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IDENTIFICATION OF NEUROPROTECTIVE GENES AGAINST ALPHA-<br />
SYNUCLEIN TOXICITY USING A CAENORHABDITIS ELEGANS<br />
PARKINSON DISEASE MODEL<br />
by<br />
SHUSEI HAMAMICHI<br />
A DISSERTATION<br />
Submitted in partial fulfillment <strong>of</strong> the requirements<br />
for the degree <strong>of</strong> Doctor <strong>of</strong> Philosophy<br />
in the Department <strong>of</strong> Biological Sciences<br />
in the Graduate School <strong>of</strong><br />
<strong>The</strong> University <strong>of</strong> Alabama<br />
TUSCALOOSA, ALABAMA<br />
2009
Copyright Shusei Hamamichi 2009<br />
ALL RIGHTS RESERVED
ABSTRACT<br />
Recent functional analyses <strong>of</strong> nine gene products linked to familial forms <strong>of</strong><br />
Parkinson disease (PD) have revealed several cellular mechanisms that are associated<br />
with PD patho<strong>genes</strong>is. For example, α-synuclein (α-syn), a primary component <strong>of</strong> Lewy<br />
bodies found in both familial and idiopathic forms <strong>of</strong> PD, has been shown to cause<br />
defects in proteasomal and lysosomal protein degradation machineries and induce<br />
mitochondrial/oxidative stress. <strong>The</strong>se findings are further supported by the fact that<br />
additional gene products are involved in the same pathways. While these studies have<br />
been invaluable to elucidate the etiology <strong>of</strong> this disease, it has been reported that<br />
monogenic forms <strong>of</strong> PD only account for 5-10% <strong>of</strong> all PD cases, indicating that multiple<br />
genetic susceptibility factors and intrinsic metabolic changes associated with aging may<br />
play a significant role. Here we report the use <strong>of</strong> an organism, Caenorhabditis elegans,<br />
to model two central PD pathological features to rapidly identify genetic components that<br />
modify α-syn misfolding in body wall muscles and neurodegeneration in DA neurons.<br />
We determined that proteins that function in lysosomal protein degradation, signal<br />
transduction, vesicle trafficking, and glycolysis, when knocked down by RNAi, enhanced<br />
α-syn misfolding. Furthermore, these components, when overexpressed, rescued DA<br />
neurons from α-syn-induced neurodegeneration, and several <strong>of</strong> them have been validated<br />
using mammalian system. Taken together, this study represents a novel set <strong>of</strong> gene<br />
products that are putative genetic susceptibility loci and potential therapeutic targets for<br />
PD.<br />
ii
LIST OF ABBREVIATIONS AND SYMBOLS<br />
6-OHDA 6-Hydroxydopamine<br />
AD Alzheimer disease<br />
ADE Anterior deirid neuron<br />
bp Base pair<br />
°C Celsius<br />
cAMP Cyclic adenosine monophosphate<br />
cDNA Complementary DNA<br />
CEP Cephalic neuron<br />
cGMP Cyclic guanosine monophosphate<br />
COR C-terminal <strong>of</strong> Roc<br />
D2 Dopamine 2<br />
D3 Dopamine 3<br />
DA Dopamine<br />
DEPC Diethylpyrocarbonate<br />
DNA Deoxyribonucleic acid<br />
DOG 2-Deoxyglucose<br />
dsRNA Double-stranded RNA<br />
E1 Ubiquitin-activating enzyme<br />
E2 Ubiquitin-conjugating enzyme<br />
iii
E3 Ubiquitin ligase<br />
ER Endoplasmic reticulum<br />
ERAD Endoplasmic reticulum-associated degradation<br />
FRAP Fluorescence recovery after photobleaching<br />
GAL4 Galactose metabolism 4<br />
GFP Green fluorescent protein<br />
GO Gene ontology<br />
HD Huntington disease<br />
HMG-CoA 3-Hydroxy-3-methyl-glutaryl-Coenzyme A<br />
hr Hour<br />
IPTG Isopropyl β-D-thiogalactoside<br />
kDa Kilodalton<br />
KOG Eukaryotic orthologous group<br />
L3 Larval stage 3<br />
L4 Larval stage 4<br />
LB Luria-Bertani<br />
L-DOPA L-3,4-Dihydroxyphenylalanine<br />
MPP+ 1-Methyl-4-phenylpyridinium<br />
MPTP 1-Methyl-4-phenyl-1,2,5,6-tetrahydropyridine<br />
mRNA messenger RNA<br />
µg Microgram<br />
iv
µl Microliter<br />
miRNA MicroRNA<br />
mg Milligram<br />
ml Milliliter<br />
mM Millimolar<br />
MAPK Mitogen-activated protein kinase<br />
MAPKK Mitogen-activated protein kinase kinase<br />
MAPKKK Mitogen-activated protein kinase kinase kinase<br />
n/a Not applicable<br />
NGM Nematode growth medium<br />
PARK Parkinson disease gene<br />
PCR Polymerase chain reaction<br />
PD Parkinson disease<br />
PDE Posterior deirid neuron<br />
RING Really interesting new gene<br />
RNA Ribonucleic acid<br />
RNAi RNA interference<br />
Roc Ras <strong>of</strong> complex<br />
ROS Reactive oxygen species<br />
rpm Revolutions per minute<br />
RT Room temperature (25 °C)<br />
v
RT-PCR Reverse transcriptase polymerase chain reaction<br />
SAGE Serial analysis <strong>of</strong> gene expression<br />
SD Standard deviation<br />
SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis<br />
SNP Single nucleotide polymorphism<br />
UPR Unfolded protein response<br />
UPS Ubiquitin-proteasome system<br />
UTR Untranslated region<br />
C. elegans Proteins<br />
AGE-1 Aging alteration 1 (phosphoinositide 3-kinase)<br />
ATGR-7 Autophagy 7<br />
BAR-1 Beta-catenin/armadillo related 1<br />
CDK-5 Cyclin dependent kinase 5<br />
CED-3 Cell death abnormality 3 (caspase)<br />
CLK-1 Clock 1 (demethoxyubiquinone hydroxylase)<br />
CMK-1 Calcium/calmodulin-dependent protein kinase 1<br />
CSNK-1 Casein kinase 1<br />
DAF-2 Abnormal dauer formation 2 (insulin receptor)<br />
DAF-16 Abnormal dauer formation 16 (forkhead Box 01A)<br />
DAT-1 Dopamine transporter 1<br />
vi
DJR-1 Oncogene DJ-1<br />
DJR-2 Oncogene DJ-1<br />
DOP-2 Dopamine receptor 2 (D2-like receptor)<br />
DPY-1 Dumpy 1 (collagen)<br />
DPY-5 Dumpy 5 (collagen)<br />
EAT-2 Eating 2 (nicotinic acetylcholine receptor)<br />
GPI-1 Glucose-6-phosphate isomerase 1<br />
HRD-1 HRD 1<br />
HRDL-1 HRD-like 1<br />
HSF-1 Heat-shock factor 1<br />
ISP-1 Rieske iron sulphur protein<br />
LRK-1 Leucine-rich repeats, Ras-like domain, kinase 1 (LRRK2)<br />
MOM-4 More <strong>of</strong> MS 4 (MAPKKK7)<br />
NHR-6 Nuclear hormone receptor family 6 (NURR1)<br />
NPR-1 Neuropeptide receptor family 1<br />
OBR-1 Oxysterol binding protein 1<br />
PDR-1 Parkinson’s disease related 1 (parkin)<br />
PINK-1 PTEN-induced putative kinase 1<br />
PMK-1 p38 MAP kinase<br />
ROL-6 Roller 6 (collagen)<br />
SMF-1 Yeast SMF homolog (divalent metal transporter)<br />
vii
TAG-278 Temporary assigned gene 278<br />
TAP-1 TAK kinase/MOM-4 binding protein<br />
TOR-2 Torsin 2 (torsinA)<br />
TRX-1 Thioredoxin 1<br />
UNC-32 Uncoordinated 32 (vacuolar proton-translocating ATPase)<br />
UNC-51 Uncoordinated 51 (unc-51-like kinase 2)<br />
UNC-54 Uncoordinated 54 (myosin class II heavy chain)<br />
UNC-75 Uncoordinated 75 (CELF/BrunoL protein)<br />
VPS-41 Vacuolar protein sorting 41<br />
YKT-6 Yeast YKT6 homolog (v-SNARE)<br />
Mammalian Proteins<br />
α-Syn Alpha synuclein<br />
ALDOA Aldolase A<br />
AMF Autocrine motility factor<br />
AMFR Autocrine motility factor receptor<br />
Amyloid-β Amyloid beta<br />
ASK1 Apoptosis signal-regulating kinase 1<br />
ATG7 Autophagy 7<br />
ATP13A2 ATPase, Type 13A2<br />
BAG5 BCL2-associated athanogene 5<br />
viii
CSNK1G3 Casein kinase 1, gamma-3<br />
CHIP C-terminus <strong>of</strong> HSC70-interacting protein<br />
Daxx Death-associated protein 6<br />
DJ-1 Oncogene DJ-1<br />
ERV29 Surfeit 4<br />
FBXW7 F-box and WD40 domain protein 7<br />
GAIP G protein <strong>alpha</strong>-interacting protein<br />
GAPDH Glyceraldehyde-3-phosphate dehydrogenase<br />
GBA Glucocerebrosidase<br />
GIGYF2 GRB10-interacting GYF protein 2<br />
GIPC GAIP C-terminus-interacting protein<br />
GPI Glucose-6-phosphate isomerase<br />
HDAC6 Histone deacetylase 6<br />
HTRA2 HTRA serine peptidase 2<br />
HSF1 Heat-shock factor 1<br />
HSP70 Heat-shock protein, 70 kDa<br />
HSPC117 Hypothetical protein 117<br />
IgG Immunoglobulin G<br />
INSR Insulin receptor<br />
LRRK2 Leucine-rich repeat kinase 2<br />
NRB54 Nuclear RNA-binding protein, 54 kDa<br />
ix
p38 Mitogen-activated protein kinase p38<br />
Pael receptor Parkin-associated endothelin receptor<br />
PDE9A Phosphodiesterase 9A<br />
PI3K Phosphoinositide 3-kinase<br />
PINK1 PTEN-induced putative kinase 1<br />
PLK2 Polo-like kinase 2<br />
PRKN Parkin<br />
PSF Polypyrimidine tract-binding protein-associated splicing factor<br />
Q82 Polyglutamine 82 containing protein<br />
RAB1A Ras-associated protein 1A<br />
RAB3A Ras-associated protein 3A<br />
RAB8A Ras-associated protein 8A<br />
RGS Regulators <strong>of</strong> G protein signaling<br />
SEC22 Secretion deficient 22<br />
SNCA Synuclein, <strong>alpha</strong><br />
SYVN1 Synoviolin 1<br />
Ub Ubiquitin<br />
Ubch7 Ubiquitin-conjugating enzyme 7<br />
Ubch8 Ubiquitin-conjugating enzyme 8<br />
UCHL Ubiquitin C-terminal hydrolase<br />
UCHL1 Ubiquitin C-terminal hydrolase 1<br />
x
USP10 Ubiquitin-specific protein 10<br />
VMAT2 Vesicular monoamine transporter 2<br />
VPS41 Vacuolar protein sorting 41<br />
XIAP X-linked inhibitor <strong>of</strong> apoptosis<br />
xi
ACKNOWLEDGMENTS<br />
First and foremost, I would like to thank Drs. Guy and Kim Caldwell, and the<br />
former and present members <strong>of</strong> the Caldwell lab. Among them, I wish to especially<br />
recognize highly motivated undergraduate students who undertook the enormous<br />
challenge <strong>of</strong> working in the PD research field. Most notably, I want to thank Renee<br />
Rivas, Adam “Deuce” Knight, Susan DeLeon, and Paige Dexter. Without their help and<br />
contribution, I guarantee that none <strong>of</strong> our projects would have worked as smoothly as<br />
they did. Furthermore, I want to thank Cody Locke for making science intellectually<br />
stimulating.<br />
Among the former and present non-undergraduates, I would like to acknowledge<br />
Dr. Laura Berkowitz, Michelle Norris, Lindsay Faircloth, and Jenny Schieltz for their<br />
assistance in many untold and underappreciated areas <strong>of</strong> research. I also want to thank<br />
the usual Wilhagans gang including Jafa Armagost, Adam “Ace” Harrington, and AJ<br />
Burdette for making my life as a graduate student more enjoyable and fulfilling. I wish<br />
all <strong>of</strong> them good luck for their future endeavors.<br />
Outside <strong>of</strong> the Caldwell lab, I would like to thank my graduate committee<br />
members, Dr. Janis O’Donnell, Dr. Katrina Ramonell, and Dr. Jianhua Zhang for their<br />
continuous support and encouragement. Furthermore, I wish to acknowledge other<br />
faculty members and staff <strong>of</strong> the Department <strong>of</strong> Biological Sciences including Dr. Martha<br />
Powell, Dr. Harriett Smith-Somerville, and many others for their assistance whenever I<br />
xii
needed it and their enthusiasm toward my research. It has been a pleasure sharing<br />
research ideas and data annually, and receiving tremendously kind responses from all <strong>of</strong><br />
you.<br />
I also would like to express my gratitude to research collaborators, Dr. Susan<br />
Lindquist (Whitehead Institute/MIT), Dr. David Standaert (UAB), Dr. Ted Dawson<br />
(Johns Hopkins University), and Dr. Antonio Miranda Vizuete (Universidad Pablo de<br />
Olavide). Thank you so much for allowing me to work on multiple influential research<br />
projects. Most notably, I would like to thank Dr. Joshua Kritzer and Dr. Chris Pacheco,<br />
both post-docs at the Lindquist lab, for believing in me.<br />
Outside <strong>of</strong> the current research world, I want to thank my parents and my brother<br />
who are currently in Japan as well as my former PIs, Dr. Hideo Nishigori (Teikyo<br />
University) and Dr. Mike Shipley (Midwestern State University). Furthermore, I wish to<br />
recognize all <strong>of</strong> my friends from all over the world. Thank you for our friendship and<br />
understanding <strong>of</strong> what we wish to achieve in our lives. I am who I am, and I do what I do<br />
because <strong>of</strong> you.<br />
Lastly, I would like to acknowledge Sylvester Stallone for making a classic<br />
movie, Rocky, the ultimate source <strong>of</strong> my inspiration while working on my PNAS article,<br />
presentation slides used for my post-doc interview at Whitehead Institute, and this current<br />
dissertation. I only wanted to go the distance, but I had never thought I would have an<br />
opportunity to go to Boston.<br />
xiii
CONTENTS<br />
ABSTRACT ............................................................................................................ ii<br />
LIST OF ABBREVIATIONS AND SYMBOLS .................................................. iii<br />
ACKNOWLEDGMENTS .................................................................................... xii<br />
LIST OF TABLES .............................................................................................. xvii<br />
LIST OF FIGURES ........................................................................................... xviii<br />
1. INTRODUCTION ...............................................................................................1<br />
a. Parkinson disease .................................................................................................1<br />
b. PD pathological feature: Lewy bodies .................................................................2<br />
c. Genetics basis <strong>of</strong> PD ............................................................................................3<br />
d. PD patho<strong>genes</strong>is: SNCA/α-syn ...........................................................................4<br />
e. PD patho<strong>genes</strong>is: proteasomal protein degradation .............................................5<br />
f. PD patho<strong>genes</strong>is: lysosomal protein degradation .................................................8<br />
g. PD patho<strong>genes</strong>is: mitochondrial/oxidative stress ..............................................10<br />
h. PD patho<strong>genes</strong>is: signaling pathways ................................................................13<br />
i. Strategy using invertebrate models <strong>of</strong> PD ..........................................................14<br />
j. C. elegans PD models .........................................................................................17<br />
k. Current studies ...................................................................................................19<br />
l. References ...........................................................................................................22<br />
m. Figure legends ...................................................................................................33<br />
xiv
2. HYPOTHESIS-BASED RNA INTERFERENCE SCREEN<br />
IDENTIFIES NEUROPROTECTIVE GENES<br />
IN A PARKINSON’S DISEASE MODEL.......................................................34<br />
a. Abstract ..............................................................................................................35<br />
b. Introduction ........................................................................................................36<br />
c. Materials and methods .......................................................................................38<br />
d. Results ................................................................................................................44<br />
e. Discussion ..........................................................................................................52<br />
f. References ..........................................................................................................58<br />
g. Figure legends ....................................................................................................97<br />
3. VALIDATION OF SUPPRESSORS OF ALPHA-SYNUCLEIN<br />
TOXICITY FROM YEAST GENETIC SCREENING ..................................100<br />
a. Abstract ............................................................................................................101<br />
b. Introduction ......................................................................................................102<br />
c. Materials and methods .....................................................................................104<br />
d. Results ..............................................................................................................106<br />
e. Discussion ........................................................................................................108<br />
f. References ........................................................................................................112<br />
g. Figure legends ..................................................................................................117<br />
4. RNAI SCREEN OF DAF-2-MODULATED AND DIFFERENTIALLY<br />
EXPRESSED GENES LINK METABOLIC ENZYMES<br />
TO NEUROPROTECTION ............................................................................119<br />
xv
a. Abstract ............................................................................................................120<br />
b. Introduction ......................................................................................................121<br />
c. Materials and methods .....................................................................................124<br />
d. Results ..............................................................................................................128<br />
e. Discussion ........................................................................................................132<br />
f. References ........................................................................................................137<br />
g. Figure legends ..................................................................................................168<br />
5. CONCLUSION ................................................................................................170<br />
a. Introduction ......................................................................................................170<br />
b. Neuroprotective mechanism <strong>of</strong> VPS41, ATG7, ULK2, and GIPC:<br />
a common pathway? ........................................................................................170<br />
c. Defining networks <strong>of</strong> <strong>neuroprotective</strong> <strong>genes</strong> by miRNAs ...............................174<br />
d. Additional PD-related studies using C. elegans ..............................................176<br />
e. Conclusion and future directions .....................................................................178<br />
f. References ........................................................................................................181<br />
g. Figure legends ..................................................................................................186<br />
xvi
LIST OF TABLES<br />
1.1. Summary <strong>of</strong> mutations in PD <strong>genes</strong> linked to PD patho<strong>genes</strong>is<br />
and their C. elegans orthologs ....................................................................... 30<br />
1.2. Summary <strong>of</strong> selected invertebrate PD models ................................................31<br />
2.1. Gene identities <strong>of</strong> the 20 top candidates isolated<br />
from RNAi screening models .........................................................................62<br />
2.2. Bioinformatic associations among gene candidates<br />
identified by RNAi .........................................................................................63<br />
2.3. Summary <strong>of</strong> the <strong>neuroprotective</strong> <strong>genes</strong> and their human homologs. ..............64<br />
2.4. Results <strong>of</strong> all <strong>genes</strong> knocked down via RNAi screening. ...............................65<br />
2.5. Summary <strong>of</strong> RNAi knockdown <strong>of</strong> the top 20 gene candidates in worms<br />
expressing Q82::GFP + TOR-2 in body wall muscle cells ............................89<br />
4.1. Summary <strong>of</strong> <strong>genes</strong> analyzed by RNAi screen. ..............................................141<br />
4.2. Summary <strong>of</strong> positive <strong>genes</strong> from RNAi screen for effectors <strong>of</strong> α-syn<br />
in the daf-2 background based on KOG and/or GO annotations. ................159<br />
xvii
LIST OF FIGURES<br />
1.1. Schematic representation <strong>of</strong> the cellular defects caused by<br />
known PD <strong>genes</strong>. ............................................................................................32<br />
2.1. RNAi knockdown <strong>of</strong> specific gene targets enhances misfolding <strong>of</strong> α-syn .....90<br />
2.2. Overexpression <strong>of</strong> candidate <strong>genes</strong> protects DA neurons from<br />
α-syn-induced degeneration ...........................................................................91<br />
2.3. An interconnectivity map ................................................................................92<br />
2.4. Expression <strong>of</strong> α-syn in worm DA neurons results in<br />
age- and dose-dependent neurodegeneration .................................................93<br />
2.5. Analysis <strong>of</strong> transgene expression in worm strains. .........................................94<br />
2.6. RNAi knockdown <strong>of</strong> the top 20 gene targets did not enhance misfolding <strong>of</strong><br />
polyglutamine aggregates ...............................................................................95<br />
2.7. Quantitative analysis <strong>of</strong> the hit rate <strong>of</strong> <strong>genes</strong> at both the primary and<br />
secondary level <strong>of</strong> RNAi screening ................................................................96<br />
3.1. RAB3A, RAB8A, PDE9A, and PLK2 protect <strong>against</strong> α-syn-induced<br />
DA neuron loss.. ...........................................................................................114<br />
3.2. PARK9 antagonizes α-syn-mediated DA neuron degeneration<br />
in C. elegans. ................................................................................................115<br />
3.3. RNAi knockdown <strong>of</strong> W08D2.5, R12E2.13, and R06F6.8 does not reduce<br />
α-syn or tor-2 mRNA expression levels ......................................................116<br />
xviii
4.1. Graphs depicting the percentage <strong>of</strong> α-syn-expressing daf-2 and/or<br />
daf-16 mutants with wildtype DA neurons. .................................................161<br />
4.2. Graph summarizing lifespan assay <strong>of</strong> N2 and daf-2 worms. ........................162<br />
4.3. daf-2 enhances degradation <strong>of</strong> α-syn::GFP fusion protein.. .........................163<br />
4.4. Graph illustrating the percentage <strong>of</strong> α-syn-expressing worms with<br />
wildtype DA after 2-deoxyglucose (DOG) treatment ..................................164<br />
4.5. Graph illustrating the percentage <strong>of</strong> 7 day-old worms with wildtype<br />
DA neurons expressing α-syn and gpi-1 or hrdl-1 ......................................165<br />
4.6. Pie chart summarizing 53 positive <strong>genes</strong> from the RNAi screening.. ..........166<br />
4.7. Diagram summarizing the DAF-2/insulin signaling pathway<br />
in C. elegans. ................................................................................................167<br />
5.1. Schematic diagram illustrating targets <strong>of</strong> mir-2/mir-43/mir-250/mir-797<br />
superfamily by miRBase (top) and TargetScan (bottom). ...........................184<br />
5.2. Schematic diagram <strong>of</strong> CEP neuronal circuitry ..............................................185<br />
xix
Parkinson disease<br />
CHAPTER ONE<br />
INTRODUCTION<br />
Parkinson disease (PD) is the second most common neurodegenerative disease,<br />
affecting approximately 1% <strong>of</strong> the population aged over 50 (Polymeropoulos et al.,<br />
1996). While the exact number <strong>of</strong> PD patients remains unclear, it is estimated that 1 to<br />
1.5 million Americans are affected with this disease, and 50,000 are newly diagnosed<br />
each year. Concurrent with medical issues associated with physical disabilities and<br />
quality <strong>of</strong> life, the financial burden is predicted to cost additional $10,349 per a PD<br />
patient annually, totaling $23 billion in the United States (Huse et al., 2005). Globally, as<br />
world population continues to increase, Dorsey et al. (2007) projected the number <strong>of</strong> PD<br />
patients from 4.1-4.6 million in 2005 to 8.7 to 9.3 million in 2030. In the light <strong>of</strong> the fact<br />
that, presently, no cure for this disease exists, identifying new diagnostic and therapeutic<br />
targets or discovering novel therapeutic strategies remains a top priority in the PD<br />
research community.<br />
Similar to Alzheimer disease (AD) and Huntington disease (HD), PD belongs to a<br />
group <strong>of</strong> movement disorders, clinically diagnosed to the individuals with muscle<br />
rigidity, tremor, bradykinesia, and postural instability. Post-mortem examination <strong>of</strong> the<br />
brains from the PD patients revealed a progressive loss <strong>of</strong> dopamine (DA) synthesizing<br />
neurons in the substantia nigra, a melanin-rich region in the basal ganglia.<br />
1
Consequently, DA neuronal death in the substantia nigra affects nigrostriatal,<br />
mesocortical, mesolimbic, and tuberoinfundibular pathways, resulting in physical<br />
impairments as well as neuropsychatric symptoms including depression, dementia, and<br />
insomnia (Lees et al., 2009). Current treatment focuses on decelerating the progression<br />
<strong>of</strong> these symptoms [e.g., by restoring DA production via L-3,4-dihydroxyphenylalanine<br />
(L-DOPA) administration]. <strong>The</strong> wide range <strong>of</strong> PD symptoms illustrates the complexity<br />
<strong>of</strong> its etiology in all facets <strong>of</strong> biological levels (e.g., molecules, neurons, nervous system,<br />
behavior, and <strong>genes</strong> vs. environment) (Lees et al., 2009; Lesage and Brice, 2009).<br />
PD pathological feature: Lewy bodies<br />
A pathological hallmark <strong>of</strong> PD at the cellular level is a formation <strong>of</strong> proteinaceous<br />
inclusions called Lewy bodies in the cytoplasm <strong>of</strong> the surviving DA neurons. <strong>The</strong> most<br />
predominant protein detected in the inclusions is a protein called α-synuclein (α-syn;<br />
PARK1/SNCA) (Spillantini et al., 1997), which is discussed in detail below.<br />
Additionally, synphilin (Murray et al., 2003), parkin (PARK2/PRKN; Schlossmacher et<br />
al., 2001), torsinA (Sharma et al., 2001), and other proteins have been found in the Lewy<br />
bodies.<br />
On the premise that these inclusion bodies, as well as reduction <strong>of</strong> the active<br />
proteins via aggregation formation, may interrupt normal cellular functions, previous<br />
research focused on identifying the components <strong>of</strong> Lewy bodies and their potential<br />
<strong>neuroprotective</strong> functions. While the <strong>neuroprotective</strong> capacities <strong>of</strong> parkin (Vercammen et<br />
2
al., 2006) and torsinA (Cao et al., 2005) have been documented, observations that the<br />
inclusion bodies are undetected in some PD cases suggests that the protein aggregates<br />
may not be the only mechanism resulting in neuronal cell death. Further supporting this<br />
view are the findings demonstrating that α-syn intermediate prot<strong>of</strong>ibrils are more<br />
neurotoxic than those found in either the monomeric or oligomerized state (Conway et<br />
al., 2001; Lashuel et al., 2002) by physically disrupting vesicular membranes (Volles et<br />
al., 2001) or inhibiting the ubiquitin-proteasome system (UPS) (Zhang et al., 2008),<br />
implying that the formation <strong>of</strong> more mature aggregates is instead <strong>neuroprotective</strong>. Taken<br />
together, although a neurodegenerative or <strong>neuroprotective</strong> role for Lewy bodies is<br />
controversial, the formation <strong>of</strong> the inclusion bodies remains as a definitive pathological<br />
feature <strong>of</strong> both familial (genetic) and sporadic (environmental) forms <strong>of</strong> PD.<br />
Genetic basis <strong>of</strong> PD<br />
While familial hereditary influences have long been documented, due to a<br />
complicated pattern <strong>of</strong> inheritance, genetic causes <strong>of</strong> this disease were unresolved until<br />
late 1990’s (Nussbaum and Polymeropoulos, 1997). Presently, nine PARK <strong>genes</strong> have<br />
been determined whereby mutations leading to modified function, altered expression<br />
level, or subcellular mislocalization are linked to PD (Table 1.1). <strong>The</strong>se <strong>genes</strong> include<br />
PARK1/SNCA (Polymeropoulos et al., 1997), PARK2/PRKN (Kitada et al., 1998),<br />
PARK5/UCHL1 (Leroy et al., 1998), PARK6/PINK1 (Valente et al., 2004), PARK7/DJ-1<br />
(Bonifati et al., 2002), PARK8/LRRK2 (Paisán-Ruíz et al., 2004), PARK9/ATP13A2<br />
3
(Ramirez et al., 2006), PARK11/GIGYF2 (Lautier et al., 2008), and PARK13/HTRA2<br />
(Strauss et al., 2005). <strong>The</strong> functional analyses <strong>of</strong> these PD-associated proteins suggest<br />
multiple defective pathways that may lead to DA neurodegeneration (Dawson and<br />
Dawson, 2003; Thomas and Beal, 2007) (Fig. 1.1).<br />
PD patho<strong>genes</strong>is: SNCA/α-syn<br />
<strong>The</strong> first PD gene discovered was SNCA/α-syn (Polymeropoulos et al., 1997),<br />
which encodes natively unfolded 140 amino-acid protein with unknown function. α-Syn<br />
has been detected in presynaptic nerve termini (Jakes et al., 1994), and shown to bind to<br />
lipids (Perrin et al., 2000). Originally identified as a non-amyloid-β component <strong>of</strong> AD<br />
amyloid (Ueda et al., 1993), α-syn, similar to amyloid-β and polyglutamine-repeat<br />
containing proteins, is prone to aggregation. Subsequent analysis <strong>of</strong> SNCA gene has<br />
revealed that multiplication <strong>of</strong> SNCA loci enhanced α-syn expression, resulting in the<br />
protein aggregation and the onset <strong>of</strong> PD (Singleton et al., 2003).<br />
Since the formation <strong>of</strong> Lewy bodies is a central pathological feature <strong>of</strong> both<br />
familial and sporadic forms <strong>of</strong> PD, most current PD research focuses on α-syn<br />
aggregation and cellular mechanisms involved in ameliorating it. For example,<br />
accumulation <strong>of</strong> α-syn has been shown to impair UPS function (Stefanis et al., 2001;<br />
Zhang et al., 2008; Nonaka et al., 2009), and α-syn is degraded by lysosomes (Webb et<br />
al., 2003; Cuervo et al., 2004). Furthermore, overexpression <strong>of</strong> α-syn blocks<br />
4
endoplasmic reticulum (ER) to Golgi trafficking (Cooper et al., 2006; Gitler et al., 2008),<br />
