Synuclein Toxicity Using a Caenorhabditis Elegans

IDENTIFICATION OF NEUROPROTECTIVE GENES AGAINST ALPHA-

SYNUCLEIN TOXICITY USING A CAENORHABDITIS ELEGANS

PARKINSON DISEASE MODEL

SHUSEI HAMAMICHI

A DISSERTATION

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biological Sciences in the Graduate School of The University of Alabama

TUSCALOOSA, ALABAMA

2009

ABSTRACT

Recent functional analyses of nine gene products linked to familial forms of

Parkinson disease (PD) have revealed several cellular mechanisms that are associated with PD pathogenesis. For example, α-synuclein ( α-syn), a primary component of Lewy bodies found in both familial and idiopathic forms of PD, has been shown to cause defects in proteasomal and lysosomal protein degradation machineries and induce mitochondrial/oxidative stress. These findings are further supported by the fact that additional gene products are involved in the same pathways. While these studies have been invaluable to elucidate the etiology of this disease, it has been reported that monogenic forms of PD only account for 5-10% of all PD cases, indicating that multiple genetic susceptibility factors and intrinsic metabolic changes associated with aging may play a significant role. Here we report the use of an organism, Caenorhabditis elegans , to model two central PD pathological features to rapidly identify genetic components that modify α-syn misfolding in body wall muscles and neurodegeneration in DA neurons.

We determined that proteins that function in lysosomal protein degradation, signal transduction, vesicle trafficking, and glycolysis, when knocked down by RNAi, enhanced

α-syn misfolding. Furthermore, these components, when overexpressed, rescued DA neurons from α-syn-induced neurodegeneration, and several of them have been validated using mammalian system. Taken together, this study represents a novel set of gene products that are putative genetic susceptibility loci and potential therapeutic targets for

PD.

LIST OF ABBREVIATIONS AND SYMBOLS

6-OHDA 6-Hydroxydopamine

AD Alzheimer disease

ADE Anterior deirid neuron bp Base pair

°C Celsius cAMP Cyclic adenosine monophosphate cDNA Complementary DNA

CEP Cephalic neuron cGMP Cyclic guanosine monophosphate

COR C-terminal of Roc

D2 Dopamine 2

D3 Dopamine 3

DA Dopamine

DEPC Diethylpyrocarbonate

DNA Deoxyribonucleic acid

DOG 2-Deoxyglucose dsRNA Double-stranded RNA

E1 Ubiquitin-activating enzyme

E2 Ubiquitin-conjugating enzyme

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E3 Ubiquitin ligase

ER Endoplasmic reticulum

ERAD Endoplasmic reticulum-associated degradation

FRAP Fluorescence recovery after photobleaching

GAL4 Galactose metabolism 4

GFP Green fluorescent protein

GO Gene ontology

HD Huntington disease

HMG-CoA 3-Hydroxy-3-methyl-glutaryl-Coenzyme A hr Hour

IPTG Isopropyl β-D-thiogalactoside kDa Kilodalton

KOG Eukaryotic orthologous group

L3 Larval stage 3

L4 Larval stage 4

LB Luria-Bertani

L-DOPA L-3,4-Dihydroxyphenylalanine

MPP+ 1-Methyl-4-phenylpyridinium

MPTP 1-Methyl-4-phenyl-1,2,5,6-tetrahydropyridine mRNA messenger RNA

g Microgram

l Microliter miRNA MicroRNA mg Milligram ml Milliliter mM Millimolar

MAPK Mitogen-activated protein kinase

MAPKK Mitogen-activated protein kinase kinase

MAPKKK Mitogen-activated protein kinase kinase kinase n/a Not applicable

NGM Nematode growth medium

PARK Parkinson disease gene

PCR Polymerase chain reaction

PD Parkinson disease

PDE Posterior deirid neuron

RING Really interesting new gene

RNA Ribonucleic acid

RNAi RNA interference

Roc Ras of complex

ROS Reactive oxygen species rpm Revolutions per minute

RT Room temperature (25 °C)

RT-PCR Reverse transcriptase polymerase chain reaction

SAGE Serial analysis of gene expression

SD Standard deviation

SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis

SNP Single nucleotide polymorphism

UPR Unfolded protein response

UPS Ubiquitin-proteasome system

UTR Untranslated region

C. elegans Proteins

AGE-1 Aging alteration 1 (phosphoinositide 3-kinase)

ATGR-7 Autophagy 7

BAR-1 Beta-catenin/armadillo related 1

CDK-5 Cyclin dependent kinase 5

CED-3 Cell death abnormality 3 (caspase)

CLK-1 Clock 1 (demethoxyubiquinone hydroxylase)

CMK-1 Calcium/calmodulin-dependent protein kinase 1

CSNK-1 Casein kinase 1

DAF-2 Abnormal dauer formation 2 (insulin receptor)

DAF-16 Abnormal dauer formation 16 (forkhead Box 01A)

DAT-1 Dopamine transporter 1

DJR-1 Oncogene DJ-1

DJR-2 Oncogene DJ-1

DOP-2 Dopamine receptor 2 (D2-like receptor)

DPY-1 Dumpy 1 (collagen)

DPY-5 Dumpy 5 (collagen)

EAT-2 Eating 2 (nicotinic acetylcholine receptor)

GPI-1 Glucose-6-phosphate isomerase 1

HRD-1 HRD 1

HRDL-1 HRD-like 1

HSF-1 Heat-shock factor 1

ISP-1 Rieske iron sulphur protein

LRK-1 Leucine-rich repeats, Ras-like domain, kinase 1 (LRRK2)

MOM-4 More of MS 4 (MAPKKK7)

NHR-6 Nuclear hormone receptor family 6 (NURR1)

NPR-1 Neuropeptide receptor family 1

OBR-1 Oxysterol binding protein 1

PDR-1 Parkinson’s disease related 1 (parkin)

PINK-1 PTEN-induced putative kinase 1

PMK-1 p38 MAP kinase

ROL-6 Roller 6 (collagen)

SMF-1 Yeast SMF homolog (divalent metal transporter)

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TAG-278 Temporary assigned gene 278

TAP-1 TAK kinase/MOM-4 binding protein

TOR-2 Torsin 2 (torsinA)

TRX-1 Thioredoxin 1

UNC-32 Uncoordinated 32 (vacuolar proton-translocating ATPase)

UNC-51 Uncoordinated 51 (unc-51-like kinase 2)

UNC-54 Uncoordinated 54 (myosin class II heavy chain)

UNC-75 Uncoordinated 75 (CELF/BrunoL protein)

VPS-41 Vacuolar protein sorting 41

YKT-6 Yeast YKT6 homolog (v-SNARE)

Mammalian Proteins

α-Syn Alpha synuclein

ALDOA Aldolase A

AMF Autocrine motility factor

AMFR Autocrine motility factor receptor

Amyloid-β Amyloid beta

ASK1 Apoptosis signal-regulating kinase 1

ATG7 Autophagy 7

ATP13A2 ATPase, Type 13A2

BAG5 BCL2-associated athanogene 5

viii

CSNK1G3 Casein kinase 1, gamma-3

CHIP C-terminus of HSC70-interacting protein

Daxx Death-associated protein 6

DJ-1 Oncogene DJ-1

ERV29 Surfeit 4

FBXW7 F-box and WD40 domain protein 7

GAIP G protein alpha-interacting protein

GAPDH Glyceraldehyde-3-phosphate dehydrogenase

GBA Glucocerebrosidase

GIGYF2 GRB10-interacting GYF protein 2

GIPC GAIP C-terminus-interacting protein

GPI Glucose-6-phosphate isomerase

HDAC6 Histone deacetylase 6

HTRA2 HTRA serine peptidase 2

HSF1 Heat-shock factor 1

HSP70 Heat-shock protein, 70 kDa

HSPC117 Hypothetical protein 117

IgG Immunoglobulin G

INSR Insulin receptor

LRRK2 Leucine-rich repeat kinase 2

NRB54 Nuclear RNA-binding protein, 54 kDa

p38 Mitogen-activated protein kinase p38

Pael receptor Parkin-associated endothelin receptor

PDE9A Phosphodiesterase 9A

PI3K Phosphoinositide 3-kinase

PINK1 PTEN-induced putative kinase 1

PLK2 Polo-like kinase 2

PRKN Parkin

PSF Polypyrimidine tract-binding protein-associated splicing factor

Q82 Polyglutamine 82 containing protein

RAB1A Ras-associated protein 1A

RAB3A Ras-associated protein 3A

RAB8A Ras-associated protein 8A

RGS Regulators of G protein signaling

SEC22 Secretion deficient 22

SNCA Synuclein, alpha

SYVN1 Synoviolin 1

Ub Ubiquitin

Ubch7 Ubiquitin-conjugating enzyme 7

Ubch8 Ubiquitin-conjugating enzyme 8

UCHL Ubiquitin C-terminal hydrolase

UCHL1 Ubiquitin C-terminal hydrolase 1

USP10 Ubiquitin-specific protein 10

VMAT2 Vesicular monoamine transporter 2

VPS41 Vacuolar protein sorting 41

XIAP X-linked inhibitor of apoptosis

ACKNOWLEDGMENTS

First and foremost, I would like to thank Drs. Guy and Kim Caldwell, and the former and present members of the Caldwell lab. Among them, I wish to especially recognize highly motivated undergraduate students who undertook the enormous challenge of working in the PD research field. Most notably, I want to thank Renee

Rivas, Adam “Deuce” Knight, Susan DeLeon, and Paige Dexter. Without their help and contribution, I guarantee that none of our projects would have worked as smoothly as they did. Furthermore, I want to thank Cody Locke for making science intellectually stimulating.

Among the former and present non-undergraduates, I would like to acknowledge

Dr. Laura Berkowitz, Michelle Norris, Lindsay Faircloth, and Jenny Schieltz for their assistance in many untold and underappreciated areas of research. I also want to thank the usual Wilhagans gang including Jafa Armagost, Adam “Ace” Harrington, and AJ

Burdette for making my life as a graduate student more enjoyable and fulfilling. I wish all of them good luck for their future endeavors.

Outside of the Caldwell lab, I would like to thank my graduate committee members, Dr. Janis O’Donnell, Dr. Katrina Ramonell, and Dr. Jianhua Zhang for their continuous support and encouragement. Furthermore, I wish to acknowledge other faculty members and staff of the Department of Biological Sciences including Dr. Martha

Powell, Dr. Harriett Smith-Somerville, and many others for their assistance whenever I

xii

needed it and their enthusiasm toward my research. It has been a pleasure sharing research ideas and data annually, and receiving tremendously kind responses from all of you.

I also would like to express my gratitude to research collaborators, Dr. Susan

Lindquist (Whitehead Institute/MIT), Dr. David Standaert (UAB), Dr. Ted Dawson

(Johns Hopkins University), and Dr. Antonio Miranda Vizuete (Universidad Pablo de

Olavide). Thank you so much for allowing me to work on multiple influential research projects. Most notably, I would like to thank Dr. Joshua Kritzer and Dr. Chris Pacheco, both post-docs at the Lindquist lab, for believing in me.

Outside of the current research world, I want to thank my parents and my brother who are currently in Japan as well as my former PIs, Dr. Hideo Nishigori (Teikyo

University) and Dr. Mike Shipley (Midwestern State University). Furthermore, I wish to recognize all of my friends from all over the world. Thank you for our friendship and understanding of what we wish to achieve in our lives. I am who I am, and I do what I do because of you.

Lastly, I would like to acknowledge Sylvester Stallone for making a classic movie, Rocky, the ultimate source of my inspiration while working on my PNAS article, presentation slides used for my post-doc interview at Whitehead Institute, and this current dissertation. I only wanted to go the distance, but I had never thought I would have an opportunity to go to Boston.

xiii

CONTENTS

ABSTRACT ...... ii

LIST OF ABBREVIATIONS AND SYMBOLS ...... iii

ACKNOWLEDGMENTS ...... xii

LIST OF TABLES ...... xvii

LIST OF FIGURES ...... xviii

1. INTRODUCTION ...... 1 a. Parkinson disease ...... 1 b. PD pathological feature: Lewy bodies ...... 2 c. Genetics basis of PD ...... 3 d. PD pathogenesis: SNCA/ α-syn ...... 4 e. PD pathogenesis: proteasomal protein degradation ...... 5 f. PD pathogenesis: lysosomal protein degradation ...... 8 g. PD pathogenesis: mitochondrial/oxidative stress ...... 10 h. PD pathogenesis: signaling pathways ...... 13 i. Strategy using invertebrate models of PD ...... 14 j. C. elegans PD models ...... 17 k. Current studies ...... 19 l. References ...... 22 m. Figure legends ...... 33

xiv

2. HYPOTHESIS-BASED RNA INTERFERENCE SCREEN IDENTIFIES NEUROPROTECTIVE GENES IN A PARKINSON’S DISEASE MODEL...... 34 a. Abstract ...... 35 b. Introduction ...... 36 c. Materials and methods ...... 38 d. Results ...... 44 e. Discussion ...... 52 f. References ...... 58 g. Figure legends ...... 97

3. VALIDATION OF SUPPRESSORS OF ALPHA-SYNUCLEIN TOXICITY FROM YEAST GENETIC SCREENING ...... 100 a. Abstract ...... 101 b. Introduction ...... 102 c. Materials and methods ...... 104 d. Results ...... 106 e. Discussion ...... 108 f. References ...... 112 g. Figure legends ...... 117

4. RNAI SCREEN OF DAF-2-MODULATED AND DIFFERENTIALLY EXPRESSED GENES LINK METABOLIC ENZYMES TO NEUROPROTECTION ...... 119

a. Abstract ...... 120 b. Introduction ...... 121 c. Materials and methods ...... 124 d. Results ...... 128 e. Discussion ...... 132 f. References ...... 137 g. Figure legends ...... 168

5. CONCLUSION ...... 170 a. Introduction ...... 170 b. Neuroprotective mechanism of VPS41, ATG7, ULK2, and GIPC:

a common pathway? ...... 170 c. Defining networks of neuroprotective genes by miRNAs ...... 174 d. Additional PD-related studies using C. elegans ...... 176 e. Conclusion and future directions ...... 178 f. References ...... 181 g. Figure legends ...... 186

xvi

LIST OF TABLES

1.1. Summary of mutations in PD genes linked to PD pathogenesis

and their C. elegans orthologs ...... 30

1.2. Summary of selected invertebrate PD models ...... 31

2.1. Gene identities of the 20 top candidates isolated

from RNAi screening models ...... 62

2.2. Bioinformatic associations among gene candidates

identified by RNAi ...... 63

2.3. Summary of the neuroprotective genes and their human homologs...... 64

2.4. Results of all genes knocked down via RNAi screening...... 65

2.5. Summary of RNAi knockdown of the top 20 gene candidates in worms

expressing Q82::GFP + TOR-2 in body wall muscle cells ...... 89

4.1. Summary of genes analyzed by RNAi screen...... 141

4.2. Summary of positive genes from RNAi screen for effectors of α-syn

in the daf-2 background based on KOG and/or GO annotations...... 159

xvii

LIST OF FIGURES

1.1. Schematic representation of the cellular defects caused by

known PD genes...... 32

2.1. RNAi knockdown of specific gene targets enhances misfolding of α-syn .....90

2.2. Overexpression of candidate genes protects DA neurons from

α-syn-induced degeneration ...... 91

2.3. An interconnectivity map ...... 92

2.4. Expression of α-syn in worm DA neurons results in

age- and dose-dependent neurodegeneration ...... 93

2.5. Analysis of transgene expression in worm strains...... 94

2.6. RNAi knockdown of the top 20 gene targets did not enhance misfolding of

polyglutamine aggregates ...... 95

2.7. Quantitative analysis of the hit rate of genes at both the primary and

secondary level of RNAi screening ...... 96

3.1. RAB3A, RAB8A, PDE9A, and PLK2 protect against α-syn-induced

DA neuron loss...... 114

3.2. PARK9 antagonizes α-syn-mediated DA neuron degeneration

in C. elegans ...... 115

3.3. RNAi knockdown of W08D2.5 , R12E2.13 , and R06F6.8 does not reduce

α-syn or tor-2 mRNA expression levels ...... 116

xviii

4.1. Graphs depicting the percentage of α-syn-expressing daf-2 and/or

daf-16 mutants with wildtype DA neurons...... 161

4.2. Graph summarizing lifespan assay of N2 and daf-2 worms...... 162

4.3. daf-2 enhances degradation of α-syn::GFP fusion protein...... 163

4.4. Graph illustrating the percentage of α-syn-expressing worms with

wildtype DA after 2-deoxyglucose (DOG) treatment ...... 164

4.5. Graph illustrating the percentage of 7 day-old worms with wildtype

DA neurons expressing α-syn and gpi-1 or hrdl-1 ...... 165

4.6. Pie chart summarizing 53 positive genes from the RNAi screening...... 166

4.7. Diagram summarizing the DAF-2/insulin signaling pathway

in C. elegans ...... 167

5.1. Schematic diagram illustrating targets of mir-2/mir-43/mir-250/mir-797

superfamily by miRBase (top) and TargetScan (bottom)...... 184

5.2. Schematic diagram of CEP neuronal circuitry ...... 185

xix CHAPTER ONE

INTRODUCTION

Parkinson disease

Parkinson disease (PD) is the second most common neurodegenerative disease, affecting approximately 1% of the population aged over 50 (Polymeropoulos et al.,

1996). While the exact number of PD patients remains unclear, it is estimated that 1 to

1.5 million Americans are affected with this disease, and 50,000 are newly diagnosed each year. Concurrent with medical issues associated with physical disabilities and quality of life, the financial burden is predicted to cost additional $10,349 per a PD patient annually, totaling $23 billion in the United States (Huse et al., 2005). Globally, as world population continues to increase, Dorsey et al. (2007) projected the number of PD patients from 4.1-4.6 million in 2005 to 8.7 to 9.3 million in 2030. In the light of the fact that, presently, no cure for this disease exists, identifying new diagnostic and therapeutic targets or discovering novel therapeutic strategies remains a top priority in the PD research community.

Similar to Alzheimer disease (AD) and Huntington disease (HD), PD belongs to a group of movement disorders, clinically diagnosed to the individuals with muscle rigidity, tremor, bradykinesia, and postural instability. Post-mortem examination of the brains from the PD patients revealed a progressive loss of dopamine (DA) synthesizing neurons in the substantia nigra , a melanin-rich region in the basal ganglia.

Consequently, DA neuronal death in the substantia nigra affects nigrostriatal, mesocortical, mesolimbic, and tuberoinfundibular pathways, resulting in physical impairments as well as neuropsychatric symptoms including depression, dementia, and insomnia (Lees et al., 2009). Current treatment focuses on decelerating the progression of these symptoms [e.g., by restoring DA production via L-3,4-dihydroxyphenylalanine

(L-DOPA) administration]. The wide range of PD symptoms illustrates the complexity of its etiology in all facets of biological levels (e.g., molecules, neurons, nervous system, behavior, and genes vs. environment) (Lees et al., 2009; Lesage and Brice, 2009).

PD pathological feature: Lewy bodies

A pathological hallmark of PD at the cellular level is a formation of proteinaceous inclusions called Lewy bodies in the cytoplasm of the surviving DA neurons. The most predominant protein detected in the inclusions is a protein called α-synuclein ( α-syn;

PARK1 /SNCA ) (Spillantini et al., 1997), which is discussed in detail below.

Additionally, synphilin (Murray et al., 2003), parkin ( PARK2/PRKN ; Schlossmacher et al., 2001), torsinA (Sharma et al., 2001), and other proteins have been found in the Lewy bodies.

On the premise that these inclusion bodies, as well as reduction of the active proteins via aggregation formation, may interrupt normal cellular functions, previous research focused on identifying the components of Lewy bodies and their potential neuroprotective functions. While the neuroprotective capacities of parkin (Vercammen et

al., 2006) and torsinA (Cao et al., 2005) have been documented, observations that the

inclusion bodies are undetected in some PD cases suggests that the protein aggregates

may not be the only mechanism resulting in neuronal cell death. Further supporting this

view are the findings demonstrating that α-syn intermediate protofibrils are more

neurotoxic than those found in either the monomeric or oligomerized state (Conway et

al., 2001; Lashuel et al., 2002) by physically disrupting vesicular membranes (Volles et

al., 2001) or inhibiting the ubiquitin-proteasome system (UPS) (Zhang et al., 2008),

implying that the formation of more mature aggregates is instead neuroprotective. Taken

together, although a neurodegenerative or neuroprotective role for Lewy bodies is

controversial, the formation of the inclusion bodies remains as a definitive pathological

feature of both familial (genetic) and sporadic (environmental) forms of PD.

Genetic basis of PD

While familial hereditary influences have long been documented, due to a

complicated pattern of inheritance, genetic causes of this disease were unresolved until

late 1990’s (Nussbaum and Polymeropoulos, 1997). Presently, nine PARK genes have

been determined whereby mutations leading to modified function, altered expression

level, or subcellular mislocalization are linked to PD (Table 1.1). These genes include

PARK1 /SNCA (Polymeropoulos et al., 1997), PARK2/PRKN (Kitada et al., 1998),

PARK5/UCHL1 (Leroy et al., 1998), PARK6/PINK1 (Valente et al., 2004), PARK7/DJ-1

(Bonifati et al., 2002), PARK8/LRRK2 (Paisán-Ruíz et al., 2004), PARK9/ATP13A2

(Ramirez et al., 2006), PARK11/GIGYF2 (Lautier et al., 2008), and PARK13/HTRA2

(Strauss et al., 2005). The functional analyses of these PD-associated proteins suggest

multiple defective pathways that may lead to DA neurodegeneration (Dawson and

Dawson, 2003; Thomas and Beal, 2007) (Fig. 1.1).

PD pathogenesis: SNCA/ α-syn

The first PD gene discovered was SNCA /α-syn (Polymeropoulos et al., 1997),

which encodes natively unfolded 140 amino-acid protein with unknown function. α-Syn has been detected in presynaptic nerve termini (Jakes et al., 1994), and shown to bind to lipids (Perrin et al., 2000). Originally identified as a non-amyloid-β component of AD amyloid (Ueda et al., 1993), α-syn, similar to amyloid-β and polyglutamine-repeat containing proteins, is prone to aggregation. Subsequent analysis of SNCA gene has revealed that multiplication of SNCA loci enhanced α-syn expression, resulting in the protein aggregation and the onset of PD (Singleton et al., 2003).

Since the formation of Lewy bodies is a central pathological feature of both familial and sporadic forms of PD, most current PD research focuses on α-syn aggregation and cellular mechanisms involved in ameliorating it. For example, accumulation of α-syn has been shown to impair UPS function (Stefanis et al., 2001;

Zhang et al., 2008; Nonaka et al., 2009), and α-syn is degraded by lysosomes (Webb et al., 2003; Cuervo et al., 2004). Furthermore, overexpression of α-syn blocks

endoplasmic reticulum (ER) to Golgi trafficking (Cooper et al., 2006; Gitler et al., 2008),

and expression of mutant α-syn induces ER stress (Smith et al., 2005). Additionally, α- syn may also be targeted to mitochondria and impair complex I function via cryptic mitochondrial targeting signal (Devi et al., 2008). As discussed below, functional analysis of six out of nine PD-associated gene products illustrate involvement of defective proteasomal and lysosomal protein degradation machineries, as well as inadequate cellular response to mitochondrial and oxidative stress in PD pathogenesis.

PD pathogenesis: proteasomal protein degradation

The most common protein degradation machinery of the cell is the UPS, which consists of a variety of proteins including the ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), ubiquitin ligases (E3), ubiquitin carboxyl-terminal hydrolases (UCHL), and proteasomal subunits. Briefly, E1, E2, and E3 are involved in the processes of activating, transferring, and binding ubiquitins (Ubs) to target proteins that are degraded by proteasomes. After proteolysis, Ubs that are attached to the degraded products are recycled by UCHL to maintain the cytoplasmic Ub pool.

Misfolded proteins, such as α-syn (Stefanis et al., 2001; Zhang et al., 2008; Nonaka et al.,

2009), polyglutamine-repeat containing gene products (Bence et al., 2001; Bennett et al.,

2007; Iwata et al., 2009), and amyloid-β (Almeida et al., 2006) have been shown to impair UPS function.

Two PD genes, parkin/PRKN (E3 ubiquitin ligase) and UCHL1 are UPS

components. Kitada et al. (1998) utilized positional cloning to identify PRKN in

Japanese PD patients. Protein sequence analysis revealed that 465 amino-acid parkin encoded moderately similar sequence to Ub at the N terminus and a RING-finger motif (a common motif in E3 ligases) at the C terminus. Since mutations in PRKN lead to autosomal recessive PD, Kitada et al. (1998) proposed that the loss of E3 ubiquitin ligase activity (i.e., loss-of-function) of PRKN as one of the primary mechanisms involved in

PD pathogenesis.

Subsequent analysis of parkin function identified its interactors including E2 ubiquitin-conjugating enzymes Ubch7 (Shimura et al., 2000) and Ubch8 (Zhang et al.,

2000), F-box/WD repeat protein FBXW7 and cullin 1 (Staropoli et al., 2003), BAG5

(Kalia et al., 2004), and its substrates such as a G protein-coupled Pael receptor (Imai et al., 2001) and p38 (Corti et al., 2003). These findings link parkin to the UPS, cell death pathway, and the cellular response to unfolded proteins. Interestingly, parkin has also been shown to interact with 22 kDa glycosylated α-syn (Shimura et al., 2001), PD-linked mutant forms of DJ-1 (Moore et al., 2005), and LRRK2 (Smith et al., 2005) suggesting a common neurodegenerative pathway in seemingly heterogeneous PD forms.

The neuroprotective function of parkin has been well documented using various animal models. Jiang et al. (2004) overexpressed parkin in human DA neuroblastoma cells (SH-SY5Y) and observed neuroprotection against DA and 6-hydroxydopamine (6-

OHDA)-induced apoptosis by decreasing reactive oxygen species (ROS) and attenuating

c-Jun N-terminal kinase and caspase 3 activities. Similarly, Hasegawa et al. (2008)

expressed an enzyme tyrosinase ( an enzyme that catalyzes both the hydroxylation of

tyrosine to L-DOPA and the subsequent conversion of L-DOPA and DA to their specific

o-quinones ) in SH-SY5Y cells to over-produce endogenous DA leading to ROS-induced apoptosis. Co-expression of wildtype parkin suppressed oxidative stress-induced cell death by enhancing the activation of c-Jun N-terminal kinase and p38. Further demonstration of the neuroprotective function of this E3 ubiquitin ligase was shown by

Petrucelli et al. (2002) whereby PD-linked mutant α-syn was expressed in mouse primary midbrain culture, and it was determined that parkin rescued these catecholaminergic neurons from α-syn toxicity.

UCHL1 , initially identified as a PD gene by Leroy et al. (1998), is not well characterized since ongoing dispute regarding UCHL1 as a PD susceptibility gene

(compare Maraganore et al., 2004 vs. Healy et al., 2006) has minimized comprehensive research efforts. Despite the controversy, Liu et al. (2002) reported that α-syn and

UCHL1 are co-localized with the synaptic vesicles, and that co-overexpression of α-syn and both wildtype and mutant UCHL1 in COS-7 cells increased accumulation of α-syn aggregates. Since overexpression of UCHL1 should enhance α-syn degradation, they proposed an alternative UCHL1 function whereby dimerization of UCHL1 exhibits ubiquitin ligase activity. Additionally, Liu et al. (2008) demonstrated that farnesylated

UCHL1 is associated with the cellular membranes, and that treatment with

farnesyltransferase inhibitor enhanced cell survival. Nevertheless, a neuroprotective mechanism for UCHL1 remains unclear.

PD pathogenesis: lysosomal protein degradation

Lysosomes are organelles that contain digestive enzymes including lipases, carbohydrases, nucleases, and proteases to break down organelles, macromolecules, and microorganisms. For instance, through activation of autophagy, mitochondria

(mitophagy) and peroxisomes (pexophagy) are degraded in the lysosomes. Interestingly, recent findings indicate that bulk of misfolded and aggregated proteins, including α-syn

(Webb et al., 2003; Cuervo et al., 2004) and polyglutamine-repeat containing proteins

(Ravikumar et al., 2004; Yamamoto et al, 2006), are degraded by lysosomes via macroautophagy and/or chaperone-mediated autophagy. Further supporting the role of lysosomal function and PD pathogenesis is the association between PD and type I

Gaucher disease (Bembi et al., 2003). Type I Gaucher disease is an autosomal recessive lysosomal storage disorder that is caused by reduced activity of glucocerebrosidase

(GBA), which is a lysosomal enzyme that catalyzes the breakdown of glucosylceramide.

While the precise neurodegenerative mechanism of defective GBA in PD is unclear, it has been postulated that the mutations may interfere with normal lysosomal function and block α-syn clearance (Goker-Alpan et al., 2008). Additionally, knockdown of cathepsin

D has been shown to enhance α-syn aggregation whereas overexpression of this

lysosomal protease promotes α-syn degradation and DA neuron survival (Qiao et al.,

2008).

Since both proteasomal and lysosomal machineries effectively degrade misfolded proteins and promote cell survival, investigating a molecular “switch” that promotes one pathway over the other is a significant research interest. Thus far, two proteins, CHIP (an ubiquitin ligase that acts as a co-chaperone for protein quality control) and HDAC6 (a microtubule-associated deacetylase) have been documented to function in this mechanism. Shin et al. (2005) examined two functional domains within CHIP, and demonstrated that while the tetratricopeptide repeat is critical for proteasomal degradation of α-syn, the U-box domain is involved in lysosomal degradation. Further,

Pandey et al. (2007) reported that impairment of the UPS led to induction of autophagy in an HDAC6-dependent manner. These findings illustrate the interconnection between these two protein degradation pathways that may indeed be compensatory.

Mutations in PD gene, ATP13A2, which encodes a lysosomal P-type ATPase lead to autosomal recessive PD. By mutation screening and linkage analysis, Ramirez et al.

