levels of Trap1 in hTrap1 expressing flies. (A) Relative mRNA levels of hTrap1 and dTrap1 in hTrap1 expressing flies. RNA was isolated from the entire flies and was reverse transcripted into cDNA. By applying real-time PCR, the mRNA levels of Trap1 were determined. The mRNA levels of dTrap1 in control flies (DaG-Gal4) were set for normalization. The mRNA levels of hTrap1 (white bars) were similar between flies expressing hTrap1WT and hTrap1D158N. Control flies (DaG-Gal4) exhibited no hTrap1 mRNA. Endogenous dTrap1 mRNA levels (black bars) did not change according to expression of hTrap1. (B) Protein levels of hTRAP1 in hTrap1 expressing flies. Protein was extracted from the whole flies and the protein levels of TRAP1 were detected by Western blot. Flies expressing hTrap1WT and hTrap1D158N presented similar levels of hTRAP1. Syntaxin was used as loading control.

3.1.1 Trap1 recues phenotypes caused by Pink1 loss-of-function in flies The most obvious phenotype of Pink1B9 flies, visible even without microscopy, is the abnormal wing posture. Compared to wild type flies, most Pink1B9 flies showed either dropped or up-held wing posture (Figure 9A), as these flies were not able to post the wings flat on their back. Quantification revealed that only 60% of 5-day-old Pink1B9 flies presented normal wing posture. In contrast about 80% of Pink1B9 flies expressing hTrap1WT displayed normal wing posture (Figure 9B). However, when hTrap1D158N was expressed, the rescuing effect was vanished. This indicates that TRAP1 rescued Pink1 loss-of-function induced abnormal wing posture. The fact that the ATP-binding deficient variant TRAP1D158N did not provide any rescue activity suggests that ATP-binding and/or that the ATPase activity of TRAP1 was required for the observed rescuing effect.

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Figure 9. Pink1B9 flies with Trap1 expression regained normal wing posture. (A) Wing posture phenotype. Healthy/normal flies position their wings flat on the back. In contrast, Pink1B9 flies display either a dropped or an up-held wing posture. (B) Quantification of wing posture phenotype. All control flies showed normal wing posture (green bar), but only 60% of Pink1B9 flies presented normal wing posture (red bar). This abnormal wing posture phenotype was rescued by ubiquitous expression of hTrap1WT (yellow bar). However, ubiquitous expression of hTrap1D158N did not affect the abnormal wing posture induced by Pink1 loss-of-function (orange bar). n > 200 flies per genotype. Fly age: 5 days. Flies were raised at 29°C. One-way ANOVA followed by the Neuman–Keuls multiple comparison test was used to determine significance. *** P < 0.001; ** P < 0.01; ns, not significant.

Chapter 3 - Results 29

Next I asked whether neuronal knock-down of Pink1 by RNAi might also cause abnormal wing posture. In a first step, I analyzed the efficiency of the Pink1-RNAi transgene. 90% of the mRNA level of Pink1 was abolished in flies with ubiquitous (DaG-Gal4) expression of Pink1-RNAi (Figure 10A). Thus, Pink1-RNAi is very efficient to achieve knocking down Pink1. Next, I silenced Pink1 in all fly neurons using the pan neural elav-Gal4. Abnormal wing posture phenotype was detected in elav>Pink1-RNAi flies at 20 days after eclosion. This suggests that low expression of Pink1 in neurons is detrimental as well. Also in this case, neuronal expression of hTrap1WT mitigated the abnormal wing posture caused by neuronal loss of Pink1. Pan neural expression of an unrelated white-RNAi did not result in any abnormal wing posture (Figure 10B).

Figure 10. Trap1 rescued the abnormal wing posture caused by neuronal loss of Pink1. (A) Efficiency of Pink1-RNAi. The mRNA level of Pink1 in DaG-Gal4>Pink1-RNAi flies was 5-10% of driver only control flies (DaG-Gal4). (B) Neuronal Pink1-knock-down flies expressing Trap1 regained the normal wing posture. The flies with neuronal knock-down of Pink1 (elav- Gal4>Pink1-RNAi) displayed abnormal wing posture (red bar), while the control flies (elav- Gal4>white-RNAi) presented normal wing posture (green bar). Flies with neuronal knock-down of Pink1 and expression of Trap1 (elav>Pink1-RNAi,hTrap1) regained normal wing posture (yellow bar). n > 200 per genotype. Fly age, 20 days. One-way ANOVA followed by the Neuman–Keuls multiple comparison test was used to determine significance. ** P < 0.01; ns, not significant.

My data strongly suggest that the abnormal wing posture phenotype induced by Pink1 loss- of-function can be rescued by TRAP1. This implies that TRAP1 acts downstream of PINK1. To test this hypothesis, I asked whether TRAP1 also rescues other Pink1B9 phenotypes.

Chapter 3 - Results 30

Thorax-indentation is another phenotype of Pink1B9 flies (Figure 11A), which was detected in 90% of Pink1B9 flies, but not in control flies. Less Pink1B9 flies with ubiquitous expression of hTrap1WT displayed abnormal thoraxes, compared to Pink1B9 flies (Figure 11B). As in previous analysis, expression of hTrap1D158N did not show any rescuing effects on this phenotype.

Negative geotaxis is an indicator of locomotor activity in Drosophila (Rhodenizer et al. 2008). It has been shown that Pink1B9 flies display disturbed locomotor ability in negative geotaxis (Park et al. 2006, Imai et al. 2010). Compared to controls, Pink1B9 flies display a strong reduction in locomotor activity. In contrast, Pink1B9 flies with expression of hTrap1WT, but not hTrap1D158N, performed significantly better in climbing analysis as compared to Pink1B9 flies (Figure 11C).

Moreover, Pink1B9 flies have also been reported to be unable to fly. Assaying the flight ability, I could confirm that more than 80% of Pink1B9 flies lost their flight ability, similar as described in Park et al. 2006. In contrast to previous findings, neither expression of hTrap1WT nor Trap1D158N caused a regain of flight ability (Figure 11D).

Abnormal wing posture, disturbed locomotor ability and loss of flight ability of Pink1B9 flies were ascribed to muscle degeneration (Park et al. 2006, Imai et al. 2010). Flies control wings by the indirect flight muscles (Figure 12A). Upward movement of the wings results indirectly from the contraction of vertical muscles within the thorax, depressing the notum (upper surface of fly thorax). Downward movement of the wings is produced indirectly by the contraction of longitudinal muscles raising the notum. Degeneration of indirect flight muscles is believed to be the reason for disability of flight and abnormal wing posture in Pink1B9 flies.

To visualize fly muscles, semi-thin transverse sections of the thorax and subsequent Toluidine blue staining were conducted. In the indirect flight muscles of Pink1B9 flies, clear vacuolization and disorganized appearing muscle fibers were observed. In contrast, Pink1B9 flies with ubiquitous expression of hTrap1WT presented well-organized muscle fibers without vacuolization, as in control flies. As for wing posture and locomotor activity, expression of hTrap1D158N was not able to rescue the muscle degeneration in Pink1B9 flies (Figure 12B).

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Figure 11. Expression of Trap1 mitigates Pink1 loss-of-function phenotypes. (A) Thorax-indentation phenotype. Wild-type flies (left) have a roundish and smooth edge on the anterior side of the dorsal surface of thorax, the so-called notum (the red arrow pointed). Pink1B9 flies (right) frequently display dints at this position of thorax (red arrow), referred as indentation. (B) Quantification of indentation phenotype. The plotted indentation index reflects the percentage of the flies showing normal thoraxes (without indentation). Pink1B9 flies showed strongest indentation phenotype (red bar). On the other hand, control flies had no indentation phenotype at all (green bar). Pink1B9 flies expressing hTrap1WT suppressed indentation (yellow bar), while expression of hTrap1D158N did not (orange bar). n=100 per genotype. Absolute values were depicted. (C) Negative geotaxis. To study the locomotor activity of flies, negative geotaxis (climbing ability) was measured. 50% of Pink1B9 flies (red bar) failed to achieve the task (8cm within 10s), while 80% of the control flies (green bar) were capable. Expression of hTrap1WT (yellow bar), but not hTrap1D158N (orange bar), in Pink1B9 flies rescued the disturbed climbing ability. (D) Flight ability. Flight index equaled the average percentage of flies that were able to escape from the vial within certain time. Less than 20% of Pink1B9 flies (red bar) escaped. A similar flight index was observed in Pink1B9 flies expressing either hTrap1WT (yellow bar) or hTrap1D158N (orange bar). In contrast, 60% of control flies (green bar) successfully escaped. (C) and (D), n > 200 flies per genotype. Fly age: 5 days. Flies were raised at 29°C. Original data of (B) and (D) were provided by Alexander J. Whitworth. One-way ANOVA followed by the Neuman–Keuls multiple comparison test was used to determine significance. *** P < 0.001; * P < 0.05; ns, not significant.

Chapter 3 - Results 32

In summary, hTrap1WT rescued phenotypes induced by Pink1 loss-of-function, including wing posture, thoracic indentation, climbing ability and muscle degeneration. Interestingly, hTrap1D158N, hTrap1 with one point mutation in one of four ATP binding sites, did not show any rescuing effect. This is in line with my hypothesis that Trap1 functions downstream of Pink1. Moreover, the ATP-binding and most likely subsequent ATP lysis by the ATPase activity of TRAP1 is required for the rescuing function.

Figure 12. Expression of Trap1 rescued the degeneration of indirect flight muscles (longitudinal muscles) in Pink1B9 flies. (A) The schematic presentation of wing/flight control by indirect flight muscles. Flies control the upward movement of wings by contracting the longitudinal indirect flight muscles. By contracting the vertical indirect flight muscles, flies control the downward movement of wings. These muscle contraction cause a deformation of the thorax and are not directly attached to the wings. Accordingly, the two groups of muscles are considered indirect flight muscles. Picture is adept from the Amateur Entomologists' Society. (B) First row, lateral sections of indirect flight muscles. Second row, dorsal/ventral sections of indirect flight muscles. Muscle fibers were well organized without vacuolization in control flies (Pink1B9/+;;DaG). In contrast, muscle fibers appeared disorganized with vacuoles in Pink1B9 flies (Pink1B9/Y;;DaG). Pink1B9 flies with ubiquitous expression of hTrap1WT, but not hTrap1D158N, regained the healthy pattern of indirect flight muscles. Semi-thin transverse sections of muscle with Toluidine blue staining. Fly age: 5 days. Flies were raised at 29°C.

Chapter 3 - Results 33

3.1.2 Expression of Trap1 mitigates mitochondrial morphology and function in Pink1 loss-of-function flies Mitochondrial dysmorphology To further explore the mechanism of muscle degeneration in Pink1B9 flies, electron microscopy was applied to visualize the muscles in detail. Pink1 B9 flies presented swollen mitochondria with fragmented cristae in indirect flight muscles. In contrast, mitochondria in control flies showed normal size and intact, densely packed cristae. Pink1B9 flies with ubiquitous expression of hTrap1WT displayed healthy mitochondria as in control flies. Expression of hTrap1D158N did not provide a rescue of altered mitochondrial morphology observed in Pink1B9 flies (Figure 13). This suggests that expression of Trap1 restores the morphology of mitochondria in indirect flight muscles in Pink1 loss-of-function flies.

Figure 13. Expression of Trap1 improved mitochondrial morphology in indirect flight muscles of Pink1B9 flies. In control flies (Pink1B9/+;;DaG), mitochondria (white arrow) appeared healthy, were of normal size, displayed densely packed cristae and showed no signs of vacuolization. In contrast, in Pink1B9 flies (Pink1B9/Y;;DaG), mitochondria were enlarged and had fragmented cristae. Ubiquitous expression of hTrap1WT, but not hTrap1D158N, in Pink1B9 flies caused a regained of the healthy morphology of mitochondria in indirect flight muscles. Second row shows a magnification of boxed areas in the first row. White asterisk mark muscle fibers.

Chapter 3 - Results 34

Original pictures were provided by Alexander J. Whitworth. Fly age: 5 days. Flies were raised at 29°C.

Mitochondrial dysmorphology usually reflects mitochondrial dysfunction. It has been reported that Pink1 loss-of-function flies represent signs of mitochondrial dysfunction, such as low levels of ATP, reduced mtDNA content and impaired mitochondrial electron transport chain (ETC) (Park et al. 2006, Clark et al. 2006, Vilain et al. 2012). Trap1 rescues mitochondrial dysfunction in Pink1 loss-of-function flies. Thus, I examined whether Trap1 also rescued mitochondrial dysfunction in Pink1B9 flies, by determining ATP levels, mtDNA content and mitochondrial ETC activity.

ATP levels The main function of mitochondria is to produce energy (ATP) to support cells. Therefore, the ATP level is an indicator of mitochondrial function. Low levels of ATP in thoraxes of Pink1 loss-of-function flies were already described (Park et al. 2006, Clark et al. 2006). I confirmed decreased ATP levels in thoraxes of Pink1B9 flies. As expected from my previous analysis (see Fig. 13), ATP levels were partly restored when hTrap1WT, but not hTrap1D158N, was ubiquitously expressed in Pink1B9 flies ( Figure 14A). The increased ATP levels and recovered mitochondrial morphology after TRAP1WT expression strongly imply a recovered mitochondrial function.