and expression <strong>of</strong> mutant α-syn induces ER stress (Smith et al., 2005). Additionally, α-<br />
syn may also be targeted to mitochondria and impair complex I function via cryptic<br />
mitochondrial targeting signal (Devi et al., 2008). As discussed below, functional<br />
analysis <strong>of</strong> six out <strong>of</strong> nine PD-associated gene products illustrate involvement <strong>of</strong><br />
defective proteasomal and lysosomal protein degradation machineries, as well as<br />
inadequate cellular response to mitochondrial and oxidative stress in PD patho<strong>genes</strong>is.<br />
PD patho<strong>genes</strong>is: proteasomal protein degradation<br />
<strong>The</strong> most common protein degradation machinery <strong>of</strong> the cell is the UPS, which<br />
consists <strong>of</strong> a variety <strong>of</strong> proteins including the ubiquitin-activating enzymes (E1),<br />
ubiquitin-conjugating enzymes (E2), ubiquitin ligases (E3), ubiquitin carboxyl-terminal<br />
hydrolases (UCHL), and proteasomal subunits. Briefly, E1, E2, and E3 are involved in<br />
the processes <strong>of</strong> activating, transferring, and binding ubiquitins (Ubs) to target proteins<br />
that are degraded by proteasomes. After proteolysis, Ubs that are attached to the<br />
degraded products are recycled by UCHL to maintain the cytoplasmic Ub pool.<br />
Misfolded proteins, such as α-syn (Stefanis et al., 2001; Zhang et al., 2008; Nonaka et al.,<br />
2009), polyglutamine-repeat containing gene products (Bence et al., 2001; Bennett et al.,<br />
2007; Iwata et al., 2009), and amyloid-β (Almeida et al., 2006) have been shown to<br />
impair UPS function.<br />
5
Two PD <strong>genes</strong>, parkin/PRKN (E3 ubiquitin ligase) and UCHL1 are UPS<br />
components. Kitada et al. (1998) utilized positional cloning to identify PRKN in<br />
Japanese PD patients. Protein sequence analysis revealed that 465 amino-acid parkin<br />
encoded moderately similar sequence to Ub at the N terminus and a RING-finger motif (a<br />
common motif in E3 ligases) at the C terminus. Since mutations in PRKN lead to<br />
autosomal recessive PD, Kitada et al. (1998) proposed that the loss <strong>of</strong> E3 ubiquitin ligase<br />
activity (i.e., loss-<strong>of</strong>-function) <strong>of</strong> PRKN as one <strong>of</strong> the primary mechanisms involved in<br />
PD patho<strong>genes</strong>is.<br />
Subsequent analysis <strong>of</strong> parkin function identified its interactors including E2<br />
ubiquitin-conjugating enzymes Ubch7 (Shimura et al., 2000) and Ubch8 (Zhang et al.,<br />
2000), F-box/WD repeat protein FBXW7 and cullin 1 (Staropoli et al., 2003), BAG5<br />
(Kalia et al., 2004), and its substrates such as a G protein-coupled Pael receptor (Imai et<br />
al., 2001) and p38 (Corti et al., 2003). <strong>The</strong>se findings link parkin to the UPS, cell death<br />
pathway, and the cellular response to unfolded proteins. Interestingly, parkin has also<br />
been shown to interact with 22 kDa glycosylated α-syn (Shimura et al., 2001), PD-linked<br />
mutant forms <strong>of</strong> DJ-1 (Moore et al., 2005), and LRRK2 (Smith et al., 2005) suggesting a<br />
common neurodegenerative pathway in seemingly heterogeneous PD forms.<br />
<strong>The</strong> <strong>neuroprotective</strong> function <strong>of</strong> parkin has been well documented using various<br />
animal models. Jiang et al. (2004) overexpressed parkin in human DA neuroblastoma<br />
cells (SH-SY5Y) and observed neuroprotection <strong>against</strong> DA and 6-hydroxydopamine (6-<br />
OHDA)-induced apoptosis by decreasing reactive oxygen species (ROS) and attenuating<br />
6
c-Jun N-terminal kinase and caspase 3 activities. Similarly, Hasegawa et al. (2008)<br />
expressed an enzyme tyrosinase (an enzyme that catalyzes both the hydroxylation <strong>of</strong><br />
tyrosine to L-DOPA and the subsequent conversion <strong>of</strong> L-DOPA and DA to their specific<br />
o-quinones) in SH-SY5Y cells to over-produce endogenous DA leading to ROS-induced<br />
apoptosis. Co-expression <strong>of</strong> wildtype parkin suppressed oxidative stress-induced cell<br />
death by enhancing the activation <strong>of</strong> c-Jun N-terminal kinase and p38. Further<br />
demonstration <strong>of</strong> the <strong>neuroprotective</strong> function <strong>of</strong> this E3 ubiquitin ligase was shown by<br />
Petrucelli et al. (2002) whereby PD-linked mutant α-syn was expressed in mouse primary<br />
midbrain culture, and it was determined that parkin rescued these catecholaminergic<br />
neurons from α-syn toxicity.<br />
UCHL1, initially identified as a PD gene by Leroy et al. (1998), is not well<br />
characterized since ongoing dispute regarding UCHL1 as a PD susceptibility gene<br />
(compare Maraganore et al., 2004 vs. Healy et al., 2006) has minimized comprehensive<br />
research efforts. Despite the controversy, Liu et al. (2002) reported that α-syn and<br />
UCHL1 are co-localized with the synaptic vesicles, and that co-overexpression <strong>of</strong> α-syn<br />
and both wildtype and mutant UCHL1 in COS-7 cells increased accumulation <strong>of</strong> α-syn<br />
aggregates. Since overexpression <strong>of</strong> UCHL1 should enhance α-syn degradation, they<br />
proposed an alternative UCHL1 function whereby dimerization <strong>of</strong> UCHL1 exhibits<br />
ubiquitin ligase activity. Additionally, Liu et al. (2008) demonstrated that farnesylated<br />
UCHL1 is associated with the cellular membranes, and that treatment with<br />
7
farnesyltransferase inhibitor enhanced cell survival. Nevertheless, a <strong>neuroprotective</strong><br />
mechanism for UCHL1 remains unclear.<br />
PD patho<strong>genes</strong>is: lysosomal protein degradation<br />
Lysosomes are organelles that contain digestive enzymes including lipases,<br />
carbohydrases, nucleases, and proteases to break down organelles, macromolecules, and<br />
microorganisms. For instance, through activation <strong>of</strong> autophagy, mitochondria<br />
(mitophagy) and peroxisomes (pexophagy) are degraded in the lysosomes. Interestingly,<br />
recent findings indicate that bulk <strong>of</strong> misfolded and aggregated proteins, including α-syn<br />
(Webb et al., 2003; Cuervo et al., 2004) and polyglutamine-repeat containing proteins<br />
(Ravikumar et al., 2004; Yamamoto et al, 2006), are degraded by lysosomes via<br />
macroautophagy and/or chaperone-mediated autophagy. Further supporting the role <strong>of</strong><br />
lysosomal function and PD patho<strong>genes</strong>is is the association between PD and type I<br />
Gaucher disease (Bembi et al., 2003). Type I Gaucher disease is an autosomal recessive<br />
lysosomal storage disorder that is caused by reduced activity <strong>of</strong> glucocerebrosidase<br />
(GBA), which is a lysosomal enzyme that catalyzes the breakdown <strong>of</strong> glucosylceramide.<br />
While the precise neurodegenerative mechanism <strong>of</strong> defective GBA in PD is unclear, it<br />
has been postulated that the mutations may interfere with normal lysosomal function and<br />
block α-syn clearance (Goker-Alpan et al., 2008). Additionally, knockdown <strong>of</strong> cathepsin<br />
D has been shown to enhance α-syn aggregation whereas overexpression <strong>of</strong> this<br />
8
lysosomal protease promotes α-syn degradation and DA neuron survival (Qiao et al.,<br />
2008).<br />
Since both proteasomal and lysosomal machineries effectively degrade misfolded<br />
proteins and promote cell survival, investigating a molecular “switch” that promotes one<br />
pathway over the other is a significant research interest. Thus far, two proteins, CHIP (an<br />
ubiquitin ligase that acts as a co-chaperone for protein quality control) and HDAC6 (a<br />
microtubule-associated deacetylase) have been documented to function in this<br />
mechanism. Shin et al. (2005) examined two functional domains within CHIP, and<br />
demonstrated that while the tetratricopeptide repeat is critical for proteasomal<br />
degradation <strong>of</strong> α-syn, the U-box domain is involved in lysosomal degradation. Further,<br />
Pandey et al. (2007) reported that impairment <strong>of</strong> the UPS led to induction <strong>of</strong> autophagy in<br />
an HDAC6-dependent manner. <strong>The</strong>se findings illustrate the interconnection between<br />
these two protein degradation pathways that may indeed be compensatory.<br />
Mutations in PD gene, ATP13A2, which encodes a lysosomal P-type ATPase lead<br />
to autosomal recessive PD. By mutation screening and linkage analysis, Ramirez et al.<br />
(2006) identified ATP13A2 from Chilean PD patients. <strong>The</strong>y examined expression pattern<br />
and subcellular localization <strong>of</strong> ATP13A2, and determined that the gene is predominantly<br />
expressed in the brain, and that while the wildtype ATP13A2 protein is localized in the<br />
lysosomes, misfolded mutant forms are retained in the ER to be subsequently degraded<br />
by proteasomes. Surprisingly, they observed approximately a 10-fold increase in<br />
ATP13A2 mRNA level in the surviving DA neurons from the substantia nigra <strong>of</strong> human<br />
9
idiopathic PD post-mortem midbrains, suggesting the potential <strong>neuroprotective</strong> function<br />
<strong>of</strong> this gene. As discussed in Chapter 3, we have shown that while knockdown <strong>of</strong> worm<br />
ATP13A2 enhances α-syn misfolding, overexpression <strong>of</strong> this gene rescues worm DA<br />
neurons from α-syn toxicity, demonstrating a novel genetic interaction between α-syn<br />
and ATP13A2 (Gitler et al., 2009).<br />
PD patho<strong>genes</strong>is: mitochondrial and oxidative stress<br />
While cellular stress induced by misfolded or aggregated proteins may shed light<br />
on the neurodegenerative mechanisms leading to PD, defects in protein degradation<br />
machinery alone cannot explain the selective loss <strong>of</strong> DA neurons. PD patho<strong>genes</strong>is<br />
consists <strong>of</strong> both genetic and environmental causes, which only 5-10% <strong>of</strong> all PD cases<br />
have been linked to genetic components. Studies on environmental PD factors have<br />
provided insights on the pathways involved in the selective DA neurodegeneration.<br />
Langston et al. (1986) studied four patients who exhibited features <strong>of</strong> clinical<br />
Parkinsonism after using a new “synthetic heroin.” Subsequent analysis <strong>of</strong> the drug<br />
components revealed 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) as a primary<br />
compound, and they proposed that MPTP might induce the selective loss <strong>of</strong> DA neurons.<br />
While MPTP was later found harmless, after crossing the blood-brain barrier, the<br />
compound is readily metabolized into toxic 1-methyl-4-phenylpyridinium (MPP+), which<br />
disrupts complex I <strong>of</strong> mitochondrial respiratory chain. Inhibition <strong>of</strong> complex I generates<br />
10
ROS, which may oxidize DA (a neurotransmitter known to be readily oxidized) to<br />
produce highly toxic DA that results in DA neurodegeneration.<br />
Two PD <strong>genes</strong>, PINK1 and HTRA2 are also linked to mitochondria. Valente et al.<br />
(2004) mapped PINK1 from an Italian family with autosomal recessive PD, and<br />
determined that PINK1 when expressed in COS-7 and SH-SY5Y cells localized to<br />
mitochondria. Furthermore, using SH-SY5Y cells overexpressing wildtype or mutant<br />
PINK1, they demonstrated that, after MG-132 (a proteasome inhibitor) treatment,<br />
wildtype PINK1 enhanced cell survival without modifying mitochondrial membrane<br />
potential whereas mutant PINK1 displayed no protection with decreased membrane<br />
potential. Taken together, these findings provided the first evidence linking a genetic<br />
cause <strong>of</strong> PD to mitochondria, further confirming the relationship between mitochondrial<br />
malfunction and PD patho<strong>genes</strong>is, and suggesting a potential <strong>neuroprotective</strong> function <strong>of</strong><br />
PINK1.<br />
Strauss et al. (2005) identified HTRA2 in German PD patients. To further validate<br />
their findings, they overexpressed wildtype and mutant HTRA2 in cell culture, and found<br />
that mutant form failed to interact with XIAP (an inhibitor <strong>of</strong> apoptosis), suggesting that<br />
misregulation <strong>of</strong> HTRA2 might be linked to PD patho<strong>genes</strong>is. Moreover, they<br />
determined that HTRA2 is predominantly localized to mitochondria by<br />
immunohistochemistry, and observed distinct mitochondrial morphological changes by<br />
electron microscopy. Focusing on mitochondrial function, they examined mitochondrial<br />
membrane potential and cell viability, and showed that mutant HTRA2 decreased the<br />
11
membrane potential and enhanced cell sensitivity to staurosporine. Intriguingly, noting<br />
that HTRA2 protease activity requires trimerization in vivo, they determined that mutant<br />
HTRA2 was able to form the protein complex with wildtype, and proposed that the<br />
mutant form may decrease protease activity without disrupting complex formation,<br />
providing a possible explanation for autosomal dominant inheritance <strong>of</strong> this gene.<br />
DJ-1, although not directly linked to mitochondrial function, was identified by<br />
Bonifati et al. (2003) while studying Dutch and Italian families with autosomal recessive<br />
PD. To characterize the <strong>neuroprotective</strong> role <strong>of</strong> DJ-1, Junn et al. (2005) transfected SH-<br />
SY5Y cells with wildtype or mutant DJ-1, and determined that overexpression <strong>of</strong><br />
wildtype protein significantly protected the cells from hydrogen peroxide, DA, and<br />
MPP+ insults, illustrating anti-oxidant properties <strong>of</strong> DJ-1. After identifying Daxx as one<br />
<strong>of</strong> the DJ-1 interactors via yeast two-hybrid screen, they also determined that wildtype<br />
DJ-1 suppressed Daxx/ASK1-induced cell death.<br />
Alternatively, Xu et al. (2005) described another <strong>neuroprotective</strong> mechanism <strong>of</strong><br />
DJ-1. <strong>The</strong>y used affinity purification and mass spectrometry to detect a nuclear RNA<br />
binding protein NRB54 and PSF as DJ-1 interactors in SH-SY5Y cells. <strong>The</strong>y showed<br />
that mutant DJ-1 exhibited reduced nuclear localization and decreased co-localization<br />
with NRB54 and PSF, readily allowing transcriptional repression by PSF. Furthermore,<br />
they examined wildtype DJ-1 in which the overexpression suppressed PSF-induced cell<br />
death as well as α-syn- and hydrogen peroxide-induced neurodegeneration. Taken<br />
12
together, although anti-oxidant activity <strong>of</strong> DJ-1 is well supported, precise pathways<br />
leading to neuroprotection need further clarification.<br />
PD patho<strong>genes</strong>is: signaling pathways<br />
Both LRRK2 and GIGYF2 are signaling components, but precisely which<br />
pathways they modify is unknown. Originally described by Paisan-Ruiz et al. (2004) by<br />
studying Spanish and British families affected by autosomal dominant PD, LRRK2<br />
encodes a 2527 amino-acid protein with leucine-rich repeat, Roc GTPase, COR,<br />
MAPKKK, and WD40 domains. Given the high frequency <strong>of</strong> G2019S mutation (in the<br />
MAPKKK domain) in autosomal dominant (Di Fonzo et al., 2005) and idiopathic (Gilks<br />
et al., 2005) PD patients, West et al. (2005) characterized LRRK2 G2019S expressed in<br />
HEK293 and SH-SY5Y cells, and determined that the mutant form exhibited<br />
significantly higher kinase activity compared to wildtype LRRK2. West et al. (2008)<br />
also analyzed 10 PD-linked LRRK2 mutations, and determined that LRRK2 GTPase<br />
activity regulates its kinase activity, and enhanced kinase activity leads to<br />
neurodegeneration.<br />
GIGYF2, similar to UCHL1, is one <strong>of</strong> the least characterized PD <strong>genes</strong> because <strong>of</strong><br />
ongoing disputes regarding the lack <strong>of</strong> evidence supporting its role in PD patho<strong>genes</strong>is<br />
(Bonifati, 2009). Focusing on one <strong>of</strong> the PARK11 microsatellite markers, D2S206, which<br />
is found in the intron <strong>of</strong> GIGYF2 coding sequence, Lautier et al. (2008) sequenced<br />
GIGYF2 gene in PD patients, and identified 7 mutations that result in single amino acid<br />
13
substitutions. While GIGYF2 mutations are uncharacterized, this gene is an attractive<br />
candidate because its interacting partner, GRB10 adaptor protein, has been shown to<br />
regulate the insulin signaling pathway (Giovannone et al., 2003), which may affect<br />
human aging (Suh et al., 2008; Willcox et al., 2008) and possibly the onset <strong>of</strong> PD (Craft<br />
and Watson, 2004).<br />
Strategy using invertebrate models <strong>of</strong> PD<br />
Both vertebrate and invertebrate models <strong>of</strong> PD have been generated and exploited,<br />
taking advantage <strong>of</strong> what each model organism <strong>of</strong>fers. Vertebrate models provide a<br />
complex neuronal circuitry as well as corresponding brain functions that most resemble<br />
humans. In contrast, while invertebrate models are not evolutionally as intricate or<br />
advanced as mammalian counterparts, their simplicity allows researchers to perform both<br />
functional and large-scale analyses in a cost-effective manner.<br />
Generally, invertebrate models have been utilized to model an aspect <strong>of</strong> the<br />
disease state (e.g., by overexpressing wildtype α-syn, expressing mutant α-syn, or<br />
treating with neurotoxins) and identify novel therapeutic targets that are subsequently<br />
validated by vertebrate models or to study genetic as well as genetic-environmental<br />
interactions in PD-associated mutant background (Table 1.2). For example,<br />
Saccharomyces cerevisiae, commonly known as baker’s yeast has been utilized for<br />
genetic and chemical screens. One major disadvantage <strong>of</strong> yeast is the fact that it is a<br />
unicellular eukaryote that does not have neurons nor synthesize DA. To this end,<br />
14
multicellular eukaryotic model organisms with DA neurons such as a nematode,<br />
Caenorhabditis elegans and the fruit fly, Drosophila melanogaster are utilized. In this<br />
section, fly and worm PD models are described below whereas yeast models are<br />
discussed in Chapter 3.<br />
A pioneering work by Feany and Bender (2000) generated a D. melanogaster PD<br />
model whereby pan-neuronal expression <strong>of</strong> wildtype and mutant α-syn resulted in the<br />
selective loss <strong>of</strong> DA neurons, presence <strong>of</strong> intraneuronal inclusions, and motor<br />
dysfunction. Subsequent analysis <strong>of</strong> α-syn patho<strong>genes</strong>is demonstrated that its toxicity is<br />
dependent on phosphorylation at serine 129 (Chen and Feany, 2005) as well as its ability<br />
to form aggregates in vivo (Periquet et al., 2007). <strong>The</strong> same model was utilized to study<br />
differential gene expression by microarray whereby <strong>genes</strong> involved in catecholamine<br />
synthesis, energy metabolism, mitochondrial function, and lipid bindings were found<br />
misregulated (Scherzer et al., 2003). Auluck et al. (2002) generated a different fly strain<br />
overexpressing wildtype and mutant α-syn using a DA neuron-specific promoter and<br />
observed DA neurodegeneration. Importantly, they also co-overexpressed human<br />
HSP70, which suppressed DA neuron death. This work, which was later confirmed by a<br />
yeast PD model whereby overexpression <strong>of</strong> yeast Ssa3 (a yeast ortholog <strong>of</strong> human<br />
HSP70) abolished α-syn-induced ROS accumulation (Flower et al., 2005), represented a<br />
groundbreaking strategy <strong>of</strong> using model organisms to identify a novel <strong>neuroprotective</strong><br />
target.<br />
15
Multiple studies have utilized mutant fly strains to identify potential<br />
<strong>neuroprotective</strong> targets, determine genetic interactions <strong>of</strong> PD-associated <strong>genes</strong>, or<br />
examine susceptibility to various forms <strong>of</strong> environmental stress. For example, using<br />
parkin mutant strains, Whitworth et al. (2005) observed the loss <strong>of</strong> DA neurons in the<br />
parkin loss-<strong>of</strong>-function background, which was reversed by overexpression <strong>of</strong> GstS1 (a<br />
fruit fly ortholog <strong>of</strong> human glutathione S-transferase). In addition, Clark et al. (2006)<br />
demonstrated the genetic interaction between PINK1 and parkin wherein overexpression<br />
<strong>of</strong> parkin rescued the male sterility and defective mitochondrial morphology that were<br />
observed in PINK1 mutants. While validation using the mammalian system is required,<br />
these findings strongly suggest that multiple PD <strong>genes</strong> may function in a common<br />
pathway. Focusing on the interplay between genetic and environmental factors,<br />
Muelener et al. (2005) reported that DJ-1 knockout flies exhibited increased<br />
susceptibility to paraquat- and rotenone-induced oxidative stress, which is consistent with<br />
the anti-oxidant role <strong>of</strong> DJ-1 in mammalian cell culture. Lastly, Chaudhuri et al. (2007)<br />
reported that paraquat-treated flies displayed DA neurodegeneration, and that variations<br />
in DA-regulating <strong>genes</strong> could modify paraquat-induced oxidative damage. Since only 5-<br />
10% <strong>of</strong> all PD cases are linked to known genetic causes, assessing the contribution <strong>of</strong><br />
environmental factors and studying the genetic-environmental interactions should provide<br />
a mechanistic insight into further elucidating PD patho<strong>genes</strong>is.<br />
16
C. elegans PD models<br />
C. elegans <strong>of</strong>fers distinct advantages similar to other model organism. For<br />
example, nematodes are approximately 1 mm long, and transparent with short generation<br />
time and lifespan. Furthermore, the worm genome sequence and neuronal circuitry are<br />
known, and mature bioinformatic databases (e.g., microarray, interactome, etc) and<br />
numerous mutant strains are accessible. Lastly, RNA interference (RNAi), a method in<br />
which a single gene is knocked down can easily be performed by feeding these worms<br />
the RNAi bacteria that produce double-stranded RNA (Kamath and Ahringer, 2003).<br />
Similar to fly PD models, worm models have been used to identify potential<br />
<strong>neuroprotective</strong> targets, study genetic interactions <strong>of</strong> PD-associated <strong>genes</strong>, or examine PD<br />
environmental factors. Cao et al. (2005) generated a worm strain overexpressing<br />
wildtype α-syn in DA neurons, and reported DA neurodegeneration. <strong>The</strong>y also co-<br />
overexpressed human torsinA, which reversed the neurotoxic effects <strong>of</strong> α-syn. TorsinA<br />
is a chaperone-like protein that, when mutated, results in another movement disorder<br />
termed early-onset primary dystonia. Similar to Auluck et al. (2002) this work<br />
represented a novel approach <strong>of</strong> utilizing model organisms to examine a <strong>neuroprotective</strong><br />
gene. Kuwahara et al. (2006) also generated worm strains overexpressing wildtype or<br />
mutant α-syn in DA neurons under the control <strong>of</strong> dat-1 (dopamine transporter) promoter.<br />
While they were unable to observe neurodegeneration, they reported accumulation <strong>of</strong> α-<br />
syn in cell bodies and dendrites, and defects in DA neuron-dependent behavior. Focusing<br />
on the worm orthologs <strong>of</strong> PD <strong>genes</strong>, multiple articles have documented uncovering the<br />
17
potential genetic interaction among different PD <strong>genes</strong>. Ved et al. (2005) reported that<br />
depletion <strong>of</strong> pdr-1/parkin and djr-1.1/DJ-1 increased the susceptibility <strong>of</strong> these mutant or<br />
RNAi-treated strains to mitochondrial stress. Moreover, Samann et al. (2009)<br />
demonstrated an antagonistic role <strong>of</strong> pink-1/PINK1 and lrk-1/LRRK2 mutations whereby<br />
the absence <strong>of</strong> worm lrk-1 suppressed mitochondrial dysfunction and defects in axonal<br />
outgrowth, two independent phenotypes, that were induced by pink-1 loss <strong>of</strong> function.<br />
Finally, both 6-OHDA (Nass et al., 2002) and MPTP/MPP+ (Braungart et al., 2004)<br />
treatment has been shown to induce DA neurodegeneration in C. elegans.<br />
Additional studies utilize large-scale methodologies conducted by either<br />
microarray or RNAi to identify modifiers <strong>of</strong> α-syn toxicity. Using transgenic strains<br />
overexpressing wildtype or mutant α-syn pan-neuronally, Vartiainen et al. (2006) studied<br />
differential gene expression by using microarray, and reported that <strong>genes</strong> involved in the<br />
UPS and mitochondrial function were up-regulated. Van Ham et al. (2008) performed<br />
genome-wide RNAi and FRAP to identify 80 genetic modifiers <strong>of</strong> α-syn misfolding and<br />
aggregation in body wall muscle cells. <strong>The</strong> positive candidates included those involved<br />
in protein quality control, vesicle trafficking, and aging. Kuwahara et al. (2008)<br />
generated worm strains overexpressing wildtype and mutant α-syn pan-neuronally, and<br />
performed RNAi <strong>against</strong> 1673 <strong>genes</strong> that are implicated in the nervous system functions.<br />
<strong>The</strong>y identified 10 positives, mostly consisting <strong>of</strong> components from the endocytic<br />
pathway that caused severe growth and motor abnormalities, suggesting that α-syn<br />
overexpression may cause defects in uptake or recycling <strong>of</strong> synaptic vesicles.<br />
18
Current studies<br />
To investigate genetic modifiers <strong>of</strong> α-syn misfolding by RNAi screen, we<br />
generated an isogenic worm strain overexpressing α-syn::GFP and TOR-2 (a worm<br />
ortholog <strong>of</strong> human TorsinA) in the body wall muscle cells. Co-overexpression <strong>of</strong> C.<br />
elegans TOR-2 provided a genetic background that allowed clear distinction between<br />
soluble vs. aggregated α-syn, and maintained the expression <strong>of</strong> α-syn::GFP at the<br />
misfolded state. We knocked down 868 genetic candidates via RNAi, and identified 20<br />
strong positives that enhanced α-syn misfolding. To verify <strong>neuroprotective</strong> capacities <strong>of</strong><br />
these positive hits, we analyzed seven <strong>genes</strong> in worm DA neurons <strong>against</strong> α-syn toxicity,<br />
and determined that five out <strong>of</strong> seven rescued DA neurons. <strong>The</strong> <strong>neuroprotective</strong> <strong>genes</strong><br />
included two autophagic components (vps-41 and atgr-7), one DA signaling protein<br />
(C35D10.2), one ER-Golgi trafficking component (F55A4.1), and one uncharacterized<br />
but evolutionarily conserved protein (F16A11.2). This study is described in detail in<br />
Chapter 2 (and published as Hamamichi et al., 2008).<br />
To validate candidate <strong>genes</strong> obtained from yeast α-syn toxicity modifier screens,<br />
we examined the <strong>neuroprotective</strong> capacities <strong>of</strong> these candidates by using our worm model<br />
<strong>of</strong> α-syn neurodegeneration (Gitler et al., 2008; 2009). Consistent with previous findings<br />
demonstrating <strong>neuroprotective</strong> function <strong>of</strong> Rab1a, overexpression <strong>of</strong> RAB3A and<br />
RAB8A rescued DA neurons. Furthermore, overexpression <strong>of</strong> W08D2.5 (a worm<br />
19
ortholog <strong>of</strong> human PARK9/ATP13A2), PLK2, and PDE9A also suppressed α-syn<br />
toxicity. In total, these six proteins demonstrated <strong>neuroprotective</strong> capacities in yeast as<br />
well as worm and mammalian DA neurons, providing additional genetic targets for PD<br />
therapy. This study is discussed in Chapter 3 (and published as Gitler et al., 2008; 2009).<br />
Lastly, to study the genetic link between aging and α-syn associated toxicity, we<br />
examined the effect <strong>of</strong> daf-2/INSR mutation on α-syn neurodegeneration and misfolding.<br />
daf-2, which encodes an insulin-like receptor is a well-characterized gene in C. elegans<br />
whereby reduced function enhances longevity and protection <strong>against</strong> various forms <strong>of</strong><br />
cellular stress (Baumeister et al., 2006). Here, we report a daf-2 strain overexpressing α-<br />
syn and GFP in DA neurons displayed a significant neuroprotection at the chronological<br />
aging stage (day 7 in both wildtype N2 and daf-2 worms) while it failed to exhibit the<br />
same rescue at the biological aging (day 20 in wildtype N2 and day 40 in daf-2 worms),<br />
demonstrating that differential gene expression in daf-2 mutant background is responsible<br />
for neuroprotection. To identify these genetic factors, we performed RNAi screen <strong>against</strong><br />
<strong>genes</strong> that are up-regulated in daf-2, and examined if knockdown might enhance α-syn<br />
misfolding. In total, we assayed 625 candidates, and identified 53 positive <strong>genes</strong>. Two<br />
<strong>genes</strong> identified from the screen, gpi-1/GPI and hrdl-1/AMFR, representing two proteins<br />
involved in the autocrine motility factor pathway, were subsequently analyzed for<br />
<strong>neuroprotective</strong> function <strong>against</strong> α-syn toxicity in DA neurons. This study is described in<br />
Chapter 4.<br />
20
Specific outcomes and future directions as a result <strong>of</strong> this dissertation research are<br />
discussed in Chapter 5. Collectively, this work further establishes C. elegans as a<br />
powerful model system for the rapid evaluation <strong>of</strong> genetic factors with the potential to<br />
influence PD. <strong>The</strong> <strong>genes</strong>, proteins, and biological pathways characterized within this<br />
research represent putative targets for therapeutic development and intervention<br />
following additional validation through mammalian models.<br />
21
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29
Table 1.1. Summary <strong>of</strong> mutations in PD <strong>genes</strong> linked to PD patho<strong>genes</strong>is and their C.<br />
elegans orthologs.<br />
PD Gene PD Protein Mutation C. elegans Ortholog E-Value<br />
PARK1 SNCA/α-syn Gain <strong>of</strong> function<br />
Multiplication<br />
n/a n/a<br />
PARK2 PRKN/parkin Loss <strong>of</strong> function pdr-1 3.4e-38<br />
PARK5 UCHL1 Loss <strong>of</strong> function ubh-1 1.2e-33<br />
PARK6 PINK1 Loss <strong>of</strong> function pink-1 7.8e-53<br />
PARK7 DJ-1 Loss <strong>of</strong> function djr-1.1<br />
1.6e-45<br />
djr-1.2<br />