(2006) identified ATP13A2 from Chilean PD patients. They examined expression pattern and subcellular localization of ATP13A2, and determined that the gene is predominantly expressed in the brain, and that while the wildtype ATP13A2 protein is localized in the lysosomes, misfolded mutant forms are retained in the ER to be subsequently degraded by proteasomes. Surprisingly, they observed approximately a 10-fold increase in

ATP13A2 mRNA level in the surviving DA neurons from the substantia nigra of human

idiopathic PD post-mortem midbrains, suggesting the potential neuroprotective function

of this gene. As discussed in Chapter 3, we have shown that while knockdown of worm

ATP13A2 enhances α-syn misfolding, overexpression of this gene rescues worm DA

neurons from α-syn toxicity, demonstrating a novel genetic interaction between α-syn and ATP13A2 (Gitler et al., 2009).

PD pathogenesis: mitochondrial and oxidative stress

While cellular stress induced by misfolded or aggregated proteins may shed light

on the neurodegenerative mechanisms leading to PD, defects in protein degradation

machinery alone cannot explain the selective loss of DA neurons. PD pathogenesis

consists of both genetic and environmental causes, which only 5-10% of all PD cases

have been linked to genetic components. Studies on environmental PD factors have

provided insights on the pathways involved in the selective DA neurodegeneration.

Langston et al. (1986) studied four patients who exhibited features of clinical

Parkinsonism after using a new “synthetic heroin.” Subsequent analysis of the drug

components revealed 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) as a primary

compound, and they proposed that MPTP might induce the selective loss of DA neurons.

While MPTP was later found harmless, after crossing the blood-brain barrier, the

compound is readily metabolized into toxic 1-methyl-4-phenylpyridinium (MPP+), which

disrupts complex I of mitochondrial respiratory chain. Inhibition of complex I generates

ROS, which may oxidize DA (a neurotransmitter known to be readily oxidized) to produce highly toxic DA that results in DA neurodegeneration.

Two PD genes, PINK1 and HTRA2 are also linked to mitochondria. Valente et al.

(2004) mapped PINK1 from an Italian family with autosomal recessive PD, and determined that PINK1 when expressed in COS-7 and SH-SY5Y cells localized to mitochondria. Furthermore, using SH-SY5Y cells overexpressing wildtype or mutant

PINK1, they demonstrated that, after MG-132 (a proteasome inhibitor) treatment, wildtype PINK1 enhanced cell survival without modifying mitochondrial membrane potential whereas mutant PINK1 displayed no protection with decreased membrane potential. Taken together, these findings provided the first evidence linking a genetic cause of PD to mitochondria, further confirming the relationship between mitochondrial malfunction and PD pathogenesis, and suggesting a potential neuroprotective function of

PINK1.

Strauss et al. (2005) identified HTRA2 in German PD patients. To further validate their findings, they overexpressed wildtype and mutant HTRA2 in cell culture, and found that mutant form failed to interact with XIAP (an inhibitor of apoptosis), suggesting that misregulation of HTRA2 might be linked to PD pathogenesis. Moreover, they determined that HTRA2 is predominantly localized to mitochondria by immunohistochemistry, and observed distinct mitochondrial morphological changes by electron microscopy. Focusing on mitochondrial function, they examined mitochondrial membrane potential and cell viability, and showed that mutant HTRA2 decreased the

membrane potential and enhanced cell sensitivity to staurosporine. Intriguingly, noting

that HTRA2 protease activity requires trimerization in vivo , they determined that mutant

HTRA2 was able to form the protein complex with wildtype, and proposed that the mutant form may decrease protease activity without disrupting complex formation, providing a possible explanation for autosomal dominant inheritance of this gene.

DJ-1, although not directly linked to mitochondrial function, was identified by

Bonifati et al. (2003) while studying Dutch and Italian families with autosomal recessive

PD. To characterize the neuroprotective role of DJ-1, Junn et al. (2005) transfected SH-

SY5Y cells with wildtype or mutant DJ-1, and determined that overexpression of wildtype protein significantly protected the cells from hydrogen peroxide, DA, and

MPP+ insults, illustrating anti-oxidant properties of DJ-1. After identifying Daxx as one of the DJ-1 interactors via yeast two-hybrid screen, they also determined that wildtype

DJ-1 suppressed Daxx/ASK1-induced cell death.

Alternatively, Xu et al. (2005) described another neuroprotective mechanism of

DJ-1. They used affinity purification and mass spectrometry to detect a nuclear RNA binding protein NRB54 and PSF as DJ-1 interactors in SH-SY5Y cells. They showed that mutant DJ-1 exhibited reduced nuclear localization and decreased co-localization with NRB54 and PSF, readily allowing transcriptional repression by PSF. Furthermore, they examined wildtype DJ-1 in which the overexpression suppressed PSF-induced cell death as well as α-syn- and hydrogen peroxide-induced neurodegeneration. Taken

together, although anti-oxidant activity of DJ-1 is well supported, precise pathways

leading to neuroprotection need further clarification.

PD pathogenesis: signaling pathways

Both LRRK2 and GIGYF2 are signaling components, but precisely which

pathways they modify is unknown. Originally described by Paisan-Ruiz et al. (2004) by studying Spanish and British families affected by autosomal dominant PD, LRRK2 encodes a 2527 amino-acid protein with leucine-rich repeat, Roc GTPase, COR,

MAPKKK, and WD40 domains. Given the high frequency of G2019S mutation (in the

MAPKKK domain) in autosomal dominant (Di Fonzo et al., 2005) and idiopathic (Gilks et al., 2005) PD patients, West et al. (2005) characterized LRRK2 G2019S expressed in

HEK293 and SH-SY5Y cells, and determined that the mutant form exhibited

significantly higher kinase activity compared to wildtype LRRK2. West et al. (2008)

also analyzed 10 PD-linked LRRK2 mutations, and determined that LRRK2 GTPase activity regulates its kinase activity, and enhanced kinase activity leads to neurodegeneration.

GIGYF2 , similar to UCHL1 , is one of the least characterized PD genes because of ongoing disputes regarding the lack of evidence supporting its role in PD pathogenesis

(Bonifati, 2009). Focusing on one of the PARK11 microsatellite markers, D2S206, which is found in the intron of GIGYF2 coding sequence, Lautier et al. (2008) sequenced

GIGYF2 gene in PD patients, and identified 7 mutations that result in single amino acid

substitutions. While GIGYF2 mutations are uncharacterized, this gene is an attractive

candidate because its interacting partner, GRB10 adaptor protein, has been shown to

regulate the insulin signaling pathway (Giovannone et al., 2003), which may affect

human aging (Suh et al., 2008; Willcox et al., 2008) and possibly the onset of PD (Craft

and Watson, 2004).

Strategy using invertebrate models of PD

Both vertebrate and invertebrate models of PD have been generated and exploited,

taking advantage of what each model organism offers. Vertebrate models provide a

complex neuronal circuitry as well as corresponding brain functions that most resemble

humans. In contrast, while invertebrate models are not evolutionally as intricate or

advanced as mammalian counterparts, their simplicity allows researchers to perform both

functional and large-scale analyses in a cost-effective manner.

Generally, invertebrate models have been utilized to model an aspect of the

disease state (e.g., by overexpressing wildtype α-syn, expressing mutant α-syn, or treating with neurotoxins) and identify novel therapeutic targets that are subsequently validated by vertebrate models or to study genetic as well as genetic-environmental interactions in PD-associated mutant background (Table 1.2). For example,

Saccharomyces cerevisiae , commonly known as baker’s yeast has been utilized for genetic and chemical screens. One major disadvantage of yeast is the fact that it is a unicellular eukaryote that does not have neurons nor synthesize DA. To this end,

multicellular eukaryotic model organisms with DA neurons such as a nematode,

Caenorhabditis elegans and the fruit fly, Drosophila melanogaster are utilized. In this section, fly and worm PD models are described below whereas yeast models are discussed in Chapter 3.

A pioneering work by Feany and Bender (2000) generated a D. melanogaster PD model whereby pan-neuronal expression of wildtype and mutant α-syn resulted in the

selective loss of DA neurons, presence of intraneuronal inclusions, and motor

dysfunction. Subsequent analysis of α-syn pathogenesis demonstrated that its toxicity is

dependent on phosphorylation at serine 129 (Chen and Feany, 2005) as well as its ability

to form aggregates in vivo (Periquet et al., 2007). The same model was utilized to study

differential gene expression by microarray whereby genes involved in catecholamine

synthesis, energy metabolism, mitochondrial function, and lipid bindings were found

misregulated (Scherzer et al., 2003). Auluck et al. (2002) generated a different fly strain

overexpressing wildtype and mutant α-syn using a DA neuron-specific promoter and

observed DA neurodegeneration. Importantly, they also co-overexpressed human

HSP70, which suppressed DA neuron death. This work, which was later confirmed by a

yeast PD model whereby overexpression of yeast Ssa3 (a yeast ortholog of human

HSP70) abolished α-syn-induced ROS accumulation (Flower et al., 2005), represented a

groundbreaking strategy of using model organisms to identify a novel neuroprotective

target.

Multiple studies have utilized mutant fly strains to identify potential

neuroprotective targets, determine genetic interactions of PD-associated genes, or

examine susceptibility to various forms of environmental stress. For example, using

parkin mutant strains, Whitworth et al. (2005) observed the loss of DA neurons in the

parkin loss-of-function background, which was reversed by overexpression of GstS1 (a

fruit fly ortholog of human glutathione S-transferase). In addition, Clark et al. (2006)

demonstrated the genetic interaction between PINK1 and parkin wherein overexpression

of parkin rescued the male sterility and defective mitochondrial morphology that were

observed in PINK1 mutants. While validation using the mammalian system is required,

these findings strongly suggest that multiple PD genes may function in a common

pathway. Focusing on the interplay between genetic and environmental factors,

Muelener et al. (2005) reported that DJ-1 knockout flies exhibited increased susceptibility to paraquat- and rotenone-induced oxidative stress, which is consistent with the anti-oxidant role of DJ-1 in mammalian cell culture. Lastly, Chaudhuri et al. (2007) reported that paraquat-treated flies displayed DA neurodegeneration, and that variations in DA-regulating genes could modify paraquat-induced oxidative damage. Since only 5-

10% of all PD cases are linked to known genetic causes, assessing the contribution of environmental factors and studying the genetic-environmental interactions should provide a mechanistic insight into further elucidating PD pathogenesis.

C. elegans PD models

C. elegans offers distinct advantages similar to other model organism. For

example, nematodes are approximately 1 mm long, and transparent with short generation time and lifespan. Furthermore, the worm genome sequence and neuronal circuitry are known, and mature bioinformatic databases (e.g., microarray, interactome, etc) and numerous mutant strains are accessible. Lastly, RNA interference (RNAi), a method in which a single gene is knocked down can easily be performed by feeding these worms the RNAi bacteria that produce double-stranded RNA (Kamath and Ahringer, 2003).

Similar to fly PD models, worm models have been used to identify potential neuroprotective targets, study genetic interactions of PD-associated genes, or examine PD environmental factors. Cao et al. (2005) generated a worm strain overexpressing wildtype α-syn in DA neurons, and reported DA neurodegeneration. They also co-

overexpressed human torsinA, which reversed the neurotoxic effects of α-syn. TorsinA is a chaperone-like protein that, when mutated, results in another movement disorder termed early-onset primary dystonia. Similar to Auluck et al. (2002) this work represented a novel approach of utilizing model organisms to examine a neuroprotective gene. Kuwahara et al. (2006) also generated worm strains overexpressing wildtype or mutant α-syn in DA neurons under the control of dat-1 (dopamine transporter) promoter.

While they were unable to observe neurodegeneration, they reported accumulation of α-

syn in cell bodies and dendrites, and defects in DA neuron-dependent behavior. Focusing

on the worm orthologs of PD genes, multiple articles have documented uncovering the

potential genetic interaction among different PD genes. Ved et al. (2005) reported that

depletion of pdr-1/parkin and djr-1.1/DJ-1 increased the susceptibility of these mutant or

RNAi-treated strains to mitochondrial stress. Moreover, Samann et al. (2009) demonstrated an antagonistic role of pink-1/PINK1 and lrk-1/LRRK2 mutations whereby

the absence of worm lrk-1 suppressed mitochondrial dysfunction and defects in axonal

outgrowth, two independent phenotypes, that were induced by pink-1 loss of function.

Finally, both 6-OHDA (Nass et al., 2002) and MPTP/MPP+ (Braungart et al., 2004)

treatment has been shown to induce DA neurodegeneration in C. elegans .

Additional studies utilize large-scale methodologies conducted by either

microarray or RNAi to identify modifiers of α-syn toxicity. Using transgenic strains

overexpressing wildtype or mutant α-syn pan-neuronally, Vartiainen et al. (2006) studied

differential gene expression by using microarray, and reported that genes involved in the

UPS and mitochondrial function were up-regulated. Van Ham et al. (2008) performed

genome-wide RNAi and FRAP to identify 80 genetic modifiers of α-syn misfolding and

aggregation in body wall muscle cells. The positive candidates included those involved

in protein quality control, vesicle trafficking, and aging. Kuwahara et al. (2008)

generated worm strains overexpressing wildtype and mutant α-syn pan-neuronally, and

performed RNAi against 1673 genes that are implicated in the nervous system functions.

They identified 10 positives, mostly consisting of components from the endocytic

pathway that caused severe growth and motor abnormalities, suggesting that α-syn

overexpression may cause defects in uptake or recycling of synaptic vesicles. 18

Current studies

To investigate genetic modifiers of α-syn misfolding by RNAi screen, we

generated an isogenic worm strain overexpressing α-syn::GFP and TOR-2 (a worm ortholog of human TorsinA) in the body wall muscle cells. Co-overexpression of C. elegans TOR-2 provided a genetic background that allowed clear distinction between soluble vs. aggregated α-syn, and maintained the expression of α-syn::GFP at the

misfolded state. We knocked down 868 genetic candidates via RNAi, and identified 20

strong positives that enhanced α-syn misfolding. To verify neuroprotective capacities of

these positive hits, we analyzed seven genes in worm DA neurons against α-syn toxicity,

and determined that five out of seven rescued DA neurons. The neuroprotective genes

included two autophagic components ( vps-41 and atgr-7), one DA signaling protein

(C35D10.2 ), one ER-Golgi trafficking component ( F55A4.1 ), and one uncharacterized

but evolutionarily conserved protein ( F16A11.2 ). This study is described in detail in

Chapter 2 (and published as Hamamichi et al., 2008).

To validate candidate genes obtained from yeast α-syn toxicity modifier screens,

we examined the neuroprotective capacities of these candidates by using our worm model

of α-syn neurodegeneration (Gitler et al., 2008; 2009). Consistent with previous findings

demonstrating neuroprotective function of Rab1a, overexpression of RAB3A and

RAB8A rescued DA neurons. Furthermore, overexpression of W08D2.5 (a worm

ortholog of human PARK9/ATP13A2 ), PLK2, and PDE9A also suppressed α-syn toxicity. In total, these six proteins demonstrated neuroprotective capacities in yeast as well as worm and mammalian DA neurons, providing additional genetic targets for PD therapy. This study is discussed in Chapter 3 (and published as Gitler et al., 2008; 2009).

Lastly, to study the genetic link between aging and α-syn associated toxicity, we examined the effect of daf-2/INSR mutation on α-syn neurodegeneration and misfolding. daf-2, which encodes an insulin-like receptor is a well-characterized gene in C. elegans whereby reduced function enhances longevity and protection against various forms of cellular stress (Baumeister et al., 2006). Here, we report a daf-2 strain overexpressing α- syn and GFP in DA neurons displayed a significant neuroprotection at the chronological aging stage (day 7 in both wildtype N2 and daf-2 worms) while it failed to exhibit the same rescue at the biological aging (day 20 in wildtype N2 and day 40 in daf-2 worms), demonstrating that differential gene expression in daf-2 mutant background is responsible for neuroprotection. To identify these genetic factors, we performed RNAi screen against genes that are up-regulated in daf-2, and examined if knockdown might enhance α-syn misfolding. In total, we assayed 625 candidates, and identified 53 positive genes. Two genes identified from the screen, gpi-1/GPI and hrdl-1/AMFR , representing two proteins involved in the autocrine motility factor pathway, were subsequently analyzed for neuroprotective function against α-syn toxicity in DA neurons. This study is described in

Chapter 4.

Specific outcomes and future directions as a result of this dissertation research are discussed in Chapter 5. Collectively, this work further establishes C. elegans as a powerful model system for the rapid evaluation of genetic factors with the potential to influence PD. The genes, proteins, and biological pathways characterized within this research represent putative targets for therapeutic development and intervention following additional validation through mammalian models.

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Table 1.1. Summary of mutations in PD genes linked to PD pathogenesis and their C. elegans orthologs. PD Gene PD Protein Mutation C. elegans Ortholog E-Value PARK1 SNCA/ α-syn Gain of function n/a n/a Multiplication PARK2 PRKN/parkin Loss of function pdr-1 3.4e-38 PARK5 UCHL1 Loss of function ubh-1 1.2e-33 PARK6 PINK1 Loss of function pink-1 7.8e-53 PARK7 DJ-1 Loss of function djr-1.1 1.6e-45 djr-1.2 8.9e-36 PARK8 LRRK2 Gain of function lrk-1 5.5e-66 PARK9 ATP13A2 Mislocalization W08D2.5 2.5e-180 PARK11 GIGYF2 Unknown n/a n/a PARK13 HTRA2 Gain of function n/a n/a n/a: Not applicable

Table 1.2. Summary of selected invertebrate PD models. Models Strategy References S. cerevisiae Overexpression of WT α-syn Genetic screens Willingham et al., 2003; Cooper et al., 2006; Gitler et al., 2009 Cytological analysis Outerio and Lindquist, 2003; Gitler et al., 2008 Overexpression of WT α-syn Cytological analysis Flower et al., 2005; Soper et and α -syn al., 2008; Liang et al., 2009 C. elegans Overexpression of WT α-syn Cytological analysis Cao et al., 2006 and/or α -syn (DA neurons) Behavioral analysis Kuwahara et al., 2006 Overexpression of WT α-syn Microarray Vartiainen et al., 2006 and/or α -syn (all neurons) RNAi Kuwahara et al., 2008 Overexpression of WT α- RNAi Van Ham et al., 2008 syn::gfp (muscles) Overexpression of WT LRRK2 Cytological analysis Saha et al., 2009 and LRRK2 (DA neurons) PD-associated mutants ( DJ-1, Mutant strain analysis Ved et al., 2005; Samann et parkin , LRRK2, or PINK1) al., 2009 Neurotoxin treatment Cytological analysis Nass et al., 2002; Braungart et al., 2004 D. melanogaster Overexpression of WT α-syn Cytological analysis Auluck et al., 2002 and/or α -syn (DA neurons) Overexpression of WT α-syn Cytological analysis Feany and Bender, 2000; and/or α -syn (all neurons) Chen and Feany, 2005; Periquet et al., 2007 Microarray Scherzer et al., 2003 Overexpression of WT LRRK2 Cytological analysis Liu et al., 2008; Venderova and LRRK2 (various neurons) et al., 2009 PD-associated mutants Mutant strain analysis Whitworth et al., 2005; Clark (parkin and/or PINK1) et al., 2006 Neurotoxin treatment Mutant strain analysis Meulener et al., 2005; Chaudhuri et al., 2007

Figure 1.1

FIGURE LEGENDS Fig. 1.1. Schematic representation of the cellular defects caused by known PD genes. Mutation or overexpression of α-syn/SNCA disrupts multiple cellular functions including ER-Golgi trafficking, proteasomal and lysosomal protein degradation, and mitochondria. Additional PD genes also affect the same cellular pathways: 1) parkin/PRKN and UCHL1, 2 proteins involved in the UPS; 2) ATP13A2, a lysosomal ATPase; 3) PINK1 and HTRA2, 2 mitochondrial components; and 4) DJ-1, a protein with anti-oxidant property. LRRK2 and GIGFY2 are proposed to modulate the MAPK and insulin signaling pathways, respectively.

CHAPTER TWO

HYPOTHESIS-BASED RNA INTERFERENCE SCREEN IDENTIFIES NEUROPROTECTIVE GENES IN A PARKINSON’S DISEASE MODEL

This work was published in Proceedings of the National Academy of Sciences of the United States of America , January, 2008 under the following citation: Hamamichi, S., Rivas, R.N., Knight, A.L., Cao, S., Caldwell, K.A., Caldwell, G.A. (2008) Proc Natl Acad Sci U S A 105,728-733. Shusei Hamamichi, Renee Rivas, and Adam Knight collected all data. Dr. Songsong Cao contributed the data shown in Fig. 2.1. Shusei Hamamichi, Dr. Kim Caldwell, and Dr. Guy Caldwell co-wrote the manuscript.

ABSTRACT

Genomic multiplication of the locus encoding human α-synuclein ( α-syn), a polypeptide with a propensity toward intracellular misfolding, results in Parkinson’s disease (PD). Here we report the results from systematic screening of nearly 900 candidate genetic targets, prioritized by bioinformatic associations to existing PD genes and pathways, via RNAi knockdown. Depletion of 20 gene products reproducibly enhanced misfolding of α-syn over the course of aging in the nematode Caenorhabditis elegans . Subsequent functional analysis of seven positive targets revealed five previously unreported gene products that significantly protect against age- and dose- dependent α-syn-induced degeneration in the dopamine (DA) neurons of transgenic worms. These include two trafficking proteins, a conserved cellular scaffold-type protein that modulates G-protein signaling, a protein of unknown function, and one gene reported to cause neurodegeneration in knockout mice. These data represent putative genetic susceptibility loci and potential therapeutic targets for PD, a movement disorder affecting approximately 2% of the population over 65 years of age.

INTRODUCTION

In the advent of complete genomic sequences and technologies for uncovering

putative protein interaction networks or whole-genome analyses, scientists have

generated many “lists” of candidate genes and proteins that can be harnessed for in-depth

analyses of cellular processes or disease states. In the nematode C. elegans, these include pioneering studies defining the protein “interactome” (Li et al., 2004), the “topology map” for global gene expression (Kim et al., 2001) and meta-analyses of predicted gene interactions (Zhong and Sternberg, 2006). Application of this nematode toward human disease research has already provided insights into the function of specific gene products linked to a variety of neurological disorders (Caldwell et al., 2003; Williams et al., 2004;

Cao et al., 2005; Cooper et al., 2006; Bates et al., 2006). Given that the average lifespan of this nematode is only 14-17 days, it has been especially useful in its application to diseases of aging (Driscoll and Gerstbrein, 2003; Kenyon, 2005). In this study, we exploited the potential predictive capacity of these C. elegans bioinformatic databases to discern genetic components and/or pathways that might represent heritable susceptibility factors for PD.

PD involves the progressive loss of DA neurons from the substantia nigra , accompanied by the accumulation of proteins into inclusions termed Lewy bodies.

Central to the formation of Lewy bodies is α-syn, a polypeptide with a propensity toward intracellular aggregation. Genomic multiplication of the wildtype α-syn locus results in

PD, indicating that overexpression of this protein alone can lead to the disease (Singleton

et al., 2003). Maintenance of DA neuron homeostasis has been hypothesized to be important for neuroprotection because an imbalance of cytosolic DA may contribute to neurotoxicity. Mechanistically, the selective loss of DA neurons in PD is very possibly due to the presence and chemical nature of DA itself. The capacity of DA for oxidation, and its effect on stabilizing toxic forms of α-syn (Conway et al., 2000), represents a

“perfect storm” in the context of the oxidative damage associated with the aging process, other potential environmental insults (i.e., heavy metals, pesticides), or differences in genetic predisposition.

Familial PD has been linked to specific genes, several of which function in cellular pathways involving the management of protein degradation and cellular stress

(Dawson and Dawson, 2003). Although most primary insights into the molecular nature of PD have thus far come via genetic analyses of familial forms of PD, there is significant evidence that implicates a combination of environmental factors as pivotal to sporadic causality (Tanner, 2003). Improvements in the diagnosis and treatment of PD will be contingent upon increased knowledge about susceptibility factors that render populations at risk.

We previously reported the establishment of a nematode model of age-dependent

α-syn-induced DA neurodegeneration that has facilitated successful identification of multiple neuroprotective factors, including those that have since been validated in other model organisms and mammals (Cao et al., 2005; Cooper et al., 2006). Here we take advantage of the experimental attributes of C. elegans to characterize a set of novel

neuroprotective gene products initially identified in a large-scale candidate gene screen for factors influencing misfolding of human α-syn in vivo by RNA interference (RNAi).

These data represent a collection of functionally delineated modifiers of α-syn-dependent misfolding and neurodegeneration that enhance our understanding of the molecular basis of PD and point toward new potential targets for therapeutic intervention.

MATERIALS AND METHODS

Nematode Strains. Nematodes were maintained using standard procedures

(Brenner, 1974). To make transgenic lines, each expression plasmid was injected into wildtype N2 (Bristol) worms at a concentration of 50 g/ml. For the RNAi screen,

UA50 [ baInl3 ; Punc-54 ::gfp , rol-6 (su1006) ], UA51 [ baInl4 ; Punc-54 :: α-syn::gfp, rol-6

(su1006) ], and UA52 [ baInl5 ; Punc-54 :: α-syn::gfp , Punc-54 ::tor-2, rol-6 (su1006) ] were integrated as previously described (Cao et al., 2005) and outcrossed at least three times to

N2 worms. The polyglutamine aggregation analysis was performed using integrated isogenic strain UA6 [UA6 ( baIn6) ] co-expressing Q82::GFP and TOR-2 (Caldwell et al.,

2003).

For neuroprotection analysis, three stable lines of either UA53 [ baEx42 ; Pdat-

1::FLAG-C35D10.2 , Punc-54 ::DsRed2 ], UA54 [ baEx43 ; Pdat-1::FLAG-C54H2.5, Punc-

54 ::DsRed2 ], UA55 [ baEx44 ; Pdat-1::FLAG-F16A11.2, Punc-54 ::DsRed2 ], UA56 [ baEx45 ;

Pdat-1::FLAG-F32A6.3, Punc-54 ::DsRed2 ], UA57 [ baEx46 ; Pdat-1::FLAG-F55A4.1, Punc-

54 ::DsRed2 ], UA58 [ baEx47 ; Pdat-1::FLAG-M7.5 , Punc-54 ::DsRed2 ], and UA59 [ baEx48 ; 38

Pdat-1::FLAG-R05D11.6 , Punc-54 ::DsRed2 ] were crossed with integrated UA44 [ baInl1 ;

Pdat-1:: α -syn high , Pdat-1::gfp ]. Briefly, male UA44 [ baInl1 ; Pdat-1:: α-syn high , Pdat-

1::gfp ] worms were generated by mating hermaphrodites with male N2 worms. GFP-

positive males were crossed with hermaphrodites overexpressing candidate genes in DA

neurons and DsRed2 in body wall muscle cells. The resulting GFP- and dsRed2-positive

hermaphrodites were individually picked, and self-fertilized until all worms displayed

GFP expression indicating homozygous expression of α-syn. These strains are

designated as follows: UA60 {[ baInl1 ; Pdat-1:: α -syn high , Pdat-1::gfp ]; [ baEx49; Pdat-

1::FLAG-C35D10.2 , Punc-54 ::DsRed2 ]}, UA62 {[ baInl1 ; Pdat-1:: α -syn high , Pdat-1::gfp ];

[baEx51 ; Pdat-1::FLAG-C54H2.5 , Punc-54 ::DsRed2 ]}, UA64 {[ baInl1 ; Pdat-1:: α -syn high ,

Pdat-1::gfp ]; [ baEx53 ; Pdat-1::FLAG-F16A11.2 , Punc-54 ::DsRed2 ]}, UA66 {[ baInl1 ; Pdat-

1:: α -syn high , Pdat-1::gfp ]; [ baEx55 ; Pdat-1::FLAG-F32A6.3 , Punc-54 ::DsRed2 ]}, UA68

{[ baInl1 ; Pdat-1:: α -syn high , Pdat-1::gfp ]; [ baEx57 ; Pdat-1::FLAG-F55A4.1 , Punc-

54 ::DsRed2 ]}, UA70 {[ baInl1 ; Pdat-1:: α -syn high , Pdat-1::gfp ]; [ baEx59 ; Pdat-1::FLAG-

M7.5 , Punc-54 ::DsRed2 ]}, and UA72 {[ baInl1 ; Pdat-1:: α -syn high , Pdat-1::gfp ]; [ baEx61 ;

Pdat-1::FLAG-R05D11.6 , Punc-54 ::DsRed2 ]}.

Plasmid Constructs. Plasmids were constructed using Gateway Technology

(Invitrogen). To generate α-syn::gfp , α-syn cDNA (a gift from Philipp Kahle, University of Tubingen, Germany) was cloned into a gfp -containing plasmid, pPD95.75 (Andy Fire,

Stanford University) by double digestion using XbaI and BamHI . Gateway entry vectors

were generated by cloning PCR-amplified α-syn::gfp , gfp as well as candidate cDNAs into pDONR201 or pDONR221 by BP reaction. The cDNAs encoding C35D10.2 ,

C54H2.5 , F16A11.2 , F55A4.1 , M7.5 , and R05D11.6 were obtained from Open

Biosystems (Huntsville, AL) while F32A6.3 was isolated from our C. elegans cDNA

library (Caldwell et al., 2006). DsRed2 was obtained from Clontech (Mountain View,

CA). An N-terminal FLAG tag sequence was added during the PCR amplification

process.

The gene fusions were shuttled from entry vectors into the Gateway destination

vector, pDEST-DAT-1 (Cao et al., 2005) or pDEST-UNC-54 via LR reaction. pDEST-

UNC-54 was generated by converting a unc-54 promoter containing plasmid, pPD30.38

(Andy Fire), using a Gateway Vector Conversion System (Invitrogen). The molecular

cloning yielded expression plasmids, Punc-54 :: α -syn::gfp, Punc-54 ::gfp, Pdat-1::FLAG-

C35D10.2 , Pdat-1::FLAG-C54H2.5 , Pdat-1::FLAG-F16A11.2 , Pdat-1::FLAG-F32A6.3, Pdat-

1::FLAG-F55A4.1 , Pdat-1::FLAG-M7.5 , Pdat-1::FLAG-R05D11.6 , and Punc-54 ::DsRed2 .

The cDNAs were verified by DNA sequencing.

Preparation of worm protein extracts and western blotting. Worm protein extracts were prepared and western blotting was performed as described previously (Cao et al., 2005). For all worm strains, 30 g/ l protein was loaded into 15% SDS PAGE gels (Bio-Rad) and detected by 1:2000 goat anti-α-syn primary antibody (Chemicon) and

1:10000 horseradish peroxidase-conjugated mouse anti-goat IgG secondary antibody

(Pierce). For detection of actin, 1:8000 mouse anti-actin primary antibody (ICN

Biochemicals) and 1:10000 horseradish peroxidase-conjugated sheep anti-mouse IgG

secondary antibody (Amersham) were utilized.