Elevated TRAP1 levels might increase ATP levels per se. In this case, increased ATP upon TRAP1WT-expression in Pink1B9 flies would be caused by an indirect effect. To test for this possibility, ATP levels were measured in flies expressing either hTrap1WT or hTrap1D158N ubiquitously (DaG-Gal4). Compared to controls, there was no significant difference in hTrap1WT or hTrap1D158N expressing flies ( Figure 14B). This means that expression of hTrap1 does not simply increase the ATP levels, but compensates the decreased ATP production caused by Pink1 loss-of-function.

Chapter 3 - Results 35

Figure 14. Expression of Trap1 restored ATP levels in thoraxes of Pink1B9 flies. (A) Compared to the control flies, Pink1B9 flies displayed low levels of the ATP. Ubiquitous expression of hTrap1WT, but not hTrap1D158N, in Pink1B9 flies restored the ATP levels. (B) Ubiquitous expressing either hTrap1WT or hTrap1D158N did not change ATP levels in thoraxes of flies. Fly age: 5 days. Flies were raised at 29°C. One-way ANOVA followed by the Neuman–Keuls multiple comparison test was used to determine significance. *** P < 0.001; * P < 0.05; ns, not significant.

Mitochondrial DNA The amount of mtDNA is regarded as an indicator of mitochondrial function. It is linked to abundance and fitness of the mitochondrial population. Declined mtDNA levels in Pink1B9 flies were reported (Park et al. 2006). The index of mtDNA level equals the amount of mitochondrial encoded cytochrome c oxidase I (COI) divided by the amount of nuclearly encoded cytochrome c oxidase X (COX). Pink1B9 flies presented very low levels of mtDNA. However, the mtDNA levels were restored by expression of hTrap1WT. On the contrary, expression of hTrap1D158N did not elevate the reduced content of mtDNA in Pink1B9 flies (Figure 15).

Chapter 3 - Results 36

Figure 15. Expression of hTrap1 restored mtDNA levels in Pink1B9 flies. mtDNA levels were reduced in Pink1B9 flies. Ubiquitous expression of hTrap1WT, but not hTrap1D158N, increased mtDNA levels in Pink1 loss-of-function flies. Fly age: 5 days. Flies were raised at 29°C. One-way ANOVA followed by the Neuman–Keuls multiple comparison test was used to determine significance. * P < 0.05; ns, not significant.

Mitochondrial Complex I The ETC is a series of protein complexes that create an electrochemical proton gradient that drives ATP synthesis. Declined mitochondrial complex I activity has been shown in Pink1 loss-of-function flies (Park et al. 2006,Vilain et al. 2012). Accordingly, I asked whether Trap1 is able to restore mitochondrial complex I activity. Thus I measured mitochondrial complex I activity in Pink1B9 flies with and without concomitant expression of Trap1.

In Pink1B9 flies, mitochondrial complex I activity was reduced compared to the control flies. However, Pink1B9 flies with ubiquitous expression of hTrap1WT regained the mitochondrial complex I activity almost to the control levels. In contrast, Pink1B9 flies with expression of hTrap1D158N presented similar mitochondrial complex I activity to Pink1B9 flies (Figure 16A).

Chapter 3 - Results 37

Figure 16. hTrap1 elevated mitochondrial complex I activity and the levels of NDUFS3 in Pink1B9 flies. (A) Plotted is mitochondrial complex I activity relative to control flies (Pink1B9/+;;DaG/+). Pink1B9 flies (Pink1B9/Y;;DaG/+) showed decreased mitochondrial complex I activity. Ubiquitous expression of hTrap1WT, but not hTrap1D158N, rescued mitochondrial complex I activity in Pink1B9 flies (8 measurements per genotype). (B) Compared to controls, Pink1B9 flies displayed a reduced abundance of NDUFS3. Pink1B9 flies with ubiquitous expression of hTrap1WT, but not hTrap1D158N, increased NDUFS3 almost to the control level. Fly age: 5 days. Flies were raised at 29°C. One-way ANOVA followed by the Neuman–Keuls multiple comparison test was used to determine significance. *** P < 0.001; ** P < 0.01; * P < 0.05; ns, not significant.

To investigate mitochondrial complex I protein abundance, the levels of one nuclear encoded mitochondrial complex I subunit, NADH dehydrogenase [ubiquinone] iron-sulfur protein 3 (NDUFS3) was assayed in Western blot. Compared to control, NDUFS3 levels were reduced in Pink1B9 flies, In contrast, Pink1B9 flies with ubiquitous expression of hTrap1WT displayed similar levels of NDUFS3 as control (Figure 16B). In agreement with the previous analyses, hTrap1D158N had no beneficial effect.

Male fertility Proper mitochondrial function is required for spermatogenesis. Male sterility due to impaired spermatogenesis and swollen nebenkern (a special mitochondrial formation in Drosophila spermatids) has been reported in Pink1B9 flies (Park et al. 2006). In my study, single Pink1B9 male flies (20 days old) were crossed with female virgins (Pink1B9/FM7, GFP) to check whether the male was fertile or sterile. Only 50% of Pink1B9 male flies were fertile. However, 80% of Pink1B9 male flies with expression of hTrap1WT were fertile (Figure 17). In contrast, Pink1B9 male flies expressing hTrap1D158N performed similar to Pink1B9 male flies. Therefore

Chapter 3 - Results 38

expression of hTrap1 also rescues the partial sterility of Pink1B9 male flies. Altogether, all my data strengthen the idea that Trap1 rescues mitochondrial dysfunction induced by Pink1 loss- of-function.

Figure 17. Trap1 rescues the sterility of Pink1 mutant male flies. Single Pink1B9 male fly at the same age (20 days old) was crossed with female virgins (Pink1B9/FM7, GFP). About 50% of Pink1B9 male flies were fertile (red bar) while roughly 80% of Pink1B9 male flies with expression of hTrap1WT were fertile (yellow bar). Expression of hTrap1D158N did not influence the sterility of Pink1B9 males. One-way ANOVA followed by the Neuman–Keuls multiple comparison test was used to determine significance. *** P < 0.001; ** P < 0.01; ns, not significant.

To summarize, Pink1 loss-of-function flies presented mitochondrial dysmorphology and dysfunction (reduced ATP production, decreased mitochondrial complex I activity, depletion of mtDNA and decreased expression of nuclear encoded NDUFS3). The mitochondrial dysmorphology and dysfunction in Pink1 loss-of-function flies attributed to the phenotypes such as impaired locomotor activity, thorax-indentation, abnormal wing posture and male sterility. Expression of hTrap1 rescued these phenotypes via enhancing the mitochondrial function. On the other hand, expression of hTrap1D158N did not present any rescuing effects in

Chapter 3 - Results 39

Pink1B9 flies. This illustrates the ATP binding and most likely subsequent ATPase activity of TRAP1 is critical for the rescuing effects.

3.1.3 Knocking down Trap1 in Pink1 loss-of-function flies cause semi-lethality Enhancing Trap1 abundance (endogenous and exogenous) in flies rescued abnormal phenotypes caused by Pink1 loss-of-function. Next I asked whether reduced expression levels of Trap1 would enhance the phenotypes induced by Pink1 loss-of-function in flies. To tackle this question, I used a P-element insertion (Trap1KG) in the Trap1 locus. This P- element insertion is known to cause a hypomorphic Trap1 allele (Butler et al., 2012).

Following the crossing scheme depicted in Figure 18A, I asked whether the Trap1KG allele would reduce the viability of hemizygous Pink1B9 males. In the F1 generation I compared the hatching rates of hemizygous Pink1B9 males with or without presence of one Trap1KG allele (Figure 18B). I found that the combination of Pink1B9 and one copy of the Trap1KG allele caused semi-lethality. This indicates that Trap1 loss-of-function enhances the effect of Pink1 loss-of-function.

I have shown that the phenotypes induced by Pink1 loss-of-function flies were mitigated by expression of hTrap1WT, such as abnormal wing posture, collapsed thorax, impaired climbing and flight ability, as well as mitochondrial dysmorphology and dysfunction. Furthermore, Trap1 loss-of-function worsened the effects caused by Pink1 loss-of-function, leading to semi-lethality in flies. Therefore, I assume that Trap1 functions downstream of Pink1.

Chapter 3 - Results 40

Figure 18. Genetic interaction of Pink1 and Trap1 loss-of-function alleles. (A) yw/Y ( y, yellow, determines cuticle pigmentation of fly; w, white, determines eye color of fly. y and w are used as visible biological makers) and yw/Y;Trap1KG male flies were crossed to Pink1B9/FM7,GFP virgin females. The resulting male offspring in the F1 generation can be either Pink1B9/Y or FM7,GFP/Y for control (upper) crosses or in combination with one copy of the Trap1KG allele, Pink1B9/Y;Trap1KG/+ or FM7,GFP/Y;Trap1KG/+, respectively (lower scheme). (B) In controls, the observed ratio of Pink1B9/Y versus FM7,GFP/Y male offspring was 35%:65%. The presence of the Trap1KG allele reduced the percentage of Pink1B9 (Pink1B9/Y;Trap1KG/+ versus FM7,GFP/Y;Trap1KG/+, 15%:85% respectively). Unpaired t test was used to determine significance. ** P < 0.01.

3.2 Trap1 functions independently of PINK1/Parkin pathway

Parkin loss-of-function flies display almost identical phenotypes to Pink1 loss-of-function flies, such as abnormal wing posture, thoracic indentation, declined locomotor ability, impaired mitochondrial morphology and function (Park et al. 2006, Clark et al. 2006, Yang et al. 2006). This suggested that the both proteins act in the same pathway. Indeed, epistatic analysis showed that overexpression of parkin rescues Pink1 loss-of-function phenotypes in flies, suggesting a function of parkin downstream of Pink1 (Park et al. 2006, Clark et al. 2006).

Though Pink1 has been reported to be involved in multiple pathways, the main function of PINK1 in vertebrate cells seems to be the quality control of the mitochondrial population via the so-called PINK1/Parkin pathway. In agreement with the analysis in flies, Pink1 is reported to function upstream of parkin in mitochondrial quality control in vertebrate cells

Chapter 3 - Results 41

(Geisler et al. 2010, Narendra et al. 2008, Narendra et al. 2010a, Chen & Dorn 2013, Jin & Youle 2013, McLelland et al. 2014).

My data suggest that expression of hTrap1 rescued almost all of the phenotypes in Pink1 loss-of-function flies, this acting downstream of Pink1. Similar rescue activities have been reported for parkin (Park et al. 2006, Clark et al. 2006). Thus, I asked whether Trap1 functions downstream of parkin in this pathway. To address this question, I examined whether Trap1 rescued the phenotypes induced by parkin loss-of-function. The park25 allele is a null mutation, accordingly homozygous park25 flies do not have any residual Parkin function (Whitworth et al. 2005, Deng et al. 2008). In this study, homozygous Park25 flies (w/Y;;Park25,DaG-Gal4/Park25) were used as parkin loss-of-function flies and termed as “Park25 flies”, while heterozygous Park25 flies (w/Y;;Park25,DaG-Gal4/TM3,Sb,e) served as the controls.

3.2.1 Trap1 does not influence phenotypes in parkin mutant flies As reported for Pink1 mutants, also parkin deficient flies display almost invariable phenotypes in adults. In close similarity to previous approaches, I applied these analyses in parkin deficient flies to examine whether Trap1 rescued parkin loss-of-function. First, I addressed abnormal wing posture. Only 40% of Park25 flies showed normal wing posture, whereas 100% of the control flies did (Figure 19A). Unlike for Pink1B9 flies, expression of hTrap1WT did not improve the wing posture in parkin deficient flies.

As reported for Pink1 deficient flies, Park25 flies displayed disturbed climbing ability as well (Figure 19B). However, expression of hTrap1 did not mitigate the impaired locomotor activity in Park25 flies (Figure 19B). The abnormal wing posture and reduced climbing ability is believed to be caused by mitochondrial dysfunction. Both Pink1 and parkin deficent flies display disorganized muscles with enlarged mitochondria harboring fragmented cristae. Thus, I analyzed muscle sections of Park25 flies. Also in this case, expression of hTrap1 did not rescue disorganized muscle fibers in thoraxes of park deficient flies (Figure 19C).

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Figure 19. Expression of Trap1 did not influence phenotypes in park25 flies. (A) Wing posture phenotype. Only 40% of Park25 flies displayed normal wing posture (red bar), while none of the control flies showed abnormal wing posture (green bar). Expression of hTrap1 did not show significant rescue of abnormal wing posture in Park25 flies (yellow bar). (B) Index of climbing ability. Park25 flies presented impaired climbing ability. Expression of hTrap1 did not improve the climbing ability in Park25 flies. (C) Fly muscle sections. Park25 flies displayed disorganized indirect flight muscle fibers in thoraxes. Expression of hTrap1 did not cause a regaining the well-organized muscle fibers in thoraxes in Park25 flies. Fly age: 5 days. Flies were raised at 29°C. One-way ANOVA followed by the Neuman–Keuls multiple comparison test was used to determine significance. *** P < 0.001; ** P < 0.01; ns, not significant.