8.9e-36<br />
PARK8 LRRK2 Gain <strong>of</strong> function lrk-1 5.5e-66<br />
PARK9 ATP13A2 Mislocalization W08D2.5 2.5e-180<br />
PARK11 GIGYF2 Unknown n/a n/a<br />
PARK13 HTRA2 Gain <strong>of</strong> function n/a n/a<br />
n/a: Not applicable<br />
30
Table 1.2. Summary <strong>of</strong> selected invertebrate PD models.<br />
Models Strategy References<br />
S. cerevisiae<br />
Overexpression <strong>of</strong> WT α-syn Genetic screens Willingham et al., 2003;<br />
Cooper et al., 2006; Gitler et<br />
al., 2009<br />
Cytological analysis Outerio and Lindquist, 2003;<br />
Overexpression <strong>of</strong> WT α-syn<br />
and ∆α-syn<br />
C. elegans<br />
31<br />
Gitler et al., 2008<br />
Cytological analysis Flower et al., 2005; Soper et<br />
al., 2008; Liang et al., 2009<br />
Overexpression <strong>of</strong> WT α-syn Cytological analysis Cao et al., 2006<br />
and/or ∆α-syn (DA neurons) Behavioral analysis Kuwahara et al., 2006<br />
Overexpression <strong>of</strong> WT α-syn Microarray<br />
Vartiainen et al., 2006<br />
and/or ∆α-syn (all neurons) RNAi<br />
Kuwahara et al., 2008<br />
Overexpression <strong>of</strong> WT αsyn::gfp<br />
(muscles)<br />
RNAi Van Ham et al., 2008<br />
Overexpression <strong>of</strong> WT LRRK2<br />
and ∆LRRK2 (DA neurons)<br />
Cytological analysis Saha et al., 2009<br />
PD-associated mutants (∆DJ-1, Mutant strain analysis Ved et al., 2005; Samann et<br />
∆parkin, ∆LRRK2, or ∆PINK1)<br />
al., 2009<br />
Neurotoxin treatment<br />
D. melanogaster<br />
Cytological analysis Nass et al., 2002; Braungart<br />
et al., 2004<br />
Overexpression <strong>of</strong> WT α-syn<br />
and/or ∆α-syn (DA neurons)<br />
Cytological analysis Auluck et al., 2002<br />
Overexpression <strong>of</strong> WT α-syn Cytological analysis Feany and Bender, 2000;<br />
and/or ∆α-syn (all neurons)<br />
Chen and Feany, 2005;<br />
Periquet et al., 2007<br />
Microarray<br />
Scherzer et al., 2003<br />
Overexpression <strong>of</strong> WT LRRK2 Cytological analysis Liu et al., 2008; Venderova<br />
and ∆LRRK2 (various neurons)<br />
et al., 2009<br />
PD-associated mutants Mutant strain analysis Whitworth et al., 2005; Clark<br />
(∆parkin and/or ∆PINK1)<br />
et al., 2006<br />
Neurotoxin treatment Mutant strain analysis Meulener et al., 2005;<br />
Chaudhuri et al., 2007
Figure 1.1<br />
32
FIGURE LEGENDS<br />
Fig. 1.1. Schematic representation <strong>of</strong> the cellular defects caused by known PD<br />
<strong>genes</strong>. Mutation or overexpression <strong>of</strong> α-syn/SNCA disrupts multiple cellular functions<br />
including ER-Golgi trafficking, proteasomal and lysosomal protein degradation, and<br />
mitochondria. Additional PD <strong>genes</strong> also affect the same cellular pathways: 1)<br />
parkin/PRKN and UCHL1, 2 proteins involved in the UPS; 2) ATP13A2, a lysosomal<br />
ATPase; 3) PINK1 and HTRA2, 2 mitochondrial components; and 4) DJ-1, a protein<br />
with anti-oxidant property. LRRK2 and GIGFY2 are proposed to modulate the MAPK<br />
and insulin signaling pathways, respectively.<br />
33
CHAPTER TWO<br />
HYPOTHESIS-BASED RNA INTERFERENCE SCREEN IDENTIFIES<br />
NEUROPROTECTIVE GENES IN A PARKINSON’S DISEASE MODEL<br />
This work was published in Proceedings <strong>of</strong> the National Academy <strong>of</strong> Sciences <strong>of</strong> the<br />
United States <strong>of</strong> America, January, 2008 under the following citation: Hamamichi, S.,<br />
Rivas, R.N., Knight, A.L., Cao, S., Caldwell, K.A., Caldwell, G.A. (2008) Proc Natl<br />
Acad Sci U S A 105,728-733. Shusei Hamamichi, Renee Rivas, and Adam Knight<br />
collected all data. Dr. Songsong Cao contributed the data shown in Fig. 2.1. Shusei<br />
Hamamichi, Dr. Kim Caldwell, and Dr. Guy Caldwell co-wrote the manuscript.<br />
34
ABSTRACT<br />
Genomic multiplication <strong>of</strong> the locus encoding human α-synuclein (α-syn), a<br />
polypeptide with a propensity toward intracellular misfolding, results in Parkinson’s<br />
disease (PD). Here we report the results from systematic screening <strong>of</strong> nearly 900<br />
candidate genetic targets, prioritized by bioinformatic associations to existing PD <strong>genes</strong><br />
and pathways, via RNAi knockdown. Depletion <strong>of</strong> 20 gene products reproducibly<br />
enhanced misfolding <strong>of</strong> α-syn over the course <strong>of</strong> aging in the nematode Caenorhabditis<br />
elegans. Subsequent functional analysis <strong>of</strong> seven positive targets revealed five<br />
previously unreported gene products that significantly protect <strong>against</strong> age- and dose-<br />
dependent α-syn-induced degeneration in the dopamine (DA) neurons <strong>of</strong> transgenic<br />
worms. <strong>The</strong>se include two trafficking proteins, a conserved cellular scaffold-type protein<br />
that modulates G-protein signaling, a protein <strong>of</strong> unknown function, and one gene reported<br />
to cause neurodegeneration in knockout mice. <strong>The</strong>se data represent putative genetic<br />
susceptibility loci and potential therapeutic targets for PD, a movement disorder affecting<br />
approximately 2% <strong>of</strong> the population over 65 years <strong>of</strong> age.<br />
35
INTRODUCTION<br />
In the advent <strong>of</strong> complete genomic sequences and technologies for uncovering<br />
putative protein interaction networks or whole-genome analyses, scientists have<br />
generated many “lists” <strong>of</strong> candidate <strong>genes</strong> and proteins that can be harnessed for in-depth<br />
analyses <strong>of</strong> cellular processes or disease states. In the nematode C. elegans, these include<br />
pioneering studies defining the protein “interactome” (Li et al., 2004), the “topology<br />
map” for global gene expression (Kim et al., 2001) and meta-analyses <strong>of</strong> predicted gene<br />
interactions (Zhong and Sternberg, 2006). Application <strong>of</strong> this nematode toward human<br />
disease research has already provided insights into the function <strong>of</strong> specific gene products<br />
linked to a variety <strong>of</strong> neurological disorders (Caldwell et al., 2003; Williams et al., 2004;<br />
Cao et al., 2005; Cooper et al., 2006; Bates et al., 2006). Given that the average lifespan<br />
<strong>of</strong> this nematode is only 14-17 days, it has been especially useful in its application to<br />
diseases <strong>of</strong> aging (Driscoll and Gerstbrein, 2003; Kenyon, 2005). In this study, we<br />
exploited the potential predictive capacity <strong>of</strong> these C. elegans bioinformatic databases to<br />
discern genetic components and/or pathways that might represent heritable susceptibility<br />
factors for PD.<br />
PD involves the progressive loss <strong>of</strong> DA neurons from the substantia nigra,<br />
accompanied by the accumulation <strong>of</strong> proteins into inclusions termed Lewy bodies.<br />
Central to the formation <strong>of</strong> Lewy bodies is α-syn, a polypeptide with a propensity toward<br />
intracellular aggregation. Genomic multiplication <strong>of</strong> the wildtype α-syn locus results in<br />
PD, indicating that overexpression <strong>of</strong> this protein alone can lead to the disease (Singleton<br />
36
et al., 2003). Maintenance <strong>of</strong> DA neuron homeostasis has been hypothesized to be<br />
important for neuroprotection because an imbalance <strong>of</strong> cytosolic DA may contribute to<br />
neurotoxicity. Mechanistically, the selective loss <strong>of</strong> DA neurons in PD is very possibly<br />
due to the presence and chemical nature <strong>of</strong> DA itself. <strong>The</strong> capacity <strong>of</strong> DA for oxidation,<br />
and its effect on stabilizing toxic forms <strong>of</strong> α-syn (Conway et al., 2000), represents a<br />
“perfect storm” in the context <strong>of</strong> the oxidative damage associated with the aging process,<br />
other potential environmental insults (i.e., heavy metals, pesticides), or differences in<br />
genetic predisposition.<br />
Familial PD has been linked to specific <strong>genes</strong>, several <strong>of</strong> which function in<br />
cellular pathways involving the management <strong>of</strong> protein degradation and cellular stress<br />
(Dawson and Dawson, 2003). Although most primary insights into the molecular nature<br />
<strong>of</strong> PD have thus far come via genetic analyses <strong>of</strong> familial forms <strong>of</strong> PD, there is significant<br />
evidence that implicates a combination <strong>of</strong> environmental factors as pivotal to sporadic<br />
causality (Tanner, 2003). Improvements in the diagnosis and treatment <strong>of</strong> PD will be<br />
contingent upon increased knowledge about susceptibility factors that render populations<br />
at risk.<br />
We previously reported the establishment <strong>of</strong> a nematode model <strong>of</strong> age-dependent<br />
α-syn-induced DA neurodegeneration that has facilitated successful <strong>identification</strong> <strong>of</strong><br />
multiple <strong>neuroprotective</strong> factors, including those that have since been validated in other<br />
model organisms and mammals (Cao et al., 2005; Cooper et al., 2006). Here we take<br />
advantage <strong>of</strong> the experimental attributes <strong>of</strong> C. elegans to characterize a set <strong>of</strong> novel<br />
37
<strong>neuroprotective</strong> gene products initially identified in a large-scale candidate gene screen<br />
for factors influencing misfolding <strong>of</strong> human α-syn in vivo by RNA interference (RNAi).<br />
<strong>The</strong>se data represent a collection <strong>of</strong> functionally delineated modifiers <strong>of</strong> α-syn-dependent<br />
misfolding and neurodegeneration that enhance our understanding <strong>of</strong> the molecular basis<br />
<strong>of</strong> PD and point toward new potential targets for therapeutic intervention.<br />
MATERIALS AND METHODS<br />
Nematode Strains. Nematodes were maintained using standard procedures<br />
(Brenner, 1974). To make transgenic lines, each expression plasmid was injected into<br />
wildtype N2 (Bristol) worms at a concentration <strong>of</strong> 50 µg/ml. For the RNAi screen,<br />
UA50 [baInl3; Punc-54::gfp, rol-6 (su1006)], UA51 [baInl4; Punc-54::α-syn::gfp, rol-6<br />
(su1006)], and UA52 [baInl5; Punc-54:: α-syn::gfp, Punc-54::tor-2, rol-6 (su1006)] were<br />
integrated as previously described (Cao et al., 2005) and outcrossed at least three times to<br />
N2 worms. <strong>The</strong> polyglutamine aggregation analysis was performed using integrated<br />
isogenic strain UA6 [UA6 (baIn6)] co-expressing Q82::GFP and TOR-2 (Caldwell et al.,<br />
2003).<br />
For neuroprotection analysis, three stable lines <strong>of</strong> either UA53 [baEx42; Pdat-<br />
1::FLAG-C35D10.2, Punc-54::DsRed2], UA54 [baEx43; Pdat-1::FLAG-C54H2.5, Punc-<br />
54::DsRed2], UA55 [baEx44; Pdat-1::FLAG-F16A11.2, Punc-54::DsRed2], UA56 [baEx45;<br />
Pdat-1::FLAG-F32A6.3, Punc-54::DsRed2], UA57 [baEx46; Pdat-1::FLAG-F55A4.1, Punc-<br />
54::DsRed2], UA58 [baEx47; Pdat-1::FLAG-M7.5, Punc-54::DsRed2], and UA59 [baEx48;<br />
38
Pdat-1::FLAG-R05D11.6, Punc-54::DsRed2] were crossed with integrated UA44 [baInl1;<br />
Pdat-1:: α-syn high, Pdat-1::gfp]. Briefly, male UA44 [baInl1; Pdat-1::α-syn high, Pdat-<br />
1::gfp] worms were generated by mating hermaphrodites with male N2 worms. GFP-<br />
positive males were crossed with hermaphrodites overexpressing candidate <strong>genes</strong> in DA<br />
neurons and DsRed2 in body wall muscle cells. <strong>The</strong> resulting GFP- and dsRed2-positive<br />
hermaphrodites were individually picked, and self-fertilized until all worms displayed<br />
GFP expression indicating homozygous expression <strong>of</strong> α-syn. <strong>The</strong>se strains are<br />
designated as follows: UA60 {[baInl1; Pdat-1:: α-syn high, Pdat-1::gfp]; [baEx49; Pdat-<br />
1::FLAG-C35D10.2, Punc-54::DsRed2]}, UA62 {[baInl1; Pdat-1:: α-syn high, Pdat-1::gfp];<br />
[baEx51; Pdat-1::FLAG-C54H2.5, Punc-54::DsRed2]}, UA64 {[baInl1; Pdat-1:: α-syn high,<br />
Pdat-1::gfp]; [baEx53; Pdat-1::FLAG-F16A11.2, Punc-54::DsRed2]}, UA66 {[baInl1; Pdat-<br />
1:: α-syn high, Pdat-1::gfp]; [baEx55; Pdat-1::FLAG-F32A6.3, Punc-54::DsRed2]}, UA68<br />
{[baInl1; Pdat-1:: α-syn high, Pdat-1::gfp]; [baEx57; Pdat-1::FLAG-F55A4.1, Punc-<br />
54::DsRed2]}, UA70 {[baInl1; Pdat-1:: α-syn high, Pdat-1::gfp]; [baEx59; Pdat-1::FLAG-<br />
M7.5, Punc-54::DsRed2]}, and UA72 {[baInl1; Pdat-1:: α-syn high, Pdat-1::gfp]; [baEx61;<br />
Pdat-1::FLAG-R05D11.6, Punc-54::DsRed2]}.<br />
Plasmid Constructs. Plasmids were constructed using Gateway Technology<br />
(Invitrogen). To generate α-syn::gfp, α-syn cDNA (a gift from Philipp Kahle, University<br />
<strong>of</strong> Tubingen, Germany) was cloned into a gfp-containing plasmid, pPD95.75 (Andy Fire,<br />
Stanford University) by double digestion using XbaI and BamHI. Gateway entry vectors<br />
39
were generated by cloning PCR-amplified α-syn::gfp, gfp as well as candidate cDNAs<br />
into pDONR201 or pDONR221 by BP reaction. <strong>The</strong> cDNAs encoding C35D10.2,<br />
C54H2.5, F16A11.2, F55A4.1, M7.5, and R05D11.6 were obtained from Open<br />
Biosystems (Huntsville, AL) while F32A6.3 was isolated from our C. elegans cDNA<br />
library (Caldwell et al., 2006). DsRed2 was obtained from Clontech (Mountain View,<br />
CA). An N-terminal FLAG tag sequence was added during the PCR amplification<br />
process.<br />
<strong>The</strong> gene fusions were shuttled from entry vectors into the Gateway destination<br />
vector, pDEST-DAT-1 (Cao et al., 2005) or pDEST-UNC-54 via LR reaction. pDEST-<br />
UNC-54 was generated by converting a unc-54 promoter containing plasmid, pPD30.38<br />
(Andy Fire), using a Gateway Vector Conversion System (Invitrogen). <strong>The</strong> molecular<br />
cloning yielded expression plasmids, Punc-54:: α-syn::gfp, Punc-54::gfp, Pdat-1::FLAG-<br />
C35D10.2, Pdat-1::FLAG-C54H2.5, Pdat-1::FLAG-F16A11.2, Pdat-1::FLAG-F32A6.3, Pdat-<br />
1::FLAG-F55A4.1, Pdat-1::FLAG-M7.5, Pdat-1::FLAG-R05D11.6, and Punc-54::DsRed2.<br />
<strong>The</strong> cDNAs were verified by DNA sequencing.<br />
Preparation <strong>of</strong> worm protein extracts and western blotting. Worm protein<br />
extracts were prepared and western blotting was performed as described previously (Cao<br />
et al., 2005). For all worm strains, 30 µg/µl protein was loaded into 15% SDS PAGE<br />
gels (Bio-Rad) and detected by 1:2000 goat anti-α-syn primary antibody (Chemicon) and<br />
1:10000 horseradish peroxidase-conjugated mouse anti-goat IgG secondary antibody<br />
(Pierce). For detection <strong>of</strong> actin, 1:8000 mouse anti-actin primary antibody (ICN<br />
40
Biochemicals) and 1:10000 horseradish peroxidase-conjugated sheep anti-mouse IgG<br />
secondary antibody (Amersham) were utilized.<br />
RNAi screen and analysis <strong>of</strong> α-syn misfolding or polyglutamine aggregation.<br />
RNAi feeding was performed as described (Kamath and Ahringer, 2003) with the<br />
following modifications. Bacterial clones leading to enhanced α-syn misfolding were<br />
tested in two trials, and the clones resulting in significant aggregation (80% <strong>of</strong> worms<br />
with increased quantity and size <strong>of</strong> α-syn aggregates) were scored as positive. For each<br />
trial, 20 worms were transferred onto a 2% agarose pad, immobilized with 2 mM<br />
levamisole, and analyzed. <strong>The</strong> identities <strong>of</strong> the top 20 positive hits from the RNAi screen<br />
were sequenced and verified. For polyglutamine aggregation analysis, 20 worms at the<br />
L3-stage were scored for aggregate number in two separate trials.<br />
RNAi feeding clones (Geneservice, Cambridge, UK) were grown for 14 hrs in LB<br />
culture with 100 mg/ml ampicillin and seeded onto NGM agar plates containing 1 mM<br />
isopropyl β-D-thiogalactoside. When the bacterial lawn was grown, five L4<br />
hermaphrodites (strain UA52) were transferred onto the plates and incubated at 25°C for<br />
44 hr. <strong>The</strong> gravid adults were then placed onto the corresponding RNAi plates and<br />
allowed to lay eggs for 9 hours, and the resulting age-synchronized worms were analyzed<br />
at the indicated stage. For polyglutamine aggregation analysis, L3-staged 20 worms were<br />
transferred onto a 2% agarose pad and immobilized with 2 mM levamisole, and the<br />
quantity <strong>of</strong> aggregates was scored. <strong>The</strong> aggregation analysis was also conducted in<br />
duplicate.<br />
41
Candidate gene analysis for neuroprotection. Synchronized embryos expressing<br />
both GFP and DsRed2 were transferred onto NGM plates, and grown at 20°C for 7 days<br />
(Lewis and Fleming, 1995). For each trial, 30 worms were transferred to a 2% agarose<br />
pad, immobilized with 2 mM levamisole, and scored. Worms were considered rescued<br />
when all four CEP and both ADE neurons were intact and had no visible signs <strong>of</strong><br />
degeneration. Each stable line was analyzed three times (for a total <strong>of</strong> 90<br />
worms/transgenic line). Three separate transgenic lines were analyzed per gene, for a<br />
total <strong>of</strong> 270 animals/gene analyzed.<br />
RNA isolation and semi-quantitative RT-PCR. Worms were harvested and snap-<br />
frozen in liquid nitrogen. After total RNA and cDNA preparation, semi-quantitative RT-<br />
PCR was performed as previously described (Locke et al., 2006). Briefly, 50 L4-staged<br />
worms were transferred into 10 µl 1:10-diluted Single Worm Lysis Buffer (10 mM Tris,<br />
pH 8.3, 50 mM KCl, 2.5 mM MgCl2, 0.45% NP-40, 0.45% Tween 20, 0.01% gelatin, and<br />
60 µg proteinase K), mixed with 100 µl TRI Reagent (Molecular Research Center), and<br />
incubated at RT for 10 min. <strong>The</strong> samples were freeze-thawed 5 times using liquid N2,<br />
vortexed with 10 µl 1-bromo-3-chloropropane (Acros Organics) for 15 sec, incubated at<br />
RT for 10 min, and centrifuged at 14500 rpm at 4°C for 15 min. <strong>The</strong> supernatant was<br />
transferred to a new RNase-free tube, mixed with 2 µl glycoblue (Ambion) and 50 µl -<br />
20°C-chilled isopropanol, and incubated at -20°C overnight. After incubation, the<br />
supernatant was centrifuged at 14500 rpm at 4°C for 15 min, and discarded. <strong>The</strong> pellet<br />
42
was washed with 100 µl RNase-free 75% ethanol, and resuspended in 10 µl DEPC-<br />
treated water. For RT-PCR using SuperScript III RT (Invitrogen) with oligo dT<br />
primers, the total RNAs were treated with amplification grade RNase-free DNase I<br />
(Invitrogen) as well as RNase H (Invitrogen) following the manufacture’s protocol. PCR<br />
was then performed using Phusion polymerase (Finnzymes). <strong>The</strong> PCR products were<br />
separated by 0.8% agarose gel electrophoresis and visualized by GelRed staining<br />
(Biotium). <strong>The</strong> following primers were designed for the PCR:<br />
cdk-5 Primer 1: 5’ ggg-gat-gat-gag-ggt-gtt-cca-agc 3’<br />
Primer 2: 5’ ggc-gac-cgg-cat-ttg-aga-tct-ctg-c 3’<br />
α-syn Primer 1: 5’ atg-gat-gta-ttc-atg-aaa-gga-ctt-tca-aag 3’<br />
Primer 2: 5’ tta-ggc-ttc-agg-ttc-gta-gtc-ttg 3’<br />
<strong>The</strong> FLAG-tagged <strong>genes</strong> were PCR amplified by using primer sequences specific to<br />
FLAG and each respective open reading frame.<br />
FLAG Primer 1: 5’ gac-tac-aag-gac-gac-gat-gac 3’<br />
C35D10.2 Primer 2: 5’ gaa-tgt-ggg-cga-aga-gca-tat-c 3’<br />
C54H2.5 Primer 2: 5’ gtc-ctc-cac-caa-cgg-caa-tg 3’<br />
F16A11.2 Primer 2: 5’ cca-gag-tga-ata-tct-gga-aga-cc 3’<br />
F55A4.1 Primer 2: 5’ caa-att-cga-gga-aat-ggt-atg-gac 3’<br />
F32A6.3 Primer 2: 5’ gag-cgg-aac-ctg-gtt-ctt-tat-g 3’<br />
M7.5 Primer 2: 5’ ggc-tcc-gag-aga-tga-tag-tgg 3’<br />
R05D11.6 Primer 2: 5’ cat-tgc-aag-aga-tgc-ctt-gag 3’<br />
43
Imaging. All fluorescence microscopy was performed using a Nikon Eclipse<br />
E800 epifluorescence microscope equipped with Endow GFP HYQ filter cube (Chroma<br />
Technology). Images were captured with a Photometrics Cool Snap CCD camera driven<br />
by MetaMorph s<strong>of</strong>tware (Universal Imaging).<br />
Statistics. Statistical analysis for neuroprotection was performed using Student’s<br />
t-test (p
and effectively discerned via RNAi screening. <strong>The</strong> reasoning behind use <strong>of</strong> the body-wall<br />
muscles was two-fold; first, these are the largest, most readily scored cell type in adult C.<br />
elegans within which to accurately judge changes in α-syn misfolding, and second, C.<br />
elegans DA neurons are recalcitrant to RNAi (Asikainen et al., 2005). Moreover, we<br />
theorized that the presence <strong>of</strong> TOR-2, a protein with chaperone activity, served to<br />
maintain overexpressed α-syn at a threshold <strong>of</strong> misfolding, thereby enabling<br />
<strong>identification</strong> <strong>of</strong> genetic factors that more readily effect the formation <strong>of</strong> misfolded<br />
oligomers, or less mature α-syn aggregates, currently considered to be the more toxic<br />
species associated with degeneration (Taylor et al., 2002; Lee et al., 2004).<br />
To investigate putative effectors <strong>of</strong> α-syn misfolding, we have systematically<br />
screened 868 genetic targets with the potential to influence PD by selecting for<br />
candidates that, when knocked down, enhanced age-associated aggregation <strong>of</strong> α-<br />
syn::GFP. We used the C. elegans orthologs <strong>of</strong> established familial PD <strong>genes</strong> as the<br />
foundation for constructing a candidate gene list (Table 2.4). <strong>The</strong> worm genome includes<br />
orthologs <strong>of</strong> all established familial PD <strong>genes</strong> (Parkin, DJ-1, PINK1, UCHL-1, LRRK2,<br />
PARK9, NURR1) with the exception <strong>of</strong> α-syn. Specific C. elegans bioinformatic datasets<br />
were subsequently mined to define hypothetical interrelationships between the worm PD<br />
orthologs and previously unrelated gene targets. For example, using the C. elegans<br />
topology map (Kim et al., 2001), we identified all gene products that are co-expressed<br />
with the worm PD orthologs within a radius <strong>of</strong> one. Additionally, we identified all gene<br />
45
products that interact with these PD orthologs, as assessed by the worm interactome (Li<br />
et al., 2004). Also included among our RNAi targets were the worm orthologs <strong>of</strong> <strong>genes</strong><br />
that were uncovered via screens for effectors <strong>of</strong> α-syn toxicity in Saccharomyces<br />
cerevisiae (Cooper et al., 2006; Willingham et al., 2003), as well as <strong>genes</strong> encoding<br />
nematode versions <strong>of</strong> proteins identified in a proteomic analysis <strong>of</strong> rotenone-induced<br />
Lewy bodies in DA neuron cell cultures (Zhou et al., 2004). We further extended our<br />
RNAi target gene set by identifying worm homologs <strong>of</strong> gene products ascribed to<br />
encompass the cellular protein degradation machinery. <strong>The</strong>se included <strong>genes</strong> annotated<br />
in Wormbase as being involved in the ubiquitin-proteasome system (UPS), unfolded-<br />
protein response (UPR), endoplasmic reticulum-associated degradation (ERAD), and<br />
autophagy. Gene candidates derived from these pathways were assessed for homology to<br />
mammals, and non-conserved <strong>genes</strong> were excluded since it has been estimated that 47%<br />
<strong>of</strong> worm <strong>genes</strong> have no visible homology to mammals (Schwarz, 2005). A table<br />
corresponding to 868 candidate <strong>genes</strong> targeted for knockdown is available in the<br />
Supplementary Material (Table 2.4). Furthermore, we have constructed a relational<br />
interconnectivity map depicting gene targets classified in more than one category (Fig.<br />
2.3).<br />
<strong>The</strong>se candidate gene targets were knocked down using RNAi, a method that is<br />
both rapidly and economically performed in C. elegans by feeding worms target-specific<br />
dsRNA-producing bacteria (Kamath et al., 2003). In total, 13% (111/868) were lethal;<br />
however, the remaining 757 <strong>genes</strong> were analyzed for accumulation <strong>of</strong> misfolded α-syn<br />
46
protein. <strong>The</strong> primary RNAi screen <strong>of</strong> adult stage worms (44-48 hrs after eggs were laid at<br />
25°C) revealed that 17% (125/757) <strong>of</strong> these gene targets enhanced aggregation <strong>of</strong> α-syn<br />
in worms co-expressing α-syn::GFP and TOR-2. <strong>The</strong> misfolded protein appeared over<br />
developmental time and was randomly distributed in the cytoplasm <strong>of</strong> the body-wall<br />
muscle cells (Fig. 2.1C, D). RNAi was performed on approximately 20-30 animals in<br />
duplicate for each gene. As would be expected, a significant number <strong>of</strong> <strong>genes</strong> that alter<br />
folding or protein degradation were identified (Table 2.4). Notably included within this<br />
collection <strong>of</strong> positives were worm orthologs <strong>of</strong> five familial PD <strong>genes</strong>: Parkin<br />
(K08E3.7/pdr-1), DJ-1 (B0432.2/djr-1.1), PINK1 (EEED8.9/pink-1), NURR1<br />
(C48D5.1/nhr-6) and PARK9/ATP13A2 (W08D2.5) (Dawson and Dawson, 2003;<br />
Ramirez et al., 2006).<br />
Since PD is a disease <strong>of</strong> aging, we reasoned gene products that play a more<br />
significant functional role in the management <strong>of</strong> α-syn misfolding or clearance would<br />
exhibit a stronger effect at an earlier age. In this regard, a secondary screen <strong>of</strong> the top<br />
125 candidates was performed in worms at the L3 larval stage <strong>of</strong> development (32-36 hrs<br />
after eggs laid). This resulted in further reduction <strong>of</strong> candidates where only about 3%<br />
(20/757) <strong>of</strong> <strong>genes</strong> enhanced misfolding <strong>of</strong> human α-syn following RNAi treatment at this<br />
earlier developmental stage (Table 2.1). Retained within this list <strong>of</strong> 20 more stringently<br />
selected hits were orthologs <strong>of</strong> known recessive PD <strong>genes</strong>, DJ-1 and PINK1, thereby<br />
representing internal validation <strong>of</strong> the screen. Another expected control for the screen,<br />
the C. elegans torsinA gene homolog, tor-2, was also recovered. A notable gene from<br />
47
this dataset was T07F12.4, a serine-threonine kinase that is homologous to UNC-51, a<br />
protein similar to yeast Atg1p, required for autophagy, that also plays a role in axon<br />
elongation (Okazaki et al., 2000; Lai and Garriga, 2004). A human ortholog <strong>of</strong> worm<br />
UNC-51, termed ULK2, was recently identified by geneticists as one <strong>of</strong> only six <strong>genes</strong><br />
that were distinguished as significant in a genome-wide association study <strong>of</strong> single-<br />
nucleotide polymorphisms within PD patients (Fung et al., 2006). <strong>The</strong> remaining 16 (<strong>of</strong><br />
20) positives encompass gene products previously unassociated with either α-syn<br />
function or PD.<br />
Our <strong>identification</strong> <strong>of</strong> gene products that influence the misfolding <strong>of</strong> α-syn does<br />
not preclude the possibility that these proteins play a more generalized role in regulating<br />
protein misfolding or degradation. Previous screens in both C. elegans and yeast have<br />
implicated various classes <strong>of</strong> gene products that influence the misfolding or clearance <strong>of</strong><br />
polyglutamine-repeat containing proteins (Willingham et al., 2005; Nollen et al., 2004).<br />
In comparing the gene sets identified in those studies to our list <strong>of</strong> 125 less stringent<br />
modifiers <strong>of</strong> α-syn misfolding, we determined that only one positive gene was shared<br />
between our datasets, the C. elegans HSF-1 protein. HSF-1 is a critical evolutionarily<br />
conserved regulator <strong>of</strong> chaperone gene expression that would be presumed to exhibit a<br />
generalized function in mediating protein misfolding.<br />
To further explore the prospect that the specific <strong>genes</strong> identified in our screen<br />
potentially act in a more generalized capacity, we used RNAi knockdown to evaluate<br />
loss-<strong>of</strong>-function associated with our strongest 20 α-syn modifiers in transgenic worms<br />
48
expressing a polyglutamine::GFP fusion protein. <strong>The</strong> results <strong>of</strong> this analysis indicate that<br />
RNAi knockdown <strong>of</strong> these targets had no significant influence on polyglutamine-<br />
dependent aggregation in vivo (Table 2.5 and Fig. 2.6). <strong>The</strong> sole exception was the TOR-<br />
2 chaperone-like protein, which served as a control in this analysis, as this protein has<br />
been shown to suppress polyglutamine aggregation in C. elegans (Caldwell et al., 2003).<br />
<strong>The</strong>se data are consistent with a previous report that demonstrated the toxicity mediated<br />
by overexpression <strong>of</strong> α-syn vs. a mutant huntingtin fragment in yeast was regulated by<br />
non-overlapping gene sets (Willingham et al., 2003). In all, this analysis demonstrates<br />
that the strongest α-syn effector <strong>genes</strong> identified through our RNAi screening do not<br />
exert their influence via a general effect on protein misfolding, but more specifically<br />
contribute to cellular pathways associated with α-syn.<br />
A distinct advantage <strong>of</strong> using C. elegans for functional investigation <strong>of</strong> gene<br />
activity is the level <strong>of</strong> accuracy that can be obtained in evaluating neurodegeneration. C.<br />
elegans has precisely 8 DA neurons, with three pairs <strong>of</strong> neurons in the anterior<br />
(designated dorsal/ventral CEPs and ADEs) and 1 pair in the posterior <strong>of</strong> the animal<br />
(PDEs). We have established that overexpression <strong>of</strong> wildtype human α-syn under the<br />
control <strong>of</strong> a DA neuron-specific promoter [Pdat-1; DA transporter] results in age- and<br />
dose-dependent neurodegeneration. We generated two separate transgenic lines <strong>of</strong><br />
animals that express α-syn at different levels, based on semi-quantitative RT-PCR<br />
analysis (Fig. 2.4A). At day 7 <strong>of</strong> adulthood, 87% <strong>of</strong> animals expressing a higher level <strong>of</strong><br />
α-syn show DA neurodegeneration (Fig. 2.4B) while 75% <strong>of</strong> animals expressing α-syn at<br />
49
lower levels display degenerative changes (data not shown). <strong>The</strong> loss <strong>of</strong> DA neurons also<br />
occurs as animals age and no degeneration (0%) is observed in control animals (Pdat-<br />
1::GFP) lacking α-syn overexpression (Fig. 2.4B). Previously, these same animals have<br />