RNAi screen and analysis of α-syn misfolding or polyglutamine aggregation.

RNAi feeding was performed as described (Kamath and Ahringer, 2003) with the following modifications. Bacterial clones leading to enhanced α-syn misfolding were

tested in two trials, and the clones resulting in significant aggregation (80% of worms

with increased quantity and size of α-syn aggregates) were scored as positive. For each

trial, 20 worms were transferred onto a 2% agarose pad, immobilized with 2 mM

levamisole, and analyzed. The identities of the top 20 positive hits from the RNAi screen

were sequenced and verified. For polyglutamine aggregation analysis, 20 worms at the

L3-stage were scored for aggregate number in two separate trials.

RNAi feeding clones (Geneservice, Cambridge, UK) were grown for 14 hrs in LB

culture with 100 mg/ml ampicillin and seeded onto NGM agar plates containing 1 mM

isopropyl β-D-thiogalactoside. When the bacterial lawn was grown, five L4

hermaphrodites (strain UA52) were transferred onto the plates and incubated at 25 °C for

44 hr. The gravid adults were then placed onto the corresponding RNAi plates and

allowed to lay eggs for 9 hours, and the resulting age-synchronized worms were analyzed

at the indicated stage. For polyglutamine aggregation analysis, L3-staged 20 worms were

transferred onto a 2% agarose pad and immobilized with 2 mM levamisole, and the

quantity of aggregates was scored. The aggregation analysis was also conducted in

duplicate. 41

Candidate gene analysis for neuroprotection. Synchronized embryos expressing both GFP and DsRed2 were transferred onto NGM plates, and grown at 20 °C for 7 days

(Lewis and Fleming, 1995). For each trial, 30 worms were transferred to a 2% agarose pad, immobilized with 2 mM levamisole, and scored. Worms were considered rescued when all four CEP and both ADE neurons were intact and had no visible signs of degeneration. Each stable line was analyzed three times (for a total of 90 worms/transgenic line). Three separate transgenic lines were analyzed per gene, for a total of 270 animals/gene analyzed.

RNA isolation and semi-quantitative RT-PCR. Worms were harvested and snap- frozen in liquid nitrogen. After total RNA and cDNA preparation, semi-quantitative RT-

PCR was performed as previously described (Locke et al., 2006). Briefly, 50 L4-staged worms were transferred into 10 l 1:10-diluted Single Worm Lysis Buffer (10 mM Tris, pH 8.3, 50 mM KCl, 2.5 mM MgCl 2, 0.45% NP-40, 0.45% Tween 20, 0.01% gelatin, and

60 g proteinase K), mixed with 100 l TRI Reagent (Molecular Research Center), and incubated at RT for 10 min. The samples were freeze-thawed 5 times using liquid N 2, vortexed with 10 l 1-bromo-3-chloropropane (Acros Organics) for 15 sec, incubated at

RT for 10 min, and centrifuged at 14500 rpm at 4 °C for 15 min. The supernatant was transferred to a new RNase-free tube, mixed with 2 l glycoblue (Ambion) and 50 l -

20 °C-chilled isopropanol, and incubated at -20 °C overnight. After incubation, the supernatant was centrifuged at 14500 rpm at 4 °C for 15 min, and discarded. The pellet

was washed with 100 l RNase-free 75% ethanol, and resuspended in 10 l DEPC- treated water. For RT-PCR using SuperScript III RT (Invitrogen) with oligo dT primers, the total RNAs were treated with amplification grade RNase-free DNase I

(Invitrogen) as well as RNase H (Invitrogen) following the manufacture’s protocol. PCR was then performed using Phusion polymerase (Finnzymes). The PCR products were separated by 0.8% agarose gel electrophoresis and visualized by GelRed staining

(Biotium). The following primers were designed for the PCR: cdk-5 Primer 1: 5’ ggg-gat-gat-gag-ggt-gtt-cca-agc 3’

Primer 2: 5’ ggc-gac-cgg-cat-ttg-aga-tct-ctg-c 3’

α-syn Primer 1: 5’ atg-gat-gta-ttc-atg-aaa-gga-ctt-tca-aag 3’

Primer 2: 5’ tta-ggc-ttc-agg-ttc-gta-gtc-ttg 3’

The FLAG-tagged genes were PCR amplified by using primer sequences specific to

FLAG and each respective open reading frame.

FLAG Primer 1: 5’ gac-tac-aag-gac-gac-gat-gac 3’

C35D10.2 Primer 2: 5’ gaa-tgt-ggg-cga-aga-gca-tat-c 3’

C54H2.5 Primer 2: 5’ gtc-ctc-cac-caa-cgg-caa-tg 3’

F16A11.2 Primer 2: 5’ cca-gag-tga-ata-tct-gga-aga-cc 3’

F55A4.1 Primer 2: 5’ caa-att-cga-gga-aat-ggt-atg-gac 3’

F32A6.3 Primer 2: 5’ gag-cgg-aac-ctg-gtt-ctt-tat-g 3’

M7.5 Primer 2: 5’ ggc-tcc-gag-aga-tga-tag-tgg 3’

R05D11.6 Primer 2: 5’ cat-tgc-aag-aga-tgc-ctt-gag 3’

Imaging. All fluorescence microscopy was performed using a Nikon Eclipse

E800 epifluorescence microscope equipped with Endow GFP HYQ filter cube (Chroma

Technology). Images were captured with a Photometrics Cool Snap CCD camera driven

by MetaMorph software (Universal Imaging).

Statistics. Statistical analysis for neuroprotection was performed using Student’s t-test ( p<0.05) to compare control worms with strains that overexpress candidate genes in

DA neurons. The Fisher Exact Test was performed using the online program at

http://home.clara.net/sisa/fisher.htm .

RESULTS

Using a fusion of human α-syn to GFP, we generated transgenic nematodes that

enabled us to evaluate the consequences of α-syn overexpression and misfolding in vivo.

This isogenic strain contains misfolded α-syn::GFP aggregates in body wall muscles that

worsen as these animals develop and age (Fig. 2.1A). Co-expression of worm torsinA

(TOR-2) ameliorated the formation of these fluorescent aggregates, thereby extending

prior observations on torsin chaperone activity (Fig. 2.1B) (McLean et al., 2002;

Caldwell et al., 2003). Expression of an intact α-syn::GFP fusion, with and without

TOR-2 co-expression, was verified by western blotting (Fig 2.1E).

Worms expressing both α-syn::GFP and TOR-2 in body wall muscles represented

a genetic background whereby subtle changes in α-syn misfolding could be reproducibly 44

and effectively discerned via RNAi screening. The reasoning behind use of the body-wall muscles was two-fold; first, these are the largest, most readily scored cell type in adult C.

elegans within which to accurately judge changes in α-syn misfolding, and second, C.

elegans DA neurons are recalcitrant to RNAi (Asikainen et al., 2005). Moreover, we

theorized that the presence of TOR-2, a protein with chaperone activity, served to

maintain overexpressed α-syn at a threshold of misfolding, thereby enabling

identification of genetic factors that more readily effect the formation of misfolded

oligomers, or less mature α-syn aggregates, currently considered to be the more toxic species associated with degeneration (Taylor et al., 2002; Lee et al., 2004).

To investigate putative effectors of α-syn misfolding, we have systematically

screened 868 genetic targets with the potential to influence PD by selecting for

candidates that, when knocked down, enhanced age-associated aggregation of α-

syn::GFP . We used the C. elegans orthologs of established familial PD genes as the

foundation for constructing a candidate gene list (Table 2.4). The worm genome includes

orthologs of all established familial PD genes ( Parkin , DJ-1, PINK1 , UCHL-1, LRRK2 ,

PARK9 , NURR1 ) with the exception of α-syn . Specific C. elegans bioinformatic datasets

were subsequently mined to define hypothetical interrelationships between the worm PD

orthologs and previously unrelated gene targets. For example, using the C. elegans topology map (Kim et al., 2001), we identified all gene products that are co-expressed with the worm PD orthologs within a radius of one. Additionally, we identified all gene

products that interact with these PD orthologs, as assessed by the worm interactome (Li et al., 2004). Also included among our RNAi targets were the worm orthologs of genes that were uncovered via screens for effectors of α-syn toxicity in Saccharomyces cerevisiae (Cooper et al., 2006; Willingham et al., 2003), as well as genes encoding nematode versions of proteins identified in a proteomic analysis of rotenone-induced

Lewy bodies in DA neuron cell cultures (Zhou et al., 2004). We further extended our

RNAi target gene set by identifying worm homologs of gene products ascribed to encompass the cellular protein degradation machinery. These included genes annotated in Wormbase as being involved in the ubiquitin-proteasome system (UPS), unfolded- protein response (UPR), endoplasmic reticulum-associated degradation (ERAD), and autophagy. Gene candidates derived from these pathways were assessed for homology to mammals, and non-conserved genes were excluded since it has been estimated that 47% of worm genes have no visible homology to mammals (Schwarz, 2005). A table corresponding to 868 candidate genes targeted for knockdown is available in the

Supplementary Material (Table 2.4). Furthermore, we have constructed a relational interconnectivity map depicting gene targets classified in more than one category (Fig.

2.3).

These candidate gene targets were knocked down using RNAi, a method that is both rapidly and economically performed in C. elegans by feeding worms target-specific dsRNA-producing bacteria (Kamath et al., 2003). In total, 13% (111/868) were lethal; however, the remaining 757 genes were analyzed for accumulation of misfolded α-syn

protein. The primary RNAi screen of adult stage worms (44-48 hrs after eggs were laid at

25 °C) revealed that 17% (125/757) of these gene targets enhanced aggregation of α-syn in worms co-expressing α-syn::GFP and TOR-2. The misfolded protein appeared over developmental time and was randomly distributed in the cytoplasm of the body-wall muscle cells (Fig. 2.1C, D). RNAi was performed on approximately 20-30 animals in duplicate for each gene. As would be expected, a significant number of genes that alter folding or protein degradation were identified (Table 2.4). Notably included within this collection of positives were worm orthologs of five familial PD genes: Parkin

(K08E3.7/pdr-1), DJ-1 (B0432.2/djr-1.1 ), PINK1 (EEED8.9/pink-1), NURR1

(C48D5.1/nhr-6) and PARK9/ATP13A2 (W08D2.5 ) (Dawson and Dawson, 2003;

Ramirez et al., 2006).

Since PD is a disease of aging, we reasoned gene products that play a more significant functional role in the management of α-syn misfolding or clearance would exhibit a stronger effect at an earlier age. In this regard, a secondary screen of the top

125 candidates was performed in worms at the L3 larval stage of development (32-36 hrs after eggs laid). This resulted in further reduction of candidates where only about 3%

(20/757) of genes enhanced misfolding of human α-syn following RNAi treatment at this earlier developmental stage (Table 2.1). Retained within this list of 20 more stringently selected hits were orthologs of known recessive PD genes, DJ-1 and PINK1 , thereby representing internal validation of the screen. Another expected control for the screen, the C. elegans torsinA gene homolog, tor-2, was also recovered. A notable gene from

this dataset was T07F12.4 , a serine-threonine kinase that is homologous to UNC-51, a protein similar to yeast Atg1p, required for autophagy, that also plays a role in axon elongation (Okazaki et al., 2000; Lai and Garriga, 2004). A human ortholog of worm

UNC-51, termed ULK2, was recently identified by geneticists as one of only six genes that were distinguished as significant in a genome-wide association study of single- nucleotide polymorphisms within PD patients (Fung et al., 2006). The remaining 16 (of

20) positives encompass gene products previously unassociated with either α-syn function or PD.

Our identification of gene products that influence the misfolding of α-syn does not preclude the possibility that these proteins play a more generalized role in regulating protein misfolding or degradation. Previous screens in both C. elegans and yeast have implicated various classes of gene products that influence the misfolding or clearance of polyglutamine-repeat containing proteins (Willingham et al., 2005; Nollen et al., 2004).

In comparing the gene sets identified in those studies to our list of 125 less stringent modifiers of α-syn misfolding, we determined that only one positive gene was shared between our datasets, the C. elegans HSF-1 protein. HSF-1 is a critical evolutionarily conserved regulator of chaperone gene expression that would be presumed to exhibit a generalized function in mediating protein misfolding.

To further explore the prospect that the specific genes identified in our screen potentially act in a more generalized capacity, we used RNAi knockdown to evaluate loss-of-function associated with our strongest 20 α-syn modifiers in transgenic worms

expressing a polyglutamine::GFP fusion protein. The results of this analysis indicate that

RNAi knockdown of these targets had no significant influence on polyglutamine-

dependent aggregation in vivo (Table 2.5 and Fig. 2.6). The sole exception was the TOR-

2 chaperone-like protein, which served as a control in this analysis, as this protein has

been shown to suppress polyglutamine aggregation in C. elegans (Caldwell et al., 2003).

These data are consistent with a previous report that demonstrated the toxicity mediated

by overexpression of α-syn vs. a mutant huntingtin fragment in yeast was regulated by

non-overlapping gene sets (Willingham et al., 2003). In all, this analysis demonstrates

that the strongest α-syn effector genes identified through our RNAi screening do not

exert their influence via a general effect on protein misfolding, but more specifically

contribute to cellular pathways associated with α-syn.

A distinct advantage of using C. elegans for functional investigation of gene

activity is the level of accuracy that can be obtained in evaluating neurodegeneration. C.

elegans has precisely 8 DA neurons, with three pairs of neurons in the anterior

(designated dorsal/ventral CEPs and ADEs) and 1 pair in the posterior of the animal

(PDEs). We have established that overexpression of wildtype human α-syn under the

control of a DA neuron-specific promoter [ Pdat-1; DA transporter] results in age- and

dose-dependent neurodegeneration. We generated two separate transgenic lines of

animals that express α-syn at different levels, based on semi-quantitative RT-PCR analysis (Fig. 2.4A). At day 7 of adulthood, 87% of animals expressing a higher level of

α-syn show DA neurodegeneration (Fig. 2.4B) while 75% of animals expressing α-syn at 49

lower levels display degenerative changes (data not shown). The loss of DA neurons also occurs as animals age and no degeneration (0%) is observed in control animals ( Pdat-

1::GFP) lacking α-syn overexpression (Fig. 2.4B). Previously, these same animals have been utilized to validate the neuroprotective capacity of both worm TOR-2 and mammalian Rab1a, a GTPase involved in ER to Golgi transport (Cao et al., 2005; Cooper et al., 2006). Here we further extend our functional characterization of genes that resulted in enhanced α-syn misfolding when depleted by RNAi by systematically testing their prospective neuroprotective potential in vivo .

Figure 2.2A depicts a classification of the 20 positives where they are displayed according to their bioinformatic associations; several of these candidates shared more than one bioinformatic relationship (Table 2.2). For example, the C. elegans open- reading frame F32A6.3 encodes a gene ( vps-41 ) that is co-expressed in microarrays with the worm ortholog of UCHL-1, contains a RING-finger motif common to E3 ligases, and is involved in autophagy (Fig. 2.2A, circle). We utilized inferred relationships between genes exhibiting such overlap to prioritize subsequent construction of transgenic animals to examine their ability to influence DA neuron survival.

Transgenic animals co-expressing cDNAs corresponding to seven prioritized positive targets from the RNAi screen were generated and crossed to isogenic lines of worms expressing α-syn in DA neurons (Cao et al., 2005; Cooper et al., 2006).

Overexpression of α-syn alone resulted in significant degeneration, where only 12.8% of worms displayed wildtype DA neurons when they were assayed at the seven day-old

stage (Fig. 2.2B-D). Two genes exhibited an insignificant level of DA neuroprotection;

these encoded an uncharacterized transcription factor ( R05D11.6 ) and a C. elegans ortholog of Erv29p ( C54H2.5 ), a vesicle-associated protein involved in ER-Golgi transport (Fig. 2.2B). Strikingly, co-expression of five out of seven candidate genes examined significantly rescued DA neurodegeneration with average wildtype worm populations from 24 to 37% (Fig. 2.2B). Worms were scored as wildtype when all six anterior DA neurons were intact (Fig. 3E, F). Expression of the all gene products tested was verified via semi-quantitative RT-PCR (Fig. 2.5). Three independent transgenic lines were scored per gene tested, with 30 animals analyzed in triplicate experimental trials. In considering published evidence that TOR-2, DJ-1, and PINK1 have all previously been shown to be neuroprotective as well (Cao et al., 2005; Menzies et al.,

2005; Petit et al., 2005; Xu et al., 2005; Zhou and Freed, 2005; Pridgeon et al., 2007), these combined results indicate that the strategy employed in our screen is highly predictive of neuroprotective genetic modifiers.

The five genes found to display significant neuroprotection ( P<0.05; Student’s t- test) in our analysis included: 1) F32A6.3 , the worm ortholog of VPS41 , a conserved vesicular protein necessary for lysosomal biogenesis; 2) C35D10.2 , classified as GIPC , a

PDZ-domain containing protein which interacts with a vesicular GTPase named RGS-

GAIP involved with G protein-coupled signaling; 3) M7.5 , an open-reading frame corresponding to an autophagy-associated regulatory gene termed ATG7 ; 4) F16A11.2 , a hypothetical protein of unknown function with high amino acid sequence identity to an

uncharacterized human gene product; 5) F55A4.1 , the worm ortholog of Sec22p, a well characterized vesicular trafficking protein in yeast. The Blast E values for the Homo sapiens orthologs of these C. elegans gene products indicate they are all highly conserved

(Table 2.3).

DISCUSSION

The key pathological hallmarks of PD include the development of α-syn containing protein inclusions and DA neurodegeneration. Although it remains unclear if mature α-syn aggregates or Lewy bodies are causative for PD, evidence suggests factors that influence the misfolding and oligomerization of this polypeptide lead to neurotoxicity (Taylor et al., 2002; Lee et al., 2004). Regardless, proteins that play a role in protecting DA neurons from the degenerative loss associated with α-syn overproduction are candidate susceptibility markers, as well as potential targets for therapeutic development. Here we have combined these distinct PD-associated phenotypic readouts to discern novel gene products with functional consequences for PD.

Among the gene products identified via this screen, a protein that demonstrated high neuroprotective capacity was C. elegans VPS41. VPS41 is highly conserved across species and has been best characterized in S. cerevisiae , where it is involved in trafficking from the trans Golgi to the vacuole, the yeast equivalent of the lysosome (Rehling et al.,

1999). Little is known about the precise function of VPS41 in mammalian systems; however, in situ hybridization predicts the VPS41 gene to be expressed in brain neurons,

with strong expression localized to the DA neurons of the substantia nigra (Lein et al.,

2007).

Evidence for lysosomal system dysfunction is emerging as a potential

consequence of α-syn cytotoxicity. α-Syn is degraded in part by the lysosomal pathway,

under the regulation of the co-chaperone CHIP (Shin et al., 2005) and mutant forms of α-

syn can block chaperone-mediated autophagy (Finkbeiner et al., 2006; Cuervo et al.,

2004). Therefore, lysosomal failure has been proposed as a mechanism underlying the

age dependence of PD (Chu and Kordower, 2007). PARK9 , a hereditary form of

parkinsonism with dementia, has been recently linked to mutation of a lysosomal ATPase

(Ramirez et al., 2006). Notably, the worm homolog of PARK9 , W08D2.5 , was uncovered in our original RNAi screen (one of 125 initial hits) where knockdown led to α-syn

aggregation. This gene product is also neuroprotective when overexpressed in DA

neurons (Hamamichi and Caldwell, manuscript in preparation).

Our identification of C. elegans ATG7 as a neuroprotective gene product is

further suggestive of a significant role for autophagy and lysosomal function in restoring

homeostatic balance to DA neurons in response to excess α-syn. ATG7 is an E1-like enzyme required for the initiation of autophagosome formation. Added validation for these worm data comes from mammals where it was shown that loss of the Atg7 gene in

mice results in neurodegeneration and that this protein may function to prevent neuronal

impairment and axonal degeneration (Komatsu et al., 2006, Komatsu et al., 2007).

Among the factors that mediate DA neuron homeostasis is the interplay of DA

production, transport, and receptor signaling. In the “classical” view of DA signaling, D2

autoreceptors modulate a putative pre-synaptic feedback mechanism resulting in a net

neuroprotective effect (Bozzi and Borrelli, 2006). As the complexities of D2 signaling

continue to be unraveled, it is critical to consider that this model does not take into

account the largely unknown impact of α-syn misfolding and overabundance associated

with PD.

Here we describe evidence indicating that GIPC (GAIP interacting protein, C

terminus), a conserved cellular scaffold-type protein, has the capacity to function in a

neuroprotective manner against α-syn-induced neurodegeneration. GIPC has been shown to interact with mammalian D2 and D3 receptors in heterologous cell cultures, where its expression appears to mediate endosomal trafficking and receptor stability

(Jeanneteau et al., 2004). GIPC was originally identified in a screen for proteins that bind to GAIP (G-alpha interacting protein) (De Vries et al., 1998), a member of the large family called Regulators of G protein Signaling (RGS), yet GAIP is the only RGS protein that binds GIPC. Overexpression of GAIP has been shown to stimulate protein degradation via G αi-mediated induction of autophagy in human intestinal cells (Ogier-

Denis et al., 1997). It is interesting to speculate that GIPC serves to modulate a presynaptic protein-coupled pathway that can somehow combat the effects of α-syn misfolding and accumulation, perhaps by a DA or DA-receptor regulated manner.

Our data demonstrating that the C. elegans F55A4.1 gene product, an ortholog of

Sec22p, is neuroprotective accentuates the importance of vesicular trafficking between the ER and Golgi as an integral process affected by α-syn. We previously hypothesized that α-syn-dependent blockage of vesicular trafficking could lead to the limitation of available monoamine vesicular transporters (i.e., VMAT2) (Cooper et al., 2006). This would theoretically result in an excess pool of cytosolic DA and contribute to selective

DA neurodegeneration. Indeed, α-syn overexpression may exacerbate this process, leading to increased cytosolic catecholamine concentration (Mosharov et al., 2006;

Caudle et al., 2007). Thus, cellular proteins, like Sec22p or those in the Rab GTPase family (i.e., Rab1) that enhance vesicular trafficking and the removal of DA from the cytosol, likely contribute to neuroprotection by relieving this α-syn-mediated blockade

(Cooper et al., 2006). While hypotheses focused on the intrinsic contributions of DA to cytotoxicity are appealing, it is important to remember that other neuronal subtypes are also susceptible in PD and that disruption of basic cellular functions has implications beyond the DA system.

The candidate gene approach may limit the ability to make generalized conclusions about all possible gene families, pathways, or non-biased gene sets that could potentially be revealed by genome-wide screening. By design, the genes pre-selected for analysis in our RNAi screen will not reveal all possible effectors of α-syn misfolding and neuroprotection in C. elegans . They are further limited by factors such as lethality and redundancy. Nevertheless, this focused strategy did not restrict the detection of 55

unexpected effectors, as evidenced by the identification of the F16A11.2 gene product,

which has not been previously linked to neuroprotection. This protein, which contains an

RNA-binding motif, has over 99% identity to an uncharacterized human gene product

that is associated with neuronal RNA-rich granules where it may be involved in transport

(Kanai et al., 2004). This is intriguing considering the recent discovery that miRNAs in

DA neurons may play a role in neurodegenerative process (Kim et al., 2007).

We contend that our a priori elimination of targets without significant homology

to mammals, as well as prioritizing targets with putative relationships to known PD genes

via our “guilt by association” bioinformatics selection strategy, significantly enhanced

our ability to identify functionally relevant effectors. For example, we determined that

genes co-expressed with known PD genes that are also components of cellular pathways

implicated in PD have a far greater likelihood of significantly effecting α-syn misfolding

(17% vs. 3% strong positives for entire gene set; Fig. 2.7A). Furthermore, we discovered that 11% (3/28) genes co-expressed with both DJ-1 and PINK1 were significantly enriched within our top 20 hits, as compared to the other top candidates that represented a

3% (17/757) hit rate ( P<0.05; Fisher exact test; Fig. 2.7B). In the era of completed genome sequences, this directed approach is applicable to other disease gene studies in model organisms and serves to accelerate the pace of gene discovery. With continued expansion in large-scale genomic/proteomic studies, established means for rapid functional validation of gene candidates will be important and necessary for reconciling

disparate datasets, such as those obtained from the initial genome-wide analyses on PD

(Perez-Tur, 2006).

Taken together, the results of this study indicate that further characterization of

the genes identified in our RNAi screen will yield additional insights into mediators of α-

syn-induced cytotoxicity. An emerging model underlying PD involves dysfunction within

a variety of intersecting pathways that maintain homeostasis via a compensatory balance

between intracellular protein trafficking and degradation systems, as well as other

signaling mechanisms triggered by stress. The manner by which α-syn impacts these mechanisms remains poorly defined and factors that influence the stability, production, and clearance of this protein likely represent effectors of disease onset and progression.

Identification of critical cellular mediators within these processes will enhance development of biomarkers and therapeutic agents to halt this disease.

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Table 2.1. Gene identities of the 20 top candidates isolated from RNAi screening. C. elegans Gene ID NCBI KOGs

B0432.2 (djr-1.1) Putative transcriptional regulator DJ-1 T05C3.5 (dnj-19) Molecular chaperone (DnaJ superfamily) C35D10.2 RGS-GAIP interacting protein GIPC C54H2.5 (sft-4) Putative cargo transport protein ERV29 EEED8.9 (pink-1) BRPK/PTEN-induced protein kinase F11H8.1 (rfl-1) NEDD8-activating complex, catalytic component UBA3 F16A11.2 Uncharacterized conserved protein F26E4.11 (hrdl-1) E3 ubiquitin ligase F32A6.3 (vps-41) Vacuolar assembly/sorting protein VPS41 F48E3.7 (acr-22) Acetylcholine receptor F55A4.1 Synaptobrevin/VAMP-like protein SEC22 F57B10.5 Emp24/gp25L/p24 family of membrane trafficking proteins F59F4.1 Acyl-CoA oxidase K11G12.4 (smf-1) Mn 2+ and Fe 2+ transporters of the NRAMP family M7.5 (atgr-7) Ubiquitin activating E1 enzyme-like protein R05D11.6 Transcription factor T07F12.4 Serine/threonine-protein kinase involved in autophagy T08D2.4 Tripartite motif-containing 32 T13A10.2 Predicted E3 ubiquitin ligase Y37A1B.13 (tor-2) ATPase of the AAA+ superfamily

Table 2.2. Bioinformatic associations among gene candidates identified by RNAi. C. elegans Gene ID Bioinformatic Component of: Association K11G12.4 (smf-1) PINK1 and DJ-1* R05D11.6 PINK1 and DJ-1* F26E4.11 (hrdl-1) PINK1 and DJ-1* ERAD C35D10.2 DJ-1** Autophagy M7.5 (atgr-7) UPS and Autophagy F32A6.3 (vps-41) UCHL-1* UPS and Autophagy

*Co-expressed in microarray topology map, as per Kim et al. (2) **Identified within same protein interactome network, as per Li et al. (1)

Table 2.3. Summary of the neuroprotective genes and their human homologs. Average % Relevance to worms with H. sapiens C. elegans wildtype NCBI KOGs Gene ID Blast e- DA % length value neurons* F32A6.3 Vacuolar assembly/sorting 36.7 + 5 5.1e -96 95.7 (vps-41) protein VSP41 RGS-GAIP interacting C35D10.2 31.5 + 1 1.1e -49 75.4 protein GIPC Ubiquitin activating E1 M7.5 (atgr-7) 31.1 + 1 7.4e -87 69.2 enzyme-like protein Uncharacterized F16A11.2 24.8 + 3 1.2e -207 99.8 conserved protein Synaptobrevin/VAMP- F55A4.1 23.7 + 3 2.3e -47 96.3 like protein SEC22 *Compared to 12.8% with a-syn alone.

Table 2.4. Results of all genes knocked down via RNAi screening. Light blue indicates 125 positives from primary RNAi screen analyzed at young adult stage. Dark blue indicates 20 positives from secondary RNAi screen analyzed at L3 stage. Gray indicates lethal genes.