3.2.2 Expression of Trap1 does not mitigate mitochondrial function/morphology in parkin mutant flies My analysis of indirect flight muscles (Figure 19C) suggests that mitochondrial function is impaired in Park25 flies. Thus, I analyzed mitochondrial function by ATP measurement and protein abundance of mitochondrial complex I. The Park25 flies had reduced ATP levels in thoraxes (Figure 20A) and low abundance of a mitochondrial complex I subunit NDUFS3 (Figure 20B). In contrast to my findings in Pink1 deficient flies, expression of hTrap1 was neither able to restore the ATP levels in thoraxes nor NDUFS3 protein levels in Park25 flies (Figure 20).

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In summary, Trap1 rescued no phenotypes induced by parkin loss-of-function, although all these phenotypes were rescued in Pink1 loss-of-function flies. Therefore, I assume that Trap1 functions downstream of Pink1, but not downstream of parkin. Trap1 acts either upstream/parallel to parkin in the Pink1/parkin pathway, or in an alternative, so far unknown pathway, parallel to the Pink1/parkin pathway.

Figure 20. Expression of Trap1 did not mitigate mitochondrial function in park25 flies. (A) ATP content. Park25 flies showed declined ATP levels as compared to control flies. Expression of hTrap1 did not rescue the lost ATP levels in Park25 flies. (B) Expression levels of NDUFS3. Park25 flies possessed low expression levels of NDUFS3 compared to control flies. Expression of hTrap1 did not change the expression levels of NDUFS3 in Park25 flies. Fly age: 5 days. Flies were raised at 29°C. One-way ANOVA followed by the Neuman–Keuls multiple comparison test was used to determine significance. ** P < 0.01; ns, not significant.

3.3 Trap1 mitigates mitochondrial morphology/function in Pink1 knock-out SH-SY5Y cells

In an attempt to test whether my findings obtained in flies can be reproduced in vertebrate cells, I tried to recapitulate my findings in cooperation with Kathrin Müller-Rischart and Konstanze F. Winklhofer (LMU, Munich). In human SH-SY5Y cells, transient silencing of either Pink1 or parkin by transfection of specific shRNAs results in mitochondrial fragmentation and reduced ATP content. Thus I asked whether these phenotypes could be rescued by co-transfection of either hTrap1WT or hTrap1D158N or hTrap1ΔMTS (TRAP1 lacking the mitochondrial target sequence). In line with my finding in flies, expression of hTrap1WT,

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but neither hTrap1D158N nor hTrap1ΔMTS, in Pink1-siRNA treated SH-SY5Y cells caused a regained normal ATP levels and restored mitochondrial morphology (Figure 21). In parkin- siRNA treated SH-SY5Y cells, there was only a mild rescuing effect after expression of hTrap1WT. hTrap1D158N or hTrap1∆MTS did not display any rescuing activity. All these findings were coherent with the data obtained in flies.

Figure 21. ATP levels and mitochondrial fragmentation in Pink1-siRNA & parkin-siRNA SH- SY5Y cells. SH-SY5Y cells treated with either Pink1-siRNA or parkin-siRNA displayed declined ATP levels (A) and fragmented mitochondria (B). Expression of hTrap1WT, but neither hTrap1D158N nor hTrap1ΔMTS, in Pink1-siRNA SH-SY5Y cells restored the ATP content and regained normal morphology of mitochondria. In contrast, in parkin-siRNA treated SH-SY5Y cells, the rescue effect by expression of hTrap1WT was not as pronounced as in Pink1-siRNA treated cells. Two-way ANOVA followed by the Bonferroni post-test was used to determine significance. *** P < 0.001; ** P < 0.01; * P < 0.05.

3.4 Trap1 and mitochondrial complex I rescue each other 3.4.1 Trap1 rescues mitochondrial complex I subunits loss-of- function My data imply that Trap1 does not function within the Pink1/parkin pathway. Thus I wondered how Trap1 acts to rescue the phenotypes caused by Pink1 loss-of-function. According to the location of TRAP1 in mitochondria and the observed rescuing effect on ETC (mitochondrial complex I acitivty and NDUSF3 abundance), as well as the rescue on overall ATP levels in Pink1 deficient flies and cells, I reasoned that TRAP1 might act in mitochondria, most likely in stabilizing the ETC. To gain more insights in possible function(s) of TRAP1, I silenced genes, known to code for proteins acting in mitochondria in flies. In a first attempt, I asked whether ubiquitous (DaG-Gal4) silencing of these genes by RNAi caused lethality. A detailed summary of genes silenced and obtained results are depicted in the appendix (Table 10). Overall, I silenced 93 genes. In brief 19 genes were silenced coding for enzymes of the Krebs cycle, 7 caused lethality;

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25 genes were silenced coding for complex I subunits, 12 caused lethality; 6 genes were silenced coding for complex II subunits, 4 caused lethality; 6 genes were silenced coding for complex III subunits, 4 caused lethality; 13 genes were silenced coding for complex IV subunits, 7 caused lethality; 24 genes were silenced coding for ATP synthase (complex V; F-type and V-type), 16 caused lethality.

In a second step, I asked whether the lethal phenotype observed after ubiquitous gene silencing could be reverted by co-expression of Trap1WT. To control for potential titration effects of Gal4, I co-expressed the inactive Trap1D158N. As a result, I found that expression of hTrap1 WT rescued the lethality caused by the silencing of three mitochondrial complex I subunits (NDUFa5, NDUFb1, NDUFS8) and one mitochondrial complex IV subunit (COX6c) (Table 9). Interestingly, expression of hTrap1D158N did not rescue the lethality after ubiquitous RNAi-mediated silencing of these genes. This suggests that the rescue effect is specific for TRAP1 and requires fully functional TRAP1 activity. Table 9. hTrap1WT rescues silencing of mitochondrial complex subunits Ubiquitous knock down of these genes caused lethality. The lethal phenotype was rescued by co- expression of hTrap1WT, but not hTrap1D158N. CG Gene Name Symbol 6439 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 5 NDUFa5 18624 NADH dehydrogenase (ubiquinone) 1 beta subcomplex 1 NDUFb1 4094 NADH dehydrogenase (ubiquinone) Fe-S protein 8 NDUFS8 14028 cytochrome c oxidase subunit 6c, cyclope COX6c

Moreover, my findings suggest that TRAP1 acts to stabilize protein complexes of the ETC, especially mitochondrial complex I. Mitochondrial complex I is the first complex of the ETC, and the main role of the ETC is to produce energy (ATP). Therefore, I measured the ATP levels in NDUFb1 silenced larvae. As expected, ubiquitous knock down of NDUFb1 caused decreased ATP levels compared to control. Larvae with ubiquitous knock down of NDUFb1 and co-expression of hTrap1WT displayed normal ATP levels (Figure 22). This reveals that Trap1 overexpression remains the ETC functional in NDUFb1 deficient situations.

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Figure 22. Trap1 restored ATP levels in L3 Larve of NDUFb1-knock-down flies. Ubiquitous knock-down of NDUFb1 resulted in reduced ATP levels (red bar) compared to controls (green bar). However, the ATP levels reached control levels when hTrap1WT was co-expressed. One- way ANOVA followed by the Neuman–Keuls multiple comparison test was used to determine significance. ** P < 0.01; ns, not significant.

3.4.2 Ndi1p (mitochondrial complex I) rescues Trap1 loss-of- function Since Trap1 rescued the lethality caused by knocking down of certain mitochondrial complex I subunits in flies, I assumed that Trap1 might protect mitochondrial complex I. To support this assumption, I analyzed whether overexpression of mitochondrial complex I could compensate Trap1 loss-of-function. I used the amorphic Trap14 allele. Homozygous Trap14 flies are viable but do not display any detectable Trap1 mRNA in qRT-PCR analysis (Figure 23A). Moreover, homozygous Trap14 have been reported to show characteristic phenotypes (Costa et al. 2013). Among these are reduced climbing activity (Figure 23B), increased sensitivity to mitochondrial complex I inhibitor Rotenone (Figure 23C) and decreased ATP levels (Figure 23D). All of these phenotypes can be explained by a reduced activity of the ETC.

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Figure 23. Phenotypes induced by Trap1 loss-of-function in Trap14 flies. (A) Expression levels of Trap1 assayed by qRT-PCR revealed the absence of detectable Trap1- transcripts in homozgous Trap14 flies. (B) Compared to heterozygous Trap14 flies (Trap14/CyO), homozygous Trap14 flies presented an impaired climbing ability. (C) Trap14 flies displayed shortened life span under 2 mM Rotenone compared to heterozygous controls (Trap14/Cyo). (D) Low ATP levels were observed in Trap14 flies. One-way ANOVA followed by the Neuman–Keuls multiple comparison test (A) and T-test (B, D) was used to determine significance. *** P < 0.001; * P < 0.05; ns, not significant.

To investigate whether normalized mitochondrial complex I could rescue Trap1 loss-of- function, Ndi1p was expressed in the neurons of homozygous Trap14 flies. In yeast, Ndi1p is the only protein that determines mitochondrial complex I function. Overexpression of Ndi1p in flies was reported to functionally bypass mitochondrial complex I. Moreover, overexpression of Ndi1p rescued phenotypes in Pink1 deficient flies (Vilain et al. 2012). Homozygous Trap14 flies were vulnerable against heat stress, and only 40% survived 24 h after heat shock treatment (Figure 24A). Compared to homozygous Trap14 flies, ectopic neuronal expression of Ndip1 resulted in survival of more Trap14 homozygous flies after heat shock (Figure 24A). This indicates that reconstitution of mitochondrial complex I function in Trap1 deficient flies at least partially rescues sensitivity to heat stress. My own and previously published data suggest that there is a decline in mitochondrial function and abundance in Trap1 deficient flies (Costa et al. 2013). Accordingly, I tested indirectly

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mitochondrial abundance by assessing the protein levels of a mitochondrial complex I subunit NDUFS3. Homozygous Trap14 flies showed low levels of NDUFS3, while Trap14 flies with neuronal expression of Ndi1p regained the levels of NDUFS3 (Figure 24B).

Figure 24. Ndi1p rescued Trap1 loss-of-function. (A) Survival of flies after induction of heat stress. Homozygous Trap14 flies were vulnerable to heat stress, indicated by low survival after heat stress (red bar). Neuronal (elav- Gal4) expression of Ndip1 (grey bar) ameliorated sensitivity to heat stress, whereas heterozygous Trap14 controls (black bar) did not show reduced survival 24 h after heat stress. (B) Determination of mitochondrial complex I subunit NDUFS3 abundance in Western blot. Homozygous Trap14 flies (Trap1[4]) display reduced abundance of NDUFS3, which was restored to control levels (elav>Ndi1p) after pan neural expression of Ndi1p (elav>Ndi1p, Trap1[4]). Fly age: 10 days. One-way ANOVA followed by the Neuman–Keuls multiple comparison test was used to determine significance. *** P < 0.001; * P < 0.05.

In summary, Trap1 rescues the phenotypes caused by Pink1 loss-of-function, which reflects that Trap1 functions downstream of Pink1. In contrast, Trap1 did not show any rescuing effects on the phenotypes induced by parkin loss-of-function. This suggested that Trap1 is not involved in mitophagy/mitochondria-derived vesicles (MDVs) controlled by the Pink1/parkin pathway. In addition, I found that Trap1 rescued the lethality and decreased ATP levels caused by knocking down certain mitochondrial complex I subunits (Table 9, Figure 22). All these data imply that TRAP1 acts on mitochondrial complex I to maintain a functional ETC. The fact that Ndi1p, bypassing mitochondrial complex I rescues Trap1 loss- of-function is in line with the assumed function of TRAP1.

Chapter 4 –Discussion 49

4 Discussion

PD is the most common movement disorder, characterized by a loss of dopaminergic neurons in the SNpc. Current therapies of PD are mainly based on exogenous replacement of dopamine. Such methods of therapies temporarily remit the symptoms, but do not impede the progression of neuronal decline. This is one reason why the efficiency of the therapies is decreasing over time. Accordingly, it is crucial to understand why dopaminergic neurons degenerate. Slowing down or preventing DA neuron degeneration would be a key step in the rational development of new therapies to ameliorate PD (progression). This requires further investigation at the genetic/molecular levels of those dying neurons. So far, there are two pathological hallmarks in dying DA neurons, LBs/LNs and impairment of mitochondrial complex I. α-Syn has been found to be the main component in LBs/LNs. Specific mutations in SNCA, the gene encoding α-Syn, as well as duplication or triplication of SNCA locus cause autosomal dominant PD (Polymeropoulos et al. 1997, Kruger et al. 1998, Singleton et al. 2003, Chartier-Harlin et al. 2004). Thus, the mechanism of α-Syn-induced toxicity is critical for understanding PD pathology. The mitochondrial chaperone TRAP1 has been found to mitigate α-Syn-induced toxicity in vitro and in vivo (Butler et al. 2012). The toxicity caused by α-Syn was decreased by overexpression of Trap1 and increased absence of Trap1. Interestingly, TRAP1 has been reported as a substrate of a serione/theronine kinase, PINK1. Mutations in Pink1 cause autosomal recessive PD (Valente et al. 2004). Overexpression of PINK1 protects cells from oxidative stress and this protection is known to dependent on TRAP1 (Pridgeon et al. 2007). These findings suggest that TRAP1 might act downstream of PINK1. Thus, I asked whether TRAP1 might act downstream of PINK1 and has a role in the etiology of PD.