been utilized to validate the <strong>neuroprotective</strong> capacity <strong>of</strong> both worm TOR-2 and<br />
mammalian Rab1a, a GTPase involved in ER to Golgi transport (Cao et al., 2005; Cooper<br />
et al., 2006). Here we further extend our functional characterization <strong>of</strong> <strong>genes</strong> that<br />
resulted in enhanced α-syn misfolding when depleted by RNAi by systematically testing<br />
their prospective <strong>neuroprotective</strong> potential in vivo.<br />
Figure 2.2A depicts a classification <strong>of</strong> the 20 positives where they are displayed<br />
according to their bioinformatic associations; several <strong>of</strong> these candidates shared more<br />
than one bioinformatic relationship (Table 2.2). For example, the C. elegans open-<br />
reading frame F32A6.3 encodes a gene (vps-41) that is co-expressed in microarrays with<br />
the worm ortholog <strong>of</strong> UCHL-1, contains a RING-finger motif common to E3 ligases, and<br />
is involved in autophagy (Fig. 2.2A, circle). We utilized inferred relationships between<br />
<strong>genes</strong> exhibiting such overlap to prioritize subsequent construction <strong>of</strong> transgenic animals<br />
to examine their ability to influence DA neuron survival.<br />
Transgenic animals co-expressing cDNAs corresponding to seven prioritized<br />
positive targets from the RNAi screen were generated and crossed to isogenic lines <strong>of</strong><br />
worms expressing α-syn in DA neurons (Cao et al., 2005; Cooper et al., 2006).<br />
Overexpression <strong>of</strong> α-syn alone resulted in significant degeneration, where only 12.8% <strong>of</strong><br />
worms displayed wildtype DA neurons when they were assayed at the seven day-old<br />
50
stage (Fig. 2.2B-D). Two <strong>genes</strong> exhibited an insignificant level <strong>of</strong> DA neuroprotection;<br />
these encoded an uncharacterized transcription factor (R05D11.6) and a C. elegans<br />
ortholog <strong>of</strong> Erv29p (C54H2.5), a vesicle-associated protein involved in ER-Golgi<br />
transport (Fig. 2.2B). Strikingly, co-expression <strong>of</strong> five out <strong>of</strong> seven candidate <strong>genes</strong><br />
examined significantly rescued DA neurodegeneration with average wildtype worm<br />
populations from 24 to 37% (Fig. 2.2B). Worms were scored as wildtype when all six<br />
anterior DA neurons were intact (Fig. 3E, F). Expression <strong>of</strong> the all gene products tested<br />
was verified via semi-quantitative RT-PCR (Fig. 2.5). Three independent transgenic<br />
lines were scored per gene tested, with 30 animals analyzed in triplicate experimental<br />
trials. In considering published evidence that TOR-2, DJ-1, and PINK1 have all<br />
previously been shown to be <strong>neuroprotective</strong> as well (Cao et al., 2005; Menzies et al.,<br />
2005; Petit et al., 2005; Xu et al., 2005; Zhou and Freed, 2005; Pridgeon et al., 2007),<br />
these combined results indicate that the strategy employed in our screen is highly<br />
predictive <strong>of</strong> <strong>neuroprotective</strong> genetic modifiers.<br />
<strong>The</strong> five <strong>genes</strong> found to display significant neuroprotection (P
uncharacterized human gene product; 5) F55A4.1, the worm ortholog <strong>of</strong> Sec22p, a well<br />
characterized vesicular trafficking protein in yeast. <strong>The</strong> Blast E values for the Homo<br />
sapiens orthologs <strong>of</strong> these C. elegans gene products indicate they are all highly conserved<br />
(Table 2.3).<br />
DISCUSSION<br />
<strong>The</strong> key pathological hallmarks <strong>of</strong> PD include the development <strong>of</strong> α-syn<br />
containing protein inclusions and DA neurodegeneration. Although it remains unclear if<br />
mature α-syn aggregates or Lewy bodies are causative for PD, evidence suggests factors<br />
that influence the misfolding and oligomerization <strong>of</strong> this polypeptide lead to<br />
neurotoxicity (Taylor et al., 2002; Lee et al., 2004). Regardless, proteins that play a role<br />
in protecting DA neurons from the degenerative loss associated with α-syn<br />
overproduction are candidate susceptibility markers, as well as potential targets for<br />
therapeutic development. Here we have combined these distinct PD-associated<br />
phenotypic readouts to discern novel gene products with functional consequences for PD.<br />
Among the gene products identified via this screen, a protein that demonstrated<br />
high <strong>neuroprotective</strong> capacity was C. elegans VPS41. VPS41 is highly conserved across<br />
species and has been best characterized in S. cerevisiae, where it is involved in trafficking<br />
from the trans Golgi to the vacuole, the yeast equivalent <strong>of</strong> the lysosome (Rehling et al.,<br />
1999). Little is known about the precise function <strong>of</strong> VPS41 in mammalian systems;<br />
however, in situ hybridization predicts the VPS41 gene to be expressed in brain neurons,<br />
52
with strong expression localized to the DA neurons <strong>of</strong> the substantia nigra (Lein et al.,<br />
2007).<br />
Evidence for lysosomal system dysfunction is emerging as a potential<br />
consequence <strong>of</strong> α-syn cytotoxicity. α-Syn is degraded in part by the lysosomal pathway,<br />
under the regulation <strong>of</strong> the co-chaperone CHIP (Shin et al., 2005) and mutant forms <strong>of</strong> α-<br />
syn can block chaperone-mediated autophagy (Finkbeiner et al., 2006; Cuervo et al.,<br />
2004). <strong>The</strong>refore, lysosomal failure has been proposed as a mechanism underlying the<br />
age dependence <strong>of</strong> PD (Chu and Kordower, 2007). PARK9, a hereditary form <strong>of</strong><br />
parkinsonism with dementia, has been recently linked to mutation <strong>of</strong> a lysosomal ATPase<br />
(Ramirez et al., 2006). Notably, the worm homolog <strong>of</strong> PARK9, W08D2.5, was uncovered<br />
in our original RNAi screen (one <strong>of</strong> 125 initial hits) where knockdown led to α-syn<br />
aggregation. This gene product is also <strong>neuroprotective</strong> when overexpressed in DA<br />
neurons (Hamamichi and Caldwell, manuscript in preparation).<br />
Our <strong>identification</strong> <strong>of</strong> C. elegans ATG7 as a <strong>neuroprotective</strong> gene product is<br />
further suggestive <strong>of</strong> a significant role for autophagy and lysosomal function in restoring<br />
homeostatic balance to DA neurons in response to excess α-syn. ATG7 is an E1-like<br />
enzyme required for the initiation <strong>of</strong> autophagosome formation. Added validation for<br />
these worm data comes from mammals where it was shown that loss <strong>of</strong> the Atg7 gene in<br />
mice results in neurodegeneration and that this protein may function to prevent neuronal<br />
impairment and axonal degeneration (Komatsu et al., 2006, Komatsu et al., 2007).<br />
53
Among the factors that mediate DA neuron homeostasis is the interplay <strong>of</strong> DA<br />
production, transport, and receptor signaling. In the “classical” view <strong>of</strong> DA signaling, D2<br />
autoreceptors modulate a putative pre-synaptic feedback mechanism resulting in a net<br />
<strong>neuroprotective</strong> effect (Bozzi and Borrelli, 2006). As the complexities <strong>of</strong> D2 signaling<br />
continue to be unraveled, it is critical to consider that this model does not take into<br />
account the largely unknown impact <strong>of</strong> α-syn misfolding and overabundance associated<br />
with PD.<br />
Here we describe evidence indicating that GIPC (GAIP interacting protein, C<br />
terminus), a conserved cellular scaffold-type protein, has the capacity to function in a<br />
<strong>neuroprotective</strong> manner <strong>against</strong> α-syn-induced neurodegeneration. GIPC has been<br />
shown to interact with mammalian D2 and D3 receptors in heterologous cell cultures,<br />
where its expression appears to mediate endosomal trafficking and receptor stability<br />
(Jeanneteau et al., 2004). GIPC was originally identified in a screen for proteins that bind<br />
to GAIP (G-<strong>alpha</strong> interacting protein) (De Vries et al., 1998), a member <strong>of</strong> the large<br />
family called Regulators <strong>of</strong> G protein Signaling (RGS), yet GAIP is the only RGS protein<br />
that binds GIPC. Overexpression <strong>of</strong> GAIP has been shown to stimulate protein<br />
degradation via Gαi-mediated induction <strong>of</strong> autophagy in human intestinal cells (Ogier-<br />
Denis et al., 1997). It is interesting to speculate that GIPC serves to modulate a pre-<br />
synaptic protein-coupled pathway that can somehow combat the effects <strong>of</strong> α-syn<br />
misfolding and accumulation, perhaps by a DA or DA-receptor regulated manner.<br />
54
Our data demonstrating that the C. elegans F55A4.1 gene product, an ortholog <strong>of</strong><br />
Sec22p, is <strong>neuroprotective</strong> accentuates the importance <strong>of</strong> vesicular trafficking between<br />
the ER and Golgi as an integral process affected by α-syn. We previously hypothesized<br />
that α-syn-dependent blockage <strong>of</strong> vesicular trafficking could lead to the limitation <strong>of</strong><br />
available monoamine vesicular transporters (i.e., VMAT2) (Cooper et al., 2006). This<br />
would theoretically result in an excess pool <strong>of</strong> cytosolic DA and contribute to selective<br />
DA neurodegeneration. Indeed, α-syn overexpression may exacerbate this process,<br />
leading to increased cytosolic catecholamine concentration (Mosharov et al., 2006;<br />
Caudle et al., 2007). Thus, cellular proteins, like Sec22p or those in the Rab GTPase<br />
family (i.e., Rab1) that enhance vesicular trafficking and the removal <strong>of</strong> DA from the<br />
cytosol, likely contribute to neuroprotection by relieving this α-syn-mediated blockade<br />
(Cooper et al., 2006). While hypotheses focused on the intrinsic contributions <strong>of</strong> DA to<br />
cytotoxicity are appealing, it is important to remember that other neuronal subtypes are<br />
also susceptible in PD and that disruption <strong>of</strong> basic cellular functions has implications<br />
beyond the DA system.<br />
<strong>The</strong> candidate gene approach may limit the ability to make generalized<br />
conclusions about all possible gene families, pathways, or non-biased gene sets that could<br />
potentially be revealed by genome-wide screening. By design, the <strong>genes</strong> pre-selected for<br />
analysis in our RNAi screen will not reveal all possible effectors <strong>of</strong> α-syn misfolding and<br />
neuroprotection in C. elegans. <strong>The</strong>y are further limited by factors such as lethality and<br />
redundancy. Nevertheless, this focused strategy did not restrict the detection <strong>of</strong><br />
55
unexpected effectors, as evidenced by the <strong>identification</strong> <strong>of</strong> the F16A11.2 gene product,<br />
which has not been previously linked to neuroprotection. This protein, which contains an<br />
RNA-binding motif, has over 99% identity to an uncharacterized human gene product<br />
that is associated with neuronal RNA-rich granules where it may be involved in transport<br />
(Kanai et al., 2004). This is intriguing considering the recent discovery that miRNAs in<br />
DA neurons may play a role in neurodegenerative process (Kim et al., 2007).<br />
We contend that our a priori elimination <strong>of</strong> targets without significant homology<br />
to mammals, as well as prioritizing targets with putative relationships to known PD <strong>genes</strong><br />
via our “guilt by association” bioinformatics selection strategy, significantly enhanced<br />
our ability to identify functionally relevant effectors. For example, we determined that<br />
<strong>genes</strong> co-expressed with known PD <strong>genes</strong> that are also components <strong>of</strong> cellular pathways<br />
implicated in PD have a far greater likelihood <strong>of</strong> significantly effecting α-syn misfolding<br />
(17% vs. 3% strong positives for entire gene set; Fig. 2.7A). Furthermore, we discovered<br />
that 11% (3/28) <strong>genes</strong> co-expressed with both DJ-1 and PINK1 were significantly<br />
enriched within our top 20 hits, as compared to the other top candidates that represented a<br />
3% (17/757) hit rate (P
disparate datasets, such as those obtained from the initial genome-wide analyses on PD<br />
(Perez-Tur, 2006).<br />
Taken together, the results <strong>of</strong> this study indicate that further characterization <strong>of</strong><br />
the <strong>genes</strong> identified in our RNAi screen will yield additional insights into mediators <strong>of</strong> α-<br />
syn-induced cytotoxicity. An emerging model underlying PD involves dysfunction within<br />
a variety <strong>of</strong> intersecting pathways that maintain homeostasis via a compensatory balance<br />
between intracellular protein trafficking and degradation systems, as well as other<br />
signaling mechanisms triggered by stress. <strong>The</strong> manner by which α-syn impacts these<br />
mechanisms remains poorly defined and factors that influence the stability, production,<br />
and clearance <strong>of</strong> this protein likely represent effectors <strong>of</strong> disease onset and progression.<br />
Identification <strong>of</strong> critical cellular mediators within these processes will enhance<br />
development <strong>of</strong> biomarkers and therapeutic agents to halt this disease.<br />
57
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Xu, J., Zhong, N., Wang, H., Elias, J.E., Kim, C.Y., Woldman, I., Pifl, C., Gygi, S.P.,<br />
Geula, C., Yanker, B.A. (2005) Hum Molec Genet 14, 1231-1241.<br />
Zhong, W. & Sternberg, P.W. (2006) Science 311, 1481-1484.<br />
Zhou, W., Freed, C.R. (2005) J Biol Chem 280, 43150-43158.<br />
Zhou, Y., Gu, G., Goodlett, D.R., Zhang, T., Pan, C., Montine, T.J., Montine, K.S.,<br />
Aebersold, R.H., Zhang, J. (2004) J Biol Chem 279, 39155-39164.<br />
61
Table 2.1. Gene identities <strong>of</strong> the 20 top candidates isolated from RNAi screening.<br />
C. elegans Gene ID NCBI KOGs<br />
B0432.2 (djr-1.1) Putative transcriptional regulator DJ-1<br />
T05C3.5 (dnj-19) Molecular chaperone (DnaJ superfamily)<br />
C35D10.2 RGS-GAIP interacting protein GIPC<br />
C54H2.5 (sft-4) Putative cargo transport protein ERV29<br />
EEED8.9 (pink-1) BRPK/PTEN-induced protein kinase<br />
F11H8.1 (rfl-1) NEDD8-activating complex, catalytic component UBA3<br />
F16A11.2 Uncharacterized conserved protein<br />
F26E4.11 (hrdl-1) E3 ubiquitin ligase<br />
F32A6.3 (vps-41) Vacuolar assembly/sorting protein VPS41<br />
F48E3.7 (acr-22) Acetylcholine receptor<br />
F55A4.1 Synaptobrevin/VAMP-like protein SEC22<br />
F57B10.5 Emp24/gp25L/p24 family <strong>of</strong> membrane trafficking proteins<br />
F59F4.1 Acyl-CoA oxidase<br />
K11G12.4 (smf-1) Mn 2+ and Fe 2+ transporters <strong>of</strong> the NRAMP family<br />
M7.5 (atgr-7) Ubiquitin activating E1 enzyme-like protein<br />
R05D11.6 Transcription factor<br />
T07F12.4 Serine/threonine-protein kinase involved in autophagy<br />
T08D2.4 Tripartite motif-containing 32<br />
T13A10.2 Predicted E3 ubiquitin ligase<br />
Y37A1B.13 (tor-2) ATPase <strong>of</strong> the AAA+ superfamily<br />
62
Table 2.2. Bioinformatic associations among gene candidates identified by RNAi.<br />
C. elegans Gene ID Bioinformatic<br />
Association<br />
Component <strong>of</strong>:<br />
K11G12.4 (smf-1) PINK1 and DJ-1*<br />
R05D11.6 PINK1 and DJ-1*<br />
F26E4.11 (hrdl-1) PINK1 and DJ-1* ERAD<br />
C35D10.2 DJ-1** Autophagy<br />
M7.5 (atgr-7) UPS and Autophagy<br />
F32A6.3 (vps-41) UCHL-1* UPS and Autophagy<br />
*Co-expressed in microarray topology map, as per Kim et al. (2)<br />
**Identified within same protein interactome network, as per Li et al. (1)<br />
63
Table 2.3. Summary <strong>of</strong> the <strong>neuroprotective</strong> <strong>genes</strong> and their human homologs.<br />
C. elegans<br />
Gene ID<br />
Average %<br />
worms with<br />
wildtype<br />
DA<br />
neurons*<br />
NCBI KOGs<br />
F32A6.3<br />
(vps-41)<br />
36.7 + 5<br />
Vacuolar assembly/sorting<br />
protein VSP41<br />
C35D10.2 31.5 + 1<br />
RGS-GAIP interacting<br />
protein GIPC<br />
M7.5 (atgr-7) 31.1 + 1<br />
Ubiquitin activating E1<br />
enzyme-like protein<br />
F16A11.2 24.8 + 3<br />
Uncharacterized<br />
conserved protein<br />
F55A4.1 23.7 + 3<br />
Synaptobrevin/VAMPlike<br />
protein SEC22<br />
*Compared to 12.8% with a-syn alone.<br />
64<br />
Blast evalue<br />
Relevance to<br />
H. sapiens<br />
5.1e -96 95.7<br />
1.1e -49 75.4<br />
7.4e -87 69.2<br />
1.2e -207 99.8<br />
2.3e -47 96.3<br />
% length
Table 2.4. Results <strong>of</strong> all <strong>genes</strong> knocked down via RNAi screening. Light blue indicates<br />
125 positives from primary RNAi screen analyzed at young adult stage. Dark blue<br />
indicates 20 positives from secondary RNAi screen analyzed at L3 stage. Gray indicates<br />
lethal <strong>genes</strong>.<br />
Name Human Homolog Outcome<br />
B0024.6 Atrial natriuretic peptide receptor A precursor<br />
B0025.1 PI3-kinase catalytic subunit type 3<br />
B0035.14 DnaJ homolog subfamily B member 12<br />
B0035.2 GNG10<br />
B0205.3 PSMD4<br />
B0250.1 60S ribosomal protein L8<br />
B0272.1 Tubulin beta-2C chain<br />
B0281.1 Centrosomal protein Cep290<br />
B0281.3 Tripartite motif-containing protein 59<br />
B0281.8 GTP-binding protein ARD-1<br />
B0303.9 Vacuolar protein sorting-associated protein 33A<br />
B0336.8 APG12 autophagy 12-like<br />
B0361.10 Synaptobrevin homolog YKT6<br />
B0393.6 Tripartite motif-containing 10 is<strong>of</strong>orm 2<br />
B0403.2 Baculoviral IAP repeat-containing protein 6<br />
B0403.4 Protein disulfide isomerase A6 precursor<br />
B0414.8 Is<strong>of</strong>orm 1 <strong>of</strong> Uncharacterized protein C11orf2<br />
B0416.4 Tripartite motif-containing 32<br />
B0432.2 Protein DJ-1<br />
B0432.5 Tyrosine hydroxylase is<strong>of</strong>orm c<br />
B0511.1 Is<strong>of</strong>orm 2 <strong>of</strong> FK506-binding protein 7 precursor<br />
B0513.2 THO complex 7 homolog<br />
B0545.1 Protein kinase C, delta<br />
B0563.7 Calmodulin<br />
B0564.2 AlkB, alkylation repair homolog 6 is<strong>of</strong>orm 2<br />
C01A2.4 Charged multivesicular body protein 2b<br />
C01B7.6 Probable E3 ubiquitin-protein ligase MYCBP2<br />
C01G10.12 GNG10<br />
C01G12.5 DHRS4<br />
C01G6.4 RING finger protein 11<br />
C01G8.2 Is<strong>of</strong>orm 1 <strong>of</strong> Battenin<br />
65
C01G8.4 DnaJ homolog subfamily C member 4<br />
C02B8.6 RING finger protein 146<br />
C02F5.9 Proteasome subunit beta type 1 precursor<br />
C04A2.7 DnaJ homolog subfamily C member 14<br />
C04E12.4 N-glycanase<br />
C04E12.5 N-glycanase<br />
C04E6.5 Ubiquitin specific protease 30<br />
C04F12.10 CAAX prenyl protease 1 homolog<br />
C04F5.1 SID1 transmembrane family member 2<br />
C04G6.1 Mitogen-activated protein kinase 7, is<strong>of</strong>orm 1<br />
C04G6.3 Splice Is<strong>of</strong>orm PLD1A <strong>of</strong> Phospholipase D1<br />
C04H5.1 TRM112-like protein<br />
C05C10.6 Phospholipase A-2-activating protein<br />
C05C8.3 FK506 binding protein 9 precursor<br />
C05D10.2 Mitogen-activated protein kinase 15<br />
C05D11.2 Vacuolar protein sorting-associated protein 16<br />
C05D9.2 Lysosomal-associated membrane protein 1<br />
C05G5.2 Proteoglycan-4 precursor<br />
C06A1.1 Transitional endoplasmic reticulum ATPase<br />
C06A5.4 Unnamed protein<br />
C06A5.8 Tripartite motif-containing 32<br />
C06A5.9 SH3 domain containing ring finger 2<br />
C06E2.3 Ubiquitin-conjugating enzyme E2-25 kDa<br />
C06E2.7 Ubiquitin-conjugating enzyme E2-25 kDa<br />
C06H2.2 START domain containing 7<br />
C07A12.4 Protein disulfide isomerase precursor<br />
C07G2.3 T-complex protein 1, epsilon subunit<br />
C08A9.8 Uncharacterized protein<br />
C08B11.1 ZYG11B protein<br />
C08B11.7 Ubiquitin carboxyl-terminal hydrolase L5<br />
C08B11.8 ALG6<br />
C08F8.2 SUV3-like protein 1<br />
C08F8.8 Orphan nuclear receptor NR2E1<br />
C08H9.1 Lysosomal protective protein precursor<br />
C09B8.6 Heat-shock protein beta-1<br />
C09D1.1 Is<strong>of</strong>orm 2 <strong>of</strong> Titin<br />
C09D4.4 FAM135A<br />
66
C09G12.9 Tumor susceptibility gene 101 protein<br />
C09G4.3 Cyclin-dependent kinases regulatory subunit 1<br />
C10A4.8 ZNF690 protein<br />
C10C6.6 Probable cation-transporting ATPase 13A1<br />
C10G11.8 26S protease regulatory subunit 4<br />
C11D2.2 Is<strong>of</strong>orm 1 <strong>of</strong> Cathepsin E precursor<br />
C11H1.3 RING finger protein 157<br />
C12C8.1 Heat shock cognate 71 kDa protein<br />
C12C8.2 Cystathionine gamma-lyase<br />
C12C8.3 Tripartite motif-containing protein 71<br />
C12D8.1 KH-type splicing regulatory protein<br />
C13B9.2 Glycerate kinase<br />
C13B9.3 Coatomer subunit delta<br />
C13C12.1 Calmodulin<br />
C14A4.4 Exosome component 10<br />
C14A4.5 Exosome complex exonuclease RRP46<br />
C14B1.1 Protein disulfide isomerase precursor<br />
C14B9.1 Alpha crystallin B chain<br />
C14B9.2 Protein disulfide isomerase A4 precursor<br />
C14B9.4 Serine/threonine-protein kinase PLK2<br />
C14F11.5 Heat-shock protein beta-1<br />
C14F5.4 Sider<strong>of</strong>lexin-2<br />
C15F1.5 Keratin-associated protein 10-6<br />
C15F1.6 Synaptic glycoprotein SC2<br />
C15F1.7 Superoxide dismutase<br />
C15H9.6 78 kDa glucose-regulated protein precursor<br />
C16A11.5 ELKS/RAB6-interacting/CAST family member 1<br />
C16A3.8 THO complex 2 is<strong>of</strong>orm 1<br />
C16C10.5 RING finger protein 121<br />
C16C10.7 RING finger protein 185<br />
C16C8.11 Is<strong>of</strong>orm 4 <strong>of</strong> Nesprin-1<br />
C16C8.12 RUN and FYVE domain-containing protein<br />
C16C8.13 Synaptonemal complex protein 1<br />
C16C8.13 Synaptonemal complex protein 1<br />
C16C8.14 Centromere protein E<br />
C16C8.4 Ubiquitin C<br />
C16C8.5 Hypothetical protein<br />
67
C17D12.3 N-glycanase<br />
C17D12.5 Ubiquitin-conjugating enzyme E2 D1<br />
C17G10.1 OGFOD1<br />
C17H11.4 Ariadne-1 protein homolog variant (Fragment)<br />
C17H11.6 E3 ubiquitin-protein ligase RNF19<br />
C18A11.1 Unnamed protein<br />
C18A3.5 Nucleolysin TIA-1 is<strong>of</strong>orm p40<br />
C18B12.4 RING finger protein 13<br />
C18E9.1 Calmodulin<br />
C18E9.10 Vesicle transport protein SFT2C<br />
C18E9.2 Translocation protein SEC62<br />
C18H9.7 43kDa acetylcholine receptor-associated protein<br />
C23G10.8 Conserved hypothetical protein<br />
C23H3.4 Serine palmitoyltransferase 1<br />
C23H5.7 Cyclic nucleotide-gated olfactory channel<br />
C25B8.3 Cathepsin B precursor<br />
C25G4.4 GMEB1<br />
C26B9.6 RING finger protein 146<br />
C26E6.11 Cob<br />
C26E6.8 NEDD8-activating enzyme E1 regulatory subunit<br />
C26F1.4 Ubiquitin-like protein FUBI<br />
C27A12.6 Protein ariadne-1 homolog<br />
C27A12.7 Protein ariadne-1 homolog<br />
C27A12.8 Protein ariadne-1 homolog<br />
C27A7.3 ENPP3<br />
C27C12.2 Early growth response protein 1<br />
C27C12.4 ET putative translation product<br />
C27F2.5 Vacuolar sorting protein SNF8<br />
C27H5.3 Fus-like protein (Fragment)<br />
C28D4.1 Retinoic acid receptor RXR-beta<br />
C28G1.1 Ubiquitin-conjugating enzyme E2-25 kDa<br />
C28G1.3 Exocyst complex component Sec15B is<strong>of</strong>orm 1<br />
C28H8.1 BCL7A<br />
C30B5.1 KIAA0953 protein<br />
C30C11.2 PSMD3<br />
C30C11.4 Heat shock protein apg-1<br />
C30F2.2 Tripartite motif-containing 32<br />
68
C32B5.13 Cathepsin H precursor<br />
C32B5.7 Cathepsin H precursor<br />
C32D5.10 E3 ubiquitin-protein ligase Topors<br />
C32D5.11 E3 ubiquitin-protein ligase Topors<br />
C32D5.9 GABA receptor-associated protein<br />
C32E8.1 GTP-binding protein ARD-1<br />
C32E8.3 Protein p25-beta<br />
C32F10.1 Oxysterol-binding protein-related protein 9<br />
C32F10.6 Retinoic acid receptor<br />
C33H5.10 FAM98B<br />
C33H5.6 WD repeat protein 82<br />
C34B7.2 SAC domain-containing protein 3<br />
C34D4.12 Peptidyl-prolyl cis-trans isomerase like 1<br />
C34D4.14 E3 ligase for inhibin receptor<br />
C34E10.4 WARS2<br />
C34E10.5 Protein arginine N-methyltransferase 5<br />
C34F11.3 AMP deaminase 2<br />
C34F6.9 Deubiquitinating enzyme 3<br />
C35B1.1 Ubiquitin-conjugating enzyme E2A<br />
C35D10.2 PDZ domain-containing protein GIPC1<br />
C36A4.8 BRCA1<br />
C36B1.4 Proteasome subunit <strong>alpha</strong> type 7<br />
C36B1.7 Dihydr<strong>of</strong>olate reductase<br />
C36E8.5 Tubulin beta-2C chain<br />
C37H5.8 Stress-70 protein, mitochondrial precursor<br />
C38D4.3 Neur<strong>of</strong>ilament heavy polypeptide<br />
C39F7.2 Tripartite motif-containing protein 67<br />
C39F7.4 Ras-related protein Rab-1A<br />
C41C4.4 IRE1 precursor<br />
C41C4.8 Transitional endoplasmic reticulum ATPase<br />
C42C1.4 Vacuolar protein sorting 8 homolog is<strong>of</strong>orm b<br />
C42D8.8 Amyloid-like protein 2 precursor<br />
C44B11.3 Tubulin <strong>alpha</strong>-3 chain<br />
C44B7.1 PSMD9<br />
C44C1.4 Vacuolar protein sorting-associated protein 45<br />
C44E4.6 Is<strong>of</strong>orm 2 <strong>of</strong> Acyl-CoA-binding protein<br />
C44H4.5 MAP3K7 interacting protein 1 is<strong>of</strong>orm beta<br />
69
C45G7.4 Tripartite motif-containing 13<br />
C47A4.1 GNGT10<br />
C47B2.2 Protein FLJ31792<br />
C47B2.3 Tubulin <strong>alpha</strong>-3 chain<br />
C47B2.4 Proteasome subunit beta type 7 precursor<br />
C47E12.3 EDEM1<br />
C47E12.5 Ubiquitin-activating enzyme E1<br />
C48D5.1 Orphan nuclear receptor NR4A2<br />
C49C3.6 Trichoplein keratin filament-binding protein<br />
C49G7.11 Protein DJ-1<br />
C50C3.5 Calmodulin-like protein 5<br />
C50E10.4 Proteoglycan-4 precursor<br />
C50E3.3 C-type lectin<br />
C50F2.6 FK506-binding protein 9 precursor<br />
C50F4.3 Cathepsin H precursor<br />
C50H11.5 12 kDa protein<br />
C52B11.5 Ras-related protein Rab-5B<br />
C52E12.4 Hypothetical protein LOC160518<br />
C52E4.1 Cathepsin B precursor<br />
C52E4.4 26S protease regulatory subunit 7<br />
C53A3.2 Hypothetical protein LOC283871<br />
C53A5.2 tRNA (guanine-N(1)-)-methyltransferase<br />
C53A5.6 IPP protein<br />
C53D5.6 RAN binding protein 5<br />
C54C6.2 Tubulin beta-2C chain<br />
C54H2.3 RING1 and YY1-binding protein<br />
C54H2.5 Surfeit locus protein 4<br />
C55A6.1 RING finger protein 146<br />
C55B6.2 DnaJ homolog subfamily C member 3<br />
C56A3.4 RNF157 protein<br />
C56C10.1 Vacuolar protein sorting-associated protein 33B<br />
C56E10.3 Nuclear pore complex-associated protein TPR<br />
C56G2.15 Putative tumor suppressor FUS2<br />
CC8.2 Protein phosphatase 1 regulatory subunit 3D<br />
CD4.6 Proteasome subunit <strong>alpha</strong> type 1<br />
D1007.5 Is<strong>of</strong>orm 1 <strong>of</strong> Transmembrane protein 39A<br />
D1009.2 Peptidyl-prolyl cis-trans isomerase G<br />
70
D1014.3 Alpha-soluble NSF attachment protein<br />
D1022.1 Ubiquitin-conjugating enzyme E2 J1<br />
D1054.8 DHRS4<br />
D2007.5 Uncharacterized protein KIAA0652<br />
D2013.8 SREBP cleavage-activating protein<br />
D2030.7 Kaiso<br />
D2030.8 Family with sequence similarity 113, member B<br />
D2085.4 Ubiquitin-protein ligase E3C<br />
D2092.4 Protein disulfide-isomerase A5 precursor<br />
EEED8.5 ATP-dependent RNA helicase DHX8<br />
EEED8.8 ADP-ribose pyrophosphatase<br />
EEED8.9 Serine/threonine-protein kinase PINK1<br />
F01E11.2 Is<strong>of</strong>orm E <strong>of</strong> Proteoglycan-4 precursor<br />
F01F1.14 n/a<br />
F01F1.8 T-complex protein 1, zeta subunit<br />
F01G4.2 3-Hydroxyacyl-CoA dehydrogenase type-2<br />
F02E8.5 Autophagy protein 16-like<br />
F02E9.7 Tartrate-resistant acid ATPase<br />
F07A11.4 Ubiquitin specific protease 19<br />
F07E5.5 ZCCHC9 protein<br />
F08B12.2 Peroxisome assembly protein 12<br />
F08C6.3 Vacuolar protein sorting-associated protein 52<br />
F08D12.1 Signal recognition particle 72 kDa protein<br />
F08F8.2 HMG-CoA reductase<br />
F08G12.4 Von Hippel-Lindau-like protein<br />
F08G12.5 Tripartite motif-containing 13<br />
F08H9.3 Alpha crystallin B chain<br />
F08H9.4 Alpha crystallin B chain<br />
F09B9.3 ER lumen protein retaining receptor 1<br />
F09C12.2 Mitogen-activated protein kinase 7, is<strong>of</strong>orm 1<br />
F09D1.1 U4/U6.U5 tri-snRNP-associated protein 2<br />
F09G2.9 Neur<strong>of</strong>ilament heavy polypeptide<br />
F10C2.5 EDEM2<br />
F10C5.1 Cell division cycle protein 23<br />
F10D11.1 SOD2<br />
F10D7.5 Neuralized-like protein<br />
F10E7.8 Is<strong>of</strong>orm 1 <strong>of</strong> Protein FAM40A<br />
71
F10E9.6 APBB1<br />
F10G7.2 SND1<br />
F10G7.8 PSMD12<br />
F10G7.9 Neur<strong>of</strong>ilament heavy polypeptide<br />
F11A10.3 Polycomb group RING finger protein 3<br />
F11H8.1 NEDD8-activating enzyme E1 catalytic subunit<br />
F12A10.5 Calmodulin<br />
F12F6.6 Protein transport protein Sec24C<br />
F12F6.7 DNA polymerase subunit delta 2<br />
F13H10.2 NUDT13<br />
F13H10.4 Mannosyl-oligosaccharide glucosidase<br />
F14D2.11 Polyubiquitin 9<br />
F14F4.3 Multidrug resistance-associated protein 5<br />
F15C11.2 Is<strong>of</strong>orm 2 <strong>of</strong> Ubiquilin-1<br />
F15D4.4 Cathepsin S precursor<br />
F15H10.3 Anaphase-promoting complex subunit 10<br />
F15H10.4 E3 ubiquitin-protein ligase RNF19<br />
F16A11.1 RSPRY1<br />
F16A11.2 UPF0027 protein C22orf28<br />
F16D3.1 Tubulin <strong>alpha</strong>-ubiquitous chain<br />
F17C11.8 Vacuolar protein sorting-associated protein 36<br />
F17E5.1 CAMK1<br />
F18C5.2 Werner syndrome ATP-dependent helicase<br />
F19B10.10 Serologically defined colon cancer antigen 8<br />
F19B10.2 Coiled-coil domain-containing protein 123<br />
F19B6.2 Ubiquitin fusion degradation protein 1 homolog<br />
F19G12.1 Tripartite motif-containing protein 2<br />
F19H8.1 Trehalose-6-phosphate synthase<br />
F20C5.6 Myosin-9<br />
F20D1.9 Mitochondrial glutamate carrier 2<br />
F21C3.3 Histidine triad nucleotide-binding protein 1<br />
F21D5.7 Signal recognition particle 54 kDa protein<br />
F21F8.2 Gastricsin precursor<br />
F21F8.3 43 kDa protein<br />
F21F8.4 43 kDa protein<br />
F21F8.7 43 kDa protein<br />
F22B5.7 Cytoskeleton-associated protein 5<br />
72
F22B7.5 DNAJA3, mitochondrial precursor<br />
F22B8.6 Cystathionine gamma-lyase<br />
F22E10.5 Choline/ethanolaminephosphotransferase<br />
F23F1.8 26S protease regulatory subunit S10B<br />
F23F12.6 26S protease regulatory subunit 6B<br />
F25B5.4 Ubiquitin C<br />
F25C8.1 Acyl-coenzyme A oxidase 1, peroxisomal<br />
F25D7.3 PRDM1<br />
F25G6.8 Signal recognition particle 14 kDa protein<br />
F25H2.8 Ubiquitin-conjugating enzyme E2 Q2<br />
F25H2.9 Proteasome subunit <strong>alpha</strong> type 5<br />
F25H5.6 39S ribosomal protein 54<br />
F26D10.3 Heat shock cognate 71 kDa protein<br />
F26D2.15 DHRS4<br />
F26E4.11 Autocrine motility factor receptor, is<strong>of</strong>orm 2<br />
F26E4.4 Cell death regulator Aven<br />
F26E4.6 Cytochrome c oxidase polypeptide VIIc<br />
F26E4.8 Tubulin <strong>alpha</strong>-3 chain<br />
F26E4.9 Cytochrome c oxidase polypeptide Vb<br />
F26F12.2 Uncharacterized protein<br />
F26F4.1 UPF0363 protein C7orf20<br />
F26F4.7 Tripartite motif-containing protein 2<br />