Name Human Homolog Outcome B0024.6 Atrial natriuretic peptide receptor A precursor B0025.1 PI3-kinase catalytic subunit type 3 B0035.14 DnaJ homolog subfamily B member 12 B0035.2 GNG10 B0205.3 PSMD4 B0250.1 60S ribosomal protein L8 B0272.1 Tubulin beta-2C chain B0281.1 Centrosomal protein Cep290 B0281.3 Tripartite motif-containing protein 59 B0281.8 GTP-binding protein ARD-1 B0303.9 Vacuolar protein sorting-associated protein 33A B0336.8 APG12 autophagy 12-like B0361.10 Synaptobrevin homolog YKT6 B0393.6 Tripartite motif-containing 10 isoform 2 B0403.2 Baculoviral IAP repeat-containing protein 6 B0403.4 Protein disulfide isomerase A6 precursor B0414.8 Isoform 1 of Uncharacterized protein C11orf2 B0416.4 Tripartite motif-containing 32 B0432.2 Protein DJ-1 B0432.5 Tyrosine hydroxylase isoform c B0511.1 Isoform 2 of FK506-binding protein 7 precursor B0513.2 THO complex 7 homolog B0545.1 Protein kinase C, delta B0563.7 Calmodulin B0564.2 AlkB, alkylation repair homolog 6 isoform 2 C01A2.4 Charged multivesicular body protein 2b C01B7.6 Probable E3 ubiquitin-protein ligase MYCBP2 C01G10.12 GNG10 C01G12.5 DHRS4 C01G6.4 RING finger protein 11 C01G8.2 Isoform 1 of Battenin

C01G8.4 DnaJ homolog subfamily C member 4 C02B8.6 RING finger protein 146 C02F5.9 Proteasome subunit beta type 1 precursor C04A2.7 DnaJ homolog subfamily C member 14 C04E12.4 N-glycanase C04E12.5 N-glycanase C04E6.5 Ubiquitin specific protease 30 C04F12.10 CAAX prenyl protease 1 homolog C04F5.1 SID1 transmembrane family member 2 C04G6.1 Mitogen-activated protein kinase 7, isoform 1 C04G6.3 Splice Isoform PLD1A of Phospholipase D1 C04H5.1 TRM112-like protein C05C10.6 Phospholipase A-2-activating protein C05C8.3 FK506 binding protein 9 precursor C05D10.2 Mitogen-activated protein kinase 15 C05D11.2 Vacuolar protein sorting-associated protein 16 C05D9.2 Lysosomal-associated membrane protein 1 C05G5.2 Proteoglycan-4 precursor C06A1.1 Transitional endoplasmic reticulum ATPase C06A5.4 Unnamed protein C06A5.8 Tripartite motif-containing 32 C06A5.9 SH3 domain containing ring finger 2 C06E2.3 Ubiquitin-conjugating enzyme E2-25 kDa C06E2.7 Ubiquitin-conjugating enzyme E2-25 kDa C06H2.2 START domain containing 7 C07A12.4 Protein disulfide isomerase precursor C07G2.3 T-complex protein 1, epsilon subunit C08A9.8 Uncharacterized protein C08B11.1 ZYG11B protein C08B11.7 Ubiquitin carboxyl-terminal hydrolase L5 C08B11.8 ALG6 C08F8.2 SUV3-like protein 1 C08F8.8 Orphan nuclear receptor NR2E1 C08H9.1 Lysosomal protective protein precursor C09B8.6 Heat-shock protein beta-1 C09D1.1 Isoform 2 of Titin C09D4.4 FAM135A

C09G12.9 Tumor susceptibility gene 101 protein C09G4.3 Cyclin-dependent kinases regulatory subunit 1 C10A4.8 ZNF690 protein C10C6.6 Probable cation-transporting ATPase 13A1 C10G11.8 26S protease regulatory subunit 4 C11D2.2 Isoform 1 of Cathepsin E precursor C11H1.3 RING finger protein 157 C12C8.1 Heat shock cognate 71 kDa protein C12C8.2 Cystathionine gamma-lyase C12C8.3 Tripartite motif-containing protein 71 C12D8.1 KH-type splicing regulatory protein C13B9.2 Glycerate kinase C13B9.3 Coatomer subunit delta C13C12.1 Calmodulin C14A4.4 Exosome component 10 C14A4.5 Exosome complex exonuclease RRP46 C14B1.1 Protein disulfide isomerase precursor C14B9.1 Alpha crystallin B chain C14B9.2 Protein disulfide isomerase A4 precursor C14B9.4 Serine/threonine-protein kinase PLK2 C14F11.5 Heat-shock protein beta-1 C14F5.4 Sideroflexin-2 C15F1.5 Keratin-associated protein 10-6 C15F1.6 Synaptic glycoprotein SC2 C15F1.7 Superoxide dismutase C15H9.6 78 kDa glucose-regulated protein precursor C16A11.5 ELKS/RAB6-interacting/CAST family member 1 C16A3.8 THO complex 2 isoform 1 C16C10.5 RING finger protein 121 C16C10.7 RING finger protein 185 C16C8.11 Isoform 4 of Nesprin-1 C16C8.12 RUN and FYVE domain-containing protein C16C8.13 Synaptonemal complex protein 1 C16C8.13 Synaptonemal complex protein 1 C16C8.14 Centromere protein E C16C8.4 Ubiquitin C C16C8.5 Hypothetical protein

C17D12.3 N-glycanase C17D12.5 Ubiquitin-conjugating enzyme E2 D1 C17G10.1 OGFOD1 C17H11.4 Ariadne-1 protein homolog variant (Fragment) C17H11.6 E3 ubiquitin-protein ligase RNF19 C18A11.1 Unnamed protein C18A3.5 Nucleolysin TIA-1 isoform p40 C18B12.4 RING finger protein 13 C18E9.1 Calmodulin C18E9.10 Vesicle transport protein SFT2C C18E9.2 Translocation protein SEC62 C18H9.7 43kDa acetylcholine receptor-associated protein C23G10.8 Conserved hypothetical protein C23H3.4 Serine palmitoyltransferase 1 C23H5.7 Cyclic nucleotide-gated olfactory channel C25B8.3 Cathepsin B precursor C25G4.4 GMEB1 C26B9.6 RING finger protein 146 C26E6.11 Cob C26E6.8 NEDD8-activating enzyme E1 regulatory subunit C26F1.4 Ubiquitin-like protein FUBI C27A12.6 Protein ariadne-1 homolog C27A12.7 Protein ariadne-1 homolog C27A12.8 Protein ariadne-1 homolog C27A7.3 ENPP3 C27C12.2 Early growth response protein 1 C27C12.4 ET putative translation product C27F2.5 Vacuolar sorting protein SNF8 C27H5.3 Fus-like protein (Fragment) C28D4.1 Retinoic acid receptor RXR-beta C28G1.1 Ubiquitin-conjugating enzyme E2-25 kDa C28G1.3 Exocyst complex component Sec15B isoform 1 C28H8.1 BCL7A C30B5.1 KIAA0953 protein C30C11.2 PSMD3 C30C11.4 Heat shock protein apg-1 C30F2.2 Tripartite motif-containing 32

C32B5.13 Cathepsin H precursor C32B5.7 Cathepsin H precursor C32D5.10 E3 ubiquitin-protein ligase Topors C32D5.11 E3 ubiquitin-protein ligase Topors C32D5.9 GABA receptor-associated protein C32E8.1 GTP-binding protein ARD-1 C32E8.3 Protein p25-beta C32F10.1 Oxysterol-binding protein-related protein 9 C32F10.6 Retinoic acid receptor C33H5.10 FAM98B C33H5.6 WD repeat protein 82 C34B7.2 SAC domain-containing protein 3 C34D4.12 Peptidyl-prolyl cis-trans isomerase like 1 C34D4.14 E3 ligase for inhibin receptor C34E10.4 WARS2 C34E10.5 Protein arginine N-methyltransferase 5 C34F11.3 AMP deaminase 2 C34F6.9 Deubiquitinating enzyme 3 C35B1.1 Ubiquitin-conjugating enzyme E2A C35D10.2 PDZ domain-containing protein GIPC1 C36A4.8 BRCA1 C36B1.4 Proteasome subunit alpha type 7 C36B1.7 Dihydrofolate reductase C36E8.5 Tubulin beta-2C chain C37H5.8 Stress-70 protein, mitochondrial precursor C38D4.3 Neurofilament heavy polypeptide C39F7.2 Tripartite motif-containing protein 67 C39F7.4 Ras-related protein Rab-1A C41C4.4 IRE1 precursor C41C4.8 Transitional endoplasmic reticulum ATPase C42C1.4 Vacuolar protein sorting 8 homolog isoform b C42D8.8 Amyloid-like protein 2 precursor C44B11.3 Tubulin alpha-3 chain C44B7.1 PSMD9 C44C1.4 Vacuolar protein sorting-associated protein 45 C44E4.6 Isoform 2 of Acyl-CoA-binding protein C44H4.5 MAP3K7 interacting protein 1 isoform beta

C45G7.4 Tripartite motif-containing 13 C47A4.1 GNGT10 C47B2.2 Protein FLJ31792 C47B2.3 Tubulin alpha-3 chain C47B2.4 Proteasome subunit beta type 7 precursor C47E12.3 EDEM1 C47E12.5 Ubiquitin-activating enzyme E1 C48D5.1 Orphan nuclear receptor NR4A2 C49C3.6 Trichoplein keratin filament-binding protein C49G7.11 Protein DJ-1 C50C3.5 Calmodulin-like protein 5 C50E10.4 Proteoglycan-4 precursor C50E3.3 C-type lectin C50F2.6 FK506-binding protein 9 precursor C50F4.3 Cathepsin H precursor C50H11.5 12 kDa protein C52B11.5 Ras-related protein Rab-5B C52E12.4 Hypothetical protein LOC160518 C52E4.1 Cathepsin B precursor C52E4.4 26S protease regulatory subunit 7 C53A3.2 Hypothetical protein LOC283871 C53A5.2 tRNA (guanine-N(1)-)-methyltransferase C53A5.6 IPP protein C53D5.6 RAN binding protein 5 C54C6.2 Tubulin beta-2C chain C54H2.3 RING1 and YY1-binding protein C54H2.5 Surfeit locus protein 4 C55A6.1 RING finger protein 146 C55B6.2 DnaJ homolog subfamily C member 3 C56A3.4 RNF157 protein C56C10.1 Vacuolar protein sorting-associated protein 33B C56E10.3 Nuclear pore complex-associated protein TPR C56G2.15 Putative tumor suppressor FUS2 CC8.2 Protein phosphatase 1 regulatory subunit 3D CD4.6 Proteasome subunit alpha type 1 D1007.5 Isoform 1 of Transmembrane protein 39A D1009.2 Peptidyl-prolyl cis-trans isomerase G

D1014.3 Alpha-soluble NSF attachment protein D1022.1 Ubiquitin-conjugating enzyme E2 J1 D1054.8 DHRS4 D2007.5 Uncharacterized protein KIAA0652 D2013.8 SREBP cleavage-activating protein D2030.7 Kaiso D2030.8 Family with sequence similarity 113, member B D2085.4 Ubiquitin-protein ligase E3C D2092.4 Protein disulfide-isomerase A5 precursor EEED8.5 ATP-dependent RNA helicase DHX8 EEED8.8 ADP-ribose pyrophosphatase EEED8.9 Serine/threonine-protein kinase PINK1 F01E11.2 Isoform E of Proteoglycan-4 precursor F01F1.14 n/a F01F1.8 T-complex protein 1, zeta subunit F01G4.2 3-Hydroxyacyl-CoA dehydrogenase type-2 F02E8.5 Autophagy protein 16-like F02E9.7 Tartrate-resistant acid ATPase F07A11.4 Ubiquitin specific protease 19 F07E5.5 ZCCHC9 protein F08B12.2 Peroxisome assembly protein 12 F08C6.3 Vacuolar protein sorting-associated protein 52 F08D12.1 Signal recognition particle 72 kDa protein F08F8.2 HMG-CoA reductase F08G12.4 Von Hippel-Lindau-like protein F08G12.5 Tripartite motif-containing 13 F08H9.3 Alpha crystallin B chain F08H9.4 Alpha crystallin B chain F09B9.3 ER lumen protein retaining receptor 1 F09C12.2 Mitogen-activated protein kinase 7, isoform 1 F09D1.1 U4/U6.U5 tri-snRNP-associated protein 2 F09G2.9 Neurofilament heavy polypeptide F10C2.5 EDEM2 F10C5.1 Cell division cycle protein 23 F10D11.1 SOD2 F10D7.5 Neuralized-like protein F10E7.8 Isoform 1 of Protein FAM40A

F10E9.6 APBB1 F10G7.2 SND1 F10G7.8 PSMD12 F10G7.9 Neurofilament heavy polypeptide F11A10.3 Polycomb group RING finger protein 3 F11H8.1 NEDD8-activating enzyme E1 catalytic subunit F12A10.5 Calmodulin F12F6.6 Protein transport protein Sec24C F12F6.7 DNA polymerase subunit delta 2 F13H10.2 NUDT13 F13H10.4 Mannosyl-oligosaccharide glucosidase F14D2.11 Polyubiquitin 9 F14F4.3 Multidrug resistance-associated protein 5 F15C11.2 Isoform 2 of Ubiquilin-1 F15D4.4 Cathepsin S precursor F15H10.3 Anaphase-promoting complex subunit 10 F15H10.4 E3 ubiquitin-protein ligase RNF19 F16A11.1 RSPRY1 F16A11.2 UPF0027 protein C22orf28 F16D3.1 Tubulin alpha-ubiquitous chain F17C11.8 Vacuolar protein sorting-associated protein 36 F17E5.1 CAMK1 F18C5.2 Werner syndrome ATP-dependent helicase F19B10.10 Serologically defined colon cancer antigen 8 F19B10.2 Coiled-coil domain-containing protein 123 F19B6.2 Ubiquitin fusion degradation protein 1 homolog F19G12.1 Tripartite motif-containing protein 2 F19H8.1 Trehalose-6-phosphate synthase F20C5.6 Myosin-9 F20D1.9 Mitochondrial glutamate carrier 2 F21C3.3 Histidine triad nucleotide-binding protein 1 F21D5.7 Signal recognition particle 54 kDa protein F21F8.2 Gastricsin precursor F21F8.3 43 kDa protein F21F8.4 43 kDa protein F21F8.7 43 kDa protein F22B5.7 Cytoskeleton-associated protein 5

F22B7.5 DNAJA3, mitochondrial precursor F22B8.6 Cystathionine gamma-lyase F22E10.5 Choline/ethanolaminephosphotransferase F23F1.8 26S protease regulatory subunit S10B F23F12.6 26S protease regulatory subunit 6B F25B5.4 Ubiquitin C F25C8.1 Acyl-coenzyme A oxidase 1, peroxisomal F25D7.3 PRDM1 F25G6.8 Signal recognition particle 14 kDa protein F25H2.8 Ubiquitin-conjugating enzyme E2 Q2 F25H2.9 Proteasome subunit alpha type 5 F25H5.6 39S ribosomal protein 54 F26D10.3 Heat shock cognate 71 kDa protein F26D2.15 DHRS4 F26E4.11 Autocrine motility factor receptor, isoform 2 F26E4.4 Cell death regulator Aven F26E4.6 Cytochrome c oxidase polypeptide VIIc F26E4.8 Tubulin alpha-3 chain F26E4.9 Cytochrome c oxidase polypeptide Vb F26F12.2 Uncharacterized protein F26F4.1 UPF0363 protein C7orf20 F26F4.7 Tripartite motif-containing protein 2 F26G5.9 PAX interacting protein 1 F26H9.6 Ras-related protein Rab-5B F26H9.7 Ubiquitin-conjugating enzyme E2 N F27B3.5 CENPE variant protein (Fragment) F28A12.4 Gastricsin precursor F28C12.5 Sphingosine 1-phosphate receptor Edg-3 F28C6.4 GPI ethanolamine phosphate transferase 2 F28H7.2 DHRS4 F29B9.6 SUMO-conjugating enzyme UBC9 F29C4.5 Ubiquitin carboxyl-terminal hydrolase 12 F29D10.4 Myosin-If F30A10.10 Ubiquitin carboxyl-terminal hydrolase 48 F30F8.8 Transcription initiation factor TFIID subunit 5 F31D4.3 FK506-binding protein 4 F31E3.5 Elongation factor 1-alpha 2

F31E8.2 Synaptotagmin-1 F32A5.3 Lysosomal protective protein precursor F32A6.3 Vacuolar protein sorting-associated protein 41 F32B5.8 Cathepsin Z precursor F32H2.4 THO complex subunit 3 F32H5.1 Cathepsin B precursor F33D11.9 GPAA1 F33H2.6 Protein FAM82B F35B3.1 Ubiquitin specific protease 11 F35D11.11 Trichohyalin F35F10.1 N-glycanase F35G12.12 PSMD5 F35G12.9 Anaphase-promoting complex subunit 11 F35H10.7 Chromosome 16 open reading frame 35 F36A2.1 Uncharacterized protein KIAA0460 F36A2.13 EDD1 protein F36D3.9 Cathepsin B precursor F36F2.3 Retinoblastoma-binding protein 6 F36H1.1 FK506-binding protein 2 precursor F37A4.1 HLA-B associated transcript 5 F37A4.5 PSMD14 F37B12.4 Ubiquitin carboxyl-terminal hydrolase 24 F37C12.1 Coiled-coil domain-containing protein 94 F37C12.14 n/a F37F2.2 Signal recognition particle 19 kDa protein F38A1.8 SRPR protein, alpha subunit F38A5.13 Zuotin-related factor 1 F38B7.5 Ubiquitin carboxyl-terminal hydrolase 29 F38C2.4 Uncharacterized protein F38E11.1 Alpha crystallin B chain F38E11.2 Alpha crystallin B chain F38E11.5 Coatomer subunit beta' F38H4.9 PPP2CB F39B2.10 DnaJ homolog subfamily A member 1 F39B2.2 Ubiquitin-conjugating enzyme E2 variant 1 F39H11.5 Proteasome subunit beta type 4 precursor F40E3.3 RIG

F40F4.5 Tubulin alpha-ubiquitous chain F40F9.8 Calmodulin F40G9.1 PSMD10 F40G9.12 Tripartite motif-containing 32 F40G9.3 Ubiquitin-conjugating enzyme E2-25 kDa F41E6.13 WIPI-2 F41E6.6 Cathepsin F precursor F41E6.9 Charged multivesicular body protein 5 F41H10.3 Conserved hypothetical protein F42A6.6 Uncharacterized protein C11orf73 F42C5.8 40S ribosomal protein S8 F42G2.5 VAPA F42G8.3 Mitogen-activated protein kinase 14 F42G8.4 Mitogen-activated protein kinase 14 F42G9.2 Peptidylprolyl isomerase B precursor F42G9.9 Microtubule-associated protein 4 isoform 2 F43C1.2 Mitogen-activated protein kinase 1 F43C9.2 Calcium-binding protein 4 F43D9.4 Alpha crystallin B chain F43E2.8 78 kDa glucose-regulated protein precursor F43G6.8 Tripartite motif-containing 32 F44B9.5 Ancient ubiquitous protein 1 precursor F44C8.10 Hepatocyte nuclear factor 4, gamma F44C8.3 Orphan nuclear receptor TR4 F44C8.9 Hepatocyte nuclear factor 4 alpha isoform f F44D12.10 Zinc finger protein 407 F44E5.4 Heat shock cognate 71 kDa protein F44E5.5 Heat shock cognate 71 kDa protein F44E7.2 Hypothetical protein LOC283871 F44F4.11 Tubulin alpha-ubiquitous chain F44G3.9 Photoreceptor-specific nuclear receptor F45E6.2 ATF-6 alpha F45G2.6 TNF receptor-associated factor 3 F45H11.2 NEDD8 precursor F45H7.6 E3 ubiquitin-protein ligase HECW1 F46C3.1 eIF-2 alpha kinase 3 F46E10.8 Ubiquitin carboxyl-terminal hydrolase L1

F46F11.4 Ubiquitin-like protein 5 F46F6.2 Serine/threonine-protein kinase N2 F47C10.7 Retinoic acid receptor RXR-alpha F47D12.4 High mobility group protein B2 F47G4.4 Katanin p80 subunit B 1 F47G9.4 Isoform 2 of Midline-2 F48C1.1 Alpha-mannosidase 2 F48E3.7 CHRNA9 F48G7.10 Protein kinase C, epsilon type F48G7.9 Protein kinase C, epsilon type F49C12.11 Coiled-coil domain-containing protein 72 F49C12.9 Ubiquitin-like protein 7 F49E12.4 18 kDa protein F49E7.1 GTPase activating protein and VPS9 domains 1 F49E8.3 Puromycin-sensitive aminopeptidase F52C6.12 Ubiquitin-conjugating enzyme E2 D4 F52C6.2 Ribosomal protein S27a F52C6.3 Ubiquitin F52C6.4 Ubiquitin F52C6.8 Kelch-like protein 28 F52D1.1 Neutral alpha-glucosidase AB precursor F52D10.3 14-3-3 protein zeta/delta F52F12.3 Mitogen-activated protein kinase kinase kinase 7 F53A2.6 Eukaryotic translation initiation factor 4E F53C11.5 Protein enabled homolog F53C3.13 Lipid phosphate phosphohydrolase 1 F53F8.1 Krueppel-like factor 3 F53F8.3 Tripartite motif-containing 32 F53G12.4 FAM133B F53G2.7 CDK-activating kinase assembly factor MAT1 F53H8.1 AP-3 complex subunit mu-1 F54A3.3 T-complex protein 1, gamma subunit F54B11.5 RING finger protein 141 F54C1.7 Calmodulin F54C9.2 STCH F54D5.8 DNAJB5 protein F54D8.2 Cytochrome c oxidase subunit Via

F54F7.5 Serine/threonine kinase 24 F54G8.4 Tripartite motif-containing protein 3 F55A11.3 E3 ubiquitin-protein ligase synoviolin precursor F55A12.8 N-acetyltransferase 10 F55A4.1 Vesicle-trafficking protein SEC22b F55B12.3 F-box/WD repeat protein 7 F55C5.7 Ribosomal protein S6 kinase delta-1 F55C5.8 Signal recognition particle 68 kDa protein F55D10.1 Lysosomal alpha-mannosidase precursor F55G1.5 Mitochondrial glutamate carrier 2 F55H2.1 Superoxide dismutase F55H2.5 Cytochrome b561 F56C9.1 PPP1CA F56D12.5 SERBP1 F56D2.2 E3 ubiquitin-protein ligase RNF14 F56D2.4 SUMO-conjugating enzyme UBC9 F56G4.2 Unnamed protein F56G4.5 N-glycanase 1 F56H1.4 26S protease regulatory subunit 6A F57B10.10 DAD1 F57B10.11 BCL2-associated athanogene F57B10.5 TMED7 F57B9.10 Proteasome 26S non-ATPase subunit 11 variant F57F5.1 Cathepsin B precursor F57F5.5 Protein kinase C, eta F58A4.10 Ubiquitin-conjugating enzyme E2 G1 F58A4.8 Tubulin gamma-1 chain F58B6.3 9 kDa protein F59B2.3 CGI-14 protein F59B2.5 Proteasome 26S non-ATPase subunit 11 variant F59D6.2 Gastricsin precursor F59D6.3 Gastricsin precursor F59E10.2 Peptidyl-prolyl cis-trans isomerase-like 2 F59E12.2 CaM kinase ID F59E12.4 Nuclear protein localization protein 4 homolog F59E12.5 Nuclear protein localization protein 4 homolog F59E12.6 Ubiquitin carboxyl-terminal hydrolase 25

F59F3.5 VEGFR-1 F59F4.1 Acyl-coenzyme A oxidase 1, peroxisomal F59G1.3 Vacuolar protein sorting-associated protein 35 H05L14.2 Golgin subfamily B member 1 H06O01.1 Protein disulfide isomerase A3 precursor H08M01.1 Golgin subfamily A member 2 H10E21.4 Calmodulin H10E21.5 RING finger protein 150 precursor H12I13.2 Ubiquitin specific protease 3 H15N14.2 Vesicle-fusing ATPase H19N07.2 Ubiquitin-specific protease 7 isoform H19N07.4 Diacylglycerol O-acyltransferase 1 H21P03.3 Sphingomyelin synthase 2 H22K11.1 Cathepsin D precursor H34C03.2 Ubiquitin carboxyl-terminal hydrolase 4 H38K22.2 DCN1-like protein 1 H38K22.3 Cytochrome b5 domain containing 2 JC8.10 Synaptojanin-1 K01A2.11 Intestinal mucin K01C8.10 T-complex protein 1, delta subunit K01C8.5 Cylicin-1 K01C8.6 39S ribosomal protein L10 K01G5.1 RING finger protein 113A K01G5.7 Tubulin beta-2C chain K02A11.1 PPP1R16A K02B12.3 Prolactin regulatory element-binding protein K02C4.3 Ubiquitin carboxyl-terminal hydrolase 25 K02E7.10 Cathepsin H precursor K02F3.10 Apolipoprotein O-like precursor K02G10.8 DnaJ homolog subfamily C member 5 K03A1.4 Calmodulin K04G2.4 Hypothetical protein K05F1.5 Leukocyte cell-derived chemotaxin 2 precursor K06H7.3 Zinc finger protein 744 K07A1.7 Headcase protein homolog K07A1.8 ERGIC-53 protein precursor K07A12.4 HBS1-like protein

K07A9.2 CAMK1 K07C11.9 Hypothetical protein DKFZp313D191 K07C5.1 Actin-like protein 2 K07D4.3 PSMD14 K07F5.12 Transmembrane protein 144 K08A8.1 Mitogen-activated protein kinase kinase 7 K08B12.5 Serine/threonine-protein kinase MRCK alpha K08B4.5 Ubiquitin-specific protease 7 isoform K08D10.2 DNAJC20, mitochondrial precursor K08D12.1 Proteasome subunit beta type 6 precursor K08E3.7 Isoform 1 of Parkin K09A9.4 Ubiquitin carboxyl-terminal hydrolase 33 K09E3.7 Mucin-2 precursor K09E4.2 Dolichyl-P-Man:Man K09F6.7 Tripartite motif-containing protein 2 K09H11.7 Hypothetical protein LOC283871 K09H9.2 DCC1 K10B2.2 Lysosomal protective protein precursor K10C2.3 Gastricsin precursor K10C3.2 Isoform 1 of Alpha-endosulfine K11E8.1 CAMK2G K11G12.4 Divalent metal transporter K12B6.8 E3 ubiquitin-protein ligase DZIP3 K12C11.2 Small ubiquitin-related modifier 1 precursor K12C11.4 Death-associated protein kinase 1 M02A10.3 E3 ubiquitin-protein ligase CBL-B M04G12.1 interferon regulatory factor 2 binding protein 2 M110.4 eIF-4-gamma 3 M116.2 Isoform GN-1L of Glycogenin-1 M117.2 14-3-3 protein zeta/delta M117.3 YWHAB M142.6 Isoform 1 of Roquin M151.3 Hook-related protein 1 M28.5 NHP2-like protein 1 M7.1 Ubiquitin-conjugating enzyme E2 D2 M7.5 Autophagy-related protein 7 R01H2.6 Ubiquitin-conjugating enzyme E2 L3

R02D3.4 Uncharacterized protein C12orf11 R02D3.5 FNTA R02E12.4 Tektin-1 R02F11.4 LRRIQ2 R03G5.1 Elongation factor 1-alpha 2 R03G5.2 MAP2K6 R04A9.2 Eukaryotic translation initiation factor 2C, 1 R05D11.6 Similar to RIKEN cDNA A430101B06 gene R05D3.1 Isoform Beta-1 of DNA topoisomerase 2-beta R05D3.4 E3 ubiquitin-protein ligase BRE1A R06C7.2 Translation initiation factor 2C R06F6.2 Vacuolar protein sorting-associated protein 11 R07B7.16 Orphan nuclear receptor NR5A2 R07E3.1 Cathepsin F precursor R07E4.1 ANKFN1 R07E5.1 GPATCH1 R07G3.3 Nucleoprotein TPR R07H5.2 Carnitine O-palmitoyltransferase 2 R07H5.3 Uncharacterized protein C3orf60 R09B3.4 NEDD8-conjugating enzyme UBE2F R09F10.1 Cathepsin F precursor R10A10.2 RING-box protein 2 R10D12.14 Grb10- interacting GYF protein 2 R10E11.2 ATP6V0C R10E11.3 Ubiquitin carboxyl-terminal hydrolase 46 R10E11.8 ATP6V0C R11A5.1 AP-3 complex subunit beta-2 R12E2.13 Stromal cell-derived factor 2 precursor R12E2.3 PSMD7 R12G8.1 Protein kinase C, eta R12H7.2 Cathepsin D precursor R13D7.7 Glutathione S-transferase pi R151.6 Derlin-2 R151.7 TRAP1 R166.2 Cleft lip and palate transmembrane protein 1 R186.3 SRPR protein, beta subunit R74.3 X-box binding protein 1

R74.4 DNAJB1 precursor T01B7.4 Peptidyl-prolyl cis-trans isomerase H T01C3.3 E3 ubiquitin-protein ligase RNF8 T01C8.1 PRKAA2 T01D1.1 Heme oxygenase 2 T01D3.3 IgGFc-binding protein precursor T01G1.1 Kinesin family member 21B T01G5.7 E3 ubiquitin-protein ligase RNF8 T01G6.1 DHRS4 T01H3.2 DKFZP434B0335 protein T02C1.1 Tripartite motif-containing protein 5 T03E6.7 Cathepsin L precursor T03F1.1 UBE1L T03F6.2 DNAJA5 T03G11.4 MAN1B1 T03G11.6 Methyltransferase like 9 isoform 1 T04A8.16 Calpain-7 T04A8.7 1,4-alpha-glucan branching enzyme T04A8.9 DnaJ homolog subfamily B member 7 T04C9.4 Cysteine and glycine-rich protein 2 T04G9.3 VIP36 precursor T04H1.9 Tubulin beta-2C chain T05A12.4 SNF2 histone linker PHD RING helicase T05C12.7 T-complex protein 1, alpha subunit T05C3.5 DnaJ homolog subfamily A member 2 T05D4.1 Fructose-bisphosphate aldolase A T05E11.3 Endoplasmin precursor T05E11.5 Minor histocompatibility antigen H13 T05E11.6 GPI-anchor transamidase precursor T05H10.1 Ubiquitin carboxyl-terminal hydrolase 47 T05H10.5 Isoform 2 of Ubiquitin conjugation factor E4 B T05H4.4 NADH-cytochrome b5 reductase T06C10.3 Proto-oncogene c-fes variant 3 T06D8.8 HSPC027 T06E8.1 AGPAT2 T06G6.4 Centromere protein F T07F12.4 Serine/threonine-protein kinase ULK2

T07G12.1 Calmodulin T08D2.4 Tripartite motif-containing 32 T08G5.5 Vam6/Vps39-like protein T09A5.11 DDOST T09B4.10 STUB1 T09B4.4 Calmodulin-like 4 isoform 1 T09E8.3 Cornichon homolog T10B11.6 Transmembrane protein 53 T10B5.5 T-complex protein 1, eta subunit T10D4.6 Tetra-peptide repeat homeobox-like protein T10F2.3 Sentrin-specific protease 1 T10H4.12 Cathepsin B precursor T11F1.8 Predicted receptor T12E12.1 Protein ariadne-2 homolog T13A10.11 S-adenosylmethionine synthetase T13A10.2 Tripartite motif-containing protein 2 T13H2.3 Plectin 1 isoform 3 T14G10.4 Unnamed protein T14G8.3 150 kDa oxygen-regulated protein precursor T16H12.2 CBF1-interacting corepressor T18H9.2 Gastricsin precursor T19B4.4 DnaJ homolog subfamily C member 15 T19E7.3 Beclin-1 T19H12.2 Acidic nuclear phosphoprotein pp32 T20B5.1 AP-2 complex subunit alpha-1 T20D4.13 N-glycanase T20D4.3 N-glycanase T20D4.5 N-glycanase T20F5.2 Proteasome subunit beta type 2 T20F5.6 Tripartite motif-containing protein 2 T20F5.7 Tripartite motif-containing 32 T21B10.7 T-complex protein 1, beta subunit T21C9.2 Vacuolar protein sorting-associated protein 54 T21D12.3 Polyglutamine-binding protein 1 T21H3.3 Calmodulin T22A3.2 Alpha crystallin B chain T22B2.1 Restin