4.1 Trap1 functions downstream of Pink1

To investigate whether Trap1 affects Pink1 loss-of-function, I expressed human Trap1 (hTrap1) in Pink1B9 flies (Pink1 loss-of-function flies). Pink1B9 flies display well characterized phenotypes, including male sterility, abnormal wing posture, collapsed thoraxes, impaired climbing ability, lack of flight ability as well as a dysmorphology and dysfunction of mitochondria, loss of dopamine content and dopaminergic neurons (Park et al. 2006, Clark et al. 2006). Ubiquitous hTrap1WT expression in a Pink1B9 background attenuated abnormal wing posture (Figure 9), collapsed thoraxes (Figure 11) and impaired locomotor ability (Figure 11). Except for the lack of the ability to fly, all other Pink1B9-induced phenotypes

Chapter 4 –Discussion 50

were at least partially rescued by expression of hTrap1. These above described phenotypes might be explained by a dysmorphology and dysfunction of mitochondria in muscles of Pink1B9 flies. In Pink1B9 flies, mitochondria in indirect flight muscles appeared swollen and displayed highly fragmented cristae. Expression of hTrap1 caused a re-gain of normal mitochondria size and densely packed cristae (Figure 13). Also the amount of mitochondrial DNA and abundance of mitochondrial complex I, as measured by NDUSF3 levels, was restored (Figure 14, Figure 15, Figure 16). In agreement with these observations, also the impaired mitochondrial function in Pink1B9 flies was rescued by expression of hTrap1. A mitochondrial dysmorphology is usually accompanied by mitochondrial dysfunction. The main function of mitochondria is to produce energy (ATP). Therefore, the ATP levels were examined. Whereas Pink1B9 flies showed low ATP levels and reduced mitochondrial complex I activity, expression of hTrap1 ameliorated these parameters of mitochondrial function.

Of note, the sterility observed in Pink1B9 males is also caused by mitochondrial dysfunction. In sperm development, mitochondria fuse to form a structure called ‘Nebenkern’. The Nebenkern is wrapped with multiple layers of mitochondrial membranes, and in later stages of sperm development, the Nebenkern elongates and splits into two mitochondrial derivatives. The proper function of these mitochondria is crucial for sperm motility. Ubiquitous hTrap1 expression restored male fertility almost to control levels (Figure 17). This indicates that not only mitochondrial defects in muscles are rescued by hTrap1.

Moreover, I found that flies with neuronal Pink1 knock-down (elav>Pink1-RNAi) developed an abnormal wing posture phenotype. Neuronal expression of hTrap1WT mitigated the abnormal wing posture caused by neuronal Pink1-RNAi (Figure 10). This further supports the idea that hTrap1 rescues phenotypes in Pink1 loss-of-function situations in many (if not all) tissues. Moreover, a parallel study basically confirmed my findings in flies. There, it has been shown that overexpression of Drosophila Trap1 (dTrap1), instead of hTrap1, rescued the phenotypes in Pink1B9 flies (Costa et al. 2013).

Altogether, these data suggest that Trap1 rescues the phenotypes caused by Pink1 loss-of- function, most likely through rescuing the mitochondrial dysmorphology and dysfunction. Mitochondrial dysfunction leads to an impaired function of muscles. Dysfunctional muscles in turn might explain abnormal motor functions, such as abnormal wing posture and impaired locomotor activity. Flight requires an extremely high amount of energy. Given the partial

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rescue observed after overexpression of hTrap1 on other phenotypes induced by Pink1 loss- of-function (e.g. wing posture, ATP levels etc.), this might explain why a partial rescue is not sufficient to restore flight ability. Even the reduced male fertility in PINK1B9 flies can be explained by mitochondrial dysfunction. In PINK1B9 males, sperm with a swollen Nebenkern are observed.

In summary, expression of hTrap1 rescues the phenotypes induced by Pink1 loss-of-function most likely by normalizing mitochondria function and integrity. With regard to the rescue effect, I suppose that TRAP1 supports mitochondria maintaining their normal morphology and function.

4.2 The rescuing effect of Trap1 on Pink1 loss-of-function requires mitochondrial location of TRAP1 and its ATPase activity

TRAP1 is a mitochondrial molecular chaperone with similarity to HSP 90, containing three main domains: a 59 amino acids N-terminal MTS, an ATPase domain with four ATP-binding sites and a C-terminal chaperone domain (Matassa et al. 2012). To explore which component of TRAP1 is involved in the rescuing effect, I introduced hTrap1D158N (with a point mutation in one ATP-binding site) and hTrap1ΔMTS (without mitochondrial target sequence). By RT- PCR, I showed that flies with ubiquitous expression of hTrap1WT and hTrap1D158N presented the same levels of hTrap1 mRNA. In addition, hTrap1 overexpression had no effect on endogenous dTrap1 mRNA abundance. Western blot analysis revealed also very similar/identical protein levels (Figure 8). These results indicate that the differences of the effects between hTrap1WT and hTrap1D158N presented in this study were due to the functional variation between two exogenous TRAP1 proteins and did not arise by difference in expression levels of endogenous dTrap1.

Sequence alignment of TRAP1 with Hsp90 family members showed striking sequence similarities in the ATP binding sites. According to this conservation in protein sequence, we suggested that Aspartic acid at position 158 is a crucial amino acid in one of the ATP-binding sites of TRAP1. A mutation in one of the four ATP binding sites of Hsp90 is known to abolish Hsp90 function (Panaretou, et al. 1998). Using this information, hTrap1 was mutagenized, replacing Aspartic acid at amino acid position 158 by Asparagine (hTrap1D158N). In contrast

Chapter 4 –Discussion 52

to hTrap1WT, expression of hTrap1D158N did not show any rescuing effect in Pink1 loss-of- function in flies (Figure 9-17).

In cooperation with Konstanze Winklhofer, Pink1 deficient SH-SY5Y cells were analyzed. Pink1 and parkin deficient cells displayed fragmented mitochondria and reduced ATP levels , both findings are indicative of a general dysfunction of mitochondria. Expression of hTrap1WT ameliorated phenotypes in Pink1, but not parkin deficient SH-SY5Y cells. Similar to the observation in Pink1 deficient flies, Trap1D158N or Trap1∆mito (Trap1 lacking the mitochondrial localization signal) did not provide any rescue effect neither on mitochondrial fragmentation nor on ATP levels (Figure 21). Therefore, the protective effects by Trap1 not only require proper ATP binding, but also require proper localization to mitochondria. Interestingly, flies expressing hTrap1D158N did not show any protective effect against. As hTrap1WT expressing flies provided protective effects towards α-SynA53T-induced toxicity, these previous findings are in agreement with the assumption that ATP binding and most likely subsequent ATP to ADP conversion is required for TRAP1 (chaperone) function in mitochondria (Butler et al. 2012).

4.3 Trap1 does not function in PINK1/Parkin pathway

Pink1 has been found to be involved in numerous pathways like oxidative-stress and calcium-induced cell death (Pridgeon et al. 2007, Plun-Favreau et al. 2007, Gandhi et al. 2009). Among these pathways, the PINK1/Parkin pathway is the best understood. It has been reported that Parkin functions downstream of PINK1 in regulating the quality of mitochondrial population by either mitophagy or mitochondria-derived vesicles (MDVs) (Park et al. 2006, Clark et al. 2006, Yang et al. 2006, Klionsky 2010, Wang et al. 2011, Geisler et al. 2010, McLelland et al. 2014). Thus, it was analyzed whether TRAP1 is able to also rescue phenotypes in Parkin-deficient flies.

Parkin loss-of-function flies display very similar/identical phenotypes compared to Pink1 loss-of-function flies (Greene et al. 2003, Park et al. 2006, Clark et al. 2006). This already suggested that the two gene products may act in a common pathway. Epistatic analysis revealed that overexpression of parkin is able to rescue phenotypes in Pink1-deficient flies. Overexpression of Pink1, however, did not rescue phenotypes observed in parkin deficient flies. This showed that Pink1 acts upstream of parkin (Clark et al. 2006, Park et al. 2006). I have shown that expression of Trap1 rescued almost all phenotypes in Pink1 deficient flies.

Chapter 4 –Discussion 53

As Pink1 acts upstream of parkin, I asked whether Trap1 also mitigates phenotypes in parkin deficient flies.

Parkin loss-of-function flies (park25) showed abnormal wing posture, disturbed climbing ability and degenerated mitochondria in flight muscles (Figure 19). However, unlike in Pink1B9 flies, expression of hTrap1 did not mitigate those phenotypes in park25 flies (Figure 19). Similar to Pink1 loss-of-function flies, parkin loss-of-function flies also display mitochondrial dysfunction. The park25 flies presented reduced ATP levels in thoraxes and low protein abundance of the mitochondrial complex I subunit NDUFS3. Expression of hTrap1 was neither able to restore the ATP level in thoraxes nor the decreased NDUFS3 protein level in park25 homozygous flies (Figure 20). In summary, none of the abnormal phenotypes in parkin loss-of-function flies were rescued by ectopic expression of hTrap1. Since the (almost) identical phenotypes were rescued in a Pink1 deficiency, I assume that Trap1 acts downstream of Pink1 but upstream or parallel to parkin.

This assumption has been further supported by my collaborator Konstanze Winklhofer. SH- SY5Y cells, treated with parkin-siRNA exhibited refrained ATP levels and fragmented mitochondria, similar to Pink1-deficient cells (Figure 21). However, in parkin-deficient SH- SY5Y cells, hTrap1-expression provided no (ATP levels) or an only mild rescue (mitochondrial fragmentation). These findings are coherent with the data obtained in flies.

Another study has shown that overexpression of dTrap1 (fly Trap1), instead of hTrap1, rescues Pink1 loss-of-function (Costa et al. 2013). However, overexpression of dTrap1 mildly suppressed some parkin loss-of-function phenotypes like thorax indentation and reduced ATP levels. Other phenotypes like locomotor ability were not suppressed. In addition to my analysis, these authors investigated the phenotypes of Trap1-deficient (Trap14) flies. Trap14 flies showed shortened life span, sensitivity to heat stress and chemicals (Paraquat, Rotenone, Antimycin), impaired climbing ability and impaired mitochondrial function. Interestingly, overexpression of parkin was able to partially restore some phenotypes of Trap14 flies. Climbing ability and protein levels of the complex I subunit NDUFS3 in Trap14 flies were partially rescued by overexpression of parkin. The authors suppose that Trap1 functions downstream of Pink1 and independent of parkin in flies (Costa et al. 2013). In summary, the reported findings and conclusions by Costa and co-workers is consistent with my data and the interpretation thereof.

Chapter 4 –Discussion 54

4.4 α-Synuclein, PINK1, TRAP1 and the mitochondrial complex I

To further investigate how Trap1 might function downstream of Pink1, an unbiased genetic screen was performed. The findings on TRAP1 function so far imply that the protein preserves mitochondrial function and/or integrity. I reasoned that expression of TRAP1 should at least partially rescue RNAi-mediated knockdown of genes known to encode proteins with mitochondrial function. To test this hypothesis, I ubiquitously knocked down such genes using RNAi and asked whether this knock down caused lethality. In total, 93 genes coding proteins either of the mitochondrial electron transport chain complexes I, II, III, IV, V or the Krebs cycle were screened. 50 of those genes caused lethality when ubiquitously silenced by RNAi (Table 10). In a second approach, I asked whether co-expression of hTrap1WT is able to rescue the lethal phenotype by RNAi-mediated silencing of these 50 genes. In parallel, co-expression of hTrap1D158N was as control, since this variant was inefficient to abolish effects of Pink1 deficiency and therefore should not rescue lethality. In this screening approach, I identified three mitochondrial complex I subunits that when silenced caused lethality and were rescued by hTrap1WT but not by hTrap1D158N expression. These mitochondrial complex I subunits were NDUFa5, NDUFb1 and NDUFS8 (Table 9). Except of the three complex I subunits, only the deficiency of one complex IV protein seemed to be compensated by TRAP1 overexpression. The lethality observed upon RNAi- mediated silencing of cytochrome c oxidase subunit 6c (cyclope) was rescued by hTrap1WT but not by hTrap1D158N. These findings suggested that TRAP1 is involved in maintaining functionality of mitochondrial complex I. To further support this hypothesis, I measured ATP levels of L3 larvae with strong ubiquitous knock down of NDUFb1. These larvae presented low ATP levels compared to the controls. However, L3 larvae with ubiquitously knock down of NDUFb1 and co-expression of hTrap1WT showed normal levels of ATP (Figure 22). Larvae with strong ubiquitous knock down of NDUFb1 usually die as pupae. The regained ATP level upon co-expression of hTrap1WT in a NDUFb1-silenced background might explain why pupal lethality was at least partially rescued by hTrap1WT and adult flies hatched from the pupal cage. Although adult flies were obtained, these flies were short lived and survived only a few days (data not shown). These support the suggestion that Trap1 functions downstream of mitochondrial complex I. In addition, TRAP1 seems to maintain mitochondria in a functional state, allowing the normal production of ATP. Since hTrap1D158N

Chapter 4 –Discussion 55

did not display any rescuing effects, I assumed that the ATP and most likely subsequent ATP- lysis by TRAP1s inherited ATPase activity is crucial for the protective function.