F26G5.9 PAX interacting protein 1<br />
F26H9.6 Ras-related protein Rab-5B<br />
F26H9.7 Ubiquitin-conjugating enzyme E2 N<br />
F27B3.5 CENPE variant protein (Fragment)<br />
F28A12.4 Gastricsin precursor<br />
F28C12.5 Sphingosine 1-phosphate receptor Edg-3<br />
F28C6.4 GPI ethanolamine phosphate transferase 2<br />
F28H7.2 DHRS4<br />
F29B9.6 SUMO-conjugating enzyme UBC9<br />
F29C4.5 Ubiquitin carboxyl-terminal hydrolase 12<br />
F29D10.4 Myosin-If<br />
F30A10.10 Ubiquitin carboxyl-terminal hydrolase 48<br />
F30F8.8 Transcription initiation factor TFIID subunit 5<br />
F31D4.3 FK506-binding protein 4<br />
F31E3.5 Elongation factor 1-<strong>alpha</strong> 2<br />
73
F31E8.2 Synaptotagmin-1<br />
F32A5.3 Lysosomal protective protein precursor<br />
F32A6.3 Vacuolar protein sorting-associated protein 41<br />
F32B5.8 Cathepsin Z precursor<br />
F32H2.4 THO complex subunit 3<br />
F32H5.1 Cathepsin B precursor<br />
F33D11.9 GPAA1<br />
F33H2.6 Protein FAM82B<br />
F35B3.1 Ubiquitin specific protease 11<br />
F35D11.11 Trichohyalin<br />
F35F10.1 N-glycanase<br />
F35G12.12 PSMD5<br />
F35G12.9 Anaphase-promoting complex subunit 11<br />
F35H10.7 Chromosome 16 open reading frame 35<br />
F36A2.1 Uncharacterized protein KIAA0460<br />
F36A2.13 EDD1 protein<br />
F36D3.9 Cathepsin B precursor<br />
F36F2.3 Retinoblastoma-binding protein 6<br />
F36H1.1 FK506-binding protein 2 precursor<br />
F37A4.1 HLA-B associated transcript 5<br />
F37A4.5 PSMD14<br />
F37B12.4 Ubiquitin carboxyl-terminal hydrolase 24<br />
F37C12.1 Coiled-coil domain-containing protein 94<br />
F37C12.14 n/a<br />
F37F2.2 Signal recognition particle 19 kDa protein<br />
F38A1.8 SRPR protein, <strong>alpha</strong> subunit<br />
F38A5.13 Zuotin-related factor 1<br />
F38B7.5 Ubiquitin carboxyl-terminal hydrolase 29<br />
F38C2.4 Uncharacterized protein<br />
F38E11.1 Alpha crystallin B chain<br />
F38E11.2 Alpha crystallin B chain<br />
F38E11.5 Coatomer subunit beta'<br />
F38H4.9 PPP2CB<br />
F39B2.10 DnaJ homolog subfamily A member 1<br />
F39B2.2 Ubiquitin-conjugating enzyme E2 variant 1<br />
F39H11.5 Proteasome subunit beta type 4 precursor<br />
F40E3.3 RIG<br />
74
F40F4.5 Tubulin <strong>alpha</strong>-ubiquitous chain<br />
F40F9.8 Calmodulin<br />
F40G9.1 PSMD10<br />
F40G9.12 Tripartite motif-containing 32<br />
F40G9.3 Ubiquitin-conjugating enzyme E2-25 kDa<br />
F41E6.13 WIPI-2<br />
F41E6.6 Cathepsin F precursor<br />
F41E6.9 Charged multivesicular body protein 5<br />
F41H10.3 Conserved hypothetical protein<br />
F42A6.6 Uncharacterized protein C11orf73<br />
F42C5.8 40S ribosomal protein S8<br />
F42G2.5 VAPA<br />
F42G8.3 Mitogen-activated protein kinase 14<br />
F42G8.4 Mitogen-activated protein kinase 14<br />
F42G9.2 Peptidylprolyl isomerase B precursor<br />
F42G9.9 Microtubule-associated protein 4 is<strong>of</strong>orm 2<br />
F43C1.2 Mitogen-activated protein kinase 1<br />
F43C9.2 Calcium-binding protein 4<br />
F43D9.4 Alpha crystallin B chain<br />
F43E2.8 78 kDa glucose-regulated protein precursor<br />
F43G6.8 Tripartite motif-containing 32<br />
F44B9.5 Ancient ubiquitous protein 1 precursor<br />
F44C8.10 Hepatocyte nuclear factor 4, gamma<br />
F44C8.3 Orphan nuclear receptor TR4<br />
F44C8.9 Hepatocyte nuclear factor 4 <strong>alpha</strong> is<strong>of</strong>orm f<br />
F44D12.10 Zinc finger protein 407<br />
F44E5.4 Heat shock cognate 71 kDa protein<br />
F44E5.5 Heat shock cognate 71 kDa protein<br />
F44E7.2 Hypothetical protein LOC283871<br />
F44F4.11 Tubulin <strong>alpha</strong>-ubiquitous chain<br />
F44G3.9 Photoreceptor-specific nuclear receptor<br />
F45E6.2 ATF-6 <strong>alpha</strong><br />
F45G2.6 TNF receptor-associated factor 3<br />
F45H11.2 NEDD8 precursor<br />
F45H7.6 E3 ubiquitin-protein ligase HECW1<br />
F46C3.1 eIF-2 <strong>alpha</strong> kinase 3<br />
F46E10.8 Ubiquitin carboxyl-terminal hydrolase L1<br />
75
F46F11.4 Ubiquitin-like protein 5<br />
F46F6.2 Serine/threonine-protein kinase N2<br />
F47C10.7 Retinoic acid receptor RXR-<strong>alpha</strong><br />
F47D12.4 High mobility group protein B2<br />
F47G4.4 Katanin p80 subunit B 1<br />
F47G9.4 Is<strong>of</strong>orm 2 <strong>of</strong> Midline-2<br />
F48C1.1 Alpha-mannosidase 2<br />
F48E3.7 CHRNA9<br />
F48G7.10 Protein kinase C, epsilon type<br />
F48G7.9 Protein kinase C, epsilon type<br />
F49C12.11 Coiled-coil domain-containing protein 72<br />
F49C12.9 Ubiquitin-like protein 7<br />
F49E12.4 18 kDa protein<br />
F49E7.1 GTPase activating protein and VPS9 domains 1<br />
F49E8.3 Puromycin-sensitive aminopeptidase<br />
F52C6.12 Ubiquitin-conjugating enzyme E2 D4<br />
F52C6.2 Ribosomal protein S27a<br />
F52C6.3 Ubiquitin<br />
F52C6.4 Ubiquitin<br />
F52C6.8 Kelch-like protein 28<br />
F52D1.1 Neutral <strong>alpha</strong>-glucosidase AB precursor<br />
F52D10.3 14-3-3 protein zeta/delta<br />
F52F12.3 Mitogen-activated protein kinase kinase kinase 7<br />
F53A2.6 Eukaryotic translation initiation factor 4E<br />
F53C11.5 Protein enabled homolog<br />
F53C3.13 Lipid phosphate phosphohydrolase 1<br />
F53F8.1 Krueppel-like factor 3<br />
F53F8.3 Tripartite motif-containing 32<br />
F53G12.4 FAM133B<br />
F53G2.7 CDK-activating kinase assembly factor MAT1<br />
F53H8.1 AP-3 complex subunit mu-1<br />
F54A3.3 T-complex protein 1, gamma subunit<br />
F54B11.5 RING finger protein 141<br />
F54C1.7 Calmodulin<br />
F54C9.2 STCH<br />
F54D5.8 DNAJB5 protein<br />
F54D8.2 Cytochrome c oxidase subunit Via<br />
76
F54F7.5 Serine/threonine kinase 24<br />
F54G8.4 Tripartite motif-containing protein 3<br />
F55A11.3 E3 ubiquitin-protein ligase synoviolin precursor<br />
F55A12.8 N-acetyltransferase 10<br />
F55A4.1 Vesicle-trafficking protein SEC22b<br />
F55B12.3 F-box/WD repeat protein 7<br />
F55C5.7 Ribosomal protein S6 kinase delta-1<br />
F55C5.8 Signal recognition particle 68 kDa protein<br />
F55D10.1 Lysosomal <strong>alpha</strong>-mannosidase precursor<br />
F55G1.5 Mitochondrial glutamate carrier 2<br />
F55H2.1 Superoxide dismutase<br />
F55H2.5 Cytochrome b561<br />
F56C9.1 PPP1CA<br />
F56D12.5 SERBP1<br />
F56D2.2 E3 ubiquitin-protein ligase RNF14<br />
F56D2.4 SUMO-conjugating enzyme UBC9<br />
F56G4.2 Unnamed protein<br />
F56G4.5 N-glycanase 1<br />
F56H1.4 26S protease regulatory subunit 6A<br />
F57B10.10 DAD1<br />
F57B10.11 BCL2-associated athanogene<br />
F57B10.5 TMED7<br />
F57B9.10 Proteasome 26S non-ATPase subunit 11 variant<br />
F57F5.1 Cathepsin B precursor<br />
F57F5.5 Protein kinase C, eta<br />
F58A4.10 Ubiquitin-conjugating enzyme E2 G1<br />
F58A4.8 Tubulin gamma-1 chain<br />
F58B6.3 9 kDa protein<br />
F59B2.3 CGI-14 protein<br />
F59B2.5 Proteasome 26S non-ATPase subunit 11 variant<br />
F59D6.2 Gastricsin precursor<br />
F59D6.3 Gastricsin precursor<br />
F59E10.2 Peptidyl-prolyl cis-trans isomerase-like 2<br />
F59E12.2 CaM kinase ID<br />
F59E12.4 Nuclear protein localization protein 4 homolog<br />
F59E12.5 Nuclear protein localization protein 4 homolog<br />
F59E12.6 Ubiquitin carboxyl-terminal hydrolase 25<br />
77
F59F3.5 VEGFR-1<br />
F59F4.1 Acyl-coenzyme A oxidase 1, peroxisomal<br />
F59G1.3 Vacuolar protein sorting-associated protein 35<br />
H05L14.2 Golgin subfamily B member 1<br />
H06O01.1 Protein disulfide isomerase A3 precursor<br />
H08M01.1 Golgin subfamily A member 2<br />
H10E21.4 Calmodulin<br />
H10E21.5 RING finger protein 150 precursor<br />
H12I13.2 Ubiquitin specific protease 3<br />
H15N14.2 Vesicle-fusing ATPase<br />
H19N07.2 Ubiquitin-specific protease 7 is<strong>of</strong>orm<br />
H19N07.4 Diacylglycerol O-acyltransferase 1<br />
H21P03.3 Sphingomyelin synthase 2<br />
H22K11.1 Cathepsin D precursor<br />
H34C03.2 Ubiquitin carboxyl-terminal hydrolase 4<br />
H38K22.2 DCN1-like protein 1<br />
H38K22.3 Cytochrome b5 domain containing 2<br />
JC8.10 Synaptojanin-1<br />
K01A2.11 Intestinal mucin<br />
K01C8.10 T-complex protein 1, delta subunit<br />
K01C8.5 Cylicin-1<br />
K01C8.6 39S ribosomal protein L10<br />
K01G5.1 RING finger protein 113A<br />
K01G5.7 Tubulin beta-2C chain<br />
K02A11.1 PPP1R16A<br />
K02B12.3 Prolactin regulatory element-binding protein<br />
K02C4.3 Ubiquitin carboxyl-terminal hydrolase 25<br />
K02E7.10 Cathepsin H precursor<br />
K02F3.10 Apolipoprotein O-like precursor<br />
K02G10.8 DnaJ homolog subfamily C member 5<br />
K03A1.4 Calmodulin<br />
K04G2.4 Hypothetical protein<br />
K05F1.5 Leukocyte cell-derived chemotaxin 2 precursor<br />
K06H7.3 Zinc finger protein 744<br />
K07A1.7 Headcase protein homolog<br />
K07A1.8 ERGIC-53 protein precursor<br />
K07A12.4 HBS1-like protein<br />
78
K07A9.2 CAMK1<br />
K07C11.9 Hypothetical protein DKFZp313D191<br />
K07C5.1 Actin-like protein 2<br />
K07D4.3 PSMD14<br />
K07F5.12 Transmembrane protein 144<br />
K08A8.1 Mitogen-activated protein kinase kinase 7<br />
K08B12.5 Serine/threonine-protein kinase MRCK <strong>alpha</strong><br />
K08B4.5 Ubiquitin-specific protease 7 is<strong>of</strong>orm<br />
K08D10.2 DNAJC20, mitochondrial precursor<br />
K08D12.1 Proteasome subunit beta type 6 precursor<br />
K08E3.7 Is<strong>of</strong>orm 1 <strong>of</strong> Parkin<br />
K09A9.4 Ubiquitin carboxyl-terminal hydrolase 33<br />
K09E3.7 Mucin-2 precursor<br />
K09E4.2 Dolichyl-P-Man:Man<br />
K09F6.7 Tripartite motif-containing protein 2<br />
K09H11.7 Hypothetical protein LOC283871<br />
K09H9.2 DCC1<br />
K10B2.2 Lysosomal protective protein precursor<br />
K10C2.3 Gastricsin precursor<br />
K10C3.2 Is<strong>of</strong>orm 1 <strong>of</strong> Alpha-endosulfine<br />
K11E8.1 CAMK2G<br />
K11G12.4 Divalent metal transporter<br />
K12B6.8 E3 ubiquitin-protein ligase DZIP3<br />
K12C11.2 Small ubiquitin-related modifier 1 precursor<br />
K12C11.4 Death-associated protein kinase 1<br />
M02A10.3 E3 ubiquitin-protein ligase CBL-B<br />
M04G12.1 interferon regulatory factor 2 binding protein 2<br />
M110.4 eIF-4-gamma 3<br />
M116.2 Is<strong>of</strong>orm GN-1L <strong>of</strong> Glycogenin-1<br />
M117.2 14-3-3 protein zeta/delta<br />
M117.3 YWHAB<br />
M142.6 Is<strong>of</strong>orm 1 <strong>of</strong> Roquin<br />
M151.3 Hook-related protein 1<br />
M28.5 NHP2-like protein 1<br />
M7.1 Ubiquitin-conjugating enzyme E2 D2<br />
M7.5 Autophagy-related protein 7<br />
R01H2.6 Ubiquitin-conjugating enzyme E2 L3<br />
79
R02D3.4 Uncharacterized protein C12orf11<br />
R02D3.5 FNTA<br />
R02E12.4 Tektin-1<br />
R02F11.4 LRRIQ2<br />
R03G5.1 Elongation factor 1-<strong>alpha</strong> 2<br />
R03G5.2 MAP2K6<br />
R04A9.2 Eukaryotic translation initiation factor 2C, 1<br />
R05D11.6 Similar to RIKEN cDNA A430101B06 gene<br />
R05D3.1 Is<strong>of</strong>orm Beta-1 <strong>of</strong> DNA topoisomerase 2-beta<br />
R05D3.4 E3 ubiquitin-protein ligase BRE1A<br />
R06C7.2 Translation initiation factor 2C<br />
R06F6.2 Vacuolar protein sorting-associated protein 11<br />
R07B7.16 Orphan nuclear receptor NR5A2<br />
R07E3.1 Cathepsin F precursor<br />
R07E4.1 ANKFN1<br />
R07E5.1 GPATCH1<br />
R07G3.3 Nucleoprotein TPR<br />
R07H5.2 Carnitine O-palmitoyltransferase 2<br />
R07H5.3 Uncharacterized protein C3orf60<br />
R09B3.4 NEDD8-conjugating enzyme UBE2F<br />
R09F10.1 Cathepsin F precursor<br />
R10A10.2 RING-box protein 2<br />
R10D12.14 Grb10- interacting GYF protein 2<br />
R10E11.2 ATP6V0C<br />
R10E11.3 Ubiquitin carboxyl-terminal hydrolase 46<br />
R10E11.8 ATP6V0C<br />
R11A5.1 AP-3 complex subunit beta-2<br />
R12E2.13 Stromal cell-derived factor 2 precursor<br />
R12E2.3 PSMD7<br />
R12G8.1 Protein kinase C, eta<br />
R12H7.2 Cathepsin D precursor<br />
R13D7.7 Glutathione S-transferase pi<br />
R151.6 Derlin-2<br />
R151.7 TRAP1<br />
R166.2 Cleft lip and palate transmembrane protein 1<br />
R186.3 SRPR protein, beta subunit<br />
R74.3 X-box binding protein 1<br />
80
R74.4 DNAJB1 precursor<br />
T01B7.4 Peptidyl-prolyl cis-trans isomerase H<br />
T01C3.3 E3 ubiquitin-protein ligase RNF8<br />
T01C8.1 PRKAA2<br />
T01D1.1 Heme oxygenase 2<br />
T01D3.3 IgGFc-binding protein precursor<br />
T01G1.1 Kinesin family member 21B<br />
T01G5.7 E3 ubiquitin-protein ligase RNF8<br />
T01G6.1 DHRS4<br />
T01H3.2 DKFZP434B0335 protein<br />
T02C1.1 Tripartite motif-containing protein 5<br />
T03E6.7 Cathepsin L precursor<br />
T03F1.1 UBE1L<br />
T03F6.2 DNAJA5<br />
T03G11.4 MAN1B1<br />
T03G11.6 Methyltransferase like 9 is<strong>of</strong>orm 1<br />
T04A8.16 Calpain-7<br />
T04A8.7 1,4-<strong>alpha</strong>-glucan branching enzyme<br />
T04A8.9 DnaJ homolog subfamily B member 7<br />
T04C9.4 Cysteine and glycine-rich protein 2<br />
T04G9.3 VIP36 precursor<br />
T04H1.9 Tubulin beta-2C chain<br />
T05A12.4 SNF2 histone linker PHD RING helicase<br />
T05C12.7 T-complex protein 1, <strong>alpha</strong> subunit<br />
T05C3.5 DnaJ homolog subfamily A member 2<br />
T05D4.1 Fructose-bisphosphate aldolase A<br />
T05E11.3 Endoplasmin precursor<br />
T05E11.5 Minor histocompatibility antigen H13<br />
T05E11.6 GPI-anchor transamidase precursor<br />
T05H10.1 Ubiquitin carboxyl-terminal hydrolase 47<br />
T05H10.5 Is<strong>of</strong>orm 2 <strong>of</strong> Ubiquitin conjugation factor E4 B<br />
T05H4.4 NADH-cytochrome b5 reductase<br />
T06C10.3 Proto-oncogene c-fes variant 3<br />
T06D8.8 HSPC027<br />
T06E8.1 AGPAT2<br />
T06G6.4 Centromere protein F<br />
T07F12.4 Serine/threonine-protein kinase ULK2<br />
81
T07G12.1 Calmodulin<br />
T08D2.4 Tripartite motif-containing 32<br />
T08G5.5 Vam6/Vps39-like protein<br />
T09A5.11 DDOST<br />
T09B4.10 STUB1<br />
T09B4.4 Calmodulin-like 4 is<strong>of</strong>orm 1<br />
T09E8.3 Cornichon homolog<br />
T10B11.6 Transmembrane protein 53<br />
T10B5.5 T-complex protein 1, eta subunit<br />
T10D4.6 Tetra-peptide repeat homeobox-like protein<br />
T10F2.3 Sentrin-specific protease 1<br />
T10H4.12 Cathepsin B precursor<br />
T11F1.8 Predicted receptor<br />
T12E12.1 Protein ariadne-2 homolog<br />
T13A10.11 S-adenosylmethionine synthetase<br />
T13A10.2 Tripartite motif-containing protein 2<br />
T13H2.3 Plectin 1 is<strong>of</strong>orm 3<br />
T14G10.4 Unnamed protein<br />
T14G8.3 150 kDa oxygen-regulated protein precursor<br />
T16H12.2 CBF1-interacting corepressor<br />
T18H9.2 Gastricsin precursor<br />
T19B4.4 DnaJ homolog subfamily C member 15<br />
T19E7.3 Beclin-1<br />
T19H12.2 Acidic nuclear phosphoprotein pp32<br />
T20B5.1 AP-2 complex subunit <strong>alpha</strong>-1<br />
T20D4.13 N-glycanase<br />
T20D4.3 N-glycanase<br />
T20D4.5 N-glycanase<br />
T20F5.2 Proteasome subunit beta type 2<br />
T20F5.6 Tripartite motif-containing protein 2<br />
T20F5.7 Tripartite motif-containing 32<br />
T21B10.7 T-complex protein 1, beta subunit<br />
T21C9.2 Vacuolar protein sorting-associated protein 54<br />
T21D12.3 Polyglutamine-binding protein 1<br />
T21H3.3 Calmodulin<br />
T22A3.2 Alpha crystallin B chain<br />
T22B2.1 Restin<br />
82
T22D1.9 PSMD2<br />
T22F3.2 Ubiquitin specific protease 42<br />
T22H9.2 Autophagy-related protein 9A<br />
T23B12.7 DnaJ homolog subfamily C member 17<br />
T23C6.5 Neuropeptide FF receptor 2<br />
T23G11.2 Glucosamine 6-phosphate N-acetyltransferase<br />
T23G11.3 Is<strong>of</strong>orm 4 <strong>of</strong> Quaking protein<br />
T23G5.2 SEC14-like protein 1<br />
T23G5.5 SLC6A2 protein<br />
T24D1.2 CROP<br />
T24H10.3 DnaJ homolog subfamily C member 9<br />
T25E12.4 Serine/threonine-protein kinase D3<br />
T25G3.4 Glycerol-3-phosphate dehydrogenase<br />
T26A5.4 ALG1<br />
T26C12.3 Ras-related protein Rap-2c precursor<br />
T27A1.5 SLC36A2<br />
T27A3.2 Ubiquitin carboxyl-terminal hydrolase 5<br />
T27A3.6 MOCS2<br />
T27A8.2 Hepatocyte nuclear factor 3-gamma<br />
T27C10.6 Leucine-rich repeat kinase 1<br />
T27D1.1 Peptidyl-prolyl cis-trans isomerase G<br />
T27E4.2 Alpha crystallin B chain<br />
T27E4.8 Alpha crystallin B chain<br />
T27E9.3 Cell division protein kinase 5<br />
T27F7.1 Charged multivesicular body protein 3<br />
T28D6.2 Tubulin <strong>alpha</strong>-6 chain<br />
T28F12.2 Homeobox protein Meis2<br />
T28H10.3 Legumain precursor<br />
VF13D12L.1 Myo-inositol 1-phosphate synthase A1<br />
VF39H2L.1 Syntaxin-17<br />
W01G7.4 SLC7A6OS<br />
W01H2.2 Hypothetical protein FLJ45999<br />
W02A11.3 RING finger protein 44<br />
W02A11.4 SUMO-activating enzyme subunit 2<br />
W02D3.10 Uncharacterized membrane protein KIAA1794<br />
W03A5.7 DNAJB6<br />
W03C9.3 Ras-related protein Rab-7<br />
83
W03F8.3 Mitochondrial translational release factor 1-like<br />
W03F8.4 TP53RK-binding protein<br />
W04G5.4 N-glycanase<br />
W04G5.9 Intestinal mucin<br />
W04H10.3 Tripartite motif protein 3<br />
W06B4.2 N-acetylglucosamine kinase<br />
W06B4.3 Vacuolar protein sorting-associated protein 18<br />
W06E11.4 SBDS<br />
W07A8.2 85 kDa Calcium-independent phospholipase A2<br />
W07A8.3 Putative tyrosine-protein phosphatase auxilin<br />
W07B3.2 Trichohyalin<br />
W07B8.1 Cathepsin B precursor<br />
W07E11.1 Dihydropyrimidine dehydrogenase [NADP+]<br />
W08D2.5 Probable cation-transporting ATPase 13A3<br />
W09C5.2 Is<strong>of</strong>orm 1 <strong>of</strong> Septin-7<br />
W09C5.8 Cytochrome c oxidase subunit IV is<strong>of</strong>orm 1<br />
W09G12.5 SRPR protein, <strong>alpha</strong> subunit<br />
W09G12.8 Fibronectin type III SPRY domain containing 2<br />
Y106G6H.12 Ubiquitin carboxyl-terminal hydrolase 29<br />
Y110A2AR.2 Ubiquitin conjugating enzyme E2, J2 is<strong>of</strong>orm 2<br />
Y110A7A.14 Proteasome subunit <strong>alpha</strong> type 4<br />
Y110A7A.19 Pentatricopeptide repeat domain 3<br />
Y113G7A.3 Protein transport protein Sec23A<br />
Y17G9B.4 40 kDa peptidyl-prolyl cis-trans isomerase<br />
Y18D10A.25 FKBP1A protein<br />
Y19D2B.1 Tubulin <strong>alpha</strong>-6 chain<br />
Y25C1A.5 Coatomer beta subunit<br />
Y37A1B.12 Torsin A precursor<br />
Y37A1B.13 Torsin A precursor<br />
Y37A1B.15 TUBA<br />
Y37A1B.2 SH3 and PX domain containing 3<br />
Y37D8A.14 Cytochrome c oxidase polypeptide Va<br />
Y37H9A.6 Bis(5'-nucleosyl)-tetraphosphatase<br />
Y38A10A.5 Calreticulin precursor<br />
Y38A8.2 Proteasome subunit beta type 3<br />
Y38E10A.8 eIF-2 <strong>alpha</strong> kinase 3<br />
Y38F1A.2 RING finger protein 170<br />
84
Y38F2AL.3 Vacuolar ATP synthase subunit C<br />
Y38F2AL.4 ATP6V0C<br />
Y38H8A.2 Tripartite motif-containing 39<br />
Y39A1C.2 Ubiquitin protein ligase E3B<br />
Y39A3CR.8 Conserved hypothetical protein<br />
Y39B6A.1 Hornerin<br />
Y39B6A.20 Gastricsin precursor<br />
Y39B6A.22 Gastricsin precursor<br />
Y39B6A.24 Gastricsin precursor<br />
Y39E4A.2 Solute carrier family 30, member 2 is<strong>of</strong>orm 1<br />
Y39E4B.1 ATP-binding cassette sub-family E member 1<br />
Y39G10AR.13 Inner centromere protein antigens 135/155kDa<br />
Y39H10A.7 Serine/threonine-protein kinase Chk1<br />
Y40D12A.2 Lysosomal protective protein precursor<br />
Y40G12A.1 Ubiquitin carboxyl-terminal hydrolase L3<br />
Y40H7A.9 Dipeptidyl-peptidase 1 precursor<br />
Y41E3.7 Golgi resident protein GCP60<br />
Y43C5B.2 Proto-oncogene tyrosine-protein kinase FER<br />
Y43E12A.3 KBTBD4<br />
Y45F10A.6 TBC1 domain family, member 9<br />
Y45F10B.8 Tripartite motif-containing 32<br />
Y45F10B.9 Tripartite motif-containing 32<br />
Y46H3A.2 Alpha crystallin B chain<br />
Y46H3A.3 Alpha crystallin B chain<br />
Y47G6A.22 3-Hydroxybutyrate dehydrogenase type 2<br />
Y47H9A.1 N-glycanase<br />
Y48A5A.1 SHQ1 homolog<br />
Y48G8AL.1 HERC4<br />
Y48G9A.11 PR domain zinc finger protein 5<br />
Y49E10.1 26S protease regulatory subunit 8<br />
Y49E10.20 Lysosome membrane protein 2<br />
Y49E10.4 Protein disulfide isomerase A6 precursor<br />
Y49F6B.9 E3 ubiquitin-protein ligase RNF19<br />
Y4C6A.3 Tripartite motif-containing 32<br />
Y51A2D.5 Proton myo-inositol cotransporter<br />
Y51H4A.17 Signal transducer and activator <strong>of</strong> transcription 6<br />
Y51H4A.8 n/a<br />
85
Y52B11C.1 PIGL<br />
Y53C10A.12 Heat shock factor protein 1<br />
Y53C10A.2 Microtubule-associated protein 1A<br />
Y53C12B.2 RNA-binding protein PNO1<br />
Y53F4B.4 NSUN5<br />
Y53H1A.2 Zinc finger protein 443<br />
Y54E10A.6 Leucine-rich repeat-containing protein 47<br />
Y54E10BL.4 DnaJ homolog subfamily C member 3<br />
Y54E10BR.1 GPI ethanolamine phosphate transferase 1<br />
Y54E10BR.2 ARF-related protein 1<br />
Y54E10BR.3 RING finger protein 126<br />
Y54E10BR.4 Phosphorylation regulatory protein HP-10<br />
Y54E2A.12 CDNA FLJ45119 fis, clone BRAWH3035914<br />
Y54E5B.4 Probable ubiquitin-conjugating enzyme E2 W<br />
Y55D9A.2 AGGF1<br />
Y55F3AM.3 RNA-binding protein 39<br />
Y55F3AM.4 Autophagy-related protein 3<br />
Y55F3AM.6 Makorin-1<br />
Y55F3AR.3 T-complex protein 1, theta subunit<br />
Y55F3BR.1 ATP-dependent RNA helicase DDX1<br />
Y55F3BR.6 Heat-shock protein beta-1<br />
Y56A3A.3 Macrophage migration inhibitory factor<br />
Y57A10A.31 Ariadne-1 protein homolog variant (Fragment)<br />
Y57G11C.3 6-Phosphogluconolactonase<br />
Y59A8B.2 Ubiquitin carboxyl-terminal hydrolase 8<br />
Y61A9LA.3 Hypothetical protein LOC55082<br />
Y63D3A.6 Translocation protein SEC63 homolog<br />
Y65B4A.2 Cathepsin B precursor<br />
Y65B4BR.4 NEDD4-like E3 ubiquitin-protein ligase WWP1<br />
Y67D8C.5 HUWE1<br />
Y69A2AR.2 Resistance to inhibitors <strong>of</strong> cholinesterase 8A<br />
Y69H2.6 AKT interacting protein<br />
Y6B3A.1 ARFGEF2<br />
Y71H2AM.5 Cytochrome C oxidase subunit Vib<br />
Y71H2AR.2 Cathepsin L2 precursor<br />
Y73C8C.3 CENPE variant protein (Fragment)<br />
Y73C8C.7 Myosin-9<br />
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Y73C8C.8 CENPE variant protein (Fragment)<br />
Y75B12B.2 PPIase, mitochondrial precursor<br />
Y75B12B.5 PPIase, mitochondrial precursor<br />
Y76A2A.2 Copper-transporting ATPase 1<br />
Y77E11A.2 CDNA FLJ30596 fis, clone BRAWH2009227<br />
Y87G2A.10 Vacuolar protein sorting-associated protein 28<br />
Y87G2A.6 PPIase domain and WD repeat protein 1<br />
Y95B8A.10 PDE8A<br />
Y97E10AR.2 Gamma-glutamyltranspeptidase 1 precursor<br />
Y97E10AR.4 HIV Tat-specific factor 1<br />
ZC155.7 Is<strong>of</strong>orm A <strong>of</strong> Syntaxin-16<br />
ZC196.6 Acidic repeat-containing protein<br />
ZC250.1 Is<strong>of</strong>orm 2 <strong>of</strong> Zonadhesin precursor<br />
ZC317.7 Proline-rich protein 12<br />
ZC328.2 Zinc finger protein 25<br />
ZC395.2 Ubiquinone biosynthesis protein COQ7<br />
ZC395.8 Dentin sialophosphoprotein preproprotein<br />
ZC455.10 FK506 binding protein 9 precursor<br />
ZC506.1 EDEM3<br />
ZC518.2 Protein transport protein Sec24B<br />
ZC97.1 Metaxin-2<br />
ZK1010.2 RMND1<br />
ZK1053.6 Solute carrier family 41, member 3 is<strong>of</strong>orm 4<br />
ZK112.2 Tripartite motif-containing 3<br />
ZK1128.7 Alpha-B-crystallin<br />
ZK1236.3 PAX interacting protein 1<br />
ZK1236.7 VGPW2523<br />
ZK1240.1 Tripartite motif-containing protein 2<br />
ZK1240.2 Tripartite motif-containing protein 2<br />
ZK1240.3 52 kDa Ro protein<br />
ZK1240.4 Ciliary dynein heavy chain 5<br />
ZK1240.6 GTP-binding protein ARD-1<br />
ZK1248.10 TBC1D2B protein<br />
ZK1320.6 GTP-binding protein ARD-1<br />
ZK20.5 PSMD8<br />
ZK218.11 Keratin associated protein 16-1<br />
ZK287.5 RING-box protein 1<br />
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ZK328.1 Ubiquitin carboxyl-terminal hydrolase 32<br />
ZK384.3 Gastricsin precursor<br />
ZK418.4 Junctional sarcoplasmic reticulum protein 1<br />
ZK430.3 Superoxide dismutase<br />
ZK520.5 40 kDa peptidyl-prolyl cis-trans isomerase<br />
ZK54.2 Trehalose-6-phosphate synthase<br />
ZK593.6 MAP1A/MAP1B light chain 3A precursor<br />
ZK632.2 Kanadaptin<br />
ZK632.6 Calnexin precursor<br />
ZK637.14 Hypothetical protein LOC51255<br />
ZK637.14 Hypothetical protein LOC51255<br />
ZK652.9 COQ5<br />
ZK666.6 ZNF254 protein<br />
ZK669.1 PTPL1-associated RhoGAP 1<br />
ZK675.2 DNA repair protein REV1<br />
ZK686.3 Tumor suppressor candidate 3<br />
ZK688.5 Mucin 5 (Fragment)<br />
ZK688.5 Mucin 5 (Fragment)<br />
ZK809.7 Peroxisome assembly factor 1<br />
ZK856.1 Cullin homolog 5<br />
ZK899.4 Tubulin <strong>alpha</strong>-6 chain<br />
ZK930.1 Phosphoinositide 3-kinase regulatory subunit 4<br />
ZK945.5 GTP-binding protein ARD-1<br />
ZK973.11 Protein disulfide-isomerase TXNDC10 precursor<br />
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Table 2.5. Summary <strong>of</strong> RNAi knockdown <strong>of</strong> the top 20 gene candidates in worms<br />
expressing Q82::GFP + TOR-2 in body wall muscle cells.<br />
C. elegans Gene ID <strong>of</strong> Average number <strong>of</strong><br />
targeted gene<br />
aggregates/worm 1, 2 + SD<br />
None (Q82::GFP + TOR-2) 35.3 + 5.5<br />
B0432.2 (djr-1.1) 38.6 + 6.1<br />
T05C3.5 (dnj-19) 34.5 + 5.5<br />
C35D10.2 35.8 + 6.7<br />
C54H2.5 (sft-4) 35.7 + 4.9<br />
EEED8.9 (pink-1) 37.3 + 7.6<br />
F11H8.1 (rfl-1) 35.9 + 7.4<br />
F16A11.2 39.2 + 7.2<br />
F26E4.11 (hrdl-1) 38.1 + 5.3<br />
F32A6.3 (vps-41) 37.8 + 7.0<br />
F48E3.7 (acr-22) 36.4 + 8.7<br />
F55A4.1 35.6 + 6.2<br />
F57B10.5 36.2 + 6.2<br />
F59F4.1 36.2 + 5.8<br />
K11G12.4 (smf-1) 37.4 + 4.2<br />
M7.5 (atgr-7) 38.5 + 5.2<br />
R05D11.6 36.4 + 6.6<br />
T07F12.4 36.1 + 5.7<br />
T08D2.4 37.9 + 4.4<br />
T13A10.2 36.1 + 6.0<br />
Y37A1B.13 (tor-2) 52.0 + 9.9 (P < 0.01) 2<br />
1<br />
Two separate RNAi experiments were performed for each gene target (n = 20 for each<br />
RNAi round).<br />
2<br />
Knockdown <strong>of</strong> tor-2 resulted in a significant increase in aggregate number, whereas the<br />
depletion <strong>of</strong> other gene products did not enhance aggregation.<br />
89
Figure 2.1<br />
90
Figure 2.2<br />
91
Figure 2.3<br />
92
Figure 2.4<br />
93
Figure 2.5<br />
94
Figure 2.6<br />
95
Figure 2.7<br />
96
FIGURE LEGENDS<br />
Figure 2.1. RNAi knockdown <strong>of</strong> specific gene targets enhances misfolding <strong>of</strong> α-<br />
syn (A-E). A. Isogenic worm strain expressing α-syn::GFP alone in body wall muscle<br />
cells <strong>of</strong> C. elegans. B. <strong>The</strong> presence <strong>of</strong> TOR-2, a protein with chaperone activity,<br />
attenuates the misfolded α-syn protein. C, D. When worms expressing α-syn::GFP +<br />
TOR-2 are exposed to candidate gene RNAi, the misfolded α-syn::GFP returns. E.<br />
Western analysis <strong>of</strong> α-syn::GFP demonstrating the presence <strong>of</strong> α-syn::GFP in worms<br />
with and without TOR-2 co-expression. α-syn antibody (Chemicon) was used to detect<br />
the α-syn::GFP fusion protein band; actin was probed for each lane as a loading control.<br />
Figure 2.2. Overexpression <strong>of</strong> candidate <strong>genes</strong> protects DA neurons from α-syn-<br />
induced degeneration. A. Schematic representation depicting the distribution <strong>of</strong> the top<br />
20 candidate <strong>genes</strong> isolated from the RNAi screen, as categorized using bioinformatic<br />
associations employed to select the gene for knockdown. Six <strong>of</strong> 20 <strong>genes</strong> were identified<br />
from 2 or more categories and are indicated with overlapping color. B. Graph depicting<br />
percentage α-syn-expressing worms with wildtype DA neurons at the 7-day adult stage<br />
<strong>of</strong> life when candidate <strong>genes</strong> are co-expressed. *P
separate isogenic worms strains expressing α-syn (low and high) have differing levels <strong>of</strong><br />
mRNA when compared to the cdk-5 control. B. Integrated transgenic line containing<br />
both Pdat-1::α-syn (high) and Pdat-1::GFP shows DA neurodegeneration over time, as<br />
animals age.<br />
Figure 2.5. Analysis <strong>of</strong> transgene expression in worm strains. Semi-quantitative<br />
RT-PCR was performed using primers to amplify cdk-5 (control), a-syn (specific to the<br />
α-syn-expressing transgenic line), and primers specific to the clones analyzed. For all<br />
primers, N2 wild type animals were used as both a positive (cdk-5) and negative control<br />
(α-syn and transgene expression). Worms expressing α-syn without candidate PD<br />
trans<strong>genes</strong> were also analyzed where cdk-5 and α-syn primers were positive controls and<br />
primers corresponding to the trans<strong>genes</strong> were negative controls. <strong>The</strong> candidate PD<br />
trans<strong>genes</strong> were amplified using gene-specific primers; all three separate transgenic lines<br />
for each clone were analyzed; α-syn primers were utilized as a negative control.<br />
Figure 2.6. RNAi knockdown <strong>of</strong> the top 20 gene targets did not enhance<br />
misfolding <strong>of</strong> polyglutamine aggregates in worms expressing Q82::GFP + TOR-2 in<br />
body wall muscle cells (A-D). A. Isogenic worm strain expressing Q82::GFP + TOR-2<br />
in C. elegans. B. When worms expressing Q82::GFP + TOR-2 are exposed to tor-2<br />
RNAi, the Q82::GFP aggregation returns. C, D. When worms expressing Q82::GFP +<br />
TOR-2 are exposed to C35D10.2 or vps-41 RNAi, Q82::GFP aggregation is not<br />
enhanced. <strong>The</strong> presence <strong>of</strong> TOR-2, a protein with chaperone activity, attenuates the<br />
misfolded polyglutamine protein, as previously reported (Caldwell et al., 2003) and<br />
RNAi knockdown reverses this effect (B).<br />
Figure 2.7. Quantitative analysis <strong>of</strong> the hit rate <strong>of</strong> <strong>genes</strong> at both the primary and<br />
secondary level <strong>of</strong> RNAi screening compared with starting <strong>genes</strong> based on specific<br />
associations. A. Candidates separated according to category (mechanisms = UPS, UPR,<br />
ERAD, or autophagy; worm bioinformatics = C. elegans microarray or interactome data;<br />
α-syn proteomic/genetics = proteomic or yeast genetic analyses). <strong>The</strong> highest hit rate<br />
98
came from <strong>genes</strong> that were co-expressed with a known PD gene and are associated with a<br />
cellular mechanism implicated in PD. B. Candidates derived from microarray co-<br />
expression data for both DJ-1 and PINK1 are highly enriched at both the primary and<br />
secondary level <strong>of</strong> RNAi screening for α-syn modifiers.<br />
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CHAPTER THREE<br />
VALIDATION OF SUPPRESSORS OF ALPHA-SYNUCLEIN TOXICITY FROM<br />
YEAST GENETIC SCREENING<br />
Work on RAB proteins was published in Proceedings <strong>of</strong> the National Academy <strong>of</strong><br />
Sciences <strong>of</strong> the United States <strong>of</strong> America, January, 2008 under the following citation:<br />
Gitler, A.D., Bevis, B.J., Shorter, J., Strathearn, K.E., Hamamichi, S., Su, L.J.,<br />
Caldwell, K.A., Caldwell, G.A., Rochet, J.C., McCaffery, J.M., Barlowe, C., and<br />
Lindquist, S. (2008) Proc Natl Acad Sci U S A 105, 145-150.<br />
Work on PARK9/ATP13A2 was published in Nature Genetics, March, 2009 under the<br />
following citation: Gitler, A.D., Chesi, A., Geddie, M.L., Strathearn, K.E., Hamamichi,<br />
S., Hill, K.J., Caldwell, K.A., Caldwell, G.A., Cooper, A.A., Rochet, J.C., and<br />
Lindquist, S. (2009) Nat Genet 41, 308-315.<br />
Shusei Hamamichi collected all C. elegans data. Shusei Hamamichi, Dr. Kim Caldwell,<br />
and Dr. Guy Caldwell co-wrote the manuscript.<br />
100
ABSTRACT<br />
Multiple Parkinson disease (PD) models including S. cerevisiae, C. elegans, and<br />
D. melanogaster have been generated and utilized to ascertain genetic modifiers <strong>of</strong> α-<br />
synuclein (α-syn) toxicity that are subsequently validated using mammalian system.<br />
