identification of neuroprotective genes against alpha - acumen - The ...

IDENTIFICATION OF NEUROPROTECTIVE GENES AGAINST ALPHA- 

SYNUCLEIN TOXICITY USING A CAENORHABDITIS ELEGANS 

PARKINSON DISEASE MODEL 

by 

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

Copyright Shusei Hamamichi 2009 

ALL RIGHTS RESERVED

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. 

ii

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 

iii

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 

µg Microgram 

iv

µl 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) 

v

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 

vi

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) 

vii

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 

ix

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 



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 

x

USP10 Ubiquitin-specific protein 10 

VMAT2 Vesicular monoamine transporter 2 

VPS41 Vacuolar protein sorting 41 

XIAP X-linked inhibitor of apoptosis 

xi

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 

xv

a. Abstract ............................................................................................................120 

b. Introduction ......................................................................................................121 

c. Materials and methods .....................................................................................124 

d. Results ..............................................................................................................128 

e. Discussion ........................................................................................................132 

f. References ........................................................................................................137 


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 


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

Parkinson disease 

CHAPTER ONE 

INTRODUCTION 

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. 

1

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 

2

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 

3

(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 

4

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. 

5

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 

6

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 

7

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 

8

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 

9

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 

10

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 

11

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 

12

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 

13

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, 

14

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. 

15

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. 

16

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 

17

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 

19

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. 

20

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. 

21

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29

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 

Multiplication 

n/a n/a 

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 

30

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; 

Overexpression of WT α-syn 

and ∆α-syn 

C. elegans 

31 

Gitler et al., 2008 

Cytological analysis Flower et al., 2005; Soper et 

al., 2008; Liang et al., 2009 

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 αsyn::gfp 

(muscles) 

RNAi Van Ham et al., 2008 

Overexpression of WT LRRK2 

and ∆LRRK2 (DA neurons) 

Cytological analysis Saha et al., 2009 

PD-associated mutants (∆DJ-1, Mutant strain analysis Ved et al., 2005; Samann et 

∆parkin, ∆LRRK2, or ∆PINK1) 

al., 2009 

Neurotoxin treatment 

D. melanogaster 

Cytological analysis Nass et al., 2002; Braungart 

et al., 2004 

Overexpression of WT α-syn 

and/or ∆α-syn (DA neurons) 

Cytological analysis Auluck et al., 2002 

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 

32

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. 

33

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. 

34

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. 

35

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 

36

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 

37

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 µg/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 

39

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 µg/µl 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 

40

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 µl 1:10-diluted Single Worm Lysis Buffer (10 mM Tris, 

pH 8.3, 50 mM KCl, 2.5 mM MgCl2, 0.45% NP-40, 0.45% Tween 20, 0.01% gelatin, and 

60 µg proteinase K), mixed with 100 µl TRI Reagent (Molecular Research Center), and 

incubated at RT for 10 min. The samples were freeze-thawed 5 times using liquid N2, 

vortexed with 10 µl 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 µl glycoblue (Ambion) and 50 µl - 

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 

42

was washed with 100 µl RNase-free 75% ethanol, and resuspended in 10 µl 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’ 

43

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

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 

45

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 

46

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 

47

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 

48

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 

50

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

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, 

52

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). 

53

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 Gαi-mediated induction of autophagy in human intestinal cells (Ogier- 

Denis et al., 1997). It is interesting to speculate that GIPC serves to modulate a pre- 

synaptic protein-coupled pathway that can somehow combat the effects of α-syn 

misfolding and accumulation, perhaps by a DA or DA-receptor regulated manner. 

54

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

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. 

57

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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 

62

Table 2.2. Bioinformatic associations among gene candidates identified by RNAi. 

C. elegans Gene ID Bioinformatic 

Association 

Component of: 

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) 

63

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

C. elegans 

Gene ID 

Average % 

worms with 

wildtype 

DA 

neurons* 

NCBI KOGs 

F32A6.3 

(vps-41) 

36.7 + 5 

Vacuolar assembly/sorting 

protein VSP41 

C35D10.2 31.5 + 1 

RGS-GAIP interacting 

protein GIPC 

M7.5 (atgr-7) 31.1 + 1 

Ubiquitin activating E1 

enzyme-like protein 

F16A11.2 24.8 + 3 

Uncharacterized 

conserved protein 

F55A4.1 23.7 + 3 

Synaptobrevin/VAMPlike 

protein SEC22 

*Compared to 12.8% with a-syn alone. 