Next I confirmed selected phenotypes reported in Trap14 (Trap1 mutant) flies by Costa et al. like the impaired climbing ability (Figure 23B). In addition, I showed that Trap14 flies were more sensitive to the heat stress. Only 40% of Trap14 flies survived 24 hours after a heat shock, while all the control flies survived (Figure 24A). Also, Trap14 flies were vulnerable when exposed to mitochondrial complex I inhibitor, Rotenone. Under Rotenone treatment, the medial survival of Trap14 flies was about 11 days, while it was 18 days for controls (Figure 23C). In addition, Trap14 flies also displayed mitochondrial dysfunction, by showing reduced levels of NDUFS3 (Figure 24B) and thoracic ATP levels (Figure 23D), which were also described by Costa et al. (Costa et al. 2013). These data suggest that in a Trap1 loss-of- function situation, the electron transport chain (ETC) is dysfunctional and most likely mitochondrial complex I is impaired. In this scenario, reconstitution of mitochondrial complex I activity should rescue the phenotypes observed in a Trap1 loss-of-function situation. A yeast protein Ndi1p bypasses mammalian function of mitochondrial complex I (Vilain et al. 2012). Pan neuronal expression of Ndi1p in Trap14 flies restored NDUFS3 abundance in fly heads (Figure 24B). Moreover, I found that neuronal Ndi1p mitigated the vulnerability of Trap14 flies to heat stress (Figure 24A). These findings indicate that, the phenotype induced by Trap1 loss-of-function might be attributed to mitochondrial complex I deficiency. Moreover, I have shown that TRAP1 rescues detrimental effects induced by silencing of certain single mitochondrial complex I subunits. Also in rat cortical overexpression of Trap1 has been also reported that provided a protection against Rotenone (Butler et al. 2012). Altogether, I assume that TRAP1 is beneficial in maintaining the integrity of mitochondrial complex I and/or is involved in correct of mitochondrial complex I subunits (Figure 25). In this way, without TRAP1, mitochondrial complex I is more vulnerable to environmental stressors, such as ROS. This would explain why Trap1 loss-of-function flies displayed mitochondrial complex I deficiency and reduced ATP levels. With certain mitochondrial complex I subunits being knocked down in flies, overexpression of TRAP1 may slow down the progress of mitochondrial complex I dysfunction.

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Figure 25 TRAP1 protects the function/integrity of mitochondrial complex I. Under stress, such as ROS, mitochondrial complex I undergoes damage. TRAP1 may repair/prevent the damage of mitochondrial complex I by refolding the subunits or remaining the integrity of the entire complex I. Without TRAP1 mitochondrial complex I is vulnerable to stress. With TRAP1 overexpression, the mitochondrial complex I seems less sensitive towards certain stress conditions.

Interestingly, Pink1 loss-of-function have been reported to cause mitochondrial complex I dysfunction. In Pink1 deficient mice or mice with Pink1 clinical mutations, decreased mitochondrial complex I activity has been reported (Morais et al. 2009). Furthermore, in flies, abnormal phenotypes induced by Pink1 loss-of-function can be rescued by Ndi1p (mitochondrial complex I), but not by AOX (CIII and IV) (Vilain et al. 2012). Moreover, flies with loss-of-function of some mitochondrial complex I subunits phenocopied Pink1 loss-of- function flies (Vilain et al. 2012). Those finding strongly suggest that Pink1 functions upstream of mitochondrial complex I. Interestingly, expression of Ndi1p failed to rescue any of the parkin mutant phenotypes, and parkin mutant flies did not show reduced activity of mitochondrial complex I (Vilain et al. 2012). These results using the yeast mitochondrial complex I equivalent Ndi1p resemble what has been observed after Trap1 expression. Expression of both, Ndi1p and Trap1 rescued Pink1 loss-of-function, but not parkin loss-of- function. These data suggest that TRAP1 and mitochondrial complex I function downstream of PINK1 and both of them act independent of Parkin. This is in agreement with the idea of an additional PINK1-involving pathway that acts independent of the classical PINK1/Parkin pathway. The PINK1/Parkin is facilitating mitochondrial quality control and is required to target dysfunctional mitochondria for degradation. In the alternative pathway involving PINK1 and TRAP1, TRAP1 seems to be involved in maintaining mitochondrial function (most likely by stabilizing complex I). In this scenario, according to TRAP1 function, the mitochondria are kept polarized. Polarized mitochondria in turn are not a target for mitophagy via the PINK1/Parkin pathway.

In sporadic PD, an impaired complex I function has been described already in 1989 (Schapira, J Neurochem.). Moreover, altered TRAP1 levels have been shown to modulate toxicity induced by -Syn in flies and in rat cortical neurons (Butler et al. 2012). How -Syn impairs complex I function remains elusive. It can only be speculated whether -Syn directly or

Chapter 4 –Discussion 57

indirectly impairs complex I function. Data showing that PINK1, Parkin and DJ-1 rescue - Syn-induced mitochondrial fragmentation support the idea that -Syn could directly impair complex I function. Most notably, the PD-causing mutations in Pink1, parkin and DJ-1 failed to rescue -Syn-induced mitochondrial fragmentation (Kamp et al. 2010). In support of a direct impairement of complex I function by -Syn would be the report by Cole et al. 2008. Here Cole and co-workers detected -Syn at mitochondrial outer membrane under acid cell condition in primary rat hippocampal neurons. Kamp et al. 2010 observed that -Syn directly binds to mitochondrial outer membrane in SH-SY5Y cells. At the mitochondrial outer membrane, -Syn seems to bind the outer mitochondrial membrane and promotes mitochondrial fission. Tight control of mitochondrial integrity by fission/fusion and mitochondrial transport seems to be important in PD etiology (Liu, et al. 2012, Chen, et al. 2009). Genetic studies in flies showed that the PINK1/Parkin pathway seems to promote mitochondrial fission to initiate mitophagy. Furthermore, Pink1 mutant phenotypes are enhanced by heterozygous mutation in the gene coding the pro fission Dynamin related protein1 (Deng et al. 2008).

On the other hand, there is evidence that -Syn is associated with the mitochondrial inner membrane. Devi et al. 2008 and Robotta, et al. 2014 showed that -Syn binds to the mitochondrial inner membrane, in human fetal dopaminergic primary neuronal cultures and human HEK293 cells, respectively.

Also an indirect effect of -Syn on mitochondira and/or complex I function is possible. For example, accumulation of -Syn is known to trigger several cellular mechanisms including oxidative stress and proteasomal stress (Branco et al. 2010). Chronic stress might impair the mitochondrial function. Especially dopaminergic neurons, which are known to have increased risk to produce reactive oxygen species (ROS) according to the synthesis of dopamine, are vulnerable towards such stressors.

Nevertheless, the findings in this and previous fly studies imply a genetic cascade in which α- Syn-induced toxicity is upstream of PINK1. TRAP1 in turn is located downstream mitochondrial complex I (Figure 26) and seems to be involved in maintaining a functional mitochondrial complex I. In this way, TRAP1 attenuates the phenotypes caused by overexpression of α-Syn, or induced by a Pink1 deficiency. In the context of PD etiology, the presented data are of importance. TRAP1 not only attenuates the effects of mutations in

Chapter 4 –Discussion 58

SNCA that cause dominantly inherited PD but also effects of mutations in Pink1 that cause recessively inherited PD. Moreover TRAP1 rescues a key element of PD pathology, namely mitochondrial complex I dysfunction. Thus, enhancing TRAP1 activity or abundance might be a reasonable approach in the future therapies of PD.

Figure 26. Putative role(s) of TRAP1 in PD. In case of an accumulation of misfolded proteins in mitochondria or upon depolarization of the organelle (∆), PINK1 is stabilized and locates to the mitochondrial outer membrane (MOM). At the MOM, PINK1 recruits Parkin to mitochondria. Parkin in turn ubiquitinates its mitochondrial target protein and initiates mitophagy. This process seems to be independent of TRAP1. In contrast, there seems to be a tight connection between PINK1 and TRAP1. TRAP1 is phosphorylated by PINK1 and both proteins co-localize in mitochondrial inner membrane (MIM) as well as in the mitochondrial intermembrane space (MIS). Here, the protective effects of PINK1 against oxidative stress-induced cell death require TRAP1. In addition, TRAP1 rescues Pink1 loss-of-function and mitochondrial complex I loss-of-function phenotypes. Moreover, TRAP1 mitigates α-Syn-induced mitochondrial fragmentation and α-Syn-dependent inhibition of mitochondrial complex I. Whether α- Syn localizes at MOM or MIM is still unclear. In summary, TRAP1 is a key element in PD.

Chapter 5 – References 59

5 References

Aarsland, D., Pahlhagen, S., Ballard, C. G., Ehrt, U. and Svenningsson, P. (2012) Depression in Parkinson disease--epidemiology, mechanisms and management. Nat Rev Neurol, 8, 35-47. Aarsland, D., Zaccai, J. and Brayne, C. (2005) A systematic review of prevalence studies of dementia in Parkinson's disease. Mov Disord, 20, 1255-1263. Alam, M. and Schmidt, W. J. (2002) Rotenone destroys dopaminergic neurons and induces parkinsonian symptoms in rats. Behavioural Brain Research, 136, 317-324. Auluck P. K., Edwin Chan H. Y. Trojanowski J. Q. et al. (2001) Chaperone Suppression of α-Synuclein Toxicity in a Drosophila Model for Parkinson's Disease. Science, 295, 865-868. Baba, M., Nakajo, S., Tu, P. H., Tomita, T., Nakaya, K., Lee, V. M., Trojanowski, J. Q. and Iwatsubo, T. (1998) Aggregation of alpha-synuclein in Lewy bodies of sporadic Parkinson's disease and dementia with Lewy bodies. Am J Pathol, 152, 879-884. Barnes, J. and David, A. S. (2001) Visual hallucinations in Parkinson's disease: a review and phenomenological survey. J Neurol Neurosurg Psychiatry, 70, 727-733. Becker, D., Richter, J., Tocilescu, M. A., Przedborski, S. and Voos, W. (2012) Pink1 kinase and its membrane potential (Deltapsi)-dependent cleavage product both localize to outer mitochondrial membrane by unique targeting mode. J Biol Chem, 287, 22969- 22987. Bender, A., Krishnan, K. J., Morris, C. M. et al. (2006) High levels of mitochondrial DNA deletions in substantia nigra neurons in aging and Parkinson disease. Nat Genet, 38, 515-517. Betarbet, R., Sherer, T. B., MacKenzie, G., Garcia-Osuna, M., Panov, A. V. and Greenamyre, J. T. (2000) Chronic systemic pesticide exposure reproduces features of Parkinson's disease. Nat Neurosci, 3, 1301-1306. Bindoff, L. A., Birch-Machin, M., Cartlidge, N. E., Parker, W. D., Jr. and Turnbull, D. M. (1989) Mitochondrial function in Parkinson's disease. Lancet, 2, 49. Blesa, J., Phani, S., Jackson-Lewis, V. and Przedborski, S. (2012) Classic and new animal models of Parkinson's disease. J Biomed Biotechnol, 2012, 845618. Bonifati, V., Rizzu, P., van Baren, M. J. et al. (2003a) Mutations in the DJ-1 gene associated with autosomal recessive early-onset parkinsonism. Science, 299, 256-259.