Here we report the analysis <strong>of</strong> eight suppressors <strong>of</strong> α-syn toxicity that were initially<br />
identified from the genome-wide yeast α-syn toxicity modifier screen using C. elegans<br />
model <strong>of</strong> α-syn-induced dopamine (DA) neurodegeneration. Consistent with previously<br />
reported <strong>neuroprotective</strong> function <strong>of</strong> Rab1a, both RAB3A (a RAB GTPase localized to<br />
the presynaptic termini) and RAB8A (a RAB GTPase involved in post-Golgi trafficking)<br />
significantly rescued worm DA neurons from α-syn toxicity. Interestingly, a gene<br />
identified from the screen encoded ypt9, a yeast ortholog <strong>of</strong> PARK9/ATP13A2. To<br />
examine genetic interaction between these two PD-associated <strong>genes</strong> in C. elegans, we<br />
determined that while RNAi knockdown <strong>of</strong> a worm ortholog <strong>of</strong> PARK9, W08D2.5<br />
enhanced α-syn misfolding in body wall muscles, overexpression <strong>of</strong> this gene in DA<br />
neurons exhibited neuroprotection <strong>against</strong> α-syn. Two additional <strong>genes</strong> from the screen,<br />
PDE9A (a phosphodiesterase) and PLK2 (a Polo-like kinase) also rescued DA neurons.<br />
This work illustrates a collaborative effort to utilize model organisms to validate positive<br />
genetic candidates from the yeast genetic screen, and represents putative genetic<br />
susceptibility factors as well as therapeutic targets for PD.<br />
101
INTRODUCTION<br />
While invertebrate disease models are not evolutionally as complex as vertebrate<br />
counterparts, these models provide unique advantages that are readily exploited as<br />
research tools. For example, these models are cost-effective for large-scale genetic or<br />
chemical screens. Furthermore, molecular and genetic manipulations allow meticulous<br />
genetic analysis that cannot be easily performed using mammalian cell culture or rodent<br />
models. Given the fact that numerous pathways and cellular functions are conserved<br />
across the species, these invertebrate model organisms cannot be neglected as they<br />
remain as valuable and informative tools. In PD research, S. cerevisiae, C. elegans, and<br />
D. melanogaster models have been generated to uncover novel therapeutic targets that<br />
ameliorate α-syn-induced toxicity. According to this experimental strategy, invertebrates<br />
model a disease state (e.g., overexpressing wildtype α-syn, DA neurodegeneration, etc),<br />
and are utilized for genetic or chemical screening, thereby eliminating countless genetic<br />
targets and pathways that may be irrelevant to PD patho<strong>genes</strong>is. Subsequently, these<br />
results are validated by mammalian system.<br />
In one yeast PD model, Willingham et al. (2003) transformed 4850 yeast deletion<br />
mutant strains with wildtype α-syn under the control <strong>of</strong> inducible promoter, and<br />
identified 86 <strong>genes</strong> that were sensitive to α-syn overexpression. Notably, among these<br />
positive candidates, 18 <strong>of</strong> them were predicted to function in lipid metabolism and<br />
vesicle-mediated transport. Outerio et al. (2003) confirmed these findings by examining<br />
enhanced accumulation <strong>of</strong> lipid droplets and defects in vesicular trafficking to yeast<br />
102
vacuoles (mammalian equivalent <strong>of</strong> lysosomes). Furthermore, subsequent ultrastructural<br />
analysis revealed that α-syn is clustered in the membranous vesicles that are co-localized<br />
with secretory and ER-Golgi transport vesicles (Soper et al., 2008).<br />
Utilizing a yeast strain expressing two copies <strong>of</strong> wildtype α-syn under the control<br />
<strong>of</strong> inducible GAL4 promoter, Cooper et al. (2006) transformed 3000 <strong>genes</strong>, and identified<br />
genetic modifiers <strong>of</strong> α-syn cytotoxicity. <strong>The</strong>y identified 34 <strong>genes</strong> that suppressed the<br />
toxicity, and 20 <strong>genes</strong> that enhanced it. One <strong>of</strong> the strong suppressors was Ypt1/RAB1A.<br />
To validate their findings in higher eukaryotic systems, mammalian Rab1a was<br />
overexpressed in PD models consisting <strong>of</strong> C. elegans, D. melanogaster, and mammalian<br />
DA neuron culture, and found <strong>neuroprotective</strong> across the species (Cooper et al., 2006).<br />
Furthermore, they determined that overexpression <strong>of</strong> α-syn blocked ER-Golgi trafficking,<br />
and proposed that the defects in vesicle trafficking may contribute to increased oxidized<br />
DA, which may lead to the selective loss <strong>of</strong> DA neurons. <strong>The</strong> cellular link between α-<br />
syn toxicity and ER homeostasis is further supported by the findings that mutant α-syn<br />
induces ER stress (Smith et al., 2005) and activation <strong>of</strong> UPR has been detected in DA<br />
neurons <strong>of</strong> the substantia nigra <strong>of</strong> PD patients (Hoozemans et al., 2007).<br />
This work represents a continuing collaborative effort to utilize various model<br />
organisms as a “pipeline” to validate positive genetic candidates from the yeast genetic<br />
screen in the higher eukaryotic systems. To expand and further confirm the<br />
<strong>neuroprotective</strong> role <strong>of</strong> RAB proteins (Cooper et al., 2006), RAB3A and RAB8A are<br />
103
tested here for their effect in suppressing DA neurodegeneration in C. elegans.<br />
Furthermore, in another study, the Lindquist lab (Whitehead Institute/MIT) screened an<br />
additional 5000 <strong>genes</strong>, and identified ypt9 (a S. cerevisiae ortholog <strong>of</strong> human<br />
PARK9/ATP13A2), a gene that, when overexpressed, suppressed α-syn cytotoxicity in<br />
yeast. To verify the uncharacterized but putative genetic interaction between α-syn and<br />
ATP13A2, we examined <strong>neuroprotective</strong> function <strong>of</strong> W08D2.5 (a C. elegans ortholog <strong>of</strong><br />
human PARK9/ATP13A2) in worm DA neurons. Additional α-syn toxicity suppressors<br />
from this latter yeast screen, SYVN1, USP10, PDE9A, PLK2, and CSNK1G3 were<br />
analyzed using our worm α-syn-induced neurodegeneration model.<br />
MATERIALS AND METHODS<br />
C. elegans experiments for RAB analysis. Nematodes were maintained following<br />
the standard procedures (Brenner, 1974). Strains UA81 {[baIn1; Pdat-1:: α-syn, Pdat-<br />
1::gfp]; [baEx67; Pdat-1::RAB3A, rol-6 (su1006)]} and UA82 {[baIn1; Pdat-1:: α-syn, Pdat-<br />
1::gfp]; [baEx68; Pdat-1::RAB3A, rol-6 (su1006)]} were generated by injecting 50 µg/ml <strong>of</strong><br />
each expression plasmid and 50 µg/ml <strong>of</strong> rol-6 into an integrated line UA44 [baIn1; Pdat-<br />
1::α-syn, Pdat-1::gfp]. For neuroprotection analysis, 3 independently isolated stable lines<br />
overexpressing mouse Rab1a as well as human RAB3A and RAB8A were analyzed as<br />
described previously (Cooper et al., 2006) with the following modification. <strong>The</strong> 6<br />
anterior DA neurons (4 CEP and 2 ADE neurons) <strong>of</strong> 30 animals/trial were scored for<br />
104
neuroprotection when the animals were 7 days old. <strong>The</strong> experiments were conducted in<br />
triplicate for each stable line (3 lines x 3 trials <strong>of</strong> 30 animals/trial=270 total animals<br />
scored).<br />
C. elegans experiments for PARK9/ATP13A2 analysis. Nematodes were<br />
maintained following the standard procedures (Brenner, 1974). RNAi and fluorescent<br />
microscopy were performed as described (Hamamichi et al., 2008) by feeding UA50<br />
[baInl3; Punc-54::α-syn::gfp, Punc-54::tor-2, rol-6 (su1006)] worms with the RNAi clones<br />
(Geneservice, Cambridge, UK) corresponding to W08D2.5 and its putative interactors,<br />
R12E2.13, and R06F6.8. RNA isolation, cDNA preparation, and semi-quantitative RT-<br />
PCR were conducted as described (Hamamichi et al., 2008) with the following<br />
modification. Total RNAs from 50 young adult control [RNAi bacteria HT115(DE3) with<br />
empty vector] and RNAi-treated worms were isolated to generate cDNAs. PCR was then<br />
performed using primers specific for amplifying cdk-5 as loading control, α-syn, and tor-<br />
2. For DA neurodegeneration analysis, strains UA51 {[baIn1; Pdat-1::α-syn, Pdat-1::gfp];<br />
[baEx42; Pdat-1::FLAG-W08D2.5, rol-6 (su1006)]} and UA108 {[vtls1; Pdat-1::gfp; rol-6<br />
(su1006)]; [baEx83; Pdat-1::FLAG-W08D2.5, Punc-54::mCherry]} were generated by<br />
injecting 50 µg/ml <strong>of</strong> each expression plasmid into integrated UA44 [baIn1; Pdat-1::α-syn,<br />
Pdat-1::gfp] as well as BY200 [vtls1; Pdat-1::gfp; rol-6 (su1006)] worms, respectively.<br />
Additionally, UA75 {[baIn1; Pdat-1::α-syn, Pdat-1::gfp]; [baEx64; Pdat-1::PDE9A, rol-6<br />
(su1006)]}, UA76 {[baIn1; Pdat-1::α-syn, Pdat-1::gfp]; [baEx65; Pdat-1::PLK2, rol-6<br />
105
(su1006)]}, UA93 {[baIn1; Pdat-1::α-syn, Pdat-1::gfp]; [baEx72; Pdat-1::CSNKIG3, rol-6<br />
(su1006)]}, UA94 {[baIn1; Pdat-1::α-syn, Pdat-1::gfp]; [baEx73; Pdat-1::SYVN1, rol-6<br />
(su1006)]}, and UA95 {[baIn1; Pdat-1::α-syn, Pdat-1::gfp]; [baEx74; Pdat-1::USP10, rol-6<br />
(su1006)]} were generated by injecting 50 µg/ml <strong>of</strong> each expression plasmid and 50<br />
µg/ml <strong>of</strong> rol-6 into an integrated line UA44 [baInl1; Pdat-1::α−syn, Pdat-1::gfp]. <strong>The</strong> 6<br />
anterior DA neurons (4 CEP and 2 ADE neurons) <strong>of</strong> thirty 7 day-old animals were scored<br />
for neuroprotection. <strong>The</strong> experiments were conducted in triplicate for each stable line (3<br />
lines x 3 trials <strong>of</strong> 30 animals/trial=270 total animals scored).<br />
RESULTS<br />
In the C. elegans α-syn neurodegeneration model, overexpression <strong>of</strong> wildtype α-<br />
syn is driven under the control <strong>of</strong> a promoter specific to DA neurons (Pdat-1; dopamine<br />
transporter). Because nematode development is invariable, any deviation from the<br />
normal number <strong>of</strong> DA neurons is easily scored. For example, 6 anterior DA neurons<br />
(CEPs and ADEs) are readily visualized in worms carrying a Pdat-1::gfp construct<br />
throughout the course <strong>of</strong> its lifespan (Berkowitz et al., 2009). Overexpression <strong>of</strong> α-syn<br />
reduced the number <strong>of</strong> worms with the wildtype number <strong>of</strong> DA neurons to approximately<br />
15% at the 7-day stage. In contrast, human RAB3A increased the rescue to 25% and<br />
human RAB8A to 40% (Fig. 3.1). <strong>The</strong>se findings further confirm the <strong>neuroprotective</strong><br />
function <strong>of</strong> RAB GTPases.<br />
106
To investigate the genetic interaction between α-syn and ATP13A2 in DA<br />
neurons, α-syn was overexpressed under the control <strong>of</strong> dopamine transporter promoter<br />
(Pdat-1), which resulted in an age-dependent progressive loss <strong>of</strong> DA neurons, with<br />
approximately 15% <strong>of</strong> animals having 6 intact anterior DA neurons at the 7-day stage<br />
(Fig. 3.2a,c). Expression <strong>of</strong> W08D2.5 (a C. elegans ATP13A2 ortholog) alone did not<br />
induce any change in the number <strong>of</strong> DA neurons (data not shown). Co-expression <strong>of</strong><br />
W08D2.5 and α-syn partially rescued this neurodegeneration in each <strong>of</strong> four independent<br />
transgenic lines (Fig. 3.2b,c).<br />
C. elegans was also used to explore the consequences <strong>of</strong> W08D2.5 loss-<strong>of</strong>-<br />
function. Unfortunately, neuronal cells <strong>of</strong> this organism are refractory to RNAi-mediated<br />
inhibition <strong>of</strong> gene expression (Asikainen et al., 2005). However, work with yeast and<br />
neuronal model systems establishes that α-syn toxicity is the result <strong>of</strong> general cellular<br />
defects to which neuronal cells are simply more sensitive. <strong>The</strong>refore, another cell type<br />
was chosen that has been extensively exploited for studies <strong>of</strong> protein homeostasis in this<br />
organism and readily affected by RNAi.<br />
Body wall muscle cells expressing a fusion protein α-syn::GFP exhibit age-<br />
dependent α-syn aggregation (Fig. 3.2d). Co-expression <strong>of</strong> TOR-2, a worm ortholog <strong>of</strong><br />
human torsinA chaperone-like protein, reduces protein misfolding and α-syn aggregation<br />
(McLean et al., 2002; Caldwell et al., 2003), provided a sensitized genetic background<br />
within which an enhancement <strong>of</strong> α-syn misfolding could be observed (Fig. 3.2e). To<br />
107
specifically target W08D2.5, we utilized RNAi to knock down the expression. This<br />
pr<strong>of</strong>oundly enhanced the misfolding <strong>of</strong> human α-syn and did so in an age-dependent<br />
manner (Fig. 3.2f) without modifying the expression levels <strong>of</strong> α-syn and tor-2 mRNAs<br />
(Fig. 3.3). <strong>The</strong>se data provide further evidence for an intimate functional interaction<br />
between α-syn and ATP13A2.<br />
In addition, five α-syn toxicity suppressor <strong>genes</strong> (yeast/human: Hrd1/SYVN1,<br />
Ubp3/USP10, Pde2/PDE9A, Cdc5/PLK2, Yck3/CSNK1G3) were also overexpressed in<br />
the worm DA neurons to test their <strong>neuroprotective</strong> capacities. While all candidates except<br />
CSNK1G rescued mammalian DA neurons, only two (PLK2 and PDE9A) suppressed α-<br />
syn-induced neurodegeneration in the nematode (Fig. 3.1). Since human <strong>genes</strong> were<br />
utilized in this study, it is conceivable that worm cellular machinery failed to properly<br />
fold and/or express these functional proteins. To address this issue, we subsequently<br />
analyzed worm hrd-1/SYVN1 and csnk-1/CSNK1G3, and determined that these <strong>genes</strong><br />
indeed are <strong>neuroprotective</strong> (unpublished data). Taken together, these findings<br />
demonstrate an experimental paradigm for using various model organisms to identify<br />
novel PD therapeutic targets.<br />
DISCUSSION<br />
Using our C. elegans model <strong>of</strong> α-syn-induced neurodegeneration, we demonstrate<br />
that overexpression <strong>of</strong> RAB3A and RAB8A rescue DA neurons. A previous study<br />
108
illustrated that α-syn overexpression blocked ER to Golgi trafficking, and as expected,<br />
co-overexpression <strong>of</strong> Rab1a, a small GTPase that regulates vesicle trafficking from the<br />
ER to the Golgi apparatus, ameliorated it (Cooper et al., 2006). In this study, additional<br />
RAB GTPases, RAB3A and RAB8A were tested. RAB3A is expressed in human<br />
neurons and found in the presynaptic termini where α-syn is also localized. Furthermore,<br />
using cell-free system with purified transport factors, α-syn was shown to disrupt<br />
docking or fusion <strong>of</strong> the vesicles to Golgi membranes, demonstrating that while α-syn<br />
does not alter budding <strong>of</strong> the vesicles from the ER, it interferes with the later stage <strong>of</strong> ER<br />
to Golgi trafficking (Gitler et al., 2008). To further confirm this observation, RAB8A,<br />
which closely resembles RAB1A, was examined since the protein functions in post-Golgi<br />
trafficking. <strong>The</strong> present work further validates that RAB proteins suppress disruption <strong>of</strong><br />
vesicle trafficking by α-syn. Furthermore, the fact that RAB proteins rescue yeast,<br />
worm, and mammalian PD models suggest that the defective vesicle trafficking is a<br />
conserved pathological feature related to α-syn toxicity.<br />
We have also demonstrated that while RNAi knockdown <strong>of</strong> W08D2.5<br />
enhanced α-syn misfolding in the body wall muscles, overexpression <strong>of</strong> this protein<br />
protected DA neurons from α-syn toxicity, indicating the conserved genetic interaction<br />
between α-syn and ATP13A2. Ramirez et al. (2006) reported that while wildtype<br />
ATP13A2 is localized in the lysosomes, misfolded mutant forms are retained in the ER to<br />
be subsequently degraded by proteasomes. Interestingly, they observed approximately<br />
109
10-fold increase in ATP13A2 mRNA level in the surviving DA neurons <strong>of</strong> human<br />
idiopathic PD post-mortem midbrains, suggesting the <strong>neuroprotective</strong> function <strong>of</strong> this<br />
protein. Lysosomal function has been implicated as one <strong>of</strong> the critical protein<br />
degradation machineries for proteolysis <strong>of</strong> misfolded and aggregated α-syn (Webb et al.,<br />
2003; Cuervo et al., 2004). Lastly, in the present study, depletion <strong>of</strong> yeast ypt9 increased<br />
the susceptibility <strong>of</strong> yeast cells to manganese toxicity, illustrating the potential genetic<br />
and environmental interaction between ATP13A2 and metal toxicity (Gitler et al., 2009)<br />
Through our collaborative efforts, we reported 5 <strong>genes</strong> (RAB3A, RAB8A,<br />
W08D2.5, PDE9A, and PLK2) with conserved <strong>neuroprotective</strong> capacities. PDE9A<br />
encodes a cyclic nucleotide phosphodiesterase that regulates signal transduction by<br />
hydrolyzing cAMP and cGMP to their monophosphates. In the classical view <strong>of</strong><br />
dopamine signaling, D2-like receptor inhibits cAMP production by inhibiting adenylate<br />
cyclase. Interestingly, α-syn overexpression in dop-2 (a C. elegans ortholog <strong>of</strong> D2-like<br />
DA receptor) deletion background enhances α-syn-induced neurodegeneration<br />
(unpublished data), suggesting the <strong>neuroprotective</strong> role <strong>of</strong> D2-like DA receptors.<br />
Furthermore, hyperactive adenylate cyclase in C. elegans has been shown to induce<br />
neurodegeneration (Korswagen et al., 1998). Collectively, these data suggest that<br />
increased cAMP level may induce DA neuronal death.<br />
Another <strong>neuroprotective</strong> target, PLK2 is expressed in mammalian neurons where<br />
the kinase is involved in maintaining homeostatic synaptic plasticity (Seeburg et al.,<br />
2008). In PD research, a recent article by Inglis et al. (2009) demonstrated that PLK2 is a<br />
110
main contributor <strong>of</strong> α-syn phosphorylation at serine 129, a potentially more toxic form <strong>of</strong><br />
α-syn. Interestingly, using our worm α-syn neurodegeneration model, overexpression <strong>of</strong><br />
PLK2 with kinase dead mutation still rescued DA neurons whereas PLK2 with disrupted<br />
polo-box domain did not affect the level <strong>of</strong> neurodegeneration (unpublished data). While<br />
the <strong>neuroprotective</strong> mechanism <strong>of</strong> PLK2 remains unclear, our data suggest that protein-<br />
protein interaction at the polo-box domain is responsible for reduced α-syn toxicity.<br />
Currently, nine <strong>genes</strong> have been associated with PD (α-syn, ATP13A2, DJ-1,<br />
GIGYF2, HTRA2, LRRK2, PINK1, PRKN, and UCHL1), accounting for 5-10% <strong>of</strong> all PD<br />
cases. This finding suggests that environmental PD susceptibility factors such as<br />
neurotoxins and heavy metals or interaction between genetic and environmental factors<br />
may play a critical role in disease onset or progression (Di Monte, 2003; Benmoyal-Segal<br />
et al., 2006). Alternatively, multiple, heterogeneous sets <strong>of</strong> <strong>genes</strong> may contribute to the<br />
etiology <strong>of</strong> this disease. To test the latter hypothesis, it will be interesting to compare our<br />
present list <strong>of</strong> <strong>genes</strong> to the results from genome-wide single nucleotide polymorphism<br />
(SNP) analysis <strong>of</strong> PD patients (Fung et al., 2006; Pankratz et al., 2009). Lastly, these<br />
identified <strong>genes</strong> are invaluable candidates for potential therapeutic intervention for PD.<br />
Taken together, this work illustrates a collaborative effort to exploit various advantages<br />
<strong>of</strong> model organisms to rapidly validate positive genetic candidates from the yeast genetic<br />
screen in the higher eukaryotic systems.<br />
111
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Figure 3.1<br />
114
Figure 3.2<br />
115
Figure 3.3<br />
116
FIGURE LEGENDS<br />
Figure 3.1. RAB3A, RAB8A, PDE9A, and PLK2 protect <strong>against</strong> α-syn-induced<br />
DA neuron loss. DA neurons <strong>of</strong> 7-day old transgenic nematodes overexpressing α-syn<br />
along with the indicated <strong>genes</strong> were analyzed [P < 0.05, Student’s t test (*)]. For each<br />
gene tested, 3 transgenic lines were analyzed; a worm was scored as WT when all six<br />
anterior DA neurons (4 CEP and 2 ADE neurons) were intact.<br />
Figure 3.2. PARK9 antagonizes α-syn-mediated DA neuron degeneration in C.<br />
elegans. Anterior DA neurons in worms expressing Pdat-1::GFP + Pdat-1::α-syn at the day<br />
7 stage. Arrowheads and arrows depict cell bodies and neuronal processes, respectively.<br />
WT worms have 6 anterior DA neurons. A. α−Syn toxicity is depicted by the loss <strong>of</strong><br />
anterior DA neurons. B. DA neurons are protected when Pdat-1::FLAG-W08D2.5 cDNA<br />
is co-expressed. C. Quantification <strong>of</strong> C. elegans PARK9 rescue <strong>of</strong> α-syn-induced<br />
neurodegeneration in 4 independent transgenic lines displaying all six anterior DA<br />
neuron. P < 0.05, Student’s t test. D. Overexpression <strong>of</strong> α-syn in Punc-54::α-syn::GFP<br />
results in misfolding and aggregation <strong>of</strong> α-syn in body wall muscle cells at the young<br />
adult stage. E. Co-overexpression <strong>of</strong> TOR-2, a protein with chaperone activity, attenuates<br />
the misfolding <strong>of</strong> the α-syn::GFP protein. F. <strong>The</strong> misfolding <strong>of</strong> α-syn::GFP is enhanced<br />
following RNAi targeting W08D2.5.<br />
Figure 3.3. RNAi knockdown <strong>of</strong> W08D2.5, R12E2.13, and R06F6.8 does not<br />
reduce α-syn or tor-2 mRNA expression levels. Semi-quantitative RT-PCR was<br />
117
performed by using primers that amplify cdk-5 (loading control), α-syn or tor-2. Control<br />
UA50 [baInl3; Punc-54::α−syn::gfp, Punc-54::tor-2, rol-6 (su1006)] worms were fed with<br />
RNAi bacteria HT115(DE3) with empty vector while RNAi-treated worms were fed with<br />
the bacteria producing the indicated dsRNA. Following total RNA isolation and cDNA<br />
preparation, semi-quantitative RT-PCR was performed by using primers specific for cdk-<br />
5 (5' ggg-gat-gat-gag-ggt-gtt-cca-agc 3' and 5' ggc-gac-cgg-cat-ttg-aga-tct-ctg-c 3'),<br />
α−syn (5' atg-gat-gta-ttc-atg-aaa-gga-ctt-tca-aag 3' and 5' tta-ggc-ttc-agg-ttc-gta-gtc-ttg<br />
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').<br />
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CHAPTER FOUR<br />
RNA INTERFERENCE SCREEN OF DAF-2-MODULATED AND<br />
DIFFERENTIALLY EXPRESSED GENES LINK METABOLIC<br />
ENZYMES TO NEUROPROTECTION<br />
This work presented in this chapter represents preliminary data from studies that are<br />
currently performed in Drs. Guy and Kim Caldwell’s laboratory. Shusei Hamamichi<br />
collected all data except Table 4.1, 4.2, and Fig. 4.5. Susan DeLeon, Adam Knight, Kyle<br />
Lee, Cody Locke, and Mike Zhang contributed the data shown in Tables 4.1 and 4.2.<br />
Jenny Schieltz contributed the data shown in Fig. 4.5. Shusei Hamamichi wrote the<br />
manuscript.<br />
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ABSTRACT<br />
Aging is a fundamental susceptibility factor <strong>of</strong> Parkinson disease (PD), wherein<br />
pathological features include progressive loss <strong>of</strong> dopamine (DA) neurons and misfolding<br />
<strong>of</strong> α-synuclein (α-syn) into proteinaceous inclusion bodies. In C. elegans, lifespan<br />
extension and increased stress resistance have been linked to the DAF-2/insulin-like<br />
signaling pathway. Here, we report the results <strong>of</strong> daf-2 worms that exhibit these<br />
pathological features to elucidate the genetic link between aging and α-syn toxicity. In<br />
DA neurons, daf-2 reduced-function mutation strikingly suppressed neurodegeneration at<br />
the chronological aging stage (day 7), but not at the mean lifespan (N2: day 20; daf-2:<br />
day 40), suggesting that differentially expressed <strong>genes</strong> in the daf-2 background are<br />
responsible for DA neuron survival. To identify such components, we screened 625<br />
<strong>genes</strong> that are either up-regulated in daf-2 or shown to modify α-syn toxicity in C.<br />
elegans to identify suppressors <strong>of</strong> α-syn misfolding. While α-syn::GFP was readily<br />
degraded in the daf-2 background, RNAi knockdown <strong>of</strong> 53 <strong>genes</strong> enhanced α-syn<br />
misfolding in vivo. Among the positives were <strong>genes</strong> involved in metabolism,<br />
transcription, and signal transduction. Two positive <strong>genes</strong>, gpi-1/GPI and hrdl-1/AMFR,<br />
both components <strong>of</strong> the autocrine motility factor pathway, rescued DA neurons from α-<br />
syn-induced neurodegeneration. Taken together, this study reveals a common pathway<br />
that links the progression <strong>of</strong> neurodegeneration and cancer, and illustrates the underlying<br />
metabolic changes associated with aging and these diseases.<br />
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INTRODUCTION<br />
Central to Parkinson disease (PD) neuropathology is a protein called α-synuclein<br />
(SNCA/α-syn) (Spillantini et al., 1997), a primary component <strong>of</strong> Lewy bodies found in<br />
both familial and idiopathic forms <strong>of</strong> PD. Currently, nine PARK <strong>genes</strong> have been<br />
identified that are implicated in synaptic function (α-syn), proteasomal protein<br />
degradation (PRKN, UCHL1), lysosomal function (ATP13A2), protection <strong>against</strong><br />
mitochondrial/oxidative stress (DJ-1, HTRA2, PINK1), and signal transduction (LRRK2,<br />
GIGYF2). While these findings provided insightful foundation for elucidating PD<br />
pathological mechanisms, highlighted by the selective loss <strong>of</strong> dopamine (DA) neurons in<br />
the substantia nigra, the monogenic forms <strong>of</strong> PD remain rare, accounting for only 5-10%<br />
<strong>of</strong> all PD cases.<br />
Environmental PD susceptibility factors such as neurotoxins and heavy metals<br />
have long been documented (Di Monte, 2003), and interaction between genetic and<br />
environmental factors may play a role in disease onset or progression (Benmoyal-Segal et<br />
al., 2006). Alternatively, multiple genetic susceptibility factors may presently be<br />
unidentified (Lesage and Brice, 2009), and combined genetic defects may induce DA<br />
neurodegeneration. In the latter case, genome-wide analysis <strong>of</strong> PD patients (Fung et al.,<br />
2006; Pankratz et al., 2009) or genetic screens using simple model organisms (Cooper et<br />
al., 2006; Hamamichi et al., 2008; Gitler et al., 2009) should provide potential genetic PD<br />
susceptibility candidates. In spite <strong>of</strong> the rigorous efforts to ascertain these factors, PD<br />
unequivocally remains as a disease <strong>of</strong> aging, affecting approximately 1% <strong>of</strong> the<br />
121
population aged over 50 (Polymeropoulos et al., 1996). While aging is widely accepted<br />
as an underlying factor <strong>of</strong> several neurodegenerative diseases, including PD, its<br />
association to PD patho<strong>genes</strong>is remains unexplored.<br />
Recently, the insulin signaling pathway has been proposed to affect human aging<br />
(Suh et al., 2008; Willcox et al., 2008) and neurodegenerative diseases (Craft and<br />
Watson, 2004). Notably, insulin resistance in Alzheimer patients has been reported<br />
whereby soluble amyloid-β oligomers modify insulin receptor distribution away from the<br />
neuronal surface (Zhao et al., 2007; De Felice et al., 2009), implicating Alzheimer<br />
disease as a form <strong>of</strong> Type 3 diabetes. In PD, Lautier et al. (2008) identified GIGYF2, a<br />
GRB10 interacting protein as corresponding to PARK11 locus. GRB10 is an adaptor<br />
protein that modulates the insulin signaling pathway (Giovannone et al., 2003). While<br />
association between GIGYF2 and PD remains controversial, loss <strong>of</strong> the insulin receptor<br />
and its mRNA in the substantia nigra <strong>of</strong> PD patients has been reported (Moroo et al.,<br />
1994; Takahashi et al., 1996), suggesting a role for insulin signaling pathway in this<br />
disease.<br />
A model organism, Caenorhabditis elegans <strong>of</strong>fers distinct advantages for<br />
studying aging. <strong>The</strong> studies on longevity via reduced insulin signaling (daf-2, Kenyon et<br />
al., 1993), caloric restriction (eat-2, Lakowski and Hekimi, 1998), and reduced<br />
mitochondrial respiration (clk-1, Felkai et al., 1999; isp-1, Feng et al., 2001) have been<br />
well characterized. Specifically, the daf-2 reduced-function mutation has been shown to<br />
enhance protection <strong>against</strong> various forms <strong>of</strong> cellular stress. <strong>The</strong>se stressors include<br />
122
thermotolerance (Lithgow et al., 1995), oxidative stress (Honda and Honda, 1999),<br />
hypoxia (Scott et al., 2002), heavy metal resistance (Barsyte et al., 2001), and pathogens<br />
(Bolm et al., 2004). Among proteotoxicity models, daf-2 has also shown cytoprotection<br />
<strong>against</strong> amyloid-β aggregation (Cohen et al., 2006; Florez-McClure et al., 2007).<br />
Previously, we established worm models in which α-syn-induced<br />
neurodegeneration in DA neurons can be monitored (Cao et al., 2006), and α-syn<br />
misfolding in body wall muscle is readily assayed by RNAi screening (Hamamichi et al.,<br />
2006). Collectively, C. elegans provides an advantageous platform with molecular,<br />
cellular, and genetic tools to discern genetic factors linking aging and PD. Here, we<br />
report the use <strong>of</strong> daf-2 mutant strains to identify genetic factors that modify PD-linked<br />
toxicity. We determined that while daf-2 reduced-function mutation significantly rescued<br />
DA neurons at chronological aging (day 7 in both wild-type N2 and daf-2 worms), the<br />
mutation had no effects at biological aging (day 20 in wild-type N2 and day 40 in daf-2<br />
worms). Since daf-2 did not ameliorate neurodegeneration at the equivalent biological<br />
aging stage, these findings suggested that differential gene expression in the daf-2 mutant<br />
background may be responsible for neuroprotection. Thus, to identify these specific<br />
<strong>neuroprotective</strong> factors, we performed an RNAi screen and identified 53 <strong>genes</strong> that when<br />
knocked down enhanced α-syn misfolding in the daf-2 background. <strong>The</strong>se <strong>genes</strong><br />
consisted <strong>of</strong> transcription factors, signaling components, and metabolic enzymes. Among<br />