64 

Blast evalue 

Relevance to 

H. sapiens 

5.1e -96 95.7 

1.1e -49 75.4 

7.4e -87 69.2 

1.2e -207 99.8 

2.3e -47 96.3 

% length

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 

65

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 

66

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 

67

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 


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 


C26E6.11 Cob 

C26E6.8 NEDD8-activating enzyme E1 regulatory subunit 

C26F1.4 Ubiquitin-like protein FUBI 

C27A12.6 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 

68

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 

69

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 

70

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 

71

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 


F21F8.2 Gastricsin precursor 

F21F8.3 43 kDa protein 



F22B5.7 Cytoskeleton-associated protein 5 

72

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 

73

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 

74

F40F4.5 Tubulin alpha-ubiquitous chain 

F40F9.8 Calmodulin 

F40G9.1 PSMD10 


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 


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 

75

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 

76

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 

77

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 

78

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 

79

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 

80

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 

81

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 



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 

82

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 

83

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 

84

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 



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 

85

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 

86

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 


ZK20.5 PSMD8 

ZK218.11 Keratin associated protein 16-1 

ZK287.5 RING-box protein 1 

87

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 


ZK973.11 Protein disulfide-isomerase TXNDC10 precursor 

88

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 

1 

Two separate RNAi experiments were performed for each gene target (n = 20 for each 

RNAi round). 

2 

Knockdown of tor-2 resulted in a significant increase in aggregate number, whereas the 

depletion of other gene products did not enhance aggregation. 

89

Figure 2.1 

90

Figure 2.2 

91

Figure 2.3 

92

Figure 2.4 

93

Figure 2.5 

94

Figure 2.6 

95

Figure 2.7 

96


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

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 

98

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. 

99

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: 


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. 

100

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. 

101

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 

102

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. 


C. elegans experiments for RAB analysis. Nematodes were maintained following 

the standard procedures (Brenner, 1974). Strains UA81 {[baIn1; Pdat-1:: α-syn, Pdat- 

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

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

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

1::α-syn, Pdat-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, Pdat-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 µg/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, Pdat-1::gfp]; [baEx64; Pdat-1::PDE9A, rol-6 

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

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

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

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

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

µg/ml of rol-6 into an integrated line UA44 [baInl1; Pdat-1::α−syn, Pdat-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 

108

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 

110

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 

114

Figure 3.2 

115

Figure 3.3 

116


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 + Pdat-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 Punc-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'). 

118

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 

121

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 

122

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. 


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

syn; Pdat-1::gfp]}. 

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

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

124

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; Pdat-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. 

125

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 µl 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 Punc-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

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 

128

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 

129

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 

130

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 


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 

133

edundancy 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 


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 


B0284.2 ROCK2 protein 

B0286.3 Multifunctional protein ADE2 

B0303.9 Vacuolar protein sorting-associated protein 33A 


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 

141

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 

142

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 



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 


F21F3.3 Protein-S-isoprenylcysteine O-methyltransferase 

F21F3.5 Neuronal acetylcholine receptor subunit beta-2 

F21F8.2 Gastricsin precursor 


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 

160

Figure 4.1 

161

Figure 4.2 

162

Figure 4.3 

163

Figure 4.4 

164

Figure 4.5 

165

Figure 4.6 

166

Figure 4.7 

167


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

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

Introduction 

CHAPTER FIVE 

CONCLUSION 

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 

171

(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 

172

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). 

173

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 

174

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. 

175

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 

176

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 


177

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 

178

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 

179

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. 

180

Ambros, V. (2001) Cell 107, 823-826. 

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Zhou, X., Babu, J.R., da Silva, S., Shu, Q., Graef, I.A., Oliver, T., Tomoda, T., Tani, T., 

Wooten, M.W., Wang, F. (2007) Proc Natl Acad Sci U S A 104, 5842-5847. 

183

Figure 5.1 

184

Figure 5.2 

185


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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