Chapter 5 – References 60

Bonifati, V., Rizzu, P., van Baren, M. J. et al. (2003b) Mutations in the DJ-1 gene associated with autosomal recessive early-onset parkinsonism. Science, 299, 256-259. Braak, H. and Del Tredici, K. (2009) Neuroanatomy and pathology of sporadic Parkinson's disease. Adv Anat Embryol Cell Biol, 201, 1-119. Braak, H., Del Tredici, K., Rub, U., de Vos, R. A., Jansen Steur, E. N. and Braak, E. (2003) Staging of brain pathology related to sporadic Parkinson's disease. Neurobiol Aging, 24, 197-211. Braak H, Ghebremedhin E, Rüb U, Bratzke H, Del Tredici K. Stages in the development of Parkinson's disease-related pathology. (2004) Cell Tissue Res, 318(1):121-34. Branco, D. M., Arduino, D. M., Esteves, A. R., Silva, D. F. F., Cardoso, S. M., & Oliveira, C. R. (2010). Cross-Talk Between Mitochondria and Proteasome in Parkinson’s Disease Pathogenesis. Frontiers in Aging Neuroscience, 2, 17. Butler, E. K., Voigt, A., Lutz, A. K. et al. (2012) The mitochondrial chaperone protein TRAP1 mitigates alpha-Synuclein toxicity. PLoS Genet, 8, e1002488. Calne, D. B. and Langston, J. W. (1983) Aetiology of Parkinson's disease. Lancet, 2, 1457- 1459. Chartier-Harlin, M. C., Kachergus, J., Roumier, C. et al. (2004) Alpha-synuclein locus duplication as a cause of familial Parkinson's disease. Lancet, 364, 1167-1169. Chen, Y. and Dorn, G. W. (2013) PINK1-Phosphorylated Mitofusin 2 Is a Parkin Receptor for Culling Damaged Mitochondria. Science, 340, 471-475. Clark, I. E., Dodson, M. W., Jiang, C., Cao, J. H., Huh, J. R., Seol, J. H., Yoo, S. J., Hay, B. A. and Guo, M. (2006) Drosophila pink1 is required for mitochondrial function and interacts genetically with parkin. Nature, 441, 1162-1166. Cola, N. B., DiEuliis D., Leo D., Mitchell D. C., Nussbaum R. L. (2008) Mitochondrial translocation of -Synuclein is promoted by intracellular acidification. Exp Cell Res, 314, 2076-2089. Costa, A. C., Loh, S. H. and Martins, L. M. (2013) Drosophila Trap1 protects against mitochondrial dysfunction in a PINK1/parkin model of Parkinson's disease. Cell Death Dis, 4, e467. Damier, P., Hirsch, E. C., Agid, Y. and Graybiel, A. M. (1999) The substantia nigra of the human brain. II. Patterns of loss of dopamine-containing neurons in Parkinson's disease. Brain, 122 ( Pt 8), 1437-1448.

Chapter 5 – References 61

Darios, F., Corti, O., Lucking, C. B. et al. (2003) Parkin prevents mitochondrial swelling and cytochrome c release in mitochondria-dependent cell death. Hum Mol Genet, 12, 517- 526. Deng, H., Dodson, M. W., Huang, H. and Guo, M. (2008) The Parkinson's disease genes pink1 and parkin promote mitochondrial fission and/or inhibit fusion in Drosophila. Proc Natl Acad Sci U S A, 105, 14503-14508. Devi, L., Raghavendran, V., Prabhu, B. M., Avadhani, N. G. and Anandatheerthavarada, H. K. (2008) Mitochondrial import and accumulation of alpha-synuclein impair complex I in human dopaminergic neuronal cultures and Parkinson disease brain. J Biol Chem, 283, 9089-9100. Doty, R. L. (2012) Olfactory dysfunction in Parkinson disease. Nat Rev Neurol, 8, 329-339. Eisbach SE, Outeiro TF. Alpha-synuclein and intracellular trafficking: impact on the spreading of Parkinson's disease pathology. J Mol Med (Berl). 2013 Jun;91(6):693- 703. Farrer, M., Chan, P., Chen, R. et al. (2001) Lewy bodies and parkinsonism in families with parkin mutations. Ann Neurol, 50, 293-300. Feany, M. B. and Bender, W. W. (2000) A Drosophila model of Parkinson's disease. Nature, 404, 394-398. Fitzgerald, J. C. and Plun-Favreau, H. (2008) Emerging pathways in genetic Parkinson's disease: autosomal-recessive genes in Parkinson's disease--a common pathway? FEBS J, 275, 5758-5766. Freundt EC, Maynard N, Clancy EK, Roy S, Bousset L, Sourigues Y, Covert M, Melki R, Kirkegaard K, Brahic M. Neuron-to-neuron transmission of α-synuclein fibrils through axonal transport. Ann Neurol. 2012 Oct;72(4):517-24. Gandhi, S., Muqit, M. M., Stanyer, L. et al. (2006) PINK1 protein in normal human brain and Parkinson's disease. Brain, 129, 1720-1731. Gandhi, S., Wood-Kaczmar, A., Yao, Z., et al. (2009) PINK1-Associated Parkinson's Disease Is Caused by Neuronal Vulnerability to Calcium-Induced Cell Death. Molecular Cell, 33, 627-638. Gegg, M. E., Cooper, J. M., Chau, K. Y., Rojo, M., Schapira, A. H. and Taanman, J. W. (2010) Mitofusin 1 and mitofusin 2 are ubiquitinated in a PINK1/parkin-dependent manner upon induction of mitophagy. Hum Mol Genet, 19, 4861-4870.

Chapter 5 – References 62

Geisler, S., Holmstrom, K. M., Skujat, D., Fiesel, F. C., Rothfuss, O. C., Kahle, P. J. and Springer, W. (2010) PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1. Nat Cell Biol, 12, 119-131. Chen, H. and Chan, D. C. (2009) Mitochondrial dynamics–fusion, fission, movement, and mitophagy–in neurodegenerative diseases. Hum. Mol. Genet.,18 (R2), R169-R176. Gibb, W. R. and Lees, A. J. (1988) The relevance of the Lewy body to the pathogenesis of idiopathic Parkinson's disease. J Neurol Neurosurg Psychiatry, 51, 745-752. Glauser, L., Sonnay, S., Stafa, K. and Moore, D. J. (2011) Parkin promotes the ubiquitination and degradation of the mitochondrial fusion factor mitofusin 1. J Neurochem, 118, 636-645. Greenamyre, J. T., Betarbet, R. and Sherer, T. B. (2003) The rotenone model of Parkinson's disease: genes, environment and mitochondria. Parkinsonism Relat Disord, 9 Suppl 2, S59-64. Greenamyre, J. T., Sherer, T. B., Betarbet, R. and Panov, A. V. (2001) Complex I and Parkinson's disease. IUBMB Life, 52, 135-141. Greene, A. W., Grenier, K., Aguileta, M. A., Muise, S., Farazifard, R., Haque, M. E., McBride, H. M., Park, D. S. and Fon, E. A. (2012) Mitochondrial processing peptidase regulates PINK1 processing, import and Parkin recruitment. EMBO Rep, 13, 378-385. Greene, J. C., Whitworth, A. J., Kuo, I., Andrews, L. A., Feany, M. B. and Pallanck, L. J. (2003) Mitochondrial pathology and apoptotic muscle degeneration in Drosophila parkin mutants. P Natl Acad Sci USA, 100, 4078-4083. Gu, M., Cooper, J. M., Taanman, J. W. and Schapira, A. H. (1998) Mitochondrial DNA transmission of the mitochondrial defect in Parkinson's disease. Ann Neurol, 44, 177- 186. Haas, R. H., Nasirian, F., Nakano, K., Ward, D., Pay, M., Hill, R. and Shults, C. W. (1995) Low platelet mitochondrial complex I and complex II/III activity in early untreated Parkinson's disease. Ann Neurol, 37, 714-722. Haque, M. E., Thomas, K. J., D'Souza, C. et al. (2008) Cytoplasmic Pink1 activity protects neurons from dopaminergic neurotoxin MPTP. Proc Natl Acad Sci U S A, 105, 1716- 1721. Hayashi, S., Wakabayashi, K., Ishikawa, A. et al. (2000) An autopsy case of autosomal- recessive juvenile parkinsonism with a homozygous exon 4 deletion in the parkin gene. Mov Disord, 15, 884-888.

Chapter 5 – References 63

Healy, D. G., Abou-Sleiman, P. M. and Wood, N. W. (2004) PINK, PANK, or PARK? A clinicians' guide to familial parkinsonism. Lancet Neurol, 3, 652-662. Hughes, A. J., Daniel, S. E. and Lees, A. J. (2001) Improved accuracy of clinical diagnosis of Lewy body Parkinson's disease. Neurology, 57, 1497-1499. Ibanez, P., Lesage, S., Lohmann, E., Thobois, S., De Michele, G., Borg, M., Agid, Y., Durr, A. and Brice, A. (2006) Mutational analysis of the PINK1 gene in early-onset parkinsonism in Europe and North Africa. Brain, 129, 686-694. Ikebe, S., Tanaka, M., Ohno, K., Sato, W., Hattori, K., Kondo, T., Mizuno, Y. and Ozawa, T. (1990) Increase of deleted mitochondrial DNA in the striatum in Parkinson's disease and senescence. Biochem Biophys Res Commun, 170, 1044-1048. Imai, Y., Kanao, T., Sawada, T. et al. (2010) The loss of PGAM5 suppresses the mitochondrial degeneration caused by inactivation of PINK1 in Drosophila. PLoS Genet, 6, e1001229. Jenner, P., Dexter, D. T., Sian, J., Schapira, A. H. and Marsden, C. D. (1992) Oxidative stress as a cause of nigral cell death in Parkinson's disease and incidental Lewy body disease. The Royal Kings and Queens Parkinson's Disease Research Group. Ann Neurol, 32 Suppl, S82-87. Jensen, P. H., Nielsen, M. S., Jakes, R., Dotti, C. G. and Goedert, M. (1998) Binding of alpha-synuclein to brain vesicles is abolished by familial Parkinson's disease mutation. J Biol Chem, 273, 26292-26294. Jin, S. M., Lazarou, M., Wang, C., Kane, L. A., Narendra, D. P. and Youle, R. J. (2010) Mitochondrial membrane potential regulates PINK1 import and proteolytic destabilization by PARL. J Cell Biol, 191, 933-942. Jin, S. M. and Youle, R. J. (2013) The accumulation of misfolded proteins in the mitochondrial matrix is sensed by PINK1 to induce PARK2/Parkin-mediated mitophagy of polarized mitochondria. Autophagy, 9. Kamp, F., Exner, N., Lutz, A. K. et al. (2010) Inhibition of mitochondrial fusion by alpha- synuclein is rescued by PINK1, Parkin and DJ-1. EMBO J, 29, 3571-3589. Kane, L. A., Lazarou, M., Fogel, A. I., Li, Y., Yamano, K., Sarraf, S. A., Banerjee, S. and Youle, R. J. (2014) PINK1 phosphorylates ubiquitin to activate Parkin E3 ubiquitin ligase activity. J Cell Biol, 205, 143-153. Kato, H., Lu, Q., Rapaport, D. and Kozjak-Pavlovic, V. (2013) Tom70 is essential for PINK1 import into mitochondria. PLoS One, 8, e58435.

Chapter 5 – References 64

Kawajiri, S., Saiki, S., Sato, S., Sato, F., Hatano, T., Eguchi, H. and Hattori, N. (2010) PINK1 is recruited to mitochondria with parkin and associates with LC3 in mitophagy. FEBS Lett, 584, 1073-1079. Kazlauskaite, A., Kondapalli, C., Gourlay, R. et al. (2014) Parkin is activated by PINK1- dependent phosphorylation of ubiquitin at Ser65. Biochem J, 460, 127-139. Kitada, T., Asakawa, S., Hattori, N., Matsumine, H., Yamamura, Y., Minoshima, S., Yokochi, M., Mizuno, Y. and Shimizu, N. (1998) Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism. Nature, 392, 605-608. Klionsky, D. J. (2010) The molecular machinery of autophagy and its role in physiology and disease. Semin Cell Dev Biol, 21, 663. Kordower JH, Chu Y, Hauser RA, Freeman TB, Olanow CW. Lewy body-like pathology in long-term embryonic nigral transplants in Parkinson's disease.Nat Med. 2008 May;14(5):504-6. Koyano, F., Okatsu, K., Kosako, H. et al. (2014) Ubiquitin is phosphorylated by PINK1 to activate parkin. Nature, 510, 162. Krige, D., Carroll, M. T., Cooper, J. M., Marsden, C. D. and Schapira, A. H. (1992) Platelet mitochondrial function in Parkinson's disease. The Royal Kings and Queens Parkinson Disease Research Group. Ann Neurol, 32, 782-788. Kruger, R., Kuhn, W., Muller, T. et al. (1998) Ala30Pro mutation in the gene encoding alpha-synuclein in Parkinson's disease. Nat Genet, 18, 106-108. Kubo, S. I., Kitami, T., Noda, S. et al. (2001) Parkin is associated with cellular vesicles. J Neurochem, 78, 42-54. Langston, J. W., Ballard, P., Tetrud, J. W. and Irwin, I. (1983) Chronic Parkinsonism in humans due to a product of meperidine-analog synthesis. Science, 219, 979-980. Langston, J. W., Irwin, I., Langston, E. B. and Forno, L. S. (1984) 1-Methyl-4- phenylpyridinium ion (MPP+): identification of a metabolite of MPTP, a toxin selective to the substantia nigra. Neurosci Lett, 48, 87-92. Lazarou, M., Jin, S. M., Kane, L. A. and Youle, R. J. (2012) Role of PINK1 binding to the TOM complex and alternate intracellular membranes in recruitment and activation of the E3 ligase Parkin. Dev Cell, 22, 320-333. Liu S, Lu B (2010) Reduction of Protein Translation and Activation of Autophagy Protect against PINK1 Pathogenesis in Drosophila melanogaster. PLoS Genet 6(12).