them, two specific genetic components <strong>of</strong> autocrine motility factor pathway (gpi-1/GPI<br />
and hrdl-1/AMFR), linked to glycolysis and cholesterol synthesis, were overexpressed in<br />
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DA neurons, and these <strong>genes</strong> rescued DA neurons from α-syn-induced<br />
neurodegeneration. This study illustrates a genetic link between metabolic changes<br />
associated with the aging processes and PD-linked toxicity.<br />
MATERIALS AND METHODS<br />
Plasmid Constructs. Plasmids were constructed using Gateway Technology<br />
(Invitrogen; Carlsbad, CA). <strong>The</strong> cDNAs encoding gpi-1 and hrdl-1 were obtained from<br />
Open Biosystems (Huntsville, AL). An N-terminal FLAG tag sequence was added<br />
during the PCR amplification process. mCherry was obtained from Clontech (Mountain<br />
View, CA). <strong>The</strong> gene fusions were shuttled from entry vectors into the Gateway<br />
destination vector, pDEST-DAT-1 (Cao et al., 2005) or pDEST-UNC-54 (Hamamichi et<br />
al., 2008). <strong>The</strong> molecular cloning yielded expression plasmids, Pdat-1::FLAG-gpi-1, Pdat-<br />
1::FLAG-hrdl-1, and Punc-54::mCherry.<br />
Generation <strong>of</strong> transgenic nematode strains. Nematodes were maintained using<br />
standard procedures (Brenner, 1974). <strong>The</strong> transgenic strains, UA132 {baInl1[Pdat-1::α-<br />
syn; Pdat-1::gfp]; baEx101[Pdat-1::gpi-1; Punc-54::mCherry]}, UA133 {baInl1[Pdat-1::α-syn;<br />
Pdat-1::gfp]; baEx102[Pdat-1::hrdl-1; Punc-54::mCherry]} were generated by directly<br />
microinjecting 50 µg/ml expression plasmids into the integrated UA44 {baInl1[Pdat-1::α-<br />
syn; Pdat-1::gfp]}.<br />
Neuroprotection analysis. For neuroprotection analysis, at least three stable lines<br />
<strong>of</strong> UA132 and UA133 were analyzed. Synchronized embryos expressing both GFP and<br />
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mCherry were transferred onto NGM plates, and grown at 20°C for 7 days. For each trial,<br />
30 worms were transferred to a 2% agarose pad, immobilized with 2 mM levamisole, and<br />
scored. Worms were considered rescued when all four CEP and both ADE neurons were<br />
intact and had no visible signs <strong>of</strong> degeneration. Each stable line was analyzed three<br />
times (for a total <strong>of</strong> 90 worms/transgenic line). Three separate transgenic lines were<br />
analyzed per gene, for a total <strong>of</strong> 270 animals/gene analyzed.<br />
Genetic crosses. Three UA44 {baInl1[Pdat-1::α-syn; Pdat-1::gfp]} males and 8<br />
DR128 [dpy-1(e1) daf-2(e1370)], DR129 [daf-2(e1370) unc-32(e189)], DR195 [dpy-<br />
5(e61) daf-16(m26)], or DR211 [daf-16(m26) unc-75(e950)] worms were transferred onto<br />
small mating plates, and incubated at 20°C. Subsequent Dpy or Unc worm expressing<br />
GFP were analyzed for DA neurodegeneration. For the RNAi, three wildtype N2<br />
(Bristol) males were crossed with eight DR128 [dpy-1(e1) daf-2(e1370)] to generate<br />
males with the daf-2 mutation without Dpy phenotype. <strong>The</strong> resulting males were then<br />
crossed with UA51 [baInl4; Punc-54::α-syn::gfp, rol-6 (su1006)] and subsequent worms,<br />
UA134 {baInl4; [Punc-54::α-syn::gfp, rol-6 (su1006)]; [dpy-1(e1) daf-2(e1370)]} with<br />
both the Dpy phenotype and α-syn::GFP, were used for RNAi screening.<br />
Preparation <strong>of</strong> worm protein extracts and western blotting. As described<br />
previously (Hamamichi et al., 2008), worm protein extracts were prepared and western<br />
blotting was performed to detect α-syn::GFP expression level in the daf-2 mutant<br />
background.<br />
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RNA isolation and semi-quantitative RT-PCR. As described previously<br />
(Hamamichi et al., 2008), RNA isolation and semi-quantitative RT-PCR were performed<br />
to detect α-syn::gfp mRNA level in the daf-2 mutant background. <strong>The</strong> following primers<br />
were designed for the PCR:<br />
cdk-5 forward primer: 5’ ggg-gat-gat-gag-ggt-gtt-cca-agc 3’<br />
reverse primer: 5’ ggc-gac-cgg-cat-ttg-aga-tct-ctg-c 3’<br />
α-syn forward primer: 5’ atg-gat-gta-ttc-atg-aaa-gga-ctt-tca-aag 3’<br />
reverse primer: 5’ tta-ggc-ttc-agg-ttc-gta-gtc-ttg 3’<br />
Lifespan assay. Lifespan assay was conducted as previously described (Kenyon et<br />
al., 1993). Briefly, age-synchronized 50 wildtype N2, UA44, and DR129 worms were<br />
grown at 20°C, and transferred daily to new NGM plates with 20 µl 10 mg/ml palmitic<br />
acid covering the edge to prevent the worms from crawling into the agar. <strong>The</strong> worms that<br />
responded to gentle touch were counted as alive. <strong>The</strong> assay was conducted in triplicate (n<br />
= 150 total for each strain).<br />
RNAi screen. <strong>The</strong> RNAi screen was performed as described previously<br />
(Hamamichi et al., 2008) except that RNAi-treated UA134 worms (daf-2 mutant<br />
background with Punc-54::α-syn::GFP) were grown at 20°C, and scored for enhanced α-<br />
syn misfolding at young adult stage. RNAi feeding clones (Geneservice, Cambridge,<br />
UK) were grown for 14 hrs in LB culture with 100 mg/ml ampicillin and seeded onto<br />
NGM agar plates containing 1 mM isopropyl β-D-thiogalactoside. When the bacterial<br />
lawn was grown, five L4 UA134 worms were transferred onto the plates and incubated at<br />
126
20°C for 72 hrs. <strong>The</strong> gravid adults were then placed onto the corresponding RNAi plates<br />
and allowed to lay eggs for 12 hrs, and the resulting age-synchronized worms were<br />
analyzed at young adult stage. For each trial, 20 worms were transferred onto a 2%<br />
agarose pad, immobilized with 2 mM levamisole, and analyzed. <strong>The</strong> RNAi clones<br />
resulting in significant aggregation (80% <strong>of</strong> worms with increased quantity and size <strong>of</strong> α-<br />
syn aggregates) were scored as positive. Bacterial clones leading to enhanced α-syn<br />
misfolding were tested in two trials.<br />
2-Deoxyglucose (DOG) analysis. Age-synchronized UA44 animals were grown at<br />
20°C, and analyzed at day 6. To minimize the effect <strong>of</strong> DOG on lifespan, 24 or 48 hrs<br />
prior to the analysis, 30 worms were transferred onto NGM plates with 1, 5, and 10 mM<br />
DOG. <strong>The</strong>se worms were transferred to a 2% agarose pad, immobilized with 2 mM<br />
levamisole, and scored for DA neurodegeneration. <strong>The</strong> experiment was performed three<br />
times for a total <strong>of</strong> 90 worms/treatment.<br />
Fluorescent microscopy. All fluorescence microscopy was performed using a<br />
Nikon Eclipse E800 epifluorescence microscope equipped with Endow GFP HYQ filter<br />
cube (Chroma Technology). Images were captured with a Photometrics Cool Snap CCD<br />
camera driven by MetaMorph s<strong>of</strong>tware (Universal Imaging).<br />
Statistics. Statistical analysis for neuroprotection was performed using the<br />
Student’s t-test (p
RESULTS<br />
To investigate how the insulin signaling pathway may modulate α-syn-induced<br />
DA neuron death, we generated daf-2 strains overexpressing α-syn and gfp under the<br />
control <strong>of</strong> dat-1 promoter (Pdat-1) using genetic crosses. Expression <strong>of</strong> GFP allows clear<br />
observation <strong>of</strong> morphological changes in 6 anterior DA neurons (4 CEPs and 2 ADEs)<br />
and rapid scoring <strong>of</strong> neurodegeneration when GFP expression is lost. At day 7<br />
(chronological aging), approximately 15% <strong>of</strong> wildtype N2 as well as dpy-1 and unc-32<br />
(additional controls for daf-2 strains with the corresponding phenotypic markers) worms<br />
displayed all 6 intact DA neurons (Fig. 4.1A), consistent with our previous reports.<br />
Strikingly, approximately 40% <strong>of</strong> daf-2 worms exhibited 6 normal DA neurons (Fig.<br />
4.1A), which is the highest neuroprotection observed by a single gene using this model.<br />
One <strong>of</strong> the well-characterized downstream components <strong>of</strong> the insulin signaling<br />
pathway is DAF-16/FKHR, which is regulated by phosphoinositide 3-kinase (AGE-<br />
1/PI3K). DAF-16 is a master regulator <strong>of</strong> various cytoprotective <strong>genes</strong> including heat<br />
shock proteins, catalases, and superoxide dismutases (Murphy et al., 2003). We<br />
examined daf-16 loss-<strong>of</strong>-function mutants overexpressing α-syn and GFP in the DA<br />
neurons. As expected, loss-<strong>of</strong>-function <strong>of</strong> daf-16 enhanced neurodegeneration (Fig.<br />
4.1B). Surprisingly, when daf-2 + daf-16 double mutants were analyzed, we still<br />
observed intermediate level <strong>of</strong> neuroprotection (Fig. 4.1C), suggesting that: 1) DAF-16 is<br />
not the sole genetic component responsible for neuroprotection, and 2) additional<br />
128
pathways downstream <strong>of</strong> DAF-2 excluding PI3K pathway may play a <strong>neuroprotective</strong><br />
role.<br />
Since daf-2 mutants live longer, we reasoned that a direct comparison between N2<br />
and daf-2 worms on the same chronological day might not be an accurate assay <strong>of</strong> daf-2-<br />
mediated neuroprotection. To determine biological aging, we performed lifespan assays<br />
whereby worms were incubated at 20°C, and transferred daily to new plates. We<br />
recorded living worms each day by counting the number <strong>of</strong> worms that responded to a<br />
gentle touch. Consistent with a previously published report (Kenyon et al., 1993), N2<br />
worms exhibited mean lifespan <strong>of</strong> day 20 while daf-2 was day 40, doubling its mean<br />
lifespan (Fig. 4.2). Surprisingly, analysis <strong>of</strong> DA neurodegeneration at mean lifespan<br />
(biological aging) revealed no daf-2-mediated neuroprotection (Fig. 4.1D). <strong>The</strong>se<br />
findings indicate that differential gene expression in daf-2 mutant background might be<br />
responsible for neuroprotection for two reasons: 1) knockout <strong>of</strong> daf-16, which encodes a<br />
transcription factor regulating cytoprotective <strong>genes</strong> enhanced neurodegeneration, and 2)<br />
daf-2 + daf-16 double mutations, which exhibit the normal lifespan similar to wildtype<br />
N2 resulted in the intermediate level <strong>of</strong> neuroprotection at the chronological aging.<br />
C. elegans <strong>of</strong>fers a distinct advantage over other animal systems for such an<br />
analysis, with exceptional genome-wide analyses <strong>of</strong> <strong>genes</strong> and proteins that are modified<br />
in the daf-2 background by using microarray, SAGE, and mass spectrometry (Murphy et<br />
al., 2003; McElwee et al., 2004; Halaschek-Weiner et al., 2005; Dong et al., 2007).<br />
Furthermore, we previously reported a large-scale RNAi screen to identify genetic factors<br />
129
that affect α-syn misfolding and subsequently α-syn-induced neurodegeneration, two<br />
common pathological features <strong>of</strong> PD (Hamamichi et al., 2008). To ascertain <strong>genes</strong> that<br />
are modified in the daf-2 background and affect α-syn misfolding, we generated a daf-2<br />
strain overexpressing α-syn::gfp in body wall muscle cells. In the N2 background, α-<br />
syn::GFP was readily misfolded and accumulated in the cytoplasm (Fig. 4.3). In contrast,<br />
depletion <strong>of</strong> daf-2 resulted in nearly complete degradation <strong>of</strong> the fusion protein (Fig. 4.3).<br />
This observation was further confirmed by western blot wherein α-syn::GFP could not be<br />
detected by using antibodies <strong>against</strong> either α-syn or GFP in the daf-2 background (Fig.<br />
4.3). While daf-2 has been shown to regulate autophagy (Melendez et al., 2003) and<br />
ameliorate amyloid-β aggregation, it was conceivable that the mutation might affect the<br />
expression level <strong>of</strong> the fusion protein. As demonstrated by semi-quantitative RT-PCR,<br />
the mutation had no effect on α-syn::gfp mRNA level, verifying that the fusion protein<br />
was degraded.<br />
To conduct the RNAi screen, we targeted 410 <strong>genes</strong> and/or proteins with clear<br />
human orthologs that are up-regulated in the daf-2 background (Murphy et al., 2003;<br />
McElwee et al., 2004; Halaschek-Weiner et al., 2005; Dong et al., 2007; Samuelson et al.,<br />
2007). Additionally, we included 90 genetic modifiers <strong>of</strong> α-syn toxicity that were<br />
identified using C. elegans (Vartiainen et al., 2006; Kuwahara et al., 2008; Van Ham et<br />
al., 2008) as well as 125 intermediate positive <strong>genes</strong> from the previous hypothesis-based<br />
RNAi screen performed in our laboratory (Hamamichi et al., 2008). In total, 625 <strong>genes</strong><br />
130
were assayed by growing the RNAi-treated worms at 20°C and analyzing them at the<br />
young adult stage. We identified 51 <strong>genes</strong> that caused lethality, and 53 positive <strong>genes</strong><br />
that, when knocked down by RNAi, enhanced α-syn misfolding (Table 4.1). Based on<br />
KOG and GO annotations, we classified 53 positive hits in the functional categories<br />
(Table 4.2). Interestingly, in contrast to our expectation that DAF-16-dependent<br />
cytoprotective <strong>genes</strong>, when knocked down by RNAi, would enhance the misfolding, the<br />
most represented category consisted <strong>of</strong> metabolic enzymes. <strong>The</strong>se metabolic enzymes<br />
comprised <strong>of</strong> approximately 20% <strong>of</strong> all positive <strong>genes</strong> (11/53).<br />
Since 11 positive <strong>genes</strong> from the RNAi screen were involved in metabolism, and<br />
4 out <strong>of</strong> 11 were glycolytic enzymes (F01F1.12/ALDOA, K10B3.7/GAPDH,<br />
K10B3.8/GAPDH, and Y87G2A.8/GPI), we asked if reduced glycolysis could enhance α-<br />
syn-induced neurodegeneration. Previous studies have reported the association between<br />
reduced energy metabolism and neurodegeneration (Mattson et al., 1999). To evaluate<br />
this, the N2 strain overexpressing α-syn and GFP in DA neurons was treated with 2-<br />
deoxyglucose (DOG) for 24 or 48 hrs prior to the analysis at day 6. DOG is a glucose<br />
analog that blocks glycolysis. Notably, both 5 and 10 mM DOG treatment enhanced<br />
neurodegeneration (Fig. 4.4), indicating that reduced glycolysis and perturbed energy<br />
metabolism could enhance DA neuronal death.<br />
Schultz et al. (2007) reported that DOG treatment as well as RNAi knockdown <strong>of</strong><br />
glucose-6-phosphate isomerase (gpi-1/GPI1) extended worm lifespan by inducing<br />
mitochondrial respiration and increasing oxidative stress. Furthermore, gpi-1, when<br />
131
knocked down by RNAi in our model also enhanced α-syn misfolding. Interestingly, in<br />
human cancer cells, GPI1 is secreted to function as autocrine motility factor (AMF)<br />
during metastasis to promote cancer cell survival (Funasaka and Raiz, 2007). <strong>The</strong> closest<br />
worm ortholog <strong>of</strong> its receptor AMFR is hrdl-1, another positive gene from the RNAi<br />
screen. To assess the <strong>neuroprotective</strong> role <strong>of</strong> autocrine motility factor components, both<br />
gpi-1 and hrdl-1 were overexpressed in worm DA neurons (Fig. 4.5). We determined<br />
that they both rescued DA neurons from α-syn-induced toxicity. <strong>The</strong>se results may<br />
indicate an inverse relationship <strong>of</strong> autocrine motility factor components whereby up-<br />
regulation in cancer cells enhances survival and down-regulation in DA neurons leads to<br />
neurodegeneration.<br />
DISCUSSION<br />
In this study, we demonstrated that daf-2 suppressed α-syn-induced<br />
neurodegeneration during chronological, but not biological aging, illustrating that daf-2-<br />
mediated neuroprotection as an indirect consequence <strong>of</strong> the mutation. Further, we<br />
systematically knocked down <strong>genes</strong> that are up-regulated in the daf-2 mutant background<br />
as well as 125 intermediate positives from our previous RNAi screen for α-syn modifiers,<br />
and identified 53 <strong>genes</strong> that when knocked down enhanced α-syn misfolding. Among<br />
them, metabolic <strong>genes</strong>, notably glycolytic enzymes were over-represented (Fig. 4.6). To<br />
decipher the role <strong>of</strong> the glycolytic pathway in neurodegeneration, we determined that<br />
132
while DOG treatment enhanced α-syn-induced neurodegeneration, overexpression <strong>of</strong> two<br />
components <strong>of</strong> the autocrine motility factor pathway, gpi-1 and hrdl-1 suppressed it.<br />
DAF-2/insulin signaling pathway via downstream components, AGE-1/PI3K and<br />
DAF-16 has been shown to regulate autophagy in C. elegans (Melendez et al., 2003).<br />
We previously examined <strong>neuroprotective</strong> capacities <strong>of</strong> two autophagic <strong>genes</strong>, vps-<br />
41/VPS41 and atgr-7/ATG7 in worm DA neurons, and reported suppression <strong>of</strong> α-syn-<br />
induced toxicity (Hamamichi et al., 2008). However, the fact that daf-16; daf-2 double<br />
mutants exhibited an intermediate level <strong>of</strong> neuroprotection during the chronological aging<br />
process suggests that a pathway downstream <strong>of</strong> DAF-2 that is separate from the PI3K<br />
pathway for this neuroprotection. One candidate pathway is the MAPK signaling<br />
pathway regulating cell death. In C. elegans, daf-2 enhances resistance <strong>against</strong> bacterial<br />
pathogens, Pseudomonas aeruginosa, Enterococcus faecalis, and Staphylococcus aureus<br />
(Garsin et al., 2003) via pmk-1/p38 (Kim et al., 2002; Troemel et al., 2006). Subsequent<br />
analysis <strong>of</strong> aging (PI3K pathway) vs. innate immunity (MAPK pathway) revealed that<br />
these two pathways are regulated in a genetically distinct manner (Evans et al., 2008).<br />
Taken together, these findings suggest that, in our model, both PI3K and MAPK<br />
pathways may induce neuroprotection by simultaneously stimulating cytoprotective<br />
mechanisms such as autophagy and suppressing cell death (Fig. 4.7). <strong>The</strong> analysis <strong>of</strong><br />
pmk-1 or ced-3 mutants should reveal a role for MAPK signaling in neurodegeneration.<br />
Our RNAi results demonstrate that knockdown <strong>of</strong> most heat shock proteins did<br />
not enhance α-syn misfolding in the daf-2 background, suggesting the functional<br />
133
edundancy <strong>of</strong> these chaperones. Surprisingly, knockdown <strong>of</strong> a single autophagic<br />
component did not affect α-syn misfolding. Given the up-regulation <strong>of</strong> various<br />
cytoprotective <strong>genes</strong> in the daf-2 background, knockdown <strong>of</strong> a single autophagic gene<br />
may be insufficient to substantially shift the cellular threshold toward α-syn<br />
accumulation. Lastly, in contrast to our previous findings wherein RNAi knockdown <strong>of</strong><br />
worm orthologs <strong>of</strong> DJ-1, NURR1, PINK1, parkin, and UCHL1 enhanced α-syn<br />
misfolding in the tor-2 overexpression background (Hamamichi et al., 2008), RNAi<br />
knockdown <strong>of</strong> these <strong>genes</strong> had no effect in the present study. While the mechanism<br />
remains unclear, it is interesting to speculate that monogenic forms <strong>of</strong> PD also requires<br />
modification <strong>of</strong> metabolic changes associated with aging processes to ultimately manifest<br />
a disease state. To this end, a combinatorial RNAi strategy can be utilized whereby the<br />
worm orthologs <strong>of</strong> PD <strong>genes</strong> as well as candidate <strong>genes</strong> are simultaneously knocked<br />
down using the UA134 strain.<br />
Among the positive <strong>genes</strong> from the RNAi screen, the most represented categories<br />
included transcription factors (7/53), signaling components (6/53), and metabolic<br />
enzymes (11/53) (Table 4.2; Fig. 4.6). Notably, three signaling components involved in<br />
the Wnt signaling were identified: 1) tap-1 (TAK kinase/MOM-4 binding protein), 2)<br />
D1069.3 (a putative β-catenin-Tcf/Lef signaling pathway component), and 3) mom-4<br />
(MAKKK7). While PD patho<strong>genes</strong>is has not been linked to the Wnt signaling pathway,<br />
C. elegans BAR-1 (β-catenin) has been shown to physically interact with DAF-16 to<br />
enhance its activity (Essers et al., 2005). Thus, the knockdown <strong>of</strong> these <strong>genes</strong> may<br />
134
enhance α-syn misfolding in a daf-16-dependent manner. Among metabolic enzymes,<br />
four <strong>genes</strong> involved in glycolysis, F01F1.12 (fructose-biphosphate aldolase), gpd-2 and<br />
gpd-3 (glyceraldehyde-3-phosphate dehydrogenases), and gpi-1 (glucose-6-phosphate<br />
isomerase) were uncovered. <strong>The</strong>se results indicate that perturbed glucose metabolism<br />
modifies α-syn misfolding.<br />
Interestingly, in a mammalian system, Belluci et al. (2008) demonstrated that<br />
glucose starvation increases α-syn aggregation and cell death in SH-SY5Y cells.<br />
Moreover, glucose hypometabolism has been reported in PD patients with SNCA<br />
duplication (Uchiyama et al., 2008), consistent with our model whereby α-syn is<br />
overexpressed in DA neurons or body wall muscles. Currently, no study has reported a<br />
direct link between α-syn-induced toxicity and hrdl-1/AMFR. AMFR is an E3 ligase,<br />
which is an ERAD component involved in degradation <strong>of</strong> HMG-CoA reductase (Song et<br />
al., 2005). Intriguingly, statins, inhibitors <strong>of</strong> HMG-CoA reductase and subsequent<br />
cholesterol synthesis have been shown to reduce α-syn aggregation in vitro (Bar-On et<br />
al., 2008). Protective effects <strong>of</strong> statins in animal models, as well as beneficial outcomes<br />
<strong>of</strong> these compounds in PD patients remain unclear, however, our results suggest a<br />
potential <strong>neuroprotective</strong> role <strong>of</strong> autocrine motility factor pathway, a connection between<br />
glucose metabolism and cholesterol synthesis.<br />
While speculative, cancer and PD may share common modifying mechanisms<br />
such as the UPS, cell cycle or other unexplored pathways (West et al., 2005; Zanetti et<br />
135
al., 2007). For example, HSF1, a master regulator <strong>of</strong> various cytoprotective <strong>genes</strong> may<br />
rescue neurons from neurodegenerative diseases, but stimulate malignant transformation<br />
and survival <strong>of</strong> cancer cells (Dai et al., 2007). It will be interesting to examine how these<br />
metabolic changes may regulate cytoprotective mechanisms by examining DAF-16::GFP<br />
activation (another master regulator <strong>of</strong> cytoprotective <strong>genes</strong>) and LGG-1::GFP<br />
localization (marker for activation <strong>of</strong> autophagy). Taken together, this study illustrates<br />
our initial step toward understanding the genetic link between cancer and<br />
neurodegeneration via metabolic changes associated with aging, and provide therapeutic<br />
metabolic pathways for PD.<br />
136
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140
Table 4.1. Summary <strong>of</strong> <strong>genes</strong> analyzed by RNAi screen. Blue indicates 53 <strong>genes</strong> when<br />
knocked down enhanced α-syn misfolding in the daf-2 background at the young adult<br />
stage. Gray indicates 51 lethal <strong>genes</strong>.<br />
Gene Human ortholog Outcome<br />
AC3.7 UDP-glucuronosyltransferase 1-6 precursor<br />
B0024.6 Atrial natriuretic peptide receptor A<br />
B0035.2 BA16L21.2.1<br />
B0041.4 60S ribosomal protein L4<br />
B0213.12 Cytochrome P450 2C8<br />
B0213.15 Cytochrome P450 2A7<br />
B0213.3 Keratin-associated protein 19-8<br />
B0213.6 Keratin-associated protein 19-8<br />
B0218.8 Mannose receptor<br />
B0228.5 Thioredoxin<br />
B0238.1 Carboxylesterase 7 precursor<br />
B0238.13 Carboxylesterase 7 precursor<br />
B0244.2 Receptor-type tyrosine-protein phosphatase<br />
B0250.1 60S ribosomal protein L8<br />
B0284.2 ROCK2 protein<br />
B0286.3 Multifunctional protein ADE2<br />
B0303.9 Vacuolar protein sorting-associated protein 33A<br />
B0336.10 60S ribosomal protein L23<br />
B0336.8 APG12 autophagy 12-like<br />
B0350.2 Ankyrin-1<br />
B0350.2 Ankyrin-1<br />
B0393.1 40S ribosomal protein SA<br />
B0395.2 Sterol O-acyltransferase 1<br />
B0432.2 RNA-binding protein regulatory subunit<br />
B0464.7 Barrier-to-autointegration factor<br />
B0513.3 Protein<br />
BE10.2 Transmembrane protein 195<br />
C01A2.3 Oxidase (Cytochrome c) assembly 1-like<br />
C01A2.4 DKFZP564O123 protein<br />
C01B7.6 Protein associated with Myc<br />
C01F1.1 General transcription factor IIF subunit 1<br />
C01G6.4 RING finger protein 11<br />
141
C02C2.3 Acetylcholine receptor<br />
C02D5.1 Isovaleryl-CoA dehydrogenase<br />
C02F4.2 Serine/threonine-protein phosphatase 2B<br />
C02G6.1 Insulin-degrading enzyme<br />
C03B1.12 Lysosome-associated membrane glycoprotein 1<br />
C03D6.3 mRNA-capping enzyme RNGTT<br />
C04F12.4 RPL14 protein<br />
C04F12.8 Dual specificity protein phosphatase 14<br />
C05D11.2 Vacuolar protein sorting-associated protein 16<br />
C05D9.1 Sorting nexin-2<br />
C05E11.1 Protein lunapark<br />
C05G6.1 Photoreceptor-specific nuclear receptor<br />
C06A12.3 UPF0279 protein C14orf129<br />
C06A5.1 Integrator complex subunit 1<br />
C06A5.8 Zinc-finger protein HT2A<br />
C06A6.5 Thioredoxin domain-containing protein 4<br />
C06B3.4 Estradiol 17-beta-dehydrogenase 12<br />
C06E2.3 Ubiquitin-conjugating enzyme E2<br />
C06E8.3 Serine/threonine-protein kinase Pim-3<br />
C06E8.5 Lipopolysaccharide-binding protein precursor<br />
C06G1.4 Dermokine gamma-1<br />
C06G8.1 Recombination activating gene 1 activating protein 1<br />
C07A12.7 TOM1-like protein 2<br />
C07A9.2 Protein BUD31 homolog<br />
C07A9.8 Bestrophin-3<br />
C07G1.5 Membrane trafficking and cell signaling protein HRS<br />
C08H9.13 43 kDa protein<br />
C08H9.14 Chitinase-3-like protein 1<br />
C08H9.6 Chitinase 3-like 2 is<strong>of</strong>orm b<br />
C09D4.4 Hypothetical protein KIAA1411<br />
C09D4.5 60S ribosomal protein L19<br />
C09G12.8 Ras-related C3 botulinum toxin substrate 1<br />
C10G11.5 Pantothenate kinase 4<br />
C10G11.8 26S protease regulatory subunit 4<br />
C10H11.5 UDP-glucuronosyltransferase 1-3 precursor<br />
C11D2.2 Cathepsin E precursor<br />
C11H1.3 RING finger protein 157<br />
142
C12D12.2 Excitatory amino acid transporter 2<br />
C12D8.10 RAC-<strong>alpha</strong> serine/threonine-protein kinase<br />
C13C4.6 Major facilitator superfamily domain-containing protein 7<br />
C14A4.14 Mitochondrial 28S ribosomal protein S22<br />
C15F1.7 Superoxide dismutase<br />
C15H11.2 Oxytocin receptor<br />
C15H9.1 NAD(P) transhydrogenase<br />
C15H9.1 NAD(P) transhydrogenase<br />
C16A3.9 40S ribosomal protein S13<br />
C16C10.7 Hypothetical protein FLJ38628<br />
C17C3.3 Acyl-coenzyme A thioesterase 8<br />
C17D12.5 Ubiquitin-conjugating enzyme E2 D1<br />
C17G1.4 SWI related protein<br />
C17H1.7 Uncharacterized protein<br />
C18D11.2 Acyl-CoA-binding domain-containing protein 4<br />
C18E9.10 Hypothetical protein FLJ90068<br />
C23G10.6 UDP-glucuronosyltransferase 1-6 precursor<br />
C23H4.2 Carboxylesterase 2 is<strong>of</strong>orm 2<br />
C24A11.8 FERM domain-containing protein 5<br />
C24A11.9 42 kDa protein<br />
C24F3.2 Dual specificity protein phosphatase 12<br />
C24G6.5 DnaJ homolog subfamily A member 2<br />
C25E10.8 IgGFc-binding protein precursor<br />
C25H3.6 Inner centromere protein antigens 135<br />
C26C6.3 Tolloid-like protein 1 precursor<br />
C27A2.2 60S ribosomal protein L22<br />
C27F2.5 Vacuolar-sorting protein SNF8<br />
C27H5.2 Centromeric protein E<br />
C28C12.7 Proactivator polypeptide precursor<br />
C28D4.1 Retinoic acid receptor RXR-beta<br />
C28H8.11 Tryptophan 2,3-dioxygenase<br />
C28H8.5 THAP domain-containing protein 4<br />
C29E4.7 Glutathione transferase omega-1<br />
C29E6.1 Mucin-2 precursor<br />
C29E6.5 Photoreceptor-specific nuclear receptor<br />
C29F9.2 MAP7 domain-containing protein 1<br />
C30C11.1 39S ribosomal protein L32<br />
143
C30G12.2 11-cis retinol dehydrogenase<br />
C30G7.1 Histone H1.0<br />
C31B8.8 Collagenase 3 precursor<br />
C31H2.1 TBC1 domain family member 24<br />
C32D5.10 Topoisomerase I<br />
C33A11.1 NF-kappa-B inhibitor <strong>alpha</strong><br />
C33A12.6 UDP-glucuronosyltransferase 2A1 precursor<br />
C33A12.7 ETHE1 protein, mitochondrial precursor<br />
C33H5.10 Hypothetical protein FLJ90386<br />
C33H5.18 Phosphatidate cytidylyltransferase 1<br />
C34B2.4 LIM domain-binding protein 3<br />
C34B2.7 Succinate dehydrogenase<br />
C34C12.2 Putative protein TPRXL<br />
C34C12.8 GrpE protein homolog 1<br />
C34C6.3 Notch homolog 2 N-terminal-like protein<br />
C34D1.2 Doublesex- and mab-3-related transcription factor 1<br />
C34D10.2 Unkempt-like<br />
C34E10.1 Sorting and assembly machinery component 50<br />
C35A5.3 Sialin<br />
C35D10.2 RGS19-interacting protein 1<br />
C36A4.8 Breast cancer 1, early onset is<strong>of</strong>orm BRCA1-delta11<br />
C36A4.9 Acyl-CoA synthetase short-chain family member 2<br />
C37H5.2 Abhydrolase domain-containing protein 4<br />
C37H5.8 Stress-70 protein, mitochondrial precursor<br />
C39F7.2 Tripartite motif protein 9<br />
C41C4.7 Cystinosin<br />
C41G7.1 Survival motor neuron protein<br />
C44E4.6 Benzodiazepine receptor ligand<br />
C44F1.3 Galectin-4<br />
C44H4.5 TAB1-like protein<br />
C45B11.3 Hydroxysteroid dehydrogenase-like protein 2<br />
C45E5.3 Chitinase-3-like protein 1 precursor<br />
C45G7.4 Ret finger protein 2<br />
C45H4.17 Cytochrome P450 2C8<br />
C46F4.2 Long-chain-fatty-acid--CoA ligase 4<br />
C46H11.2 Putative dimethylaniline monooxygenase 6<br />
C47A4.1 BA16L21.2.1<br />
144
C47B2.2 n/a<br />
C47C12.6 Amiloride-sensitive sodium channel subunit<br />
C48D5.1 Orphan nuclear receptor NURR1<br />
C49H3.11 40S ribosomal protein S2<br />
C50E3.3 C-type lectin<br />
C50F7.10 Cytosolic beta-glucosidase<br />
C50H11.1 Acyl-CoA synthetase family member 3<br />
C53B7.3 LTBP1<br />
C54D1.3 TRAF3-interacting protein 1<br />
C54D10.1 Uncharacterized protein C6orf168<br />
C54D10.10 Tissue factor pathway inhibitor 2<br />
C54D10.2 Uncharacterized protein C6orf168<br />
C54D10.7 Trichohyalin<br />
C54G4.1 Ribosomal protein S6 kinase <strong>alpha</strong>-5<br />
C54H2.5 Surfeit locus protein 4<br />
C55A6.6 Carbonyl reductase [NADPH] 3<br />
C55B6.2 P58<br />
C55C3.4 Proto-oncogene tyrosine-protein kinase FER<br />
C56A3.4 n/a<br />
C56E10.3 Nucleoprotein TPR<br />
C56G2.15 N-acetyltransferase 6<br />
C56G3.1 Somatostatin receptor type 2<br />
CC8.2 Protein phosphatase 1 regulatory subunit 3E<br />
CD4.4 Vacuolar protein sorting-associated protein 37B<br />
D1022.1 Ubiquitin-conjugating enzyme E2 J1<br />
D1022.1 Ubiquitin-conjugating enzyme E2 J1<br />
D1054.8 Peroxisomal short-chain alcohol dehydrogenase<br />
D1069.3 Hyccin<br />
D2007.4 39S ribosomal protein L18<br />
D2030.5 Methylmalonyl-CoA epimerase<br />