T22D1.9 PSMD2 T22F3.2 Ubiquitin specific protease 42 T22H9.2 Autophagy-related protein 9A T23B12.7 DnaJ homolog subfamily C member 17 T23C6.5 Neuropeptide FF receptor 2 T23G11.2 Glucosamine 6-phosphate N-acetyltransferase T23G11.3 Isoform 4 of Quaking protein T23G5.2 SEC14-like protein 1 T23G5.5 SLC6A2 protein T24D1.2 CROP T24H10.3 DnaJ homolog subfamily C member 9 T25E12.4 Serine/threonine-protein kinase D3 T25G3.4 Glycerol-3-phosphate dehydrogenase T26A5.4 ALG1 T26C12.3 Ras-related protein Rap-2c precursor T27A1.5 SLC36A2 T27A3.2 Ubiquitin carboxyl-terminal hydrolase 5 T27A3.6 MOCS2 T27A8.2 Hepatocyte nuclear factor 3-gamma T27C10.6 Leucine-rich repeat kinase 1 T27D1.1 Peptidyl-prolyl cis-trans isomerase G T27E4.2 Alpha crystallin B chain T27E4.8 Alpha crystallin B chain T27E9.3 Cell division protein kinase 5 T27F7.1 Charged multivesicular body protein 3 T28D6.2 Tubulin alpha-6 chain T28F12.2 Homeobox protein Meis2 T28H10.3 Legumain precursor VF13D12L.1 Myo-inositol 1-phosphate synthase A1 VF39H2L.1 Syntaxin-17 W01G7.4 SLC7A6OS W01H2.2 Hypothetical protein FLJ45999 W02A11.3 RING finger protein 44 W02A11.4 SUMO-activating enzyme subunit 2 W02D3.10 Uncharacterized membrane protein KIAA1794 W03A5.7 DNAJB6 W03C9.3 Ras-related protein Rab-7

W03F8.3 Mitochondrial translational release factor 1-like W03F8.4 TP53RK-binding protein W04G5.4 N-glycanase W04G5.9 Intestinal mucin W04H10.3 Tripartite motif protein 3 W06B4.2 N-acetylglucosamine kinase W06B4.3 Vacuolar protein sorting-associated protein 18 W06E11.4 SBDS W07A8.2 85 kDa Calcium-independent phospholipase A2 W07A8.3 Putative tyrosine-protein phosphatase auxilin W07B3.2 Trichohyalin W07B8.1 Cathepsin B precursor W07E11.1 Dihydropyrimidine dehydrogenase [NADP+] W08D2.5 Probable cation-transporting ATPase 13A3 W09C5.2 Isoform 1 of Septin-7 W09C5.8 Cytochrome c oxidase subunit IV isoform 1 W09G12.5 SRPR protein, alpha subunit W09G12.8 Fibronectin type III SPRY domain containing 2 Y106G6H.12 Ubiquitin carboxyl-terminal hydrolase 29 Y110A2AR.2 Ubiquitin conjugating enzyme E2, J2 isoform 2 Y110A7A.14 Proteasome subunit alpha type 4 Y110A7A.19 Pentatricopeptide repeat domain 3 Y113G7A.3 Protein transport protein Sec23A Y17G9B.4 40 kDa peptidyl-prolyl cis-trans isomerase Y18D10A.25 FKBP1A protein Y19D2B.1 Tubulin alpha-6 chain Y25C1A.5 Coatomer beta subunit Y37A1B.12 Torsin A precursor Y37A1B.13 Torsin A precursor Y37A1B.15 TUBA Y37A1B.2 SH3 and PX domain containing 3 Y37D8A.14 Cytochrome c oxidase polypeptide Va Y37H9A.6 Bis(5'-nucleosyl)-tetraphosphatase Y38A10A.5 Calreticulin precursor Y38A8.2 Proteasome subunit beta type 3 Y38E10A.8 eIF-2 alpha kinase 3 Y38F1A.2 RING finger protein 170

Y38F2AL.3 Vacuolar ATP synthase subunit C Y38F2AL.4 ATP6V0C Y38H8A.2 Tripartite motif-containing 39 Y39A1C.2 Ubiquitin protein ligase E3B Y39A3CR.8 Conserved hypothetical protein Y39B6A.1 Hornerin Y39B6A.20 Gastricsin precursor Y39B6A.22 Gastricsin precursor Y39B6A.24 Gastricsin precursor Y39E4A.2 Solute carrier family 30, member 2 isoform 1 Y39E4B.1 ATP-binding cassette sub-family E member 1 Y39G10AR.13 Inner centromere protein antigens 135/155kDa Y39H10A.7 Serine/threonine-protein kinase Chk1 Y40D12A.2 Lysosomal protective protein precursor Y40G12A.1 Ubiquitin carboxyl-terminal hydrolase L3 Y40H7A.9 Dipeptidyl-peptidase 1 precursor Y41E3.7 Golgi resident protein GCP60 Y43C5B.2 Proto-oncogene tyrosine-protein kinase FER Y43E12A.3 KBTBD4 Y45F10A.6 TBC1 domain family, member 9 Y45F10B.8 Tripartite motif-containing 32 Y45F10B.9 Tripartite motif-containing 32 Y46H3A.2 Alpha crystallin B chain Y46H3A.3 Alpha crystallin B chain Y47G6A.22 3-Hydroxybutyrate dehydrogenase type 2 Y47H9A.1 N-glycanase Y48A5A.1 SHQ1 homolog Y48G8AL.1 HERC4 Y48G9A.11 PR domain zinc finger protein 5 Y49E10.1 26S protease regulatory subunit 8 Y49E10.20 Lysosome membrane protein 2 Y49E10.4 Protein disulfide isomerase A6 precursor Y49F6B.9 E3 ubiquitin-protein ligase RNF19 Y4C6A.3 Tripartite motif-containing 32 Y51A2D.5 Proton myo-inositol cotransporter Y51H4A.17 Signal transducer and activator of transcription 6 Y51H4A.8 n/a

Y52B11C.1 PIGL Y53C10A.12 Heat shock factor protein 1 Y53C10A.2 Microtubule-associated protein 1A Y53C12B.2 RNA-binding protein PNO1 Y53F4B.4 NSUN5 Y53H1A.2 Zinc finger protein 443 Y54E10A.6 Leucine-rich repeat-containing protein 47 Y54E10BL.4 DnaJ homolog subfamily C member 3 Y54E10BR.1 GPI ethanolamine phosphate transferase 1 Y54E10BR.2 ARF-related protein 1 Y54E10BR.3 RING finger protein 126 Y54E10BR.4 Phosphorylation regulatory protein HP-10 Y54E2A.12 CDNA FLJ45119 fis, clone BRAWH3035914 Y54E5B.4 Probable ubiquitin-conjugating enzyme E2 W Y55D9A.2 AGGF1 Y55F3AM.3 RNA-binding protein 39 Y55F3AM.4 Autophagy-related protein 3 Y55F3AM.6 Makorin-1 Y55F3AR.3 T-complex protein 1, theta subunit Y55F3BR.1 ATP-dependent RNA helicase DDX1 Y55F3BR.6 Heat-shock protein beta-1 Y56A3A.3 Macrophage migration inhibitory factor Y57A10A.31 Ariadne-1 protein homolog variant (Fragment) Y57G11C.3 6-Phosphogluconolactonase Y59A8B.2 Ubiquitin carboxyl-terminal hydrolase 8 Y61A9LA.3 Hypothetical protein LOC55082 Y63D3A.6 Translocation protein SEC63 homolog Y65B4A.2 Cathepsin B precursor Y65B4BR.4 NEDD4-like E3 ubiquitin-protein ligase WWP1 Y67D8C.5 HUWE1 Y69A2AR.2 Resistance to inhibitors of cholinesterase 8A Y69H2.6 AKT interacting protein Y6B3A.1 ARFGEF2 Y71H2AM.5 Cytochrome C oxidase subunit Vib Y71H2AR.2 Cathepsin L2 precursor Y73C8C.3 CENPE variant protein (Fragment) Y73C8C.7 Myosin-9

Y73C8C.8 CENPE variant protein (Fragment) Y75B12B.2 PPIase, mitochondrial precursor Y75B12B.5 PPIase, mitochondrial precursor Y76A2A.2 Copper-transporting ATPase 1 Y77E11A.2 CDNA FLJ30596 fis, clone BRAWH2009227 Y87G2A.10 Vacuolar protein sorting-associated protein 28 Y87G2A.6 PPIase domain and WD repeat protein 1 Y95B8A.10 PDE8A Y97E10AR.2 Gamma-glutamyltranspeptidase 1 precursor Y97E10AR.4 HIV Tat-specific factor 1 ZC155.7 Isoform A of Syntaxin-16 ZC196.6 Acidic repeat-containing protein ZC250.1 Isoform 2 of Zonadhesin precursor ZC317.7 Proline-rich protein 12 ZC328.2 Zinc finger protein 25 ZC395.2 Ubiquinone biosynthesis protein COQ7 ZC395.8 Dentin sialophosphoprotein preproprotein ZC455.10 FK506 binding protein 9 precursor ZC506.1 EDEM3 ZC518.2 Protein transport protein Sec24B ZC97.1 Metaxin-2 ZK1010.2 RMND1 ZK1053.6 Solute carrier family 41, member 3 isoform 4 ZK112.2 Tripartite motif-containing 3 ZK1128.7 Alpha-B-crystallin ZK1236.3 PAX interacting protein 1 ZK1236.7 VGPW2523 ZK1240.1 Tripartite motif-containing protein 2 ZK1240.2 Tripartite motif-containing protein 2 ZK1240.3 52 kDa Ro protein ZK1240.4 Ciliary dynein heavy chain 5 ZK1240.6 GTP-binding protein ARD-1 ZK1248.10 TBC1D2B protein ZK1320.6 GTP-binding protein ARD-1 ZK20.5 PSMD8 ZK218.11 Keratin associated protein 16-1 ZK287.5 RING-box protein 1

ZK328.1 Ubiquitin carboxyl-terminal hydrolase 32 ZK384.3 Gastricsin precursor ZK418.4 Junctional sarcoplasmic reticulum protein 1 ZK430.3 Superoxide dismutase ZK520.5 40 kDa peptidyl-prolyl cis-trans isomerase ZK54.2 Trehalose-6-phosphate synthase ZK593.6 MAP1A/MAP1B light chain 3A precursor ZK632.2 Kanadaptin ZK632.6 Calnexin precursor ZK637.14 Hypothetical protein LOC51255 ZK637.14 Hypothetical protein LOC51255 ZK652.9 COQ5 ZK666.6 ZNF254 protein ZK669.1 PTPL1-associated RhoGAP 1 ZK675.2 DNA repair protein REV1 ZK686.3 Tumor suppressor candidate 3 ZK688.5 Mucin 5 (Fragment) ZK688.5 Mucin 5 (Fragment) ZK809.7 Peroxisome assembly factor 1 ZK856.1 Cullin homolog 5 ZK899.4 Tubulin alpha-6 chain ZK930.1 Phosphoinositide 3-kinase regulatory subunit 4 ZK945.5 GTP-binding protein ARD-1 ZK973.11 Protein disulfide-isomerase TXNDC10 precursor

Table 2.5. Summary of RNAi knockdown of the top 20 gene candidates in worms expressing Q82::GFP + TOR-2 in body wall muscle cells. C. elegans Gene ID of Average number of targeted gene aggregates/worm 1, 2 + SD None (Q82::GFP + TOR-2) 35.3 + 5.5 B0432.2 (djr-1.1) 38.6 + 6.1 T05C3.5 (dnj-19) 34.5 + 5.5 C35D10.2 35.8 + 6.7 C54H2.5 (sft-4) 35.7 + 4.9 EEED8.9 (pink-1) 37.3 + 7.6 F11H8.1 (rfl-1) 35.9 + 7.4 F16A11.2 39.2 + 7.2 F26E4.11 (hrdl-1) 38.1 + 5.3 F32A6.3 (vps-41) 37.8 + 7.0 F48E3.7 (acr-22) 36.4 + 8.7 F55A4.1 35.6 + 6.2 F57B10.5 36.2 + 6.2 F59F4.1 36.2 + 5.8 K11G12.4 (smf-1) 37.4 + 4.2 M7.5 (atgr-7) 38.5 + 5.2 R05D11.6 36.4 + 6.6 T07F12.4 36.1 + 5.7 T08D2.4 37.9 + 4.4 T13A10.2 36.1 + 6.0 Y37A1B.13 (tor-2) 52.0 + 9.9 ( P < 0.01) 2 1Two separate RNAi experiments were performed for each gene target (n = 20 for each RNAi round). 2Knockdown of tor-2 resulted in a significant increase in aggregate number, whereas the depletion of other gene products did not enhance aggregation.

Figure 2.1

Figure 2.2

Figure 2.3

Figure 2.4

Figure 2.5

Figure 2.6

Figure 2.7

FIGURE LEGENDS Figure 2.1. RNAi knockdown of specific gene targets enhances misfolding of α- syn (A-E). A. Isogenic worm strain expressing α-syn::GFP alone in body wall muscle cells of C. elegans . B. The presence of TOR-2, a protein with chaperone activity, attenuates the misfolded α-syn protein. C, D. When worms expressing α-syn::GFP + TOR-2 are exposed to candidate gene RNAi, the misfolded α-syn::GFP returns. E. Western analysis of α-syn::GFP demonstrating the presence of α-syn::GFP in worms with and without TOR-2 co-expression. α-syn antibody (Chemicon) was used to detect the α-syn::GFP fusion protein band; actin was probed for each lane as a loading control. Figure 2.2. Overexpression of candidate genes protects DA neurons from α-syn- induced degeneration. A. Schematic representation depicting the distribution of the top 20 candidate genes isolated from the RNAi screen, as categorized using bioinformatic associations employed to select the gene for knockdown. Six of 20 genes were identified from 2 or more categories and are indicated with overlapping color. B. Graph depicting percentage α-syn-expressing worms with wildtype DA neurons at the 7-day adult stage of life when candidate genes are co-expressed. * P<0.05; student t-test. C – F. Worm DA neurons at low (C, E) and high (D, F) magnification. C, D. At the 7-day stage, most worms expressing a-syn are missing anterior DA neurons. Note the presence of only 2 of 4 CEP DA neurons. E, F. Overexpression of C. elegans VPS41 protects worms from DA neurodegeneration, whereby worms display all 4 CEP neurons. Worm population analysis revealed that 36.7% of worms overexpressing VPS41 were wildtype, compared with only 12.8% of the α-syn only control worms. Figure 2.3. An interconnectivity map summarizing the relationships between 17% (125/757) of the genes knocked down via RNAi that were non-lethal and classified in more than one bioinformatic category or cellular pathway. Figure 2.4. Expression of α-syn in worm DA neurons results in age- and dose- dependent neurodegeneration. A. Semi-quantitative RT-PCR demonstrating that two

separate isogenic worms strains expressing α-syn (low and high) have differing levels of mRNA when compared to the cdk-5 control. B. Integrated transgenic line containing both Pdat-1:: α-syn (high) and Pdat-1::GFP shows DA neurodegeneration over time, as animals age. Figure 2.5. Analysis of transgene expression in worm strains. Semi-quantitative RT-PCR was performed using primers to amplify cdk-5 (control), a-syn (specific to the α-syn-expressing transgenic line), and primers specific to the clones analyzed. For all primers, N2 wild type animals were used as both a positive ( cdk-5) and negative control (α-syn and transgene expression). Worms expressing α-syn without candidate PD transgenes were also analyzed where cdk-5 and α-syn primers were positive controls and primers corresponding to the transgenes were negative controls. The candidate PD transgenes were amplified using gene-specific primers; all three separate transgenic lines for each clone were analyzed; α-syn primers were utilized as a negative control. Figure 2.6. RNAi knockdown of the top 20 gene targets did not enhance misfolding of polyglutamine aggregates in worms expressing Q82::GFP + TOR-2 in body wall muscle cells (A-D). A. Isogenic worm strain expressing Q82::GFP + TOR-2 in C. elegans . B. When worms expressing Q82::GFP + TOR-2 are exposed to tor-2 RNAi, the Q82::GFP aggregation returns. C, D. When worms expressing Q82::GFP + TOR-2 are exposed to C35D10.2 or vps-41 RNAi, Q82::GFP aggregation is not enhanced. The presence of TOR-2, a protein with chaperone activity, attenuates the misfolded polyglutamine protein, as previously reported (Caldwell et al., 2003) and RNAi knockdown reverses this effect (B). Figure 2.7. Quantitative analysis of the hit rate of genes at both the primary and secondary level of RNAi screening compared with starting genes based on specific associations. A. Candidates separated according to category (mechanisms = UPS, UPR, ERAD, or autophagy; worm bioinformatics = C. elegans microarray or interactome data; α-syn proteomic/genetics = proteomic or yeast genetic analyses). The highest hit rate

came from genes that were co-expressed with a known PD gene and are associated with a cellular mechanism implicated in PD. B. Candidates derived from microarray co- expression data for both DJ-1 and PINK1 are highly enriched at both the primary and secondary level of RNAi screening for α-syn modifiers.

CHAPTER THREE

VALIDATION OF SUPPRESSORS OF ALPHA-SYNUCLEIN TOXICITY FROM YEAST GENETIC SCREENING

Work on RAB proteins was published in Proceedings of the National Academy of Sciences of the United States of America , January, 2008 under the following citation: Gitler, A.D ., Bevis, B.J ., Shorter, J ., Strathearn, K.E ., Hamamichi, S ., Su, L.J ., Caldwell, K.A ., Caldwell, G.A ., Rochet, J.C ., McCaffery, J.M ., Barlowe, C ., and Lindquist, S. (2008) Proc Natl Acad Sci U S A 105, 145-150.

Work on PARK9/ATP13A2 was published in Nature Genetics , March, 2009 under the following citation: Gitler, A.D ., Chesi, A ., Geddie, M.L ., Strathearn, K.E ., Hamamichi, S., Hill, K.J ., Caldwell, K.A ., Caldwell, G.A ., Cooper, A.A ., Rochet, J.C ., and Lindquist, S. (2009) Nat Genet 41, 308-315.

Shusei Hamamichi collected all C. elegans data. Shusei Hamamichi, Dr. Kim Caldwell, and Dr. Guy Caldwell co-wrote the manuscript.

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ABSTRACT

Multiple Parkinson disease (PD) models including S. cerevisiae , C. elegans , and

D. melanogaster have been generated and utilized to ascertain genetic modifiers of α- synuclein (α -syn) toxicity that are subsequently validated using mammalian system.

Here we report the analysis of eight suppressors of α-syn toxicity that were initially identified from the genome-wide yeast α-syn toxicity modifier screen using C. elegans model of α-syn-induced dopamine (DA) neurodegeneration. Consistent with previously reported neuroprotective function of Rab1a, both RAB3A (a RAB GTPase localized to the presynaptic termini) and RAB8A (a RAB GTPase involved in post-Golgi trafficking) significantly rescued worm DA neurons from α-syn toxicity. Interestingly, a gene identified from the screen encoded ypt9 , a yeast ortholog of PARK9/ATP13A2. To examine genetic interaction between these two PD-associated genes in C. elegans , we

determined that while RNAi knockdown of a worm ortholog of PARK9, W08D2.5

enhanced α-syn misfolding in body wall muscles, overexpression of this gene in DA

neurons exhibited neuroprotection against α-syn. Two additional genes from the screen,

PDE9A (a phosphodiesterase) and PLK2 (a Polo-like kinase) also rescued DA neurons.

This work illustrates a collaborative effort to utilize model organisms to validate positive

genetic candidates from the yeast genetic screen, and represents putative genetic

susceptibility factors as well as therapeutic targets for PD.

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INTRODUCTION

While invertebrate disease models are not evolutionally as complex as vertebrate counterparts, these models provide unique advantages that are readily exploited as research tools. For example, these models are cost-effective for large-scale genetic or chemical screens. Furthermore, molecular and genetic manipulations allow meticulous genetic analysis that cannot be easily performed using mammalian cell culture or rodent models. Given the fact that numerous pathways and cellular functions are conserved across the species, these invertebrate model organisms cannot be neglected as they remain as valuable and informative tools. In PD research, S. cerevisiae , C. elegans , and

D. melanogaster models have been generated to uncover novel therapeutic targets that ameliorate α-syn-induced toxicity. According to this experimental strategy, invertebrates model a disease state (e.g., overexpressing wildtype α-syn, DA neurodegeneration, etc), and are utilized for genetic or chemical screening, thereby eliminating countless genetic targets and pathways that may be irrelevant to PD pathogenesis. Subsequently, these results are validated by mammalian system.

In one yeast PD model, Willingham et al. (2003) transformed 4850 yeast deletion mutant strains with wildtype α-syn under the control of inducible promoter, and identified 86 genes that were sensitive to α-syn overexpression. Notably, among these positive candidates, 18 of them were predicted to function in lipid metabolism and vesicle-mediated transport. Outerio et al. (2003) confirmed these findings by examining enhanced accumulation of lipid droplets and defects in vesicular trafficking to yeast

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vacuoles (mammalian equivalent of lysosomes). Furthermore, subsequent ultrastructural

analysis revealed that α-syn is clustered in the membranous vesicles that are co-localized with secretory and ER-Golgi transport vesicles (Soper et al., 2008).

Utilizing a yeast strain expressing two copies of wildtype α-syn under the control

of inducible GAL4 promoter, Cooper et al. (2006) transformed 3000 genes, and identified genetic modifiers of α-syn cytotoxicity. They identified 34 genes that suppressed the

toxicity, and 20 genes that enhanced it. One of the strong suppressors was Ypt1/RAB1A.

To validate their findings in higher eukaryotic systems, mammalian Rab1a was

overexpressed in PD models consisting of C. elegans , D. melanogaster , and mammalian

DA neuron culture, and found neuroprotective across the species (Cooper et al., 2006).

Furthermore, they determined that overexpression of α-syn blocked ER-Golgi trafficking, and proposed that the defects in vesicle trafficking may contribute to increased oxidized

DA, which may lead to the selective loss of DA neurons. The cellular link between α- syn toxicity and ER homeostasis is further supported by the findings that mutant α-syn

induces ER stress (Smith et al., 2005) and activation of UPR has been detected in DA

neurons of the substantia nigra of PD patients (Hoozemans et al., 2007).

This work represents a continuing collaborative effort to utilize various model

organisms as a “pipeline” to validate positive genetic candidates from the yeast genetic

screen in the higher eukaryotic systems. To expand and further confirm the

neuroprotective role of RAB proteins (Cooper et al., 2006), RAB3A and RAB8A are

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tested here for their effect in suppressing DA neurodegeneration in C. elegans .

Furthermore, in another study, the Lindquist lab (Whitehead Institute/MIT) screened an additional 5000 genes, and identified ypt9 (a S. cerevisiae ortholog of human

PARK9/ATP13A2 ), a gene that, when overexpressed, suppressed α-syn cytotoxicity in yeast. To verify the uncharacterized but putative genetic interaction between α-syn and

ATP13A2 , we examined neuroprotective function of W08D2.5 (a C. elegans ortholog of human PARK9/ATP13A2 ) in worm DA neurons. Additional α-syn toxicity suppressors from this latter yeast screen, SYVN1, USP10, PDE9A, PLK2, and CSNK1G3 were analyzed using our worm α-syn-induced neurodegeneration model.

MATERIALS AND METHODS

C. elegans experiments for RAB analysis. Nematodes were maintained following the standard procedures (Brenner, 1974). Strains UA81 {[ baIn1 ; Pdat-1:: α -syn, P dat-

1:: gfp ]; [ baEx67 ; Pdat-1:: RAB3A , rol-6 (su1006) ]} and UA82 {[ baIn1 ; Pdat-1:: α -syn, P dat-

1:: gfp ]; [ baEx68 ; Pdat-1:: RAB3A , rol-6 (su1006) ]} were generated by injecting 50 g/ml of

each expression plasmid and 50 g/ml of rol-6 into an integrated line UA44 [ baIn1 ; Pdat-

1:: α-syn, P dat-1:: gfp ]. For neuroprotection analysis, 3 independently isolated stable lines overexpressing mouse Rab1a as well as human RAB3A and RAB8A were analyzed as described previously (Cooper et al., 2006) with the following modification. The 6 anterior DA neurons (4 CEP and 2 ADE neurons) of 30 animals/trial were scored for

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neuroprotection when the animals were 7 days old. The experiments were conducted in

triplicate for each stable line (3 lines x 3 trials of 30 animals/trial=270 total animals

scored).

C. elegans experiments for PARK9/ATP13A2 analysis. Nematodes were

maintained following the standard procedures (Brenner, 1974). RNAi and fluorescent

microscopy were performed as described (Hamamichi et al., 2008) by feeding UA50

[baInl3 ; Punc-54 :: α-syn :: gfp , Punc-54 :: tor-2, rol-6 (su1006) ] worms with the RNAi clones

(Geneservice, Cambridge, UK) corresponding to W08D2.5 and its putative interactors,

R12E2.13 , and R06F6.8 . RNA isolation, cDNA preparation, and semi-quantitative RT-

PCR were conducted as described (Hamamichi et al., 2008) with the following modification. Total RNAs from 50 young adult control [RNAi bacteria HT115(DE3) with empty vector] and RNAi-treated worms were isolated to generate cDNAs. PCR was then performed using primers specific for amplifying cdk-5 as loading control, α-syn , and tor-

2. For DA neurodegeneration analysis, strains UA51 {[ baIn1 ; Pdat-1:: α-syn, P dat-1:: gfp ];

[baEx42 ; Pdat-1:: FLAG-W08D2.5 , rol-6 (su1006) ]} and UA108 {[vtls1 ; Pdat-1::gfp; rol-6

(su1006) ]; [baEx83 ; Pdat-1:: FLAG-W08D2.5 , Punc-54 ::mCherry ]} were generated by

injecting 50 g/ml of each expression plasmid into integrated UA44 [ baIn1 ; Pdat-1:: α-syn,

Pdat-1:: gfp ] as well as BY200 [ vtls1 ; Pdat-1::gfp; rol-6 (su1006) ] worms, respectively.

Additionally, UA75 {[ baIn1 ; Pdat-1:: α-syn, P dat-1:: gfp ]; [baEx64 ; Pdat-1:: PDE9A , rol-6

(su1006) ]}, UA76 {[ baIn1 ; Pdat-1:: α-syn, P dat-1:: gfp ]; [baEx65 ; Pdat-1:: PLK2 , rol-6

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(su1006) ]}, UA93 {[ baIn1 ; Pdat-1:: α-syn, P dat-1:: gfp ]; [baEx72 ; Pdat-1:: CSNKIG3 , rol-6

(su1006) ]}, UA94 {[ baIn1 ; Pdat-1:: α-syn, P dat-1:: gfp ]; [baEx73 ; Pdat-1:: SYVN1 , rol-6

(su1006) ]}, and UA95 {[ baIn1 ; Pdat-1:: α-syn, P dat-1:: gfp ]; [baEx74 ; Pdat-1:: USP10 , rol-6

(su1006) ]} were generated by injecting 50 g/ml of each expression plasmid and 50

g/ml of rol-6 into an integrated line UA44 [baInl1 ; Pdat-1:: α−syn, P dat-1:: gfp ]. The 6

anterior DA neurons (4 CEP and 2 ADE neurons) of thirty 7 day-old animals were scored

for neuroprotection. The experiments were conducted in triplicate for each stable line (3

lines x 3 trials of 30 animals/trial=270 total animals scored).

RESULTS

In the C. elegans α-syn neurodegeneration model, overexpression of wildtype α-

syn is driven under the control of a promoter specific to DA neurons ( Pdat-1; dopamine

transporter). Because nematode development is invariable, any deviation from the

normal number of DA neurons is easily scored. For example, 6 anterior DA neurons

(CEPs and ADEs) are readily visualized in worms carrying a Pdat-1:: gfp construct

throughout the course of its lifespan (Berkowitz et al., 2009). Overexpression of α-syn

reduced the number of worms with the wildtype number of DA neurons to approximately

15% at the 7-day stage. In contrast, human RAB3A increased the rescue to 25% and

human RAB8A to 40% (Fig. 3.1). These findings further confirm the neuroprotective

function of RAB GTPases.

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To investigate the genetic interaction between α-syn and ATP13A2 in DA

neurons, α-syn was overexpressed under the control of dopamine transporter promoter

(Pdat-1), which resulted in an age-dependent progressive loss of DA neurons, with

approximately 15% of animals having 6 intact anterior DA neurons at the 7-day stage

(Fig. 3.2a,c). Expression of W08D2.5 (a C. elegans ATP13A2 ortholog) alone did not induce any change in the number of DA neurons (data not shown). Co-expression of

W08D2.5 and α-syn partially rescued this neurodegeneration in each of four independent transgenic lines (Fig. 3.2b,c).

C. elegans was also used to explore the consequences of W08D2.5 loss-of- function. Unfortunately, neuronal cells of this organism are refractory to RNAi-mediated inhibition of gene expression (Asikainen et al., 2005). However, work with yeast and neuronal model systems establishes that α-syn toxicity is the result of general cellular defects to which neuronal cells are simply more sensitive. Therefore, another cell type was chosen that has been extensively exploited for studies of protein homeostasis in this organism and readily affected by RNAi.

Body wall muscle cells expressing a fusion protein α-syn::GFP exhibit age- dependent α-syn aggregation (Fig. 3.2d). Co-expression of TOR-2, a worm ortholog of human torsinA chaperone-like protein, reduces protein misfolding and α-syn aggregation

(McLean et al., 2002; Caldwell et al., 2003), provided a sensitized genetic background within which an enhancement of α-syn misfolding could be observed (Fig. 3.2e). To

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specifically target W08D2.5 , we utilized RNAi to knock down the expression. This

profoundly enhanced the misfolding of human α-syn and did so in an age-dependent

manner (Fig. 3.2f) without modifying the expression levels of α-syn and tor-2 mRNAs

(Fig. 3.3). These data provide further evidence for an intimate functional interaction

between α-syn and ATP13A2.