Chapter 5 – References 65

Liu S, Sawada T, Lee S, Yu W, Silverio G, et al. (2012) Parkinson's Disease–Associated Kinase PINK1 Regulates Miro Protein Level and Axonal Transport of Mitochondria. PLoS Genet 8(3). Lucking, C. B., Durr, A., Bonifati, V. et al. (2000) Association between early-onset Parkinson's disease and mutations in the parkin gene. N Engl J Med, 342, 1560-1567. Maraganore, Demetrius M., Lesnick, Timothy G., Elbaz, Alexis et al. (2004) UCHL1 is a Parkinson's disease susceptibility gene. Annals of neurology, 55, 512-521 Maroteaux, L., Campanelli, J. T. and Scheller, R. H. (1988) Synuclein: a neuron-specific protein localized to the nucleus and presynaptic nerve terminal. J Neurosci, 8, 2804- 2815. Marsden, C. D. and Parkes, J. D. (1977) Success and problems of long-term levodopa therapy in Parkinson's disease. Lancet, 1, 345-349. Matassa, D. S., Amoroso, M. R., Maddalena, F., Landriscina, M. and Esposito, F. (2012) New insights into TRAP1 pathway. Am J Cancer Res, 2, 235-248. McLelland, G. L., Soubannier, V., Chen, C. X., McBride, H. M. and Fon, E. A. (2014) Parkin and PINK1 function in a vesicular trafficking pathway regulating mitochondrial quality control. Embo Journal, 33, 282-295. Menzies, F. M., Yenisetti, S. C. and Min, K. T. (2005) Roles of Drosophila DJ-1 in survival of dopaminergic neurons and oxidative stress. Curr Biol, 15, 1578-1582. Michiorri, S., Gelmetti, V., Giarda, E. et al. (2010) The Parkinson-associated protein PINK1 interacts with Beclin1 and promotes autophagy. Cell Death Differ, 17, 962-974. Morais, V. A., Verstreken, P., Roethig, A. et al. (2009) Parkinson's disease mutations in PINK1 result in decreased Complex I activity and deficient synaptic function. Embo Molecular Medicine, 1, 99-111. Narendra, D., Kane, L. A., Hauser, D. N., Fearnley, I. M. and Youle, R. J. (2010a) p62/SQSTM1 is required for Parkin-induced mitochondrial clustering but not mitophagy; VDAC1 is dispensable for both. Autophagy, 6, 1090-1106. Narendra, D., Tanaka, A., Suen, D. F. and Youle, R. J. (2008) Parkin is recruited selectively to impaired mitochondria and promotes their autophagy. J Cell Biol, 183, 795-803. Narendra, D. P., Jin, S. M., Tanaka, A., Suen, D. F., Gautier, C. A., Shen, J., Cookson, M. R. and Youle, R. J. (2010b) PINK1 is selectively stabilized on impaired mitochondria to activate Parkin. PLoS Biol, 8, e1000298.

Chapter 5 – References 66

Obeso, J. A., Rodriguez-Oroz, M. C., Rodriguez, M., Lanciego, J. L., Artieda, J., Gonzalo, N. and Olanow, C. W. (2000) Pathophysiology of the basal ganglia in Parkinson's disease. Trends Neurosci, 23, S8-19. Pakkenberg, B., Moller, A., Gundersen, H. J., Mouritzen Dam, A. and Pakkenberg, H. (1991) The absolute number of nerve cells in substantia nigra in normal subjects and in patients with Parkinson's disease estimated with an unbiased stereological method. J Neurol Neurosurg Psychiatry, 54, 30-33. Panaretou B, Prodromou C, Roe SM, O'Brien R, Ladbury JE, Piper PW, Pearl LH. ATP binding and hydrolysis are essential to the function of the Hsp90 molecular chaperone in vivo. EMBO J. 1998;17(16):4829-4836. Panov, A., Dikalov, S., Shalbuyeva, N., Taylor, G., Sherer, T. and Greenamyre, J. T. (2005) Rotenone model of Parkinson disease: multiple brain mitochondria dysfunctions after short term systemic rotenone intoxication. J Biol Chem, 280, 42026-42035. Park, J., Lee, S. B., Lee, S. et al. (2006) Mitochondrial dysfunction in Drosophila PINK1 mutants is complemented by parkin. Nature, 441, 1157-1161. Parker, W. D., Jr., Boyson, S. J. and Parks, J. K. (1989) Abnormalities of the electron transport chain in idiopathic Parkinson's disease. Ann Neurol, 26, 719-723. Parker, W. D., Jr., Parks, J. K. and Swerdlow, R. H. (2008) Complex I deficiency in Parkinson's disease frontal cortex. Brain Res, 1189, 215-218. Peeraully, T., Yong, M. H., Chokroverty, S. and Tan, E. K. (2012) Sleep and Parkinson's disease: a review of case-control polysomnography studies. Mov Disord, 27, 1729- 1737. Polymeropoulos, M. H., Lavedan, C., Leroy, E. et al. (1997) Mutation in the alpha-synuclein gene identified in families with Parkinson's disease. Science, 276, 2045-2047. Poole, A. C., Thomas, R. E., Yu, S., Vincow, E. S. and Pallanck, L. (2010) The mitochondrial fusion-promoting factor mitofusin is a substrate of the PINK1/parkin pathway. PLoS One, 5, e10054. Plun-Favreau, H., Klupsch, K., Moisoi, N., et al. (2007) The mitochondrial protease HtrA2 is regulated by Parkinson's disease-associated kinase PINK1. Nature cell biology, 9, 1243-52. Pramstaller, P. P., Schlossmacher, M. G., Jacques, T. S. et al. (2005) Lewy body Parkinson's disease in a large pedigree with 77 Parkin mutation carriers. Ann Neurol, 58, 411-422.

Chapter 5 – References 67

Pridgeon, J. W., Olzmann, J. A., Chin, L. S. and Li, L. (2007) PINK1 protects against oxidative stress by phosphorylating mitochondrial chaperone TRAP1. PLoS Biol, 5, e172. Ramirez, A., Heimbach, A., Gruendemann, J. et al. (2006) Hereditary parkinsonism with dementia is caused by mutations in ATP13A2, encoding a lysosomal type 5 P-type ATPase. Nature Genetics, 38, 1184-1191. Rey NL, Petit GH, Bousset L, Melki R, Brundin P. Transfer of human α-synuclein from the olfactory bulb to interconnected brain regions in mice. Acta Neuropathol. 2013 Aug 8. Rhodenizer, D., Martin, I., Bhandari, P., Pletcher, S. D. and Grotewiel, M. (2008) Genetic and environmental factors impact age-related impairment of negative geotaxis in Drosophila by altering age-dependent climbing speed. Exp Gerontol, 43, 739-748. Robotta M., Gerding H. R., Vogel A., Hauser K., Schildknecht S., Karreman C., Leist M., Subramaniam V., Drescher M. (2014) ChemBioChem, 15, 2499-2502. Schapira, A. H. (2008) Mitochondria in the aetiology and pathogenesis of Parkinson's disease. Lancet Neurol, 7, 97-109. Schapira, A. H., Cooper, J. M., Dexter, D., Clark, J. B., Jenner, P. and Marsden, C. D. (1990) Mitochondrial complex I deficiency in Parkinson's disease. J Neurochem, 54, 823-827. Schapira, A. H., Cooper, J. M., Dexter, D., Jenner, P., Clark, J. B. and Marsden, C. D. (1989) Mitochondrial complex I deficiency in Parkinson's disease. Lancet, 1, 1269. Sherer, T. B., Betarbet, R. and Greenamyre, J. T. (2002) Environment, mitochondria, and Parkinson's disease. Neuroscientist, 8, 192-197. Sherer, T. B., Betarbet, R., Testa, C. M. et al. (2003a) Mechanism of toxicity in rotenone models of Parkinson's disease. J Neurosci, 23, 10756-10764. Sherer, T. B., Kim, J. H., Betarbet, R. and Greenamyre, J. T. (2003b) Subcutaneous rotenone exposure causes highly selective dopaminergic degeneration and alpha-synuclein aggregation. Exp Neurol, 179, 9-16. Shimura, H., Hattori, N., Kubo, S. et al. (2000) Familial Parkinson disease gene product, parkin, is a ubiquitin-protein ligase. Nat Genet, 25, 302-305. Shoffner, J. M., Watts, R. L., Juncos, J. L., Torroni, A. and Wallace, D. C. (1991) Mitochondrial oxidative phosphorylation defects in Parkinson's disease. Ann Neurol, 30, 332-339. Shulman, J. M., De Jager, P. L. and Feany, M. B. (2011) Parkinson's disease: genetics and pathogenesis. Annu Rev Pathol, 6, 193-222.

Chapter 5 – References 68

Singleton, A. B., Farrer, M., Johnson, J. et al. (2003) alpha-Synuclein locus triplication causes Parkinson's disease. Science, 302, 841. Spillantini, M. G., Divane, A. and Goedert, M. (1995) Assignment of human alpha-synuclein (SNCA) and beta-synuclein (SNCB) genes to chromosomes 4q21 and 5q35. Genomics, 27, 379-381. Spillantini, M. G., Schmidt, M. L., Lee, V. M., Trojanowski, J. Q., Jakes, R. and Goedert, M. (1997) Alpha-synuclein in Lewy bodies. Nature, 388, 839-840. Sterky, F. H., Hoffman, A. F., Milenkovic, D. et al. (2012) Altered dopamine metabolism and increased vulnerability to MPTP in mice with partial deficiency of mitochondrial complex I in dopamine neurons. Hum Mol Genet, 21, 1078-1089. Strauss, K. M., Martins, L. M., Plun-Favreau, H. et al. (2005) Loss of function mutations in the gene encoding Omi/HtrA2 in Parkinson's disease. Hum Mol Genet, 14, 2099-2111. Taipa R, Pinho J, Melo-Pires M. Clinico-Pathological. (2012) Correlations of the Most Common Neurodegenerative Dementias. Frontiers in Neurology, 3, 68. Taira, T., Saito, Y., Niki, T., Iguchi-Ariga, S. M., Takahashi, K. and Ariga, H. (2004) DJ-1 has a role in antioxidative stress to prevent cell death. EMBO Rep, 5, 213-218. Tan, E. K. and Skipper, L. M. (2007) Pathogenic mutations in Parkinson disease. Hum Mutat, 28, 641-653. Tanaka, A., Cleland, M. M., Xu, S., Narendra, D. P., Suen, D. F., Karbowski, M. and Youle, R. J. (2010) Proteasome and p97 mediate mitophagy and degradation of mitofusins induced by Parkin. J Cell Biol, 191, 1367-1380. Valente, E. M., Abou-Sleiman, P. M., Caputo, V. et al. (2004) Hereditary early-onset Parkinson's disease caused by mutations in PINK1. Science, 304, 1158-1160. Vilain, S., Esposito, G., Haddad, D. et al. (2012) The Yeast Complex I Equivalent NADH Dehydrogenase Rescues pink1 Mutants. Plos Genetics, 8. Vives-Bauza, C., Zhou, C., Huang, Y. et al. (2010) PINK1-dependent recruitment of Parkin to mitochondria in mitophagy. Proc Natl Acad Sci U S A, 107, 378-383. Wang, X., Winter, D., Ashrafi, G. et al. (2011) PINK1 and Parkin target Miro for phosphorylation and degradation to arrest mitochondrial motility. Cell, 147, 893-906. Wasner, G. and Deuschl, G. (2012) Pains in Parkinson disease--many syndromes under one umbrella. Nat Rev Neurol, 8, 284-294. Weihofen, A., Ostaszewski, B., Minami, Y. and Selkoe, D. J. (2008) Pink1 Parkinson mutations, the Cdc37/Hsp90 chaperones and Parkin all influence the maturation or subcellular distribution of Pink1. Hum Mol Genet, 17, 602-616.

Chapter 5 – References 69

Whitworth, A. J., Theodore, D. A., Greene, J. C., Benes, H., Wes, P. D. and Pallanck, L. J. (2005) Increased glutathione S-transferase activity rescues dopaminergic neuron loss in a Drosophila model of Parkinson's disease. Proc Natl Acad Sci U S A, 102, 8024- 8029. Wood-Kaczmar, A., Gandhi, S. and Wood, N. W. (2006) Understanding the molecular causes of Parkinson's disease. Trends Mol Med, 12, 521-528. Xiong, H., Wang, D., Chen, L. et al. (2009) Parkin, PINK1, and DJ-1 form a ubiquitin E3 ligase complex promoting unfolded protein degradation. J Clin Invest, 119, 650-660. Yamano, K. and Youle, R. J. (2013) PINK1 is degraded through the N-end rule pathway. Autophagy, 9. Yang, Y., Gehrke, S., Imai, Y. et al. (2006) Mitochondrial pathology and muscle and dopaminergic neuron degeneration caused by inactivation of Drosophila Pink1 is rescued by Parkin. Proc Natl Acad Sci U S A, 103, 10793-10798. Yoshino, H., Nakagawa-Hattori, Y., Kondo, T. and Mizuno, Y. (1992) Mitochondrial complex I and II activities of lymphocytes and platelets in Parkinson's disease. J Neural Transm Park Dis Dement Sect, 4, 27-34. Zhang, L., Karsten, P., Hamm, S. et al. (2013) TRAP1 rescues PINK1 loss-of-function phenotypes. Hum Mol Genet, 22, 2829-2841. Zhou, C., Huang, Y., Shao, Y., May, J., Prou, D., Perier, C., Dauer, W., Schon, E. A. and Przedborski, S. (2008) The kinase domain of mitochondrial PINK1 faces the cytoplasm. Proc Natl Acad Sci U S A, 105, 12022-12027. Zimprich, A., Biskup, S., Leitner, P. et al. (2004) Mutations in LRRK2 cause autosomal- dominant Parkinsonism with pleomorphic pathology. Neuron, 44, 601-607.