D2085.5 Protein KIAA1219<br />
D2089.1 Splicing factor, arginine/serine-rich 12<br />
DY3.2 Lamin-B1<br />
E04A4.8 60S ribosomal protein L18a<br />
EEED8.9 Serine/threonine kinase PINK1<br />
F01F1.12 Fructose-bisphosphate aldolase A<br />
F01G12.5 163 kDa protein<br />
145
F02A9.4 Methylcrotonoyl-CoA carboxylase beta chain<br />
F02C12.5 Thromboxane A synthase 1<br />
F02E8.3 AP-2 complex subunit sigma-1<br />
F08B1.1 Dual specificity protein phosphatase 16<br />
F08C6.4 Erythrocyte band 7 integral membrane protein<br />
F08G12.5 Tripartite motif protein 31<br />
F08G2.5 Dentin sialophosphoprotein preproprotein<br />
F08H9.3 Alpha B crystallin fragment 4<br />
F08H9.4 Alpha-crystallin B chain<br />
F08H9.9 CUB and sushi domain-containing protein 3<br />
F09B12.3 Putative phospholipase B-like 2 precursor<br />
F09F7.7 Alkylated DNA repair protein alkB homolog 4<br />
F09G8.3 Mitochondrial ribosomal protein S9<br />
F10B5.1 60S ribosomal protein L10<br />
F10C2.5 Putative <strong>alpha</strong>-mannosidase C20orf31<br />
F10D11.6 Bactericidal permeability-increasing protein precursor<br />
F10D2.11 UDP-glucuronosyltransferase 1-9 precursor<br />
F10D2.5 2-hydroxyacylsphingosine 1-beta-galactosyltransferase<br />
F10D2.9 Acyl-CoA desaturase<br />
F10F2.2 Phosphoribosylformylglycinamidine synthase<br />
F10G7.2 EBNA-2 co-activator<br />
F10G8.6 Nucleotide-binding protein 1<br />
F11A5.12 Estradiol 17-beta-dehydrogenase 12<br />
F11E6.5 Elongation <strong>of</strong> very long chain fatty acids protein 3<br />
F11G11.2 Glutathione-requiring prostaglandin D synthase<br />
F11H8.1 Ubiquitin-activating enzyme E1C<br />
F12A10.7 Keratin, type I cytoskeletal 10<br />
F13D11.4 3-HSD 1 protein<br />
F13E6.4 65 kDa Yes-associated protein<br />
F13G3.5 Inositol monophosphatase<br />
F13H8.5 Mediator <strong>of</strong> RNA polymerase II transcription subunit 15<br />
F14H12.4 Serine/threonine-protein kinase 3<br />
F15A4.8 43 kDa protein<br />
F15D4.4 Cathepsin S precursor<br />
F16A11.2 Hypothetical protein<br />
F17B5.1 33 kDa protein<br />
F17C11.8 Vacuolar protein-sorting-associated protein 36<br />
146
F18A1.5 Replication protein A<br />
F18E3.7 DDO-1 <strong>of</strong> D-aspartate oxidase<br />
F18H3.5 Cell division protein kinase 6<br />
F20B6.8 Homeodomain-interacting protein kinase 1<br />
F20D1.9 Mitochondrial glutamate carrier 2<br />
F20D6.11 Apoptosis-inducing factor 3<br />
F20H11.2 Protein strawberry notch homolog 1<br />
F20H11.3 Malate dehydrogenase<br />
F21C3.3 Histidine triad nucleotide-binding protein 1<br />
F21D5.7 Signal recognition particle 54 kDa protein<br />
F21F3.3 Protein-S-isoprenylcysteine O-methyltransferase<br />
F21F3.5 Neuronal acetylcholine receptor subunit beta-2<br />
F21F8.2 Gastricsin precursor<br />
F21F8.7 43 kDa protein<br />
F25B4.1 Aminomethyltransferase<br />
F25D7.3 PR domain containing 1, with ZNF domain is<strong>of</strong>orm 1<br />
F25H2.9 Proteasome subunit <strong>alpha</strong> type-5<br />
F26D10.3 Heat shock cognate 71 kDa protein<br />
F26D12.1 Forkhead box P4<br />
F26E4.11 Autocrine motility factor receptor, is<strong>of</strong>orm 2<br />
F27C8.1 Large neutral amino acids transporter small subunit 1<br />
F28A12.4 Gastricsin precursor<br />
F28B12.3 Serine/threonine-protein kinase VRK1<br />
F28D1.9 Long-chain fatty acid transport protein 4<br />
F28F8.2 Acyl-CoA synthetase family member 2<br />
F28H1.2 Transgelin-2<br />
F28H1.4 Plasmolipin<br />
F29B9.6 SUMO-conjugating enzyme UBC9<br />
F29F11.2 UDP-glucuronosyltransferase 1-8 precursor<br />
F30A10.10 Ubiquitin carboxyl-terminal hydrolase 48<br />
F30A10.6 Phosphatidylinositide phosphatase SAC1<br />
F31F4.7 UDP-glucuronosyltransferase 1-6 precursor<br />
F32A5.1 Transcriptional adapter 2-beta<br />
F32A5.5 Aquaporin-10<br />
F32A5.7 U6 snRNA-associated Sm-like protein LSm4<br />
F32A6.3 Vacuolar assembly protein VPS41<br />
F32B6.1 HNF4-Alpha-3 <strong>of</strong> Hepatocyte nuclear factor 4-<strong>alpha</strong><br />
147
F32D1.10 DNA replication licensing factor MCM7<br />
F35B12.4 Tissue factor pathway inhibitor 2<br />
F35C8.2 Dual specificity MAPKK6<br />
F35D11.11 Trichohyalin<br />
F35G12.10 ATP synthase subunit b<br />
F35H10.7 CGTHBA protein<br />
F36D3.9 n/a<br />
F37A4.5 26S proteasome non-ATPase regulatory subunit 14<br />
F37B1.4 Glutathione-requiring prostaglandin D synthase<br />
F37B1.5 Glutathione-requiring prostaglandin D synthase<br />
F38B2.4 Adenylate kinase isoenzyme 1<br />
F38B6.4 Trifunctional purine biosynthetic protein adenosine-3<br />
F38B7.1 Butyrate response factor 2<br />
F38E11.1 Alpha B crystallin fragment 4<br />
F38E11.2 Alpha B crystallin fragment 4<br />
F39H11.2 TATA-box-binding protein<br />
F40F11.1 40S ribosomal protein S11<br />
F40G9.11 Max-like protein X<br />
F41B5.4 Cytochrome P450 2J2<br />
F41E6.4 SMEK homolog 1<br />
F41E6.5 Hydroxyacid oxidase 1<br />
F41E7.1 NHEDC1<br />
F41F3.4 Collagen <strong>alpha</strong>-1(III) chain precursor<br />
F41H10.7 Elongation <strong>of</strong> very long chain fatty acids protein 3<br />
F42A10.4 Elongation factor 2 kinase<br />
F42C5.8 25 kDa protein<br />
F42G2.5 VAMP-associated protein A<br />
F43D9.4 Alpha-crystallin B chain<br />
F43E2.8 78 kDa glucose-regulated protein precursor<br />
F43G6.8 GTP-binding protein ARD-1<br />
F44B9.3 Cyclin K is<strong>of</strong>orm 1<br />
F44C8.3 Orphan nuclear receptor TR4<br />
F44C8.9 Photoreceptor-specific nuclear receptor<br />
F44G3.9 Photoreceptor-specific nuclear receptor<br />
F45E1.6 Histone H3.3<br />
F45E6.2 Cyclic AMP-dependent transcription factor ATF-6 <strong>alpha</strong><br />
F46B6.8 Gastric triacylglycerol lipase precursor<br />
148
F46E10.10 Malate dehydrogenase<br />
F46E10.8 UCHL1<br />
F46G11.3 Cyclin G-associated kinase<br />
F46H5.3 Creatine kinase M-type<br />
F46H5.7 Coiled-coil domain-containing protein FLJ36144<br />
F47G4.4 katanin p80 subunit B 1<br />
F47H4.10 S-phase kinase-associated protein 1<br />
F48E3.7 Neuronal acetylcholine receptor protein<br />
F49B2.6 Leucyl-cystinyl aminopeptidase<br />
F49E11.9 Cysteine-rich secretory protein 2<br />
F52C12.2 UPF0293 protein C16orf42<br />
F52C6.8 BTB/POZ domain containing protein 5<br />
F52F12.3 Mitogen-activated protein kinase kinase kinase 7<br />
F52H3.5 Tetratricopeptide repeat protein 36<br />
F52H3.7 Galectin-9<br />
F53A3.3 40S ribosomal protein S15a<br />
F53A9.1 Histidine-rich glycoprotein precursor<br />
F53B3.6 U1 small nuclear ribonucleoprotein 70 kDa<br />
F53B6.2 ADAMTS-like 1 is<strong>of</strong>orm 4 precursor<br />
F53C3.12 Beta,beta-carotene 15,15'-monooxygenase<br />
F53G12.6 Proto-oncogene tyrosine-protein kinase FER<br />
F54B11.3 Putative uncharacterized protein<br />
F54C1.7 Calmodulin<br />
F54H12.1 Aconitate hydratase<br />
F55A12.4 Retinol dehydrogenase 16<br />
F55A4.1 Vesicle trafficking protein SEC22b<br />
F55B12.4 tRNA-nucleotidyltransferase 1<br />
F55E10.7 Somatostatin receptor type 2<br />
F55F3.1 5'-AMP-activated protein kinase subunit beta-2<br />
F55G1.5 Mitochondrial glutamate carrier 2<br />
F55H2.2 Vacuolar proton pump subunit D<br />
F55H2.5 Cytochrome b561<br />
F56C11.2 Patched is<strong>of</strong>orm S<br />
F56C9.1 Serine/threonine-protein phosphatase PP1<br />
F56E10.4 40S ribosomal protein S27<br />
F57B1.7 Doublesex- and mab-3-related transcription factor C<br />
F57B10.5 Hypothetical protein FLJ90481<br />
149
F57C2.5 Adipocyte plasma membrane-associated protein<br />
F57H12.7 Putative uncharacterized protein Nbla00487<br />
F58A3.1 LIM domain-binding protein 1<br />
F58A4.10 26 kDa protein<br />
F59A6.6 Ribonuclease H1<br />
F59F3.5 Vascular endothelial growth factor receptor 1<br />
F59F4.1 Acyl-coenzyme A oxidase 1<br />
F59F4.4 1-acylglycerol-3-phosphate O-acyltransferase 2<br />
H03A11.1 family with sequence similarity 20, member C<br />
H10D18.2 Cysteine-rich secretory protein 2 precursor<br />
H14N18.1 BAG family molecular chaperone regulator 2<br />
H16D19.1 Mannose receptor<br />
H25P06.2 Cell division protein kinase 9<br />
H28G03.1 29 kDa protein<br />
K01A6.2 Guanylate kinase<br />
K01C8.1 Serine racemase<br />
K01G12.3 Acidic nuclear phosphoprotein 32<br />
K04A8.5 Lipase member M precursor<br />
K04D7.3 4-aminobutyrate aminotransferase<br />
K04F1.15 Aldehyde dehydrogenase<br />
K05D4.4 Cytochrome P450 2J2<br />
K05F1.3 Medium-chain specific acyl-CoA dehydrogenase<br />
K07A1.7 Headcase protein homolog<br />
K07A3.1 Fructose 1,6-bisphosphatase<br />
K07A3.2 Niemann-Pick C1 protein precursor<br />
K07B1.4 Monoacylglycerol O-acyltransferase 1<br />
K07C11.4 Carboxylesterase 2 is<strong>of</strong>orm 1<br />
K07C6.3 Cytochrome P450 2C8<br />
K07C6.4 Cytochrome P450 2U1<br />
K07E1.1 1,2-dihydroxy-3-keto-5-methylthiopentene dioxygenase<br />
K07E3.3 C-1-tetrahydr<strong>of</strong>olate synthase, cytoplasmic<br />
K07E3.8 Membrane-associated progesterone receptor<br />
K08B4.6 Cystatin-D precursor<br />
K08E3.7 Parkin<br />
K08F4.11 Glutathione-requiring prostaglandin D synthase<br />
K08F4.7 Glutathione-requiring prostaglandin D synthase<br />
K08F4.9 Orphan short-chain dehydrogenase/reductase<br />
150
K08H10.1 Dentin sialophosphoprotein preproprotein<br />
K09A11.2 Cytochrome P450 2C8<br />
K09A11.4 Cytochrome P450 2D6<br />
K09C4.5 SLC2A14<br />
K09H9.6 Suppressor <strong>of</strong> SWI4 1 homolog<br />
K10B2.2 Lysosomal protective protein precursor<br />
K10B3.7 Glyceraldehyde-3-phosphate dehydrogenase<br />
K10B3.8 Glyceraldehyde-3-phosphate dehydrogenase<br />
K10B3.9 Putative uncharacterized protein DKFZp564G0422<br />
K10C2.4 Fumarylacetoacetase<br />
K10H10.2 Cystathionine beta-synthase<br />
K10H10.5 34 kDa protein<br />
K11D2.2 Acid ceramidase precursor<br />
K11G12.4 Natural resistance-associated macrophage protein 2<br />
K11G9.5 Sialin<br />
K11G9.6 Metallothionein-3<br />
K12D12.2 Nuclear pore complex protein Nup205<br />
K12D12.3 Collagen <strong>alpha</strong>-3(IX) chain precursor<br />
K12G11.3 Alcohol dehydrogenase 4<br />
K12G11.4 Alcohol dehydrogenase 4<br />
M01E5.5 DNA topoisomerase 1<br />
M01F1.4 Hypothetical protein LOC23731<br />
M02D8.4 Asparagine synthetase<br />
M03C11.5 ATP-dependent metalloprotease YME1L1<br />
M03F4.7 Calumenin precursor<br />
M04B2.1 Uncharacterized protein FOXP2<br />
M04G12.2 Cathepsin Z precursor<br />
M142.2 Collagen <strong>alpha</strong>-6(VI) chain precursor<br />
M151.3 Girdin<br />
M4.2 Pumilio homolog 2<br />
M57.2 Geranylgeranyl transferase<br />
M7.5 E1-like protein<br />
R02F11.4 RIKEN cDNA 2810403B08 gene<br />
R03E9.1 MAX interactor 1 is<strong>of</strong>orm b<br />
R03G5.2 Dual specificity MAPKK6<br />
R03H10.7 Replication protein A 70 kDa DNA-binding subunit<br />
R04D3.1 Cytochrome P450 2J2<br />
151
R05D11.6 Transcription factor<br />
R05D3.4 ring finger protein 20<br />
R05D8.7 Peroxisomal short-chain alcohol dehydrogenase<br />
R05F9.1 BTB/POZ domain-containing protein 10<br />
R05F9.13 14 kDa protein<br />
R07B7.15 NR1H3 protein<br />
R07E4.1 Hypothetical protein FLJ38335<br />
R09B3.4 NEDD8-conjugating enzyme UBE2F<br />
R09B3.5 Protein mago nashi homolog 2<br />
R09B5.6 Hydroxyacyl-coenzyme A dehydrogenase<br />
R09D1.12 Alpha-type platelet-derived growth factor receptor<br />
R09D1.2 43 kDa protein<br />
R102.4 17 kDa protein<br />
R107.7 Glutathione S-transferase P<br />
R10H10.2 Kelch-like 20<br />
R11A5.4 Mitochondrial phosphoenolpyruvate carboxykinase 2<br />
R11A8.4 NAD-dependent deacetylase sirtuin-1<br />
R12A1.4 Liver carboxylesterase 1 precursor<br />
R12B2.5 Mediator <strong>of</strong> RNA polymerase II transcription subunit 15<br />
R12E2.13 Stromal cell-derived factor 2 precursor<br />
R12H7.2 Cathepsin D precursor<br />
R13D7.7 Glutathione S-transferase P<br />
R144.4 WAS/WASL interacting protein<br />
R151.6 Derlin-2<br />
R151.7 Heat shock protein 75 kDa<br />
R53.5 25 kDa protein<br />
R53.7 5'-AMP-activated protein kinase<br />
T01B11.2 Alanine--glyoxylate aminotransferase 2-like 1<br />
T01B6.3 5'-AMP-activated protein kinase subunit gamma-1<br />
T01D1.4 1,2-dihydroxy-3-keto-5-methylthiopentene dioxygenase<br />
T01G5.7 Ubiquitin ligase protein RNF8<br />
T01G9.4 Nucleoporin NUP85<br />
T01H3.2 DKFZP434B0335 protein<br />
T02B5.1 Liver carboxylesterase 1 precursor<br />
T02B5.1 Liver carboxylesterase 1 precursor<br />
T04H1.2 GTP-binding protein 2<br />
T04H1.9 Tubulin beta-2C chain<br />
152
T05A10.5 Cysteine-rich secretory protein 2 precursor<br />
T05A12.4 Helicase-like protein<br />
T05F1.11 Nucleoredoxin-like protein 2<br />
T05G5.9 GRIP and coiled-coil domain-containing 2<br />
T06A4.1 Carboxypeptidase A2 precursor<br />
T06G6.8 Mucin-5AC precursor (Fragment)<br />
T07D10.4 Mannose receptor<br />
T07D3.7 Eukaryotic translation initiation factor 2C 4<br />
T07F12.4 ULK2 protein<br />
T08B2.10 40S ribosomal protein S17<br />
T08B2.8 Uncharacterized protein MRPL23 (Fragment)<br />
T08D2.4 Zinc-finger protein HT2A<br />
T08H10.1 Aldo-keto reductase family 1 member B10<br />
T09A12.2 Glutathione peroxidase<br />
T09E8.3 Cornichon homolog<br />
T10B11.1 Pterin-4-<strong>alpha</strong>-carbinolamine dehydratase<br />
T10B9.1 Cytochrome P450 3A5<br />
T10B9.2 Cytochrome P450 3A5<br />
T10F2.2 Mitochondrial ornithine transporter 1<br />
T10F2.3 73 kDa protein<br />
T10H4.11 Cytochrome P450 2F1<br />
T11F1.8 n/a<br />
T13A10.11 S-adenosylmethionine synthetase is<strong>of</strong>orm type-1<br />
T13A10.2 Tripartite motif protein 2<br />
T13B5.3 ACPP protein<br />
T13H2.3 Plectin 7<br />
T14F9.1 Vacuolar proton pump subunit H<br />
T14F9.3 Beta-hexosaminidase subunit <strong>alpha</strong> precursor<br />
T17H7.1 Collagen <strong>alpha</strong>-1(III) chain precursor<br />
T19B10.1 Cytochrome P450 4V2<br />
T19C3.5 Bactericidal permeability-increasing protein<br />
T19D2.1 ADAMTS-18 precursor<br />
T19H12.10 UDP-glucuronosyltransferase 3A2 precursor<br />
T20B5.1 AP-2 complex subunit <strong>alpha</strong>-1<br />
T20D3.5 Mitochondrial substrate carrier family protein<br />
T20F5.2 Proteasome subunit beta type-2<br />
T21D12.9 Membrane glycoprotein LIG-1<br />
153
T22B11.2 Galactosyltransferases<br />
T22B7.1 Transcription factor SOX-5<br />
T22B7.7 Acyl-CoA thioesterase<br />
T22F3.11 Sialin<br />
T22G5.6 Fatty acid-binding protein<br />
T22H2.5 Uncharacterized protein PLSCR1 (Fragment)<br />
T23B12.3 Mitochondrial 28S ribosomal protein S2<br />
T23F2.1 Alpha-1,3-mannosyltransferase ALG2<br />
T23G7.2 Putative metallophosphoesterase FLJ45032<br />
T23G7.3 Pin2-interacting protein X1<br />
T23H2.2 Synaptotagmin-4<br />
T23H4.3 Meprin A subunit beta precursor<br />
T24H7.1 Prohibitin-2<br />
T24H7.5 Probable phospholipid-transporting ATPase VB<br />
T25B9.1 2-amino-3-ketobutyrate coenzyme A ligase<br />
T25B9.2 Serine/threonine-protein phosphatase PP1<br />
T25G3.4 Glycerol-3-phosphate dehydrogenase<br />
T27A3.6 Molybdenum c<strong>of</strong>actor synthesis protein 2<br />
T27C4.4 Metastasis-associated protein MTA3<br />
T27E4.2 Alpha-crystallin B chain<br />
T27E4.8 Alpha-crystallin B chain<br />
T27F7.1 Charged multivesicular body protein 3<br />
T28D9.3 Lipid phosphate phosphohydrolase 1<br />
T28F2.2 COMM domain-containing protein 4<br />
T28F4.5 Death-associated protein 1<br />
VC5.3 Centromeric protein E<br />
VZK822l.1 Acyl-CoA desaturase<br />
W01A11.1 Epoxide hydrolase 1<br />
W01A8.2 UPF0235 protein C15orf40<br />
W01B11.2 Solute carrier family 26 member 6<br />
W01G7.4 Hypothetical protein<br />
W02A11.2 Vacuolar protein-sorting-associated protein 25<br />
W02A11.3 Ring finger protein 44<br />
W02H5.8 Dihydroxyacetone kinase<br />
W03C9.3 Ras-related protein Rab-7a<br />
W03C9.3 Ras-related protein Rab-7a<br />
W03G9.1 Sodium- and chloride-dependent creatine transporter 1<br />
154
W03G9.6 Platelet-activating factor acetylhydrolase 2<br />
W04H10.3 Tripartite motif protein 3<br />
W05B5.2 Orexin receptor type 2<br />
W05G11.6 Mitochondrial phosphoenolpyruvate carboxykinase<br />
W05H9.1 Uncharacterized protein ENSP00000383081<br />
W06D12.5 Potassium channel subfamily K member 1<br />
W06D4.1 Homogentisate 1,2-dioxygenase<br />
W06E11.4 Ribosome maturation protein SBDS<br />
W06F12.1 Nemo like kinase<br />
W06H3.1 Mitochondrial inner membrane protein<br />
W07E11.1 Dihydropyrimidine dehydrogenase<br />
W07G4.4 56 kDa protein<br />
W08D2.4 Fatty acid desaturase 2<br />
W08D2.5 Probable cation-transporting ATPase 13A3<br />
W09C2.3 Plasma membrane calcium-transporting ATPase 3<br />
W10C8.5 Creatine kinase<br />
W10G6.3 Lamin-B1<br />
Y105E8A.16 40S ribosomal protein S20<br />
Y106G6E.6 Casein kinase I is<strong>of</strong>orm gamma-3<br />
Y106G6H.3 60S ribosomal protein L30<br />
Y110A2AR.2 n/a<br />
Y116A8C.35 U2 small nuclear RNA auxillary factor 1<br />
Y15E3A.1 Orphan nuclear receptor NR6A1<br />
Y17G7B.5 MCM2<br />
Y17G7B.7 Triosephosphate isomerase<br />
Y17G9B.4 40 kDa peptidyl-prolyl cis-trans isomerase<br />
Y24D9A.4 60S ribosomal protein L7a<br />
Y32H12A.3 Dehydrogenase/reductase SDR family member 1<br />
Y32H12A.8 WD repeat-containing protein 24<br />
Y37A1B.2 Hypothetical protein MGC32065<br />
Y37E11B.5 tRNA-dihydrouridine synthase 3-like<br />
Y37H9A.6 Bis(5'-nucleosyl)-tetraphosphatase<br />
Y38E10A.4 Deleted in malignant brain tumors 1 protein<br />
Y38F1A.2 Hypothetical protein<br />
Y38H6C.17 Proton-coupled amino acid transporter 2<br />
Y40B10A.2 O-methyltransferase<br />
Y40B10A.6 O-methyltransferase<br />
155
Y40D12A.2 Lysosomal protective protein precursor<br />
Y40G12A.1 UCHL3<br />
Y41D4B.5 40S ribosomal protein S28<br />
Y42G9A.4 Mevalonate kinase<br />
Y43C5A.3 Heterogeneous nuclear ribonucleoproteins A2/B1<br />
Y43C5B.2 Proto-oncogene tyrosine-protein kinase FER<br />
Y43F8A.3 Arylacetamide deacetylase-like 1<br />
Y45F10B.9 Zinc-finger protein HT2A<br />
Y45G12C.2 Glutathione S-transferase P<br />
Y46H3A.3 Alpha-crystallin B chain<br />
Y47G6A.18 Golgi phosphoprotein 3<br />
Y48B6A.2 60S ribosomal protein L37a<br />
Y48G1A.6 L3MBTL2<br />
Y48G8AL.8 60S ribosomal protein L17<br />
Y48G9A.10 Carnitine O-palmitoyltransferase I<br />
Y49E10.20 Lysosome membrane protein 2<br />
Y49G5A.1 Eppin precursor<br />
Y4C6A.3 Tripartite motif protein 2<br />
Y4C6B.4 Solute carrier family 17 (sodium phosphate), member 1<br />
Y4C6B.5 Proton-coupled folate transporter<br />
Y53C10A.12 Heat shock factor protein 1<br />
Y53C12A.2 Excitatory amino acid transporter 2<br />
Y53F4B.32 Glutathione-requiring prostaglandin D synthase<br />
Y53F4B.33 Glutathione-requiring prostaglandin D synthase<br />
Y53F4B.4 Methyltransferase-like protein 2<br />
Y53G8B.2 2-acylglycerol O-acyltransferase 2<br />
Y54E10BR.3 Hypothetical protein FLJ20552<br />
Y54E2A.12 Predicted GTPase activator protein<br />
Y54E2A.2 Uncharacterized protein C19orf61<br />
Y54G11A.6 Catalase<br />
Y55B1AR.1 Uncharacterized protein LGALS9<br />
Y55D9A.2 Angiogenic factor VG5Q<br />
Y55F3AM.3 RNA-binding protein 39<br />
Y55F3BR.1 ATP-dependent helicase DDX1<br />
Y56A3A.1 CCR4-NOT transcription complex subunit 3<br />
Y56A3A.33 Exonuclease GOR<br />
Y57G11C.24 Epidermal growth factor receptor kinase substrate 8<br />
156
Y58G8A.1 Acetylcholine receptor subunit beta precursor<br />
Y61A9LA.3 Arginine and glutamate-rich protein 1<br />
Y65B4A.2 n/a<br />
Y65B4A.3 Uncharacterized conserved protein<br />
Y65B4BR.4 NEDD4-like E3 ubiquitin-protein ligase WWP1<br />
Y66H1B.4 Sphingosine-1-phosphate lyase 1<br />
Y67A6A.2 HNF4-Alpha-3 <strong>of</strong> Hepatocyte nuclear factor 4-<strong>alpha</strong><br />
Y69H2.3 IgGFc-binding protein precursor<br />
Y6B3B.10 LAG1 longevity assurance homolog 1<br />
Y71G12B.4 Charged multivesicular body protein 6<br />
Y71H10A.1 6-phosph<strong>of</strong>ructokinase<br />
Y71H2AR.2 Cathepsin L2 precursor<br />
Y75B12B.2 Peptidyl-prolyl cis-trans isomerase<br />
Y76A2A.2 Copper-transporting ATPase 1<br />
Y80D3A.5 Cytochrome P450 4V2<br />
Y87G2A.8 Glucose-6-phosphate isomerase<br />
Y9C9A.16 Sulfide:quinone oxidoreductase<br />
ZC196.2 cDNA FLJ78586<br />
ZC250.3 UDP-N-acetylglucosamine transporter<br />
ZC395.8 Dentin sialophosphoprotein precursor<br />
ZC434.2 40S ribosomal protein S7<br />
ZC518.3 CCR4-NOT transcription complex subunit 6-like<br />
ZK1073.1 Protein NDRG1<br />
ZK270.1 Band 4.1-like protein 3<br />
ZK270.2 Niemann-Pick C1 protein precursor<br />
ZK384.1 Peptidase inhibitor 16 precursor<br />
ZK384.2 Glioma patho<strong>genes</strong>is-related protein 1<br />
ZK384.3 Gastricsin precursor<br />
ZK430.3 Superoxide dismutase<br />
ZK54.2 Trehalose-6-phosphate synthase component<br />
ZK550.6 Phytanoyl-CoA dioxygenase, peroxisomal precursor<br />
ZK593.7 U6 snRNA-associated Sm-like protein LSm7<br />
ZK596.1 Keratin, type I cytoskeletal 9<br />
ZK632.2 Solute carrier family 4 (anion exchanger)<br />
ZK652.4 60S ribosomal protein L35<br />
ZK816.5 Dehydrogenase/reductase SDR family member 1<br />
ZK829.7 Sorbitol dehydrogenase<br />
157
ZK973.5 Neuronal acetylcholine receptor subunit <strong>alpha</strong>-2<br />
158
Table 4.2. Summary <strong>of</strong> positive <strong>genes</strong> from RNAi screen for effectors <strong>of</strong> α-syn in the<br />
daf-2 background based on KOG and/or GO annotations.<br />
Category Gene Human ortholog E-value<br />
ERAD F26E4.11 (hrdl-1) AMFR 1.90E-39<br />
T27E4.2 (hsp-16.11) Alpha crystallin 2.00E-14<br />
Y38E10A.4 (clec-8) Glycoprotein 340 2.50E-12<br />
ER-Golgi C54H2.5 (sft-4) ERV29 7.80E-88<br />
trafficking F55A4.1 SEC22b 2.30E-47<br />
F57B10.5 emp24/gp25L/p24 2.70E-58<br />
Glutathione- C06A6.5 ERp44 8.30E-75<br />
related C29E4.7 (gsto-1) Glutathione S-transferase 2.00E-32<br />
C54D10.2 C6orf168 8.10E-22<br />
Glycosylation F10C2.5 Glycosyl hydrolase 3.30E-149<br />
T22F3.11 Sialin 7.80E-25<br />
Lysosomal F21F8.7 (asp-6) Aspartyl protease 5.00E-51<br />
function T13B5.3 ACPP 1.80E-38<br />
Y71H2AR.2 Cathepsin L 8.00E-33<br />
Metabolism F01F1.12 ALDOA 2.70E-117<br />
F25B4.1 Aminomethyl transferase 6.90E-101<br />
F38B6.4 GARS/AIRS 1.90E-208<br />
K07C6.3 (cyp-35B2) Cytochrome P450 2C8 4.50E-58<br />
T07D3.7 (alg-2) eIF-2C 0<br />
K10B3.7 (gpd-3) GAPDH 2.80E-136<br />
K10B3.8 (gpd-2) GAPDH 2.80E-136<br />
K12G11.4 (sodh-2) Alcohol dehydrogenase 6.90E-20<br />
T10H4.11 (cyp-34A2) Cytochrome P450 2F1 1.90E-57<br />
Y87G2A.8 (gpi-1) GPI 2.80E-207<br />
W07E11.1 Glutamate synthase 1.80E-11<br />
Signaling C02C2.3 (cup-4) Acetylcholine receptor 2.80E-16<br />
Components C44H4.5 (tap-1) TAB1-like protein 9.50E-12<br />
D1069.3 DRCTNNB1A 2.10E-10<br />
F52F12.3 (mom-4) MAPKKK7 1.20E-38<br />
159
R02F11.4 Protein phosphatase 1 2.50E-18<br />
T01B6.3 AMPK, gamma subunit 1.60E-18<br />
Transcription C03D6.3 (cel-1) RNGTT 1.70E-113<br />
F40G9.11 (mxl-2) BIGMAX 1.40E-15<br />
F44C8.3 (nhr-18) Nuclear receptor TR4 2.80E-14<br />
R03E9.1 (mdl-1) MAX interactor 1 1.40E-17<br />
T05A12.4 Helicase-like protein 5.10E-52<br />
Y45F10B.9 Zinc-finger protein HT2A 8.40E-07<br />
ZC395.8 DSPP 1.60E-06<br />
Transporter F20D1.9 SLC25A18 2.20E-71<br />
T19C3.5 BPI/LBP/CETP 3.60E-17<br />
Y76A2A.2 (cua-1) Cation transport ATPase 3.70E-261<br />
UPS T01G5.7 RNF8 7.60E-08<br />
Others C29F9.2 MAP7D1 8.00E-06<br />
C54D10.10 TFPI 3.40E-13<br />
F01G12.5 (let-2) Collagen 0<br />
F53B6.2 ADAMTSL1 1.90E-101<br />
T23H4.3 (nas-5) Meprin A metalloprotease 4.60E-31<br />
Y105E8A.16 (rps-20) 40S ribosomal protein S20 1.50E-41<br />
Uncharacterized C54D10.7 (dct-3) Uncharacterized protein 7.90E-30<br />
Protein F35H10.7 CGTHBA protein 4.30E-25<br />
H03A11.1 Uncharacterized protein 5.20E-82<br />
R05F9.1 Uncharacterized protein 1.40E-95<br />
W05H9.1 Uncharacterized protein 6.30E-16<br />
160
Figure 4.1<br />
161
Figure 4.2<br />
162
Figure 4.3<br />
163
Figure 4.4<br />
164
Figure 4.5<br />
165
Figure 4.6<br />
166
Figure 4.7<br />
167
FIGURE LEGENDS<br />
Figure 4.1. Graphs depicting the percentage <strong>of</strong> α-syn-expressing daf-2 and/or daf-<br />
16 mutants with wildtype DA neurons. A. At the chronological aging stage, daf-2<br />
significantly protected DA neurons. B. At the chronological aging stage, daf-16, which<br />
transcribes various cytoprotective <strong>genes</strong> enhanced DA neurodegeneration. C. At the<br />
chronological aging stage, daf-2 + daf-16 double mutation exhibited an intermediate level<br />
<strong>of</strong> neuroprotection. D. At the biological aging stage, daf-2 did not rescue DA neurons<br />
from α-syn-induced neurodegeneration, indicating that differential gene expression in the<br />
daf-2 background is responsible for neuroprotection. **P
Figure 4.4. Graph illustrating the percentage <strong>of</strong> α-syn-expressing worms with<br />
wildtype DA neurons 24 or 48 hrs after 1, 5, and 10 mM 2-deoxyglucose (DOG)<br />
treatment. DOG treatment enhanced DA neurodegeneration <strong>of</strong> 6 day-old animals in a<br />
concentration-dependent manner. **P
Introduction<br />
CHAPTER FIVE<br />
CONCLUSION<br />
Generation <strong>of</strong> transgenic C. elegans overexpressing α-syn::GFP in body wall<br />
muscles, as well as α-syn and GFP in DA neurons, enabled us to ascertain genetic factors<br />
that modified α-syn misfolding and α-syn-induced neurodegeneration, respectively.<br />
Using a combination <strong>of</strong> bioinformatics, RNAi, mutant analysis, and transgene<br />
overexpression, this experimental paradigm allowed us to determine genetic targets as<br />
well as cellular mechanisms that ameliorate α-syn toxicity. Further, many <strong>genes</strong><br />
identified from our studies are <strong>neuroprotective</strong> across species boundaries, strongly<br />
demonstrating C. elegans as a powerful model organism for studying PD. Based on our<br />
findings, we have embarked on the following research projects that are currently<br />
conducted in the Caldwell laboratory.<br />
Neuroprotective mechanism <strong>of</strong> VPS41, ATG7, ULK2, and GIPC: a common pathway?<br />
As described in Chapter 2, based on the hypothesis-based RNAi screen and<br />
subsequent neuroprotection analysis <strong>of</strong> candidate <strong>genes</strong>, 5 specific <strong>genes</strong> (vps-41/VPS41,<br />
atgr-7/ATG7, C35D10.2/GIPC, F55A4.1/SEC22, and F16A11.2/HSPC117) suppressed<br />
α-syn-induced neurodegeneration. Among them, an autophagic component, VPS-<br />
41/VPS41 exhibited the strongest neuroprotection. VPS41 is a well-characterized protein<br />
170
in yeast but not in humans. In yeast, Vps41 is required for trafficking <strong>of</strong> the vesicles to<br />
the vacuoles (mammalian equivalent <strong>of</strong> lysosomes) (Radisky et al., 1997) via the alkaline<br />
phosphatase pathway through its interaction with the AP-3 adaptor protein complex<br />
(Rehling et al., 1999; Darsow et al., 2001). Interestingly, LaGrassa and Ungermann<br />
(2005) reported that activity <strong>of</strong> Vps41 is regulated by yeast casein kinase, Ypt7. From a<br />
separate study, we have demonstrated that overexpression <strong>of</strong> worm casein kinase csnk-1<br />
protects DA neurons from α-syn toxicity. Given the fact that VPS41 is a highly<br />
conserved protein across the species, the protein may share similar functions in humans.<br />
One <strong>of</strong> the on-going research projects in our laboratory, in collaboration with<br />
David Standaert at UAB, is a characterization and validation <strong>of</strong> human VPS41 as a<br />
prospective therapeutic target for PD using the worm α-syn-induced neurodegeneration<br />
model and mammalian cell culture models <strong>of</strong> PD. Importantly, overexpression <strong>of</strong> human<br />
VPS41 protected mammalian cells from neurotoxin-induced toxicity, validating our<br />
previous observation on the <strong>neuroprotective</strong> role <strong>of</strong> worm VPS-41 (Ruan et al.,<br />
manuscript in revision). To further characterize its <strong>neuroprotective</strong> mechanism,<br />
structure-function analysis <strong>of</strong> human VPS41 in C. elegans has revealed that WD40 and<br />
clathrin heavy chain domains are essential for <strong>neuroprotective</strong> function. While not<br />
limited to VPS41, genome-wide analysis <strong>of</strong> single nucleotide polymorphisms (SNPs) in<br />
PD patients should be informative to confirm VPS41 as a genetic PD susceptibility factor.<br />
For example, SNPs have already been found in a gene called ULK2 (Fung et al., 2006),<br />
the worm ortholog <strong>of</strong> which was identified among the top 20 hits from our RNAi screen<br />
171
(Chapter 2). We have determined that unc-51/ULK2 loss-<strong>of</strong>-function mutation enhanced<br />
α-syn toxicity in worm DA neurons (unpublished data).<br />
Additionally, atgr-7/ATG7 is another key autophagic gene that rescued DA<br />
neurons from α-syn toxicity, further confirming autophagy as one <strong>of</strong> the <strong>neuroprotective</strong><br />
cellular mechanisms. ATG7 encodes an ubiquitin-activating enzyme E1-like protein that<br />
plays a critical role in the activation <strong>of</strong> autophagy. Importantly, Komatsu et al. (2006;<br />
2007) demonstrated that the loss <strong>of</strong> Atg7 in mice, which enhanced neurodegeneration in<br />
the central nervous system, was required for maintenance <strong>of</strong> axonal homeostasis as well<br />
as proper behavior. <strong>The</strong>se findings suggest that, similar to VPS41, ATG7 in mammals<br />
may also serve a <strong>neuroprotective</strong> role <strong>against</strong> α-syn-induced neurodegeneration.<br />
Interestingly, not only VPS41 and ATG7 are involved in autophagy, these<br />
proteins contain RING finger (commonly found in E3 ligases) and E1-like protein-<br />
activating enzyme Gsa7p/Apg7p domains, respectively. By physically interacting with<br />
ubiquitin, this association suggests VPS41 and ATG7 may function in a common<br />
pathway such as the UPS or endocytosis (Polo et al., 2002). While the functional link<br />
between these proteins and the UPS cannot be dismissed presently, evidence suggests<br />
that VPS41 and ATG7 may contribute in endocytosis. For example, both endocytic and<br />
autophagic pathways have been shown to converge at the late endosomes to eventually<br />
form late autophagosomes or mature lysosomes (Yi and Tang, 1999). Moreover, as<br />
described above, we have demonstrated that loss <strong>of</strong> unc-51/ULK2, another autophagic<br />
component, enhanced α-syn-induced neurodegeneration. ULK2 is a serine/threonine<br />
172
kinase that has also been shown to regulate axon growth through endocytosis <strong>of</strong> nerve<br />
growth factor in mouse (Zhou et al., 2007). Lastly, in C. elegans, RNAi knockdown <strong>of</strong><br />
endocytic <strong>genes</strong> enhanced growth defects and motor abnormalities that were induced by<br />
overexpression <strong>of</strong> pan-neuronal α-syn (Kuwahara et al., 2008).<br />
Consistent with this view is the <strong>identification</strong> <strong>of</strong> C35D10.2/GIPC as a<br />
<strong>neuroprotective</strong> gene (Chapter 2). GIPC encodes a scaffold protein that regulates cell<br />
surface receptor including β1-adrenergic receptor (Hu et al., 2002), D2-like DA receptor<br />
(Jeanneteau et al., 2004), and insulin-like growth factor 1 receptor (Booth et al., 2002).<br />
Since dop-2 (a C. elegans ortholog <strong>of</strong> D2-like DA receptor) knockout in our model<br />
enhanced α-syn-induced neurodegeneration (unpublished data), GIPC may modulate DA<br />
signaling and promote DA neuron survival by blocking cAMP synthesis, similar to