In addition, five α-syn toxicity suppressor genes (yeast/human: Hrd1/SYVN1,

Ubp3/USP10, Pde2/PDE9A, Cdc5/PLK2, Yck3/CSNK1G3 ) were also overexpressed in the worm DA neurons to test their neuroprotective capacities. While all candidates except

CSNK1G rescued mammalian DA neurons, only two ( PLK2 and PDE9A ) suppressed α- syn-induced neurodegeneration in the nematode (Fig. 3.1). Since human genes were utilized in this study, it is conceivable that worm cellular machinery failed to properly fold and/or express these functional proteins. To address this issue, we subsequently analyzed worm hrd-1/SYVN1 and csnk-1/CSNK1G3 , and determined that these genes indeed are neuroprotective (unpublished data). Taken together, these findings demonstrate an experimental paradigm for using various model organisms to identify novel PD therapeutic targets.

DISCUSSION

Using our C. elegans model of α-syn-induced neurodegeneration, we demonstrate that overexpression of RAB3A and RAB8A rescue DA neurons. A previous study

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illustrated that α-syn overexpression blocked ER to Golgi trafficking, and as expected,

co-overexpression of Rab1a, a small GTPase that regulates vesicle trafficking from the

ER to the Golgi apparatus, ameliorated it (Cooper et al., 2006). In this study, additional

RAB GTPases, RAB3A and RAB8A were tested. RAB3A is expressed in human

neurons and found in the presynaptic termini where α-syn is also localized. Furthermore,

using cell-free system with purified transport factors, α-syn was shown to disrupt docking or fusion of the vesicles to Golgi membranes, demonstrating that while α-syn

does not alter budding of the vesicles from the ER, it interferes with the later stage of ER

to Golgi trafficking (Gitler et al., 2008). To further confirm this observation, RAB8A,

which closely resembles RAB1A, was examined since the protein functions in post-Golgi

trafficking. The present work further validates that RAB proteins suppress disruption of

vesicle trafficking by α-syn. Furthermore, the fact that RAB proteins rescue yeast,

worm, and mammalian PD models suggest that the defective vesicle trafficking is a

conserved pathological feature related to α-syn toxicity.

We have also demonstrated that while RNAi knockdown of W08D2.5

enhanced α-syn misfolding in the body wall muscles, overexpression of this protein

protected DA neurons from α-syn toxicity, indicating the conserved genetic interaction

between α-syn and ATP13A2 . Ramirez et al. (2006) reported that while wildtype

ATP13A2 is localized in the lysosomes, misfolded mutant forms are retained in the ER to be subsequently degraded by proteasomes. Interestingly, they observed approximately

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10-fold increase in ATP13A2 mRNA level in the surviving DA neurons of human idiopathic PD post-mortem midbrains, suggesting the neuroprotective function of this protein. Lysosomal function has been implicated as one of the critical protein degradation machineries for proteolysis of misfolded and aggregated α-syn (Webb et al.,

2003; Cuervo et al., 2004). Lastly, in the present study, depletion of yeast ypt9 increased the susceptibility of yeast cells to manganese toxicity, illustrating the potential genetic and environmental interaction between ATP13A2 and metal toxicity (Gitler et al., 2009)

Through our collaborative efforts, we reported 5 genes ( RAB3A, RAB8A,

W08D2.5, PDE9A, and PLK2 ) with conserved neuroprotective capacities. PDE9A encodes a cyclic nucleotide phosphodiesterase that regulates signal transduction by hydrolyzing cAMP and cGMP to their monophosphates. In the classical view of dopamine signaling, D2-like receptor inhibits cAMP production by inhibiting adenylate cyclase. Interestingly, α-syn overexpression in dop-2 (a C. elegans ortholog of D2-like

DA receptor) deletion background enhances α-syn-induced neurodegeneration

(unpublished data), suggesting the neuroprotective role of D2-like DA receptors.

Furthermore, hyperactive adenylate cyclase in C. elegans has been shown to induce neurodegeneration (Korswagen et al., 1998). Collectively, these data suggest that increased cAMP level may induce DA neuronal death.

Another neuroprotective target, PLK2 is expressed in mammalian neurons where the kinase is involved in maintaining homeostatic synaptic plasticity (Seeburg et al.,

2008). In PD research, a recent article by Inglis et al. (2009) demonstrated that PLK2 is a

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main contributor of α-syn phosphorylation at serine 129, a potentially more toxic form of

α-syn. Interestingly, using our worm α-syn neurodegeneration model, overexpression of

PLK2 with kinase dead mutation still rescued DA neurons whereas PLK2 with disrupted

polo-box domain did not affect the level of neurodegeneration (unpublished data). While

the neuroprotective mechanism of PLK2 remains unclear, our data suggest that protein-

protein interaction at the polo-box domain is responsible for reduced α-syn toxicity.

Currently, nine genes have been associated with PD ( α-syn , ATP13A2, DJ-1,

GIGYF2, HTRA2 , LRRK2 , PINK1, PRKN , and UCHL1 ), accounting for 5-10% of all PD

cases. This finding suggests that environmental PD susceptibility factors such as

neurotoxins and heavy metals or interaction between genetic and environmental factors

may play a critical role in disease onset or progression (Di Monte, 2003; Benmoyal-Segal

et al., 2006). Alternatively, multiple, heterogeneous sets of genes may contribute to the

etiology of this disease. To test the latter hypothesis, it will be interesting to compare our

present list of genes to the results from genome-wide single nucleotide polymorphism

(SNP) analysis of PD patients (Fung et al., 2006; Pankratz et al., 2009). Lastly, these

identified genes are invaluable candidates for potential therapeutic intervention for PD.

Taken together, this work illustrates a collaborative effort to exploit various advantages

of model organisms to rapidly validate positive genetic candidates from the yeast genetic

screen in the higher eukaryotic systems.

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Figure 3.1

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Figure 3.2

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Figure 3.3

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FIGURE LEGENDS

Figure 3.1. RAB3A, RAB8A, PDE9A, and PLK2 protect against α-syn-induced

DA neuron loss. DA neurons of 7-day old transgenic nematodes overexpressing α-syn along with the indicated genes were analyzed [ P < 0.05, Student’s t test ( *)]. For each gene tested, 3 transgenic lines were analyzed; a worm was scored as WT when all six anterior DA neurons (4 CEP and 2 ADE neurons) were intact.

Figure 3.2. PARK9 antagonizes α-syn-mediated DA neuron degeneration in C. elegans . Anterior DA neurons in worms expressing Pdat-1::GFP + P dat-1:: α-syn at the day

7 stage. Arrowheads and arrows depict cell bodies and neuronal processes, respectively.

WT worms have 6 anterior DA neurons. A. α− Syn toxicity is depicted by the loss of anterior DA neurons. B. DA neurons are protected when Pdat-1::FLAG-W08D2.5 cDNA is co-expressed. C. Quantification of C. elegans PARK9 rescue of α-syn-induced neurodegeneration in 4 independent transgenic lines displaying all six anterior DA neuron. P < 0.05, Student’s t test. D. Overexpression of α-syn in P unc-54 :: α-syn::GFP results in misfolding and aggregation of α-syn in body wall muscle cells at the young adult stage. E. Co-overexpression of TOR-2, a protein with chaperone activity, attenuates the misfolding of the α-syn::GFP protein. F. The misfolding of α-syn::GFP is enhanced following RNAi targeting W08D2.5 .

Figure 3.3. RNAi knockdown of W08D2.5 , R12E2.13 , and R06F6.8 does not reduce α-syn or tor-2 mRNA expression levels. Semi-quantitative RT-PCR was

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performed by using primers that amplify cdk-5 (loading control), α-syn or tor-2. Control

UA50 [ baInl3 ; Punc-54 :: α− syn :: gfp , Punc-54 :: tor-2, rol-6 (su1006) ] worms were fed with

RNAi bacteria HT115(DE3) with empty vector while RNAi-treated worms were fed with the bacteria producing the indicated dsRNA. Following total RNA isolation and cDNA preparation, semi-quantitative RT-PCR was performed by using primers specific for cdk-

5 (5' ggg-gat-gat-gag-ggt-gtt-cca-agc 3' and 5' ggc-gac-cgg-cat-ttg-aga-tct-ctg-c 3'),

α− syn (5' atg-gat-gta-ttc-atg-aaa-gga-ctt-tca-aag 3' and 5' tta-ggc-ttc-agg-ttc-gta-gtc-ttg

3'), and tor-2 (5' caa-tta-tca-tgc-gtt-ata-caa-ag 3'; and 5' cat-tcc-act-tcg-ata-agt-att-g 3').

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CHAPTER FOUR

RNA INTERFERENCE SCREEN OF DAF-2-MODULATED AND DIFFERENTIALLY EXPRESSED GENES LINK METABOLIC ENZYMES TO NEUROPROTECTION

This work presented in this chapter represents preliminary data from studies that are currently performed in Drs. Guy and Kim Caldwell’s laboratory. Shusei Hamamichi collected all data except Table 4.1, 4.2, and Fig. 4.5. Susan DeLeon, Adam Knight, Kyle Lee, Cody Locke, and Mike Zhang contributed the data shown in Tables 4.1 and 4.2. Jenny Schieltz contributed the data shown in Fig. 4.5. Shusei Hamamichi wrote the manuscript.

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ABSTRACT

Aging is a fundamental susceptibility factor of Parkinson disease (PD), wherein

pathological features include progressive loss of dopamine (DA) neurons and misfolding

of α-synuclein ( α-syn) into proteinaceous inclusion bodies. In C. elegans , lifespan extension and increased stress resistance have been linked to the DAF-2/insulin-like signaling pathway. Here, we report the results of daf-2 worms that exhibit these

pathological features to elucidate the genetic link between aging and α-syn toxicity. In

DA neurons, daf-2 reduced-function mutation strikingly suppressed neurodegeneration at

the chronological aging stage (day 7), but not at the mean lifespan (N2: day 20; daf-2:

day 40), suggesting that differentially expressed genes in the daf-2 background are

responsible for DA neuron survival. To identify such components, we screened 625

genes that are either up-regulated in daf-2 or shown to modify α-syn toxicity in C.

elegans to identify suppressors of α-syn misfolding. While α-syn::GFP was readily

degraded in the daf-2 background, RNAi knockdown of 53 genes enhanced α-syn

misfolding in vivo . Among the positives were genes involved in metabolism,

transcription, and signal transduction. Two positive genes, gpi-1/GPI and hrdl-1/AMFR ,

both components of the autocrine motility factor pathway, rescued DA neurons from α-

syn-induced neurodegeneration. Taken together, this study reveals a common pathway

that links the progression of neurodegeneration and cancer, and illustrates the underlying

metabolic changes associated with aging and these diseases.

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INTRODUCTION

Central to Parkinson disease (PD) neuropathology is a protein called α-synuclein

(SNCA/ α-syn) (Spillantini et al., 1997), a primary component of Lewy bodies found in

both familial and idiopathic forms of PD. Currently, nine PARK genes have been

identified that are implicated in synaptic function ( α-syn ), proteasomal protein

degradation ( PRKN , UCHL1 ), lysosomal function ( ATP13A2 ), protection against

mitochondrial/oxidative stress ( DJ-1, HTRA2 , PINK1 ), and signal transduction ( LRRK2 ,

GIGYF2 ). While these findings provided insightful foundation for elucidating PD pathological mechanisms, highlighted by the selective loss of dopamine (DA) neurons in the substantia nigra, the monogenic forms of PD remain rare, accounting for only 5-10%

of all PD cases.

Environmental PD susceptibility factors such as neurotoxins and heavy metals

have long been documented (Di Monte, 2003), and interaction between genetic and

environmental factors may play a role in disease onset or progression (Benmoyal-Segal et

al., 2006). Alternatively, multiple genetic susceptibility factors may presently be

unidentified (Lesage and Brice, 2009), and combined genetic defects may induce DA

neurodegeneration. In the latter case, genome-wide analysis of PD patients (Fung et al.,

2006; Pankratz et al., 2009) or genetic screens using simple model organisms (Cooper et

al., 2006; Hamamichi et al., 2008; Gitler et al., 2009) should provide potential genetic PD

susceptibility candidates. In spite of the rigorous efforts to ascertain these factors, PD

unequivocally remains as a disease of aging, affecting approximately 1% of the

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population aged over 50 (Polymeropoulos et al., 1996). While aging is widely accepted

as an underlying factor of several neurodegenerative diseases, including PD, its

association to PD pathogenesis remains unexplored.

Recently, the insulin signaling pathway has been proposed to affect human aging

(Suh et al., 2008; Willcox et al., 2008) and neurodegenerative diseases (Craft and

Watson, 2004). Notably, insulin resistance in Alzheimer patients has been reported

whereby soluble amyloid-β oligomers modify insulin receptor distribution away from the neuronal surface (Zhao et al., 2007; De Felice et al., 2009), implicating Alzheimer disease as a form of Type 3 diabetes. In PD, Lautier et al. (2008) identified GIGYF2 , a

GRB10 interacting protein as corresponding to PARK11 locus. GRB10 is an adaptor

protein that modulates the insulin signaling pathway (Giovannone et al., 2003). While

association between GIGYF2 and PD remains controversial, loss of the insulin receptor and its mRNA in the substantia nigra of PD patients has been reported (Moroo et al.,

1994; Takahashi et al., 1996), suggesting a role for insulin signaling pathway in this disease.

A model organism, Caenorhabditis elegans offers distinct advantages for studying aging. The studies on longevity via reduced insulin signaling ( daf-2, Kenyon et al., 1993), caloric restriction ( eat-2, Lakowski and Hekimi, 1998), and reduced mitochondrial respiration ( clk-1, Felkai et al., 1999; isp-1, Feng et al., 2001) have been well characterized. Specifically, the daf-2 reduced-function mutation has been shown to enhance protection against various forms of cellular stress. These stressors include

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thermotolerance (Lithgow et al., 1995), oxidative stress (Honda and Honda, 1999), hypoxia (Scott et al., 2002), heavy metal resistance (Barsyte et al., 2001), and pathogens

(Bolm et al., 2004). Among proteotoxicity models, daf-2 has also shown cytoprotection against amyloid-β aggregation (Cohen et al., 2006; Florez-McClure et al., 2007).

Previously, we established worm models in which α-syn-induced neurodegeneration in DA neurons can be monitored (Cao et al., 2006), and α-syn misfolding in body wall muscle is readily assayed by RNAi screening (Hamamichi et al.,

2006). Collectively, C. elegans provides an advantageous platform with molecular, cellular, and genetic tools to discern genetic factors linking aging and PD. Here, we report the use of daf-2 mutant strains to identify genetic factors that modify PD-linked toxicity. We determined that while daf-2 reduced-function mutation significantly rescued

DA neurons at chronological aging (day 7 in both wild-type N2 and daf-2 worms), the mutation had no effects at biological aging (day 20 in wild-type N2 and day 40 in daf-2 worms). Since daf-2 did not ameliorate neurodegeneration at the equivalent biological aging stage, these findings suggested that differential gene expression in the daf-2 mutant background may be responsible for neuroprotection. Thus, to identify these specific neuroprotective factors, we performed an RNAi screen and identified 53 genes that when knocked down enhanced α-syn misfolding in the daf-2 background. These genes consisted of transcription factors, signaling components, and metabolic enzymes. Among them, two specific genetic components of autocrine motility factor pathway ( gpi-1/GPI and hrdl-1/AMFR ), linked to glycolysis and cholesterol synthesis, were overexpressed in

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DA neurons, and these genes rescued DA neurons from α-syn-induced

neurodegeneration. This study illustrates a genetic link between metabolic changes

associated with the aging processes and PD-linked toxicity.

MATERIALS AND METHODS

Plasmid Constructs. Plasmids were constructed using Gateway Technology

(Invitrogen; Carlsbad, CA). The cDNAs encoding gpi-1 and hrdl-1 were obtained from

Open Biosystems (Huntsville, AL). An N-terminal FLAG tag sequence was added

during the PCR amplification process. mCherry was obtained from Clontech (Mountain

View, CA). The gene fusions were shuttled from entry vectors into the Gateway

destination vector, pDEST-DAT-1 (Cao et al., 2005) or pDEST-UNC-54 (Hamamichi et

al., 2008). The molecular cloning yielded expression plasmids, Pdat-1::FLAG-gpi-1, Pdat-

1::FLAG-hrdl-1, and Punc-54 ::mCherry .

Generation of transgenic nematode strains. Nematodes were maintained using standard procedures (Brenner, 1974). The transgenic strains, UA132 { baInl1 [Pdat-1:: α-

syn ; Pdat-1::gfp ]; baEx101 [Pdat-1::gpi-1; Punc-54 ::mCherry ]}, UA133 { baInl1 [Pdat-1:: α-syn ;

Pdat-1::gfp ]; baEx102 [Pdat-1::hrdl-1; Punc-54 ::mCherry ]} were generated by directly

microinjecting 50 g/ml expression plasmids into the integrated UA44 { baInl1 [Pdat-1:: α-

syn; P dat-1::gfp ]}.

Neuroprotection analysis . For neuroprotection analysis, at least three stable lines

of UA132 and UA133 were analyzed. Synchronized embryos expressing both GFP and

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mCherry were transferred onto NGM plates, and grown at 20 °C for 7 days. For each trial,

30 worms were transferred to a 2% agarose pad, immobilized with 2 mM levamisole, and scored. Worms were considered rescued when all four CEP and both ADE neurons were intact and had no visible signs of degeneration. Each stable line was analyzed three times (for a total of 90 worms/transgenic line). Three separate transgenic lines were analyzed per gene, for a total of 270 animals/gene analyzed.

Genetic crosses. Three UA44 { baInl1 [Pdat-1:: α-syn; P dat-1::gfp ]} males and 8

DR128 [ dpy-1(e1 ) daf-2(e1370 )], DR129 [ daf-2(e1370 ) unc-32 (e189 )], DR195 [ dpy-

5(e61 ) daf-16 (m26 )], or DR211 [ daf-16 (m26 ) unc-75 (e950 )] worms were transferred onto small mating plates, and incubated at 20 °C. Subsequent Dpy or Unc worm expressing

GFP were analyzed for DA neurodegeneration. For the RNAi, three wildtype N2

(Bristol) males were crossed with eight DR128 [ dpy-1(e1 ) daf-2(e1370 )] to generate males with the daf-2 mutation without Dpy phenotype. The resulting males were then crossed with UA51 [ baInl4 ; Punc-54 :: α-syn::gfp, rol-6 (su1006) ] and subsequent worms,

UA134 {baInl4 ; [ Punc-54 :: α-syn::gfp, rol-6 (su1006) ]; [dpy-1(e1 ) daf-2(e1370 )]} with

both the Dpy phenotype and α-syn::GFP, were used for RNAi screening.

Preparation of worm protein extracts and western blotting. As described

previously (Hamamichi et al., 2008), worm protein extracts were prepared and western

blotting was performed to detect α-syn::GFP expression level in the daf-2 mutant background.

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RNA isolation and semi-quantitative RT-PCR. As described previously

(Hamamichi et al., 2008), RNA isolation and semi-quantitative RT-PCR were performed

to detect α-syn::gfp mRNA level in the daf-2 mutant background. The following primers were designed for the PCR: cdk-5 forward primer: 5’ ggg-gat-gat-gag-ggt-gtt-cca-agc 3’

reverse primer: 5’ ggc-gac-cgg-cat-ttg-aga-tct-ctg-c 3’

α-syn forward primer: 5’ atg-gat-gta-ttc-atg-aaa-gga-ctt-tca-aag 3’

reverse primer: 5’ tta-ggc-ttc-agg-ttc-gta-gtc-ttg 3’

Lifespan assay. Lifespan assay was conducted as previously described (Kenyon et al., 1993). Briefly, age-synchronized 50 wildtype N2, UA44, and DR129 worms were grown at 20 °C, and transferred daily to new NGM plates with 20 l 10 mg/ml palmitic acid covering the edge to prevent the worms from crawling into the agar. The worms that responded to gentle touch were counted as alive. The assay was conducted in triplicate (n

= 150 total for each strain).

RNAi screen. The RNAi screen was performed as described previously

(Hamamichi et al., 2008) except that RNAi-treated UA134 worms ( daf-2 mutant background with P unc-54 :: α-syn::GFP) were grown at 20 °C, and scored for enhanced α- syn misfolding at young adult stage. RNAi feeding clones (Geneservice, Cambridge,

UK) were grown for 14 hrs in LB culture with 100 mg/ml ampicillin and seeded onto

NGM agar plates containing 1 mM isopropyl β-D-thiogalactoside. When the bacterial lawn was grown, five L4 UA134 worms were transferred onto the plates and incubated at 126

20 °C for 72 hrs. The gravid adults were then placed onto the corresponding RNAi plates and allowed to lay eggs for 12 hrs, and the resulting age-synchronized worms were analyzed at young adult stage. For each trial, 20 worms were transferred onto a 2% agarose pad, immobilized with 2 mM levamisole, and analyzed. The RNAi clones resulting in significant aggregation (80% of worms with increased quantity and size of α- syn aggregates) were scored as positive. Bacterial clones leading to enhanced α-syn misfolding were tested in two trials.

2-Deoxyglucose (DOG) analysis. Age-synchronized UA44 animals were grown at

20 °C, and analyzed at day 6. To minimize the effect of DOG on lifespan, 24 or 48 hrs prior to the analysis, 30 worms were transferred onto NGM plates with 1, 5, and 10 mM

DOG. These worms were transferred to a 2% agarose pad, immobilized with 2 mM levamisole, and scored for DA neurodegeneration. The experiment was performed three times for a total of 90 worms/treatment.

Fluorescent microscopy. All fluorescence microscopy was performed using a

Nikon Eclipse E800 epifluorescence microscope equipped with Endow GFP HYQ filter cube (Chroma Technology). Images were captured with a Photometrics Cool Snap CCD camera driven by MetaMorph software (Universal Imaging).

Statistics. Statistical analysis for neuroprotection was performed using the

Student’s t-test ( p<0.05) to compare control worms with strains that overexpress candidate genes in DA neurons.

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RESULTS

To investigate how the insulin signaling pathway may modulate α-syn-induced

DA neuron death, we generated daf-2 strains overexpressing α-syn and gfp under the control of dat-1 promoter ( Pdat-1) using genetic crosses. Expression of GFP allows clear observation of morphological changes in 6 anterior DA neurons (4 CEPs and 2 ADEs) and rapid scoring of neurodegeneration when GFP expression is lost. At day 7

(chronological aging), approximately 15% of wildtype N2 as well as dpy-1 and unc-32

(additional controls for daf-2 strains with the corresponding phenotypic markers) worms displayed all 6 intact DA neurons (Fig. 4.1A), consistent with our previous reports.

Strikingly, approximately 40% of daf-2 worms exhibited 6 normal DA neurons (Fig.

4.1A), which is the highest neuroprotection observed by a single gene using this model.

One of the well-characterized downstream components of the insulin signaling pathway is DAF-16/FKHR, which is regulated by phosphoinositide 3-kinase (AGE-

1/PI3K). DAF-16 is a master regulator of various cytoprotective genes including heat shock proteins, catalases, and superoxide dismutases (Murphy et al., 2003). We examined daf-16 loss-of-function mutants overexpressing α-syn and GFP in the DA neurons. As expected, loss-of-function of daf-16 enhanced neurodegeneration (Fig.

4.1B). Surprisingly, when daf-2 + daf-16 double mutants were analyzed, we still observed intermediate level of neuroprotection (Fig. 4.1C), suggesting that: 1) DAF-16 is not the sole genetic component responsible for neuroprotection, and 2) additional

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pathways downstream of DAF-2 excluding PI3K pathway may play a neuroprotective

role.

Since daf-2 mutants live longer, we reasoned that a direct comparison between N2

and daf-2 worms on the same chronological day might not be an accurate assay of daf-2- mediated neuroprotection. To determine biological aging, we performed lifespan assays whereby worms were incubated at 20 °C, and transferred daily to new plates. We

recorded living worms each day by counting the number of worms that responded to a

gentle touch. Consistent with a previously published report (Kenyon et al., 1993), N2

worms exhibited mean lifespan of day 20 while daf-2 was day 40, doubling its mean

lifespan (Fig. 4.2). Surprisingly, analysis of DA neurodegeneration at mean lifespan

(biological aging) revealed no daf-2-mediated neuroprotection (Fig. 4.1D). These findings indicate that differential gene expression in daf-2 mutant background might be responsible for neuroprotection for two reasons: 1) knockout of daf-16 , which encodes a transcription factor regulating cytoprotective genes enhanced neurodegeneration, and 2) daf-2 + daf-16 double mutations, which exhibit the normal lifespan similar to wildtype

N2 resulted in the intermediate level of neuroprotection at the chronological aging.

C. elegans offers a distinct advantage over other animal systems for such an analysis, with exceptional genome-wide analyses of genes and proteins that are modified in the daf-2 background by using microarray, SAGE, and mass spectrometry (Murphy et al., 2003; McElwee et al., 2004; Halaschek-Weiner et al., 2005; Dong et al., 2007).

Furthermore, we previously reported a large-scale RNAi screen to identify genetic factors

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that affect α-syn misfolding and subsequently α-syn-induced neurodegeneration, two

common pathological features of PD (Hamamichi et al., 2008). To ascertain genes that

are modified in the daf-2 background and affect α-syn misfolding, we generated a daf-2

strain overexpressing α-syn::gfp in body wall muscle cells. In the N2 background, α-

syn::GFP was readily misfolded and accumulated in the cytoplasm (Fig. 4.3). In contrast,

depletion of daf-2 resulted in nearly complete degradation of the fusion protein (Fig. 4.3).

This observation was further confirmed by western blot wherein α-syn::GFP could not be

detected by using antibodies against either α-syn or GFP in the daf-2 background (Fig.

4.3). While daf-2 has been shown to regulate autophagy (Melendez et al., 2003) and

ameliorate amyloid-β aggregation, it was conceivable that the mutation might affect the

expression level of the fusion protein. As demonstrated by semi-quantitative RT-PCR,

the mutation had no effect on α-syn::gfp mRNA level, verifying that the fusion protein was degraded.

To conduct the RNAi screen, we targeted 410 genes and/or proteins with clear human orthologs that are up-regulated in the daf-2 background (Murphy et al., 2003;

McElwee et al., 2004; Halaschek-Weiner et al., 2005; Dong et al., 2007; Samuelson et al.,

2007). Additionally, we included 90 genetic modifiers of α-syn toxicity that were

identified using C. elegans (Vartiainen et al., 2006; Kuwahara et al., 2008; Van Ham et al., 2008) as well as 125 intermediate positive genes from the previous hypothesis-based

RNAi screen performed in our laboratory (Hamamichi et al., 2008). In total, 625 genes

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were assayed by growing the RNAi-treated worms at 20 °C and analyzing them at the young adult stage. We identified 51 genes that caused lethality, and 53 positive genes that, when knocked down by RNAi, enhanced α-syn misfolding (Table 4.1). Based on

KOG and GO annotations, we classified 53 positive hits in the functional categories

(Table 4.2). Interestingly, in contrast to our expectation that DAF-16-dependent cytoprotective genes, when knocked down by RNAi, would enhance the misfolding, the most represented category consisted of metabolic enzymes. These metabolic enzymes comprised of approximately 20% of all positive genes (11/53).

Since 11 positive genes from the RNAi screen were involved in metabolism, and

4 out of 11 were glycolytic enzymes ( F01F1.12/ALDOA , K10B3.7/GAPDH ,

K10B3.8/GAPDH , and Y87G2A.8/GPI ), we asked if reduced glycolysis could enhance α- syn-induced neurodegeneration. Previous studies have reported the association between reduced energy metabolism and neurodegeneration (Mattson et al., 1999). To evaluate this, the N2 strain overexpressing α-syn and GFP in DA neurons was treated with 2- deoxyglucose (DOG) for 24 or 48 hrs prior to the analysis at day 6. DOG is a glucose analog that blocks glycolysis. Notably, both 5 and 10 mM DOG treatment enhanced neurodegeneration (Fig. 4.4), indicating that reduced glycolysis and perturbed energy metabolism could enhance DA neuronal death.

Schultz et al. (2007) reported that DOG treatment as well as RNAi knockdown of glucose-6-phosphate isomerase ( gpi-1/GPI1 ) extended worm lifespan by inducing mitochondrial respiration and increasing oxidative stress. Furthermore, gpi-1, when

131

knocked down by RNAi in our model also enhanced α-syn misfolding. Interestingly, in

human cancer cells, GPI1 is secreted to function as autocrine motility factor (AMF)

during metastasis to promote cancer cell survival (Funasaka and Raiz, 2007). The closest

worm ortholog of its receptor AMFR is hrdl-1, another positive gene from the RNAi

screen. To assess the neuroprotective role of autocrine motility factor components, both

gpi-1 and hrdl-1 were overexpressed in worm DA neurons (Fig. 4.5). We determined

that they both rescued DA neurons from α-syn-induced toxicity. These results may indicate an inverse relationship of autocrine motility factor components whereby up- regulation in cancer cells enhances survival and down-regulation in DA neurons leads to neurodegeneration.

DISCUSSION

In this study, we demonstrated that daf-2 suppressed α-syn-induced

neurodegeneration during chronological, but not biological aging, illustrating that daf-2-

mediated neuroprotection as an indirect consequence of the mutation. Further, we

systematically knocked down genes that are up-regulated in the daf-2 mutant background as well as 125 intermediate positives from our previous RNAi screen for α-syn modifiers, and identified 53 genes that when knocked down enhanced α-syn misfolding. Among them, metabolic genes, notably glycolytic enzymes were over-represented (Fig. 4.6). To decipher the role of the glycolytic pathway in neurodegeneration, we determined that

132

while DOG treatment enhanced α-syn-induced neurodegeneration, overexpression of two components of the autocrine motility factor pathway, gpi-1 and hrdl-1 suppressed it.

DAF-2/insulin signaling pathway via downstream components, AGE-1/PI3K and

DAF-16 has been shown to regulate autophagy in C. elegans (Melendez et al., 2003).

We previously examined neuroprotective capacities of two autophagic genes, vps-

41/VPS41 and atgr-7/ATG7 in worm DA neurons, and reported suppression of α-syn-

induced toxicity (Hamamichi et al., 2008). However, the fact that daf-16 ; daf-2 double mutants exhibited an intermediate level of neuroprotection during the chronological aging process suggests that a pathway downstream of DAF-2 that is separate from the PI3K pathway for this neuroprotection. One candidate pathway is the MAPK signaling pathway regulating cell death. In C. elegans , daf-2 enhances resistance against bacterial pathogens, Pseudomonas aeruginosa , Enterococcus faecalis , and Staphylococcus aureus

(Garsin et al., 2003) via pmk-1/p38 (Kim et al., 2002; Troemel et al., 2006). Subsequent

analysis of aging (PI3K pathway) vs. innate immunity (MAPK pathway) revealed that

these two pathways are regulated in a genetically distinct manner (Evans et al., 2008).