Appendix 70

Appendix

Table 10. A genetic screen on mitochondrially functional proteins by lethality CG, CG (Computed Gene) number. Column ‘A’, ubiquitous expression of accordin RNAi would cause lethality (l) or viable (v) of the progenies. Column ‘B’, ubiquitous expression of accordin RNAi of the ‘l’ candidates and hTrap1WT co-expressed would cause viable, ‘yes’ would be marked, and if cause lethal, ‘no’ would be noted. CG A B Name/Orthology Symbol Category 2-oxoglutarate dehydrogenase E1 component, Neural 11661 l no Nc73EF conserved at 73EF 2-oxoglutarate dehydrogenase E2 component 5075 v - (dihydrolipoamide succinyltransferase) 4706 v - aconitate hydratase 1 / homoaconitase 9244 l no aconitate hydratase 1 / homoaconitase, Aconitase Acon 8322 l no ATP citrate lyase ATPCL 3944 l no citrate synthase kdn 14740 v - citrate synthase 4095 v - fumarate hydratase, class II 6140 v - fumarate hydratase, class II TCA Cycle 12233 l no isocitrate dehydrogenase (NAD+) l(1)G0156 5261 v - Malate dehydrogenase 1 Mdh1 7998 v - Malate dehydrogenase 2 Mdh2 17725 v - Phosphoenolpyruvate carboxykinase Pepck 1516 v - pyruvate carboxylase 7010 l no pyruvate dehydrogenase E1 component subunit alpha l(1)G0334 8808 v - Pyruvate dehydrogenase kinase Pdk 6255 l no succinyl-CoA synthetase alpha subunit 10622 v - succinyl-CoA synthetase beta subunit Sucb 11963 v - succinyl-CoA synthetase beta subunit, skpA associated protein skap 6463 l no NADH dehydrogenase 34439 v - NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 1 NDUFa1 9350 v - NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 11 NDUFa11 3214 v - NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 12 NDUFa12 3483 v - NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 13 NDUFa13 32230 v - NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 4 NDUFa4 Complex I 6439 l yes NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 5 NDUFa5 NADH 7712 l no NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 6 NDUFa6 dehydrogena se 6020 l no NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 9 NDUFa9 18624 l yes NADH dehydrogenase (ubiquinone) 1 beta subcomplex 1 NDUFb1 NDUFb10, 8844 l no NADH dehydrogenase (ubiquinone) 1 beta subcomplex 10 Pdsw 6008 l no NADH dehydrogenase (ubiquinone) 1 beta subcomplex 11 NDUFb11 10320 l no NADH dehydrogenase (ubiquinone) 1 beta subcomplex 3 NDUFb3 5389 v - NADH dehydrogenase (ubiquinone) 1 beta subcomplex 7 NDUFb7

Appendix 71

12400 v - NADH dehydrogenase (ubiquinone) 1 subcomplex unknown 2 NDUFc2 11913 v - NADH dehydrogenase (ubiquinone) Fe-S protein 2 NDUFS2 11455 v - NADH dehydrogenase (ubiquinone) Fe-S protein 5 NDUFS5 8680 l no NADH dehydrogenase (ubiquinone) Fe-S protein 6 NDUFS6 2014 v - NADH dehydrogenase (ubiquinone) Fe-S protein 7 NDUFS7 9172 v - NADH dehydrogenase (ubiquinone) Fe-S protein 7 NDUFS7 4094 l yes NADH dehydrogenase (ubiquinone) Fe-S protein 8 NDUFS8 8102 v - NADH dehydrogenase (ubiquinone) flavoprotein 1 NDUFV1 9140 l no NADH dehydrogenase (ubiquinone) flavoprotein 1 NDUFV1 11423 v - NADH dehydrogenase (ubiquinone) flavoprotein 1 NDUFV1 5548 l no NADH dehydrogenase (ubiquinone) flavoprotein 2 NDUFV2 succinate dehydrogenase (ubiquinone) cytochrome b560 6485 l no SdhC subunit succinate dehydrogenase (ubiquinone) cytochrome b560 6666 l no SdhC subunit, Succinate dehydrogenase C 5703 l no succinate dehydrogenase (ubiquinone) flavoprotein subunit SdhA Succinate dehydrogena 5718 v - succinate dehydrogenase (ubiquinone) flavoprotein subunit se 7211 v - succinate dehydrogenase (ubiquinone) iron-sulfur subunit SdhB 7349 v - succinate dehydrogenase (ubiquinone) iron-sulfur subunit 3321 l no Succinate dehydrogenase B SdhB 4169 l no ubiquinol-cytochrome c reductase core subunit 2 QCR2 4769 l no ubiquinol-cytochrome c reductase cytochrome c1 subunit Cyt 1 14482 v - ubiquinol-cytochrome c reductase subunit 10 QCR10 Complex III Cytochrome 3612 l no ubiquinol-cytochrome c reductase subunit 7 QCR7 bc1 complex 7580 v - ubiquinol-cytochrome c reductase subunit 8 QCR8 8764 l no ubiquinol-cytochrome c reductase subunit 9, oxen QCR9, ox 31648 v - cytochrome c oxidase subunit 11 COX11 3861 l no cytochrome c oxidase subunit 15 COX15 9065 v - cytochrome c oxidase subunit 17 COX17 10664 l no cytochrome c oxidase subunit 4 CoIV 11015 l no cytochrome c oxidase subunit 5b CoVb 11043 v - cytochrome c oxidase subunit 5b CoVb Complex IV 14077 v - cytochrome c oxidase subunit 6a COX6a cytochrome levy, 17280 v - cytochrome c oxidase subunit 6a c oxidase COX6a 14235 l no cytochrome c oxidase subunit 6b CoVIb cype, 14028 l yes cytochrome c oxidase subunit 6c, cyclope COX6c 9603 l no cytochrome c oxidase subunit 7a COX7a 2249 v - cytochrome c oxidase subunit 7c CoVIIc 14724 l Cytochrome c oxidase subunit Va CoVa V-type H+-transporting ATPase 16kDa proteolipid subunit, Complex V, 32089 l no Vha16-2 Vacuolar H[+] ATPase subunit 16-2 ATP V-type H+-transporting ATPase 16kDa proteolipid subunit, synthase, V- 32090 v - Vha16-3 Vacuolar H[+] ATPase subunit 16-3 type

Appendix 72

V-type H+-transporting ATPase 16kDa proteolipid subunit, 9013 v - Vha16-4 Vacuolar H[+] ATPase subunit 16-4 V-type H+-transporting ATPase 21kDa proteolipid subunit, VhaPPA1- 7007 l no Vacuolar H[+] ATPase subunit PPA1-1 1 V-type H+-transporting ATPase 21kDa proteolipid subunit, VhaPPA1- 7026 l no Vacuolar H[+] ATPase subunit PPA1-2 2 V-type H+-transporting ATPase subunit A, V-ATPase 69 kDa 3803 l no Vha68-2 subunit 2 V-type H+-transporting ATPase subunit A, Vacuolar H[+] 12403 l no Vha68-1 ATPase subunit 68-1 V-type H+-transporting ATPase subunit A, Vacuolar H[+] 5037 l no Vha68-3 ATPase subunit 68-3 V-type H+-transporting ATPase subunit AC39, Vacuolar H[+] VhaAC39- 2934 l no ATPase subunit AC39-1 1 V-type H+-transporting ATPase subunit B, Vacuolar H[+]- 17369 l no Vha55 ATPase 55kD B subunit V-type H+-transporting ATPase subunit D, Vacuolar H[+] 8186 l no Vha36-1 ATPase subunit 36-1 V-type H+-transporting ATPase subunit H, Vacuolar H[+] VhaM9.7- 14909 v - ATPase subunit M9.7-d d V-type H+-transporting ATPase subunit I, Vacuolar H[+] 12602 v - Vha100-5 ATPase subunit 100-5 1088 l no Vacuolar H[+]-ATPase 26kD E subunit Vha26 F-type H+-transporting ATPase oligomycin sensitivity 4307 l no Oscp conferral protein, Oligomycin sensitivity-conferring protein F-type H+-transporting ATPase subunit 6,ATPase coupling ATPsyn- 4412 l no factor 6 Cf6 F-type H+-transporting ATPase subunit b, ATP synthase, 8189 l no ATPsyn-b subunit b 5362 l no F-type H+-transporting ATPase subunit beta Complex V F-type H+-transporting ATPase subunit beta, ATP synthase- ATPsyn- 11154 v - ATP beta beta synthase, F- 3446 v - F-type H+-transporting ATPase subunit e type 31477 v - F-type H+-transporting ATPase subunit epsilon 9032 l no F-type H+-transporting ATPase subunit epsilon, stunted sun 4692 l no F-type H+-transporting ATPase subunit f F-type H+-transporting ATPase subunit gamma, ATP ATPsyn- 7610 v - synthase-gamma chain gamma

Chapter 6 – Appendix 73

Curriculum Vitae

Family name, First name: Zhang, Li (张力) Place of birth: Chang Chun, Jilin Province, P.R.China Date of birh: 21.05.1984 Nationality: Chinese Gender: Female

Education

RWTH Aachen, Aachen, Germany (11. 2010 - 2015) PhD. Department of Neurology, University Medical Centre, RWTH Aachen. Research on the mechanisms of Parkinson’s Disease via Drosophila melanogaster model, especially focus on mitochondrial dysfunction. University of Regensburg, Regensburg, Germany (09. 2008 – 10. 2010) Master Degree of Science. Experimental & Clinical Neuroscience (Elite network of Bavaria). Master Thesis: Analysis of mitochondrial function in an alpha-synuclein Parkinson-model in Drosophila. National Huaqiao University Quanzhou, China (09. 2003 – 07. 2007) Bachelor Degree of Science, Biotechnology. Completed 72 courses on biology, chemistry, physics, mathematics, programming, English, et al. GPA 4.207 out of 5, ranked No.1 in the major. Bachelor Thesis: The mechanism of proline dependent heat-shock resistance in self- flocculating yeast. University of Jilin Changchun, China (02 - 03, 2007) Visiting student. School of Basic Medical Science Biological Products of China National Biotechnology Group Changchun, China (06 - 07, 2006) Internship.

Publication

Zhang L, Karsten P, Hamm S, Pogson JH, Müller-Rischart AK, Exner N, Haass C, Whitworth AJ, Winklhofer KF, Schulz JB, Voigt A.. TRAP1 rescues PINK1 loss-of-function phenotypes Hum Mol Genet. 2013 Jul 15;22(14):2829-2841. Epub 2013 Mar 21

Navarro, J, Heßner, S, Yenisetti, S, Bayersdorfer, F, Zhang, L, Voigt, A, Schneuwly, S, Botella, J. Analysis of dopaminergic neuronal dysfunction in genetic and toxin-induced models of Parkinson’s disease in Drosophila. Journal of Neurochemistry. 2014 Jul 10. doi: 10.1111/jnc.12818

Professional Conferences

Regional Drosophila Meeting 2014, Heidelberg, Germany (05. 2014) Oral presentation. Trap1, a new player in Parkinson’s disease ISN-ASN Meeting 2013 Cancun, Mexico (04. 2013) Poster Presentation. TRAP1 rescues PINK1 loss-of-function phenotypes Regional Drosophila Meeting 2012 Osnabrück, Germany (10. 2012) Oral presentation. a chaperone protein Trap1 rescues PINK1 loss-of-function phenotypes and mitochondrial dysfunction in vivo, 8th FENS Forum of Neuroscience Barcelona, Spain (07. 2012)

Chapter 6 – Appendix 74

Poster Presentation. Trap1 miti ates α-Synuclein-induced toxicity and rescues PINK1 loss-of- function phenotypes in vivo The European Conference on Visual Perception (ECVP) Regensburg, Germany (08. 2009) 8th Goettingen Meeting of the German Neuroscience Society Goettingen, Germany (03. 2009)

Professional Membership

International Society for Neurochemistry Federation of European Neuroscience Societies Elite network of Bavaria, Germany

Awards

Travel grant (1200 USD, ISN-ASN Meeting 2013) Annually First-class scholarship (award top 5% student of the department), Huaqiao University, China “Extraordinarily Excellent Student” (award top 0.5% student of university), Huaqiao University, China Winner of Experimental Skill Competition, Huaqiao University, China Once City Physics Competition and twice City Olympic Mathematics Competition, Changchun, China

Activities

Writing, published book ”Tan Fan Chen Gon Zhe De Zu Ji”, ISNB 978-7-206-05709, (title translation: Trace the Footprints of the Greats). Painting. Was a student leader for years.