Taken together, these findings suggest that, in our model, both PI3K and MAPK

pathways may induce neuroprotection by simultaneously stimulating cytoprotective

mechanisms such as autophagy and suppressing cell death (Fig. 4.7). The analysis of

pmk-1 or ced-3 mutants should reveal a role for MAPK signaling in neurodegeneration.

Our RNAi results demonstrate that knockdown of most heat shock proteins did

not enhance α-syn misfolding in the daf-2 background, suggesting the functional

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redundancy of these chaperones. Surprisingly, knockdown of a single autophagic

component did not affect α-syn misfolding. Given the up-regulation of various cytoprotective genes in the daf-2 background, knockdown of a single autophagic gene may be insufficient to substantially shift the cellular threshold toward α-syn

accumulation. Lastly, in contrast to our previous findings wherein RNAi knockdown of

worm orthologs of DJ-1, NURR1, PINK1, parkin , and UCHL1 enhanced α-syn

misfolding in the tor-2 overexpression background (Hamamichi et al., 2008), RNAi

knockdown of these genes had no effect in the present study. While the mechanism

remains unclear, it is interesting to speculate that monogenic forms of PD also requires

modification of metabolic changes associated with aging processes to ultimately manifest

a disease state. To this end, a combinatorial RNAi strategy can be utilized whereby the

worm orthologs of PD genes as well as candidate genes are simultaneously knocked

down using the UA134 strain.

Among the positive genes from the RNAi screen, the most represented categories

included transcription factors (7/53), signaling components (6/53), and metabolic

enzymes (11/53) (Table 4.2; Fig. 4.6). Notably, three signaling components involved in

the Wnt signaling were identified: 1) tap-1 (TAK kinase/MOM-4 binding protein), 2)

D1069.3 (a putative β -catenin-Tcf/Lef signaling pathway component), and 3) mom-4

(MAKKK7). While PD pathogenesis has not been linked to the Wnt signaling pathway,

C. elegans BAR-1 ( β-catenin) has been shown to physically interact with DAF-16 to

enhance its activity (Essers et al., 2005). Thus, the knockdown of these genes may 134

enhance α-syn misfolding in a daf-16 -dependent manner. Among metabolic enzymes, four genes involved in glycolysis, F01F1.12 (fructose-biphosphate aldolase), gpd-2 and gpd-3 (glyceraldehyde-3-phosphate dehydrogenases), and gpi-1 (glucose-6-phosphate isomerase) were uncovered. These results indicate that perturbed glucose metabolism modifies α-syn misfolding.

Interestingly, in a mammalian system, Belluci et al. (2008) demonstrated that glucose starvation increases α-syn aggregation and cell death in SH-SY5Y cells.

Moreover, glucose hypometabolism has been reported in PD patients with SNCA duplication (Uchiyama et al., 2008), consistent with our model whereby α-syn is overexpressed in DA neurons or body wall muscles. Currently, no study has reported a direct link between α-syn-induced toxicity and hrdl-1/AMFR . AMFR is an E3 ligase, which is an ERAD component involved in degradation of HMG-CoA reductase (Song et al., 2005). Intriguingly, statins, inhibitors of HMG-CoA reductase and subsequent cholesterol synthesis have been shown to reduce α-syn aggregation in vitro (Bar-On et al., 2008). Protective effects of statins in animal models, as well as beneficial outcomes of these compounds in PD patients remain unclear, however, our results suggest a potential neuroprotective role of autocrine motility factor pathway, a connection between glucose metabolism and cholesterol synthesis.

While speculative, cancer and PD may share common modifying mechanisms such as the UPS, cell cycle or other unexplored pathways (West et al., 2005; Zanetti et

135

al., 2007). For example, HSF1, a master regulator of various cytoprotective genes may rescue neurons from neurodegenerative diseases, but stimulate malignant transformation and survival of cancer cells (Dai et al., 2007). It will be interesting to examine how these metabolic changes may regulate cytoprotective mechanisms by examining DAF-16::GFP activation (another master regulator of cytoprotective genes) and LGG-1::GFP localization (marker for activation of autophagy). Taken together, this study illustrates our initial step toward understanding the genetic link between cancer and neurodegeneration via metabolic changes associated with aging, and provide therapeutic metabolic pathways for PD.

136

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Table 4.1. Summary of genes analyzed by RNAi screen. Blue indicates 53 genes when knocked down enhanced α-syn misfolding in the daf-2 background at the young adult stage. Gray indicates 51 lethal genes.

Gene Human ortholog Outcome AC3.7 UDP-glucuronosyltransferase 1-6 precursor B0024.6 Atrial natriuretic peptide receptor A B0035.2 BA16L21.2.1 B0041.4 60S ribosomal protein L4 B0213.12 Cytochrome P450 2C8 B0213.15 Cytochrome P450 2A7 B0213.3 Keratin-associated protein 19-8 B0213.6 Keratin-associated protein 19-8 B0218.8 Mannose receptor B0228.5 Thioredoxin B0238.1 Carboxylesterase 7 precursor B0238.13 Carboxylesterase 7 precursor B0244.2 Receptor-type tyrosine-protein phosphatase B0250.1 60S ribosomal protein L8 B0284.2 ROCK2 protein B0286.3 Multifunctional protein ADE2 B0303.9 Vacuolar protein sorting-associated protein 33A B0336.10 60S ribosomal protein L23 B0336.8 APG12 autophagy 12-like B0350.2 Ankyrin-1 B0350.2 Ankyrin-1 B0393.1 40S ribosomal protein SA B0395.2 Sterol O-acyltransferase 1 B0432.2 RNA-binding protein regulatory subunit B0464.7 Barrier-to-autointegration factor B0513.3 Protein BE10.2 Transmembrane protein 195 C01A2.3 Oxidase (Cytochrome c) assembly 1-like C01A2.4 DKFZP564O123 protein C01B7.6 Protein associated with Myc C01F1.1 General transcription factor IIF subunit 1 C01G6.4 RING finger protein 11

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C02C2.3 Acetylcholine receptor C02D5.1 Isovaleryl-CoA dehydrogenase C02F4.2 Serine/threonine-protein phosphatase 2B C02G6.1 Insulin-degrading enzyme C03B1.12 Lysosome-associated membrane glycoprotein 1 C03D6.3 mRNA-capping enzyme RNGTT C04F12.4 RPL14 protein C04F12.8 Dual specificity protein phosphatase 14 C05D11.2 Vacuolar protein sorting-associated protein 16 C05D9.1 Sorting nexin-2 C05E11.1 Protein lunapark C05G6.1 Photoreceptor-specific nuclear receptor C06A12.3 UPF0279 protein C14orf129 C06A5.1 Integrator complex subunit 1 C06A5.8 Zinc-finger protein HT2A C06A6.5 Thioredoxin domain-containing protein 4 C06B3.4 Estradiol 17-beta-dehydrogenase 12 C06E2.3 Ubiquitin-conjugating enzyme E2 C06E8.3 Serine/threonine-protein kinase Pim-3 C06E8.5 Lipopolysaccharide-binding protein precursor C06G1.4 Dermokine gamma-1 C06G8.1 Recombination activating gene 1 activating protein 1 C07A12.7 TOM1-like protein 2 C07A9.2 Protein BUD31 homolog C07A9.8 Bestrophin-3 C07G1.5 Membrane trafficking and cell signaling protein HRS C08H9.13 43 kDa protein C08H9.14 Chitinase-3-like protein 1 C08H9.6 Chitinase 3-like 2 isoform b C09D4.4 Hypothetical protein KIAA1411 C09D4.5 60S ribosomal protein L19 C09G12.8 Ras-related C3 botulinum toxin substrate 1 C10G11.5 Pantothenate kinase 4 C10G11.8 26S protease regulatory subunit 4 C10H11.5 UDP-glucuronosyltransferase 1-3 precursor C11D2.2 Cathepsin E precursor C11H1.3 RING finger protein 157

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C12D12.2 Excitatory amino acid transporter 2 C12D8.10 RAC-alpha serine/threonine-protein kinase C13C4.6 Major facilitator superfamily domain-containing protein 7 C14A4.14 Mitochondrial 28S ribosomal protein S22 C15F1.7 Superoxide dismutase C15H11.2 Oxytocin receptor C15H9.1 NAD(P) transhydrogenase C15H9.1 NAD(P) transhydrogenase C16A3.9 40S ribosomal protein S13 C16C10.7 Hypothetical protein FLJ38628 C17C3.3 Acyl-coenzyme A thioesterase 8 C17D12.5 Ubiquitin-conjugating enzyme E2 D1 C17G1.4 SWI related protein C17H1.7 Uncharacterized protein C18D11.2 Acyl-CoA-binding domain-containing protein 4 C18E9.10 Hypothetical protein FLJ90068 C23G10.6 UDP-glucuronosyltransferase 1-6 precursor C23H4.2 Carboxylesterase 2 isoform 2 C24A11.8 FERM domain-containing protein 5 C24A11.9 42 kDa protein C24F3.2 Dual specificity protein phosphatase 12 C24G6.5 DnaJ homolog subfamily A member 2 C25E10.8 IgGFc-binding protein precursor C25H3.6 Inner centromere protein antigens 135 C26C6.3 Tolloid-like protein 1 precursor C27A2.2 60S ribosomal protein L22 C27F2.5 Vacuolar-sorting protein SNF8 C27H5.2 Centromeric protein E C28C12.7 Proactivator polypeptide precursor C28D4.1 Retinoic acid receptor RXR-beta C28H8.11 Tryptophan 2,3-dioxygenase C28H8.5 THAP domain-containing protein 4 C29E4.7 Glutathione transferase omega-1 C29E6.1 Mucin-2 precursor C29E6.5 Photoreceptor-specific nuclear receptor C29F9.2 MAP7 domain-containing protein 1 C30C11.1 39S ribosomal protein L32

143

C30G12.2 11-cis retinol dehydrogenase C30G7.1 Histone H1.0 C31B8.8 Collagenase 3 precursor C31H2.1 TBC1 domain family member 24 C32D5.10 Topoisomerase I C33A11.1 NF-kappa-B inhibitor alpha C33A12.6 UDP-glucuronosyltransferase 2A1 precursor C33A12.7 ETHE1 protein, mitochondrial precursor C33H5.10 Hypothetical protein FLJ90386 C33H5.18 Phosphatidate cytidylyltransferase 1 C34B2.4 LIM domain-binding protein 3 C34B2.7 Succinate dehydrogenase C34C12.2 Putative protein TPRXL C34C12.8 GrpE protein homolog 1 C34C6.3 Notch homolog 2 N-terminal-like protein C34D1.2 Doublesex- and mab-3-related transcription factor 1 C34D10.2 Unkempt-like C34E10.1 Sorting and assembly machinery component 50 C35A5.3 Sialin C35D10.2 RGS19-interacting protein 1 C36A4.8 Breast cancer 1, early onset isoform BRCA1-delta11 C36A4.9 Acyl-CoA synthetase short-chain family member 2 C37H5.2 Abhydrolase domain-containing protein 4 C37H5.8 Stress-70 protein, mitochondrial precursor C39F7.2 Tripartite motif protein 9 C41C4.7 Cystinosin C41G7.1 Survival motor neuron protein C44E4.6 Benzodiazepine receptor ligand C44F1.3 Galectin-4 C44H4.5 TAB1-like protein C45B11.3 Hydroxysteroid dehydrogenase-like protein 2 C45E5.3 Chitinase-3-like protein 1 precursor C45G7.4 Ret finger protein 2 C45H4.17 Cytochrome P450 2C8 C46F4.2 Long-chain-fatty-acid--CoA ligase 4 C46H11.2 Putative dimethylaniline monooxygenase 6 C47A4.1 BA16L21.2.1

144

C47B2.2 n/a C47C12.6 Amiloride-sensitive sodium channel subunit C48D5.1 Orphan nuclear receptor NURR1 C49H3.11 40S ribosomal protein S2 C50E3.3 C-type lectin C50F7.10 Cytosolic beta-glucosidase C50H11.1 Acyl-CoA synthetase family member 3 C53B7.3 LTBP1 C54D1.3 TRAF3-interacting protein 1 C54D10.1 Uncharacterized protein C6orf168 C54D10.10 Tissue factor pathway inhibitor 2 C54D10.2 Uncharacterized protein C6orf168 C54D10.7 Trichohyalin C54G4.1 Ribosomal protein S6 kinase alpha-5 C54H2.5 Surfeit locus protein 4 C55A6.6 Carbonyl reductase [NADPH] 3 C55B6.2 P58 C55C3.4 Proto-oncogene tyrosine-protein kinase FER C56A3.4 n/a C56E10.3 Nucleoprotein TPR C56G2.15 N-acetyltransferase 6 C56G3.1 Somatostatin receptor type 2 CC8.2 Protein phosphatase 1 regulatory subunit 3E CD4.4 Vacuolar protein sorting-associated protein 37B D1022.1 Ubiquitin-conjugating enzyme E2 J1 D1022.1 Ubiquitin-conjugating enzyme E2 J1 D1054.8 Peroxisomal short-chain alcohol dehydrogenase D1069.3 Hyccin D2007.4 39S ribosomal protein L18 D2030.5 Methylmalonyl-CoA epimerase D2085.5 Protein KIAA1219 D2089.1 Splicing factor, arginine/serine-rich 12 DY3.2 Lamin-B1 E04A4.8 60S ribosomal protein L18a EEED8.9 Serine/threonine kinase PINK1 F01F1.12 Fructose-bisphosphate aldolase A F01G12.5 163 kDa protein

145

F02A9.4 Methylcrotonoyl-CoA carboxylase beta chain F02C12.5 Thromboxane A synthase 1 F02E8.3 AP-2 complex subunit sigma-1 F08B1.1 Dual specificity protein phosphatase 16 F08C6.4 Erythrocyte band 7 integral membrane protein F08G12.5 Tripartite motif protein 31 F08G2.5 Dentin sialophosphoprotein preproprotein F08H9.3 Alpha B crystallin fragment 4 F08H9.4 Alpha-crystallin B chain F08H9.9 CUB and sushi domain-containing protein 3 F09B12.3 Putative phospholipase B-like 2 precursor F09F7.7 Alkylated DNA repair protein alkB homolog 4 F09G8.3 Mitochondrial ribosomal protein S9 F10B5.1 60S ribosomal protein L10 F10C2.5 Putative alpha-mannosidase C20orf31 F10D11.6 Bactericidal permeability-increasing protein precursor F10D2.11 UDP-glucuronosyltransferase 1-9 precursor F10D2.5 2-hydroxyacylsphingosine 1-beta-galactosyltransferase F10D2.9 Acyl-CoA desaturase F10F2.2 Phosphoribosylformylglycinamidine synthase F10G7.2 EBNA-2 co-activator F10G8.6 Nucleotide-binding protein 1 F11A5.12 Estradiol 17-beta-dehydrogenase 12 F11E6.5 Elongation of very long chain fatty acids protein 3 F11G11.2 Glutathione-requiring prostaglandin D synthase F11H8.1 Ubiquitin-activating enzyme E1C F12A10.7 Keratin, type I cytoskeletal 10 F13D11.4 3-HSD 1 protein F13E6.4 65 kDa Yes-associated protein F13G3.5 Inositol monophosphatase F13H8.5 Mediator of RNA polymerase II transcription subunit 15 F14H12.4 Serine/threonine-protein kinase 3 F15A4.8 43 kDa protein F15D4.4 Cathepsin S precursor F16A11.2 Hypothetical protein F17B5.1 33 kDa protein F17C11.8 Vacuolar protein-sorting-associated protein 36

146

F18A1.5 Replication protein A F18E3.7 DDO-1 of D-aspartate oxidase F18H3.5 Cell division protein kinase 6 F20B6.8 Homeodomain-interacting protein kinase 1 F20D1.9 Mitochondrial glutamate carrier 2 F20D6.11 Apoptosis-inducing factor 3 F20H11.2 Protein strawberry notch homolog 1 F20H11.3 Malate dehydrogenase F21C3.3 Histidine triad nucleotide-binding protein 1 F21D5.7 Signal recognition particle 54 kDa protein F21F3.3 Protein-S-isoprenylcysteine O-methyltransferase F21F3.5 Neuronal acetylcholine receptor subunit beta-2 F21F8.2 Gastricsin precursor F21F8.7 43 kDa protein F25B4.1 Aminomethyltransferase F25D7.3 PR domain containing 1, with ZNF domain isoform 1 F25H2.9 Proteasome subunit alpha type-5 F26D10.3 Heat shock cognate 71 kDa protein F26D12.1 Forkhead box P4 F26E4.11 Autocrine motility factor receptor, isoform 2 F27C8.1 Large neutral amino acids transporter small subunit 1 F28A12.4 Gastricsin precursor F28B12.3 Serine/threonine-protein kinase VRK1 F28D1.9 Long-chain fatty acid transport protein 4 F28F8.2 Acyl-CoA synthetase family member 2 F28H1.2 Transgelin-2 F28H1.4 Plasmolipin F29B9.6 SUMO-conjugating enzyme UBC9 F29F11.2 UDP-glucuronosyltransferase 1-8 precursor F30A10.10 Ubiquitin carboxyl-terminal hydrolase 48 F30A10.6 Phosphatidylinositide phosphatase SAC1 F31F4.7 UDP-glucuronosyltransferase 1-6 precursor F32A5.1 Transcriptional adapter 2-beta F32A5.5 Aquaporin-10 F32A5.7 U6 snRNA-associated Sm-like protein LSm4 F32A6.3 Vacuolar assembly protein VPS41 F32B6.1 HNF4-Alpha-3 of Hepatocyte nuclear factor 4-alpha

147

F32D1.10 DNA replication licensing factor MCM7 F35B12.4 Tissue factor pathway inhibitor 2 F35C8.2 Dual specificity MAPKK6 F35D11.11 Trichohyalin F35G12.10 ATP synthase subunit b F35H10.7 CGTHBA protein F36D3.9 n/a F37A4.5 26S proteasome non-ATPase regulatory subunit 14 F37B1.4 Glutathione-requiring prostaglandin D synthase F37B1.5 Glutathione-requiring prostaglandin D synthase F38B2.4 Adenylate kinase isoenzyme 1 F38B6.4 Trifunctional purine biosynthetic protein adenosine-3 F38B7.1 Butyrate response factor 2 F38E11.1 Alpha B crystallin fragment 4 F38E11.2 Alpha B crystallin fragment 4 F39H11.2 TATA-box-binding protein F40F11.1 40S ribosomal protein S11 F40G9.11 Max-like protein X F41B5.4 Cytochrome P450 2J2 F41E6.4 SMEK homolog 1 F41E6.5 Hydroxyacid oxidase 1 F41E7.1 NHEDC1 F41F3.4 Collagen alpha-1(III) chain precursor F41H10.7 Elongation of very long chain fatty acids protein 3 F42A10.4 Elongation factor 2 kinase F42C5.8 25 kDa protein F42G2.5 VAMP-associated protein A F43D9.4 Alpha-crystallin B chain F43E2.8 78 kDa glucose-regulated protein precursor F43G6.8 GTP-binding protein ARD-1 F44B9.3 Cyclin K isoform 1 F44C8.3 Orphan nuclear receptor TR4 F44C8.9 Photoreceptor-specific nuclear receptor F44G3.9 Photoreceptor-specific nuclear receptor F45E1.6 Histone H3.3 F45E6.2 Cyclic AMP-dependent transcription factor ATF-6 alpha F46B6.8 Gastric triacylglycerol lipase precursor

148

F46E10.10 Malate dehydrogenase F46E10.8 UCHL1 F46G11.3 Cyclin G-associated kinase F46H5.3 Creatine kinase M-type F46H5.7 Coiled-coil domain-containing protein FLJ36144 F47G4.4 katanin p80 subunit B 1 F47H4.10 S-phase kinase-associated protein 1 F48E3.7 Neuronal acetylcholine receptor protein F49B2.6 Leucyl-cystinyl aminopeptidase F49E11.9 Cysteine-rich secretory protein 2 F52C12.2 UPF0293 protein C16orf42 F52C6.8 BTB/POZ domain containing protein 5 F52F12.3 Mitogen-activated protein kinase kinase kinase 7 F52H3.5 Tetratricopeptide repeat protein 36 F52H3.7 Galectin-9 F53A3.3 40S ribosomal protein S15a F53A9.1 Histidine-rich glycoprotein precursor F53B3.6 U1 small nuclear ribonucleoprotein 70 kDa F53B6.2 ADAMTS-like 1 isoform 4 precursor F53C3.12 Beta,beta-carotene 15,15'-monooxygenase F53G12.6 Proto-oncogene tyrosine-protein kinase FER F54B11.3 Putative uncharacterized protein F54C1.7 Calmodulin F54H12.1 Aconitate hydratase F55A12.4 Retinol dehydrogenase 16 F55A4.1 Vesicle trafficking protein SEC22b F55B12.4 tRNA-nucleotidyltransferase 1 F55E10.7 Somatostatin receptor type 2 F55F3.1 5'-AMP-activated protein kinase subunit beta-2 F55G1.5 Mitochondrial glutamate carrier 2 F55H2.2 Vacuolar proton pump subunit D F55H2.5 Cytochrome b561 F56C11.2 Patched isoform S F56C9.1 Serine/threonine-protein phosphatase PP1 F56E10.4 40S ribosomal protein S27 F57B1.7 Doublesex- and mab-3-related transcription factor C F57B10.5 Hypothetical protein FLJ90481

149

F57C2.5 Adipocyte plasma membrane-associated protein F57H12.7 Putative uncharacterized protein Nbla00487 F58A3.1 LIM domain-binding protein 1 F58A4.10 26 kDa protein F59A6.6 Ribonuclease H1 F59F3.5 Vascular endothelial growth factor receptor 1 F59F4.1 Acyl-coenzyme A oxidase 1 F59F4.4 1-acylglycerol-3-phosphate O-acyltransferase 2 H03A11.1 family with sequence similarity 20, member C H10D18.2 Cysteine-rich secretory protein 2 precursor H14N18.1 BAG family molecular chaperone regulator 2 H16D19.1 Mannose receptor H25P06.2 Cell division protein kinase 9 H28G03.1 29 kDa protein K01A6.2 Guanylate kinase K01C8.1 Serine racemase K01G12.3 Acidic nuclear phosphoprotein 32 K04A8.5 Lipase member M precursor K04D7.3 4-aminobutyrate aminotransferase K04F1.15 Aldehyde dehydrogenase K05D4.4 Cytochrome P450 2J2 K05F1.3 Medium-chain specific acyl-CoA dehydrogenase K07A1.7 Headcase protein homolog K07A3.1 Fructose 1,6-bisphosphatase K07A3.2 Niemann-Pick C1 protein precursor K07B1.4 Monoacylglycerol O-acyltransferase 1 K07C11.4 Carboxylesterase 2 isoform 1 K07C6.3 Cytochrome P450 2C8 K07C6.4 Cytochrome P450 2U1 K07E1.1 1,2-dihydroxy-3-keto-5-methylthiopentene dioxygenase K07E3.3 C-1-tetrahydrofolate synthase, cytoplasmic K07E3.8 Membrane-associated progesterone receptor K08B4.6 Cystatin-D precursor K08E3.7 Parkin K08F4.11 Glutathione-requiring prostaglandin D synthase K08F4.7 Glutathione-requiring prostaglandin D synthase K08F4.9 Orphan short-chain dehydrogenase/reductase

150

K08H10.1 Dentin sialophosphoprotein preproprotein K09A11.2 Cytochrome P450 2C8 K09A11.4 Cytochrome P450 2D6 K09C4.5 SLC2A14 K09H9.6 Suppressor of SWI4 1 homolog K10B2.2 Lysosomal protective protein precursor K10B3.7 Glyceraldehyde-3-phosphate dehydrogenase K10B3.8 Glyceraldehyde-3-phosphate dehydrogenase K10B3.9 Putative uncharacterized protein DKFZp564G0422 K10C2.4 Fumarylacetoacetase K10H10.2 Cystathionine beta-synthase K10H10.5 34 kDa protein K11D2.2 Acid ceramidase precursor K11G12.4 Natural resistance-associated macrophage protein 2 K11G9.5 Sialin K11G9.6 Metallothionein-3 K12D12.2 Nuclear pore complex protein Nup205 K12D12.3 Collagen alpha-3(IX) chain precursor K12G11.3 Alcohol dehydrogenase 4 K12G11.4 Alcohol dehydrogenase 4 M01E5.5 DNA topoisomerase 1 M01F1.4 Hypothetical protein LOC23731 M02D8.4 Asparagine synthetase M03C11.5 ATP-dependent metalloprotease YME1L1 M03F4.7 Calumenin precursor M04B2.1 Uncharacterized protein FOXP2 M04G12.2 Cathepsin Z precursor M142.2 Collagen alpha-6(VI) chain precursor M151.3 Girdin M4.2 Pumilio homolog 2 M57.2 Geranylgeranyl transferase M7.5 E1-like protein R02F11.4 RIKEN cDNA 2810403B08 gene R03E9.1 MAX interactor 1 isoform b R03G5.2 Dual specificity MAPKK6 R03H10.7 Replication protein A 70 kDa DNA-binding subunit R04D3.1 Cytochrome P450 2J2

151

R05D11.6 Transcription factor R05D3.4 ring finger protein 20 R05D8.7 Peroxisomal short-chain alcohol dehydrogenase R05F9.1 BTB/POZ domain-containing protein 10 R05F9.13 14 kDa protein R07B7.15 NR1H3 protein R07E4.1 Hypothetical protein FLJ38335 R09B3.4 NEDD8-conjugating enzyme UBE2F R09B3.5 Protein mago nashi homolog 2 R09B5.6 Hydroxyacyl-coenzyme A dehydrogenase R09D1.12 Alpha-type platelet-derived growth factor receptor R09D1.2 43 kDa protein R102.4 17 kDa protein R107.7 Glutathione S-transferase P R10H10.2 Kelch-like 20 R11A5.4 Mitochondrial phosphoenolpyruvate carboxykinase 2 R11A8.4 NAD-dependent deacetylase sirtuin-1 R12A1.4 Liver carboxylesterase 1 precursor R12B2.5 Mediator of RNA polymerase II transcription subunit 15 R12E2.13 Stromal cell-derived factor 2 precursor R12H7.2 Cathepsin D precursor R13D7.7 Glutathione S-transferase P R144.4 WAS/WASL interacting protein R151.6 Derlin-2 R151.7 Heat shock protein 75 kDa R53.5 25 kDa protein R53.7 5'-AMP-activated protein kinase T01B11.2 Alanine--glyoxylate aminotransferase 2-like 1 T01B6.3 5'-AMP-activated protein kinase subunit gamma-1 T01D1.4 1,2-dihydroxy-3-keto-5-methylthiopentene dioxygenase T01G5.7 Ubiquitin ligase protein RNF8 T01G9.4 Nucleoporin NUP85 T01H3.2 DKFZP434B0335 protein T02B5.1 Liver carboxylesterase 1 precursor T02B5.1 Liver carboxylesterase 1 precursor T04H1.2 GTP-binding protein 2 T04H1.9 Tubulin beta-2C chain

152

T05A10.5 Cysteine-rich secretory protein 2 precursor T05A12.4 Helicase-like protein T05F1.11 Nucleoredoxin-like protein 2 T05G5.9 GRIP and coiled-coil domain-containing 2 T06A4.1 Carboxypeptidase A2 precursor T06G6.8 Mucin-5AC precursor (Fragment) T07D10.4 Mannose receptor T07D3.7 Eukaryotic translation initiation factor 2C 4 T07F12.4 ULK2 protein T08B2.10 40S ribosomal protein S17 T08B2.8 Uncharacterized protein MRPL23 (Fragment) T08D2.4 Zinc-finger protein HT2A T08H10.1 Aldo-keto reductase family 1 member B10 T09A12.2 Glutathione peroxidase T09E8.3 Cornichon homolog T10B11.1 Pterin-4-alpha-carbinolamine dehydratase T10B9.1 Cytochrome P450 3A5 T10B9.2 Cytochrome P450 3A5 T10F2.2 Mitochondrial ornithine transporter 1 T10F2.3 73 kDa protein T10H4.11 Cytochrome P450 2F1 T11F1.8 n/a T13A10.11 S-adenosylmethionine synthetase isoform type-1 T13A10.2 Tripartite motif protein 2 T13B5.3 ACPP protein T13H2.3 Plectin 7 T14F9.1 Vacuolar proton pump subunit H T14F9.3 Beta-hexosaminidase subunit alpha precursor T17H7.1 Collagen alpha-1(III) chain precursor T19B10.1 Cytochrome P450 4V2 T19C3.5 Bactericidal permeability-increasing protein T19D2.1 ADAMTS-18 precursor T19H12.10 UDP-glucuronosyltransferase 3A2 precursor T20B5.1 AP-2 complex subunit alpha-1 T20D3.5 Mitochondrial substrate carrier family protein T20F5.2 Proteasome subunit beta type-2 T21D12.9 Membrane glycoprotein LIG-1

153

T22B11.2 Galactosyltransferases T22B7.1 Transcription factor SOX-5 T22B7.7 Acyl-CoA thioesterase T22F3.11 Sialin T22G5.6 Fatty acid-binding protein T22H2.5 Uncharacterized protein PLSCR1 (Fragment) T23B12.3 Mitochondrial 28S ribosomal protein S2 T23F2.1 Alpha-1,3-mannosyltransferase ALG2 T23G7.2 Putative metallophosphoesterase FLJ45032 T23G7.3 Pin2-interacting protein X1 T23H2.2 Synaptotagmin-4 T23H4.3 Meprin A subunit beta precursor T24H7.1 Prohibitin-2 T24H7.5 Probable phospholipid-transporting ATPase VB T25B9.1 2-amino-3-ketobutyrate coenzyme A ligase T25B9.2 Serine/threonine-protein phosphatase PP1 T25G3.4 Glycerol-3-phosphate dehydrogenase T27A3.6 Molybdenum cofactor synthesis protein 2 T27C4.4 Metastasis-associated protein MTA3 T27E4.2 Alpha-crystallin B chain T27E4.8 Alpha-crystallin B chain T27F7.1 Charged multivesicular body protein 3 T28D9.3 Lipid phosphate phosphohydrolase 1 T28F2.2 COMM domain-containing protein 4 T28F4.5 Death-associated protein 1 VC5.3 Centromeric protein E VZK822l.1 Acyl-CoA desaturase W01A11.1 Epoxide hydrolase 1 W01A8.2 UPF0235 protein C15orf40 W01B11.2 Solute carrier family 26 member 6 W01G7.4 Hypothetical protein W02A11.2 Vacuolar protein-sorting-associated protein 25 W02A11.3 Ring finger protein 44 W02H5.8 Dihydroxyacetone kinase W03C9.3 Ras-related protein Rab-7a W03C9.3 Ras-related protein Rab-7a W03G9.1 Sodium- and chloride-dependent creatine transporter 1

154

W03G9.6 Platelet-activating factor acetylhydrolase 2 W04H10.3 Tripartite motif protein 3 W05B5.2 Orexin receptor type 2 W05G11.6 Mitochondrial phosphoenolpyruvate carboxykinase W05H9.1 Uncharacterized protein ENSP00000383081 W06D12.5 Potassium channel subfamily K member 1 W06D4.1 Homogentisate 1,2-dioxygenase W06E11.4 Ribosome maturation protein SBDS W06F12.1 Nemo like kinase W06H3.1 Mitochondrial inner membrane protein W07E11.1 Dihydropyrimidine dehydrogenase W07G4.4 56 kDa protein W08D2.4 Fatty acid desaturase 2 W08D2.5 Probable cation-transporting ATPase 13A3 W09C2.3 Plasma membrane calcium-transporting ATPase 3 W10C8.5 Creatine kinase W10G6.3 Lamin-B1 Y105E8A.16 40S ribosomal protein S20 Y106G6E.6 Casein kinase I isoform gamma-3 Y106G6H.3 60S ribosomal protein L30 Y110A2AR.2 n/a Y116A8C.35 U2 small nuclear RNA auxillary factor 1 Y15E3A.1 Orphan nuclear receptor NR6A1 Y17G7B.5 MCM2 Y17G7B.7 Triosephosphate isomerase Y17G9B.4 40 kDa peptidyl-prolyl cis-trans isomerase Y24D9A.4 60S ribosomal protein L7a Y32H12A.3 Dehydrogenase/reductase SDR family member 1 Y32H12A.8 WD repeat-containing protein 24 Y37A1B.2 Hypothetical protein MGC32065 Y37E11B.5 tRNA-dihydrouridine synthase 3-like Y37H9A.6 Bis(5'-nucleosyl)-tetraphosphatase Y38E10A.4 Deleted in malignant brain tumors 1 protein Y38F1A.2 Hypothetical protein Y38H6C.17 Proton-coupled amino acid transporter 2 Y40B10A.2 O-methyltransferase Y40B10A.6 O-methyltransferase

155

Y40D12A.2 Lysosomal protective protein precursor Y40G12A.1 UCHL3 Y41D4B.5 40S ribosomal protein S28 Y42G9A.4 Mevalonate kinase Y43C5A.3 Heterogeneous nuclear ribonucleoproteins A2/B1 Y43C5B.2 Proto-oncogene tyrosine-protein kinase FER Y43F8A.3 Arylacetamide deacetylase-like 1 Y45F10B.9 Zinc-finger protein HT2A Y45G12C.2 Glutathione S-transferase P Y46H3A.3 Alpha-crystallin B chain Y47G6A.18 Golgi phosphoprotein 3 Y48B6A.2 60S ribosomal protein L37a Y48G1A.6 L3MBTL2 Y48G8AL.8 60S ribosomal protein L17 Y48G9A.10 Carnitine O-palmitoyltransferase I Y49E10.20 Lysosome membrane protein 2 Y49G5A.1 Eppin precursor Y4C6A.3 Tripartite motif protein 2 Y4C6B.4 Solute carrier family 17 (sodium phosphate), member 1 Y4C6B.5 Proton-coupled folate transporter Y53C10A.12 Heat shock factor protein 1 Y53C12A.2 Excitatory amino acid transporter 2 Y53F4B.32 Glutathione-requiring prostaglandin D synthase Y53F4B.33 Glutathione-requiring prostaglandin D synthase Y53F4B.4 Methyltransferase-like protein 2 Y53G8B.2 2-acylglycerol O-acyltransferase 2 Y54E10BR.3 Hypothetical protein FLJ20552 Y54E2A.12 Predicted GTPase activator protein Y54E2A.2 Uncharacterized protein C19orf61 Y54G11A.6 Catalase Y55B1AR.1 Uncharacterized protein LGALS9 Y55D9A.2 Angiogenic factor VG5Q Y55F3AM.3 RNA-binding protein 39 Y55F3BR.1 ATP-dependent helicase DDX1 Y56A3A.1 CCR4-NOT transcription complex subunit 3 Y56A3A.33 Exonuclease GOR Y57G11C.24 Epidermal growth factor receptor kinase substrate 8

156

Y58G8A.1 Acetylcholine receptor subunit beta precursor Y61A9LA.3 Arginine and glutamate-rich protein 1 Y65B4A.2 n/a Y65B4A.3 Uncharacterized conserved protein Y65B4BR.4 NEDD4-like E3 ubiquitin-protein ligase WWP1 Y66H1B.4 Sphingosine-1-phosphate lyase 1 Y67A6A.2 HNF4-Alpha-3 of Hepatocyte nuclear factor 4-alpha Y69H2.3 IgGFc-binding protein precursor Y6B3B.10 LAG1 longevity assurance homolog 1 Y71G12B.4 Charged multivesicular body protein 6 Y71H10A.1 6-phosphofructokinase Y71H2AR.2 Cathepsin L2 precursor Y75B12B.2 Peptidyl-prolyl cis-trans isomerase Y76A2A.2 Copper-transporting ATPase 1 Y80D3A.5 Cytochrome P450 4V2 Y87G2A.8 Glucose-6-phosphate isomerase Y9C9A.16 Sulfide:quinone oxidoreductase ZC196.2 cDNA FLJ78586 ZC250.3 UDP-N-acetylglucosamine transporter ZC395.8 Dentin sialophosphoprotein precursor ZC434.2 40S ribosomal protein S7 ZC518.3 CCR4-NOT transcription complex subunit 6-like ZK1073.1 Protein NDRG1 ZK270.1 Band 4.1-like protein 3 ZK270.2 Niemann-Pick C1 protein precursor ZK384.1 Peptidase inhibitor 16 precursor ZK384.2 Glioma pathogenesis-related protein 1 ZK384.3 Gastricsin precursor ZK430.3 Superoxide dismutase ZK54.2 Trehalose-6-phosphate synthase component ZK550.6 Phytanoyl-CoA dioxygenase, peroxisomal precursor ZK593.7 U6 snRNA-associated Sm-like protein LSm7 ZK596.1 Keratin, type I cytoskeletal 9 ZK632.2 Solute carrier family 4 (anion exchanger) ZK652.4 60S ribosomal protein L35 ZK816.5 Dehydrogenase/reductase SDR family member 1 ZK829.7 Sorbitol dehydrogenase

157

ZK973.5 Neuronal acetylcholine receptor subunit alpha-2

158

Table 4.2. Summary of positive genes from RNAi screen for effectors of α-syn in the daf-2 background based on KOG and/or GO annotations. Category Gene Human ortholog E-value ERAD F26E4.11 (hrdl-1) AMFR 1.90E-39 T27E4.2 (hsp-16.11) Alpha crystallin 2.00E-14 Y38E10A.4 (clec-8) Glycoprotein 340 2.50E-12 ER-Golgi C54H2.5 (sft-4) ERV29 7.80E-88 trafficking F55A4.1 SEC22b 2.30E-47 F57B10.5 emp24/gp25L/p24 2.70E-58 Glutathione- C06A6.5 ERp44 8.30E-75 related C29E4.7 (gsto-1) Glutathione S-transferase 2.00E-32 C54D10.2 C6orf168 8.10E-22 Glycosylation F10C2.5 Glycosyl hydrolase 3.30E-149 T22F3.11 Sialin 7.80E-25 Lysosomal F21F8.7 (asp-6) Aspartyl protease 5.00E-51 function T13B5.3 ACPP 1.80E-38 Y71H2AR.2 Cathepsin L 8.00E-33 Metabolism F01F1.12 ALDOA 2.70E-117 F25B4.1 Aminomethyl transferase 6.90E-101 F38B6.4 GARS/AIRS 1.90E-208 K07C6.3 (cyp-35B2) Cytochrome P450 2C8 4.50E-58 T07D3.7 (alg-2) eIF-2C 0 K10B3.7 (gpd-3) GAPDH 2.80E-136 K10B3.8 (gpd-2) GAPDH 2.80E-136 K12G11.4 (sodh-2) Alcohol dehydrogenase 6.90E-20 T10H4.11 (cyp-34A2) Cytochrome P450 2F1 1.90E-57 Y87G2A.8 (gpi-1) GPI 2.80E-207 W07E11.1 Glutamate synthase 1.80E-11 Signaling C02C2.3 (cup-4) Acetylcholine receptor 2.80E-16 Components C44H4.5 (tap-1) TAB1-like protein 9.50E-12 D1069.3 DRCTNNB1A 2.10E-10 F52F12.3 (mom-4) MAPKKK7 1.20E-38

159

R02F11.4 Protein phosphatase 1 2.50E-18 T01B6.3 AMPK, gamma subunit 1.60E-18 Transcription C03D6.3 (cel-1) RNGTT 1.70E-113 F40G9.11 (mxl-2) BIGMAX 1.40E-15 F44C8.3 (nhr-18) Nuclear receptor TR4 2.80E-14 R03E9.1 (mdl-1) MAX interactor 1 1.40E-17 T05A12.4 Helicase-like protein 5.10E-52 Y45F10B.9 Zinc-finger protein HT2A 8.40E-07 ZC395.8 DSPP 1.60E-06 Transporter F20D1.9 SLC25A18 2.20E-71 T19C3.5 BPI/LBP/CETP 3.60E-17 Y76A2A.2 (cua-1) Cation transport ATPase 3.70E-261 UPS T01G5.7 RNF8 7.60E-08 Others C29F9.2 MAP7D1 8.00E-06 C54D10.10 TFPI 3.40E-13 F01G12.5 (let-2) Collagen 0 F53B6.2 ADAMTSL1 1.90E-101 T23H4.3 (nas-5) Meprin A metalloprotease 4.60E-31 Y105E8A.16 (rps-20) 40S ribosomal protein S20 1.50E-41 Uncharacterized C54D10.7 (dct-3) Uncharacterized protein 7.90E-30 Protein F35H10.7 CGTHBA protein 4.30E-25 H03A11.1 Uncharacterized protein 5.20E-82 R05F9.1 Uncharacterized protein 1.40E-95 W05H9.1 Uncharacterized protein 6.30E-16

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Figure 4.1

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Figure 4.2

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Figure 4.3

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Figure 4.4

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Figure 4.5

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Figure 4.6

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Figure 4.7

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FIGURE LEGENDS

Figure 4.1. Graphs depicting the percentage of α-syn-expressing daf-2 and/or daf-

16 mutants with wildtype DA neurons. A. At the chronological aging stage, daf-2

significantly protected DA neurons. B. At the chronological aging stage, daf-16 , which

transcribes various cytoprotective genes enhanced DA neurodegeneration. C. At the

chronological aging stage, daf-2 + daf-16 double mutation exhibited an intermediate level

of neuroprotection. D. At the biological aging stage, daf-2 did not rescue DA neurons from α-syn-induced neurodegeneration, indicating that differential gene expression in the daf-2 background is responsible for neuroprotection. **P<0.05; Student’s t-test.

Figure 4.2. Graph summarizing lifespan assay of N2 and daf-2 worms. Wildtype

N2 worms exhibited mean lifespan of day 20 (Blue). Similarly, overexpression of α-syn in DA neurons did not affect lifespan (Red). daf-2; unc-32 mutants exhibited mean lifespan of day 40 (Green) as previously published (Kenyon et al., 1993).

Figure 4.3. daf-2 enhances degradation of α-syn::GFP fusion protein. A. In N2 background, accumulation of α-syn::GFP is readily observable in the cytoplasm of body wall muscle cells. B. In daf-2 background, the fusion protein is readily degraded. This strain was used for the RNAi screen. C. With RNAi knockdown of daf-16 , return of α- syn::GFP aggregates is observed in the daf-2 background. D. Semi-quantitative RT-PCR demonstrates that daf-2 does not affect mRNA level of α-syn::gfp . E. Western blot

confirms the degradation of the fusion protein in the daf-2 background.

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Figure 4.4. Graph illustrating the percentage of α-syn-expressing worms with wildtype DA neurons 24 or 48 hrs after 1, 5, and 10 mM 2-deoxyglucose (DOG) treatment. DOG treatment enhanced DA neurodegeneration of 6 day-old animals in a concentration-dependent manner. **P<0.05; Student’s t-test.

Figure 4.5. Graph illustrating the percentage of 7 day-old worms with wildtype

DA neurons expressing α-syn and gpi-1 or hrdl-1, two genes involved in the autocrine motility factor pathway. Both proteins significantly protected DA neurons from α-syn- induced neurodegeneration. **P<0.05; Student’s t-test.

Figure 4.6. Pie chart summarizing 53 positive genes from the RNAi screening.

Functions of these gene products were assigned based on KOG and/or GO annotations.

Notably, metabolic enzymes (11/53; 21%), transcription factors (7/53; 13%), and signaling components (6/53; 11%) were over-represented.

Figure 4.7. Diagram summarizing the DAF-2/insulin signaling pathway in C. elegans . Straight line indicates the pathway studied in worms, and dotted line indicates the pathway identified in mammals. DAF-2 negatively regulates DAF-16 via an AGE-1- dependent manner. DAF-16 then modulates autophagy or protein synthesis.

Furthermore, DAF-2 may also regulate cell death through the MAPK signaling pathway.

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CHAPTER FIVE

CONCLUSION

Introduction

Generation of transgenic C. elegans overexpressing α-syn::GFP in body wall

muscles, as well as α-syn and GFP in DA neurons, enabled us to ascertain genetic factors that modified α-syn misfolding and α-syn-induced neurodegeneration, respectively.

Using a combination of bioinformatics, RNAi, mutant analysis, and transgene

overexpression, this experimental paradigm allowed us to determine genetic targets as

well as cellular mechanisms that ameliorate α-syn toxicity. Further, many genes

identified from our studies are neuroprotective across species boundaries, strongly

demonstrating C. elegans as a powerful model organism for studying PD. Based on our

findings, we have embarked on the following research projects that are currently

conducted in the Caldwell laboratory.

Neuroprotective mechanism of VPS41, ATG7, ULK2, and GIPC: a common pathway?

As described in Chapter 2, based on the hypothesis-based RNAi screen and

subsequent neuroprotection analysis of candidate genes, 5 specific genes (vps-41/VPS41 ,

atgr-7/ATG7 , C35D10.2/GIPC , F55A4.1/SEC22 , and F16A11.2/HSPC117 ) suppressed

α-syn-induced neurodegeneration. Among them, an autophagic component, VPS-

41/VPS41 exhibited the strongest neuroprotection. VPS41 is a well-characterized protein 170

in yeast but not in humans. In yeast, Vps41 is required for trafficking of the vesicles to the vacuoles (mammalian equivalent of lysosomes) (Radisky et al., 1997) via the alkaline phosphatase pathway through its interaction with the AP-3 adaptor protein complex

(Rehling et al., 1999; Darsow et al., 2001). Interestingly, LaGrassa and Ungermann

(2005) reported that activity of Vps41 is regulated by yeast casein kinase, Ypt7. From a separate study, we have demonstrated that overexpression of worm casein kinase csnk-1 protects DA neurons from α-syn toxicity. Given the fact that VPS41 is a highly conserved protein across the species, the protein may share similar functions in humans.

One of the on-going research projects in our laboratory, in collaboration with

David Standaert at UAB, is a characterization and validation of human VPS41 as a prospective therapeutic target for PD using the worm α-syn-induced neurodegeneration model and mammalian cell culture models of PD. Importantly, overexpression of human

VPS41 protected mammalian cells from neurotoxin-induced toxicity, validating our previous observation on the neuroprotective role of worm VPS-41 (Ruan et al., manuscript in revision). To further characterize its neuroprotective mechanism, structure-function analysis of human VPS41 in C. elegans has revealed that WD40 and clathrin heavy chain domains are essential for neuroprotective function. While not limited to VPS41, genome-wide analysis of single nucleotide polymorphisms (SNPs) in

PD patients should be informative to confirm VPS41 as a genetic PD susceptibility factor.

For example, SNPs have already been found in a gene called ULK2 (Fung et al., 2006), the worm ortholog of which was identified among the top 20 hits from our RNAi screen

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(Chapter 2). We have determined that unc-51/ULK2 loss-of-function mutation enhanced

α-syn toxicity in worm DA neurons (unpublished data).

Additionally, atgr-7/ATG7 is another key autophagic gene that rescued DA neurons from α-syn toxicity, further confirming autophagy as one of the neuroprotective cellular mechanisms. ATG7 encodes an ubiquitin-activating enzyme E1-like protein that plays a critical role in the activation of autophagy. Importantly, Komatsu et al. (2006;

2007) demonstrated that the loss of Atg7 in mice, which enhanced neurodegeneration in the central nervous system, was required for maintenance of axonal homeostasis as well as proper behavior. These findings suggest that, similar to VPS41, ATG7 in mammals may also serve a neuroprotective role against α-syn-induced neurodegeneration.

Interestingly, not only VPS41 and ATG7 are involved in autophagy, these proteins contain RING finger (commonly found in E3 ligases) and E1-like protein- activating enzyme Gsa7p/Apg7p domains, respectively. By physically interacting with ubiquitin, this association suggests VPS41 and ATG7 may function in a common pathway such as the UPS or endocytosis (Polo et al., 2002). While the functional link between these proteins and the UPS cannot be dismissed presently, evidence suggests that VPS41 and ATG7 may contribute in endocytosis. For example, both endocytic and autophagic pathways have been shown to converge at the late endosomes to eventually form late autophagosomes or mature lysosomes (Yi and Tang, 1999). Moreover, as described above, we have demonstrated that loss of unc-51/ULK2 , another autophagic component, enhanced α-syn-induced neurodegeneration. ULK2 is a serine/threonine

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kinase that has also been shown to regulate axon growth through endocytosis of nerve growth factor in mouse (Zhou et al., 2007). Lastly, in C. elegans , RNAi knockdown of endocytic genes enhanced growth defects and motor abnormalities that were induced by overexpression of pan-neuronal α-syn (Kuwahara et al., 2008).

Consistent with this view is the identification of C35D10.2/GIPC as a neuroprotective gene (Chapter 2). GIPC encodes a scaffold protein that regulates cell surface receptor including β1-adrenergic receptor (Hu et al., 2002), D2-like DA receptor

(Jeanneteau et al., 2004), and insulin-like growth factor 1 receptor (Booth et al., 2002).

Since dop-2 (a C. elegans ortholog of D2-like DA receptor) knockout in our model enhanced α-syn-induced neurodegeneration (unpublished data), GIPC may modulate DA signaling and promote DA neuron survival by blocking cAMP synthesis, similar to overexpression of PDE9A wherein cAMP is hydrolyzed. Alternatively, since G-protein signaling has been shown to activate autophagy (Ogier-Denis et al., 2000), it is conceivable that overexpression of GIPC may also stimulate this pathway. If VPS41,

ATG7, ULK2, and GIPC functionally converge as the components of endocytic and autophagic pathways, then it will be intriguing to examine if these proteins enhance degradation of α-syn found in the synaptic termini and/or degradation of presumably neurodegenerative receptors (e.g., D1-like receptor that stimulates cAMP synthesis).

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Defining Networks of neuroprotective genes by miRNAs

miRNAs are 21-23 bp long, single-stranded RNAs that regulate gene expression

(Ambros, 2001). Initially transcribed as approximately 1000 bp primary miRNAs, these molecules are further processed by the RISC complex to form mature miRNAs (Moss,

2002). In mammalian neurons, miRNAs have been shown to regulate neuronal differentiation, neurite outgrowth, survival, and synaptic formation, thus dysregulation of miRNAs may enhance susceptibility to neurodegenerative diseases (Bushati and Cohen,

2008; Hebert and De Strooper, 2009). In PD, Kim et al. (2007) reported that mir-133b is

deficient in the midbrain of PD patients. Further, Junn et al. (2009) reported that mir-7

suppresses α-syn expression by directly binding to 3’UTR of α-syn mRNA.

Collectively, the study of miRNAs represents an exciting field that is yet fully explored,

especially with respect to its significance for disease etiology.

As described in Chapter 3, in collaboration with Susan Lindquist at Whitehead

Institute/MIT, we have identified potential therapeutic targets that suppress α-syn toxicity

in multiple model organisms. These candidates include the gene products that function in

ER-Golgi trafficking, protein phosphorylation, ubiquitination, etc. Based on these yeast

α-syn toxicity modifiers, as well as our list of neuroprotective genes, we utilized

bioinformatic databases (miRBase and TargetScan) to mine worm miRNAs that are

predicted to target worm putative neuroprotective genes. Despite the significant number

of targets affected by a single miRNA, only a few miRNAs are predicted to target more

than five genes implicated in neuroprotection. Most notably, mir-797 , which is part of

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mir-2/mir-43/mir-250/mir-797 superfamily may regulate as many as six genes that are

predicted or shown to protect DA neurons (Fig. 5.1). For example, according to

miRBase, mir-797 may regulate ykt-6, cmk-1, csnk-1, tag-278, hrd-1, and obr-1. Among

them, we have shown that csnk-1 and hrd-1 rescue DA neurons from α-syn toxicity.

Based on the bioinformatic associations, we examined the effect of mir-2 or mir-

797 knockout on α-syn-induced neurodegeneration, and determined that the knockout of

these miRNAs enhanced neuroprotection (unpublished data). Given the role of miRNAs

in suppressing the translation of their targets, these data suggest that, under mir-2 and

mir-797 knockout backgrounds, neuroprotective genes may be up-regulated. To confirm

this, we are currently performing quantitative real-time PCR of these targets in miRNA

knockout backgrounds. While genetic analysis and quantitative real-time PCR provide

evidence for the correlation between miRNAs and α-syn-induced neurodegeneration, these results do not indicate that the observed neuroprotection is specific to the cellular changes in DA neurons. To further validate the interaction between miRNAs and α-syn toxicity in DA neurons, we generated transgenic strains that overexpress these miRNAs selectively in DA neurons. Thus far, we have analyzed the worms that overexpress a transgene encoding mir-2 primary miRNA (encompassing approximately 500 bp both up- stream and down-stream of the miRNA) under the control of dat-1 (dopamine transporter) promoter. Interestingly, overexpression of mir-2 enhanced neurodegeneration

(unpublished data), suggesting that mir-2 modifies gene expression and subsequently impacts a cellular threshold against α-syn toxicity in DA neurons.

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While speculative, it is interesting to note that the worm F16A11.2 gene and mir-2 are co-expressed in a C. elegans operon unit. F16A11.2/HSPC117 encodes an uncharacterized conserved protein that was identified in mRNA granules in humans, and might be involved in mRNA transport in neurons (Kanai et al., 2004). Since overexpression of F16A11.2 and knockout of mir-2 enhance neuroprotection, this RNA- binding protein may physically interact with mir-2 to regulate the miRNA activity.

Additional PD-related studies using C. elegans

As described in Chapters 2-4, our worm α-syn neurodegeneration model has been utilized to verify putative neuroprotective genes, but it has also been extensively used to identify novel compounds that protect DA neurons. For example, we have explored chemical space by studying two cyclic peptides that rescue DA neurons from α-syn toxicity (Kritzer et al., 2009). Interestingly, these cyclic peptides resemble a common sequence found in thioredoxin, which suggests that these compounds may be neuroprotective via reduction of oxidative stress. Furthermore, we have identified several chemicals that suppress α-syn-induced neurodegeneration and this effect is conserved across species from yeast, to worms, and to rat neurons (Su et al., unpublished data).

Focusing on genetic interactions among different PD genes, we have generated djr-1.1/DJ-1, djr-1.2/DJ-1, pdr-1/parkin , pink-1/PINK1 , and ubh-1/UCHL1 worm mutant strains overexpressing α-syn and GFP in DA neurons, and determined that the depletion

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of djr-1.1 and djr-1.2 enhance α-syn toxicity (unpublished data). These results suggest

that in our model, protection against α-syn-induced oxidative stress may be a key feature

for rescuing DA neurons. Further supporting this view is the result demonstrating that α-

syn-induced neurodegeneration is enhanced in trx-1 (a C. elegans ortholog of thioredoxin) knockout background (unpublished data). It will be interesting to further explore the association between α-syn-induced neurodegeneration and oxidative stress by using neurotoxins (e.g., 6-OHDA, MPTP/MPP+) or overexpressing proteins with anti- oxidant properties.

We have also generated transgenic worms overexpressing wildtype or mutant

LRRK2 and GFP in DA neurons, and determined that the overexpression of LRRK2

G2019S enhanced DA neurodegeneration (unpublished data). Both C. elegans (Saha et al., 2009) and D. melanogaster (Liu et al., 2008; Venderova et al., 2009) models of

LRRK2 G2019S-induced neurodegeneration have been created showing LRRK2 G2019S more neurodegenerative than wildtype LRRK2. Using our LRRK2 G2019S neurodegeneration model, we have also identified kinase inhibitors that rescue DA neurons in worms, mammalian cell culture, and mouse primary neurons (unpublished data). Since increased kinase activity of LRRK2 is linked to enhanced toxicity (West et al., 2005), it will be intriguing to examine downstream components of LRRK2 (e.g.,

MAPKK, MAPK, etc). In C. elegans , the MAPK signaling has been shown to regulate cell death, therefore, pmk-1/p38 mutant strain may suppress LRRK2 G2019S-induced neurodegeneration.

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Lastly, one of the 20 positive genes from the hypothesis-based RNAi screen

(Chapter 2) is smf-1/SLC11A2 . smf-1 encodes a divalent metal transporter that is predicted to transfer iron, manganese, and other divalent metals. In C. elegans , smf-1 is predicted to be co-expressed with djr-1.1 and pink-1. Consistent with the RNAi data indicating that knockdown of smf-1 enhanced α-syn misfolding, we have also demonstrated that smf-1 knockout enhances α-syn toxicity in worm DA neurons

(unpublished data). Since manganese exposure has been linked to PD pathological features, and more recently through ATP13A2 protein (as mentioned in Chapter 3), it will be interesting to examine how decreased manganese uptake in smf -1 background enhances α-syn toxicity. For example, expression pattern or subcellular localization of smf-1 will significantly facilitate our understanding of PD pathogenesis and manganese toxicity.

Conclusion and future directions

The experimental strategy we have employed using transgenic C. elegans to model primary pathological features of PD in vivo has enabled us to identify various and novel factors that modify α-syn misfolding and neurodegeneration. While vertebrate/mammalian models are the most suitable research tools for studying human diseases, generation of a degenerative model using α-syn in mammalian cell cultures has been difficult because overexpression of α-syn kills these cells, and rodent models are

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expensive, since they require high maintenance. Therefore, invertebrate PD models,

admittedly imperfect, remain both valuable and informative tools for discerning genetic, chemical, and environmental modifiers of PD pathogenesis. Moreover, as described in

Chapter 4, these invertebrate animals with short lifespans readily allow us to examine the effect of longevity on aging-related diseases, including PD.

While C. elegans will continue to be a model organism of choice for genetic and chemical screens, another distinct advantage of this organism is a well-defined neuronal circuitry (White et al., 1986). As we begin to address PD pathogenesis beyond DA neurons, C. elegans offers accuracy and precision in studying neuronal connection unmatched in other model organisms. For instance, we know that CEP neurons (worm

DA neurons) receive signals from ADE (another set of worm DA neurons), ALM, OLL,

RIH, RIS, and URB (Fig. 5.2). Furthermore, CEP neurons send signals to AVE, IL1,

OLL, OLQ, RIC, RMD, RMG, RMH, URA, and URB while forming gap junctions with

OLQ and RIH. It will be interesting to examine how neurodegenerative genes affect synaptic plasticity and how DA neurodegeneration may influence synaptic transmission to or from other surrounding neurons (Desplats et al., 2009).

In addition, since neurodegeneration is also linked to inflammation, it will be interesting to determine which neuronal types respond to DA neurodegeneration via inflammatory signals. For example, Styer et al. (2008) reported that npr-1 suppresses

innate immune response in C. elegans . Interestingly, npr-1 is expressed in OLQ neurons,

which form synapses as well as gap functions with CEP neurons. If DA neuron death is

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enhanced by inflammatory signals, then npr-1 knockout may further promote neurodegeneration. Conversely, if NPR-1 is overexpressed in the neurons surrounding

CEP and ADE, then this may lead to neuroprotection.

Taken together, the experimental outcomes, current and future directions collectively described herein provide a substantial scaffold on which further advances pertaining to therapeutic strategies and cellular mechanisms associated with PD can be built. Moreover, the capacity to more rapidly ascertain factors influencing neurodegeneration using the nematode system accelerates the pace toward defining novel neuroprotective strategies that may translate to the clinic. With available tools, techniques, and databases, C. elegans is deemed to offer immeasurable opportunities for studying PD and other neurodegenerative diseases.

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Figure 5.1

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Figure 5.2

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FIGURE LEGENDS Figure 5.1. Schematic diagram illustrating predicted targets of mir-2/mir-43/mir- 250/mir-797 superfamily by miRBase (top) and TargetScan (bottom). According to miRBase, mir-797 regulates 6 genes including ykt-6, cmk-1, csnk-1, tag-278, hrd-1, and obr-1. Among them, csnk-1 and hrd-1 have been shown to display neuroprotective capacities against α-syn-induced neurodegeneration. Figure 5.2. Schematic diagram of CEP neuronal circuitry. CEP receives signal from 6 neurons, sends signal to 10 neurons, and forms gap junctions with 2 neurons. A marker for DA neurons, dat-1 (dopamine transporter) is expressed in both CEP and ADE neurons. Notice npr-1, which suppresses innate immune response in C. elegans is expressed in OLQ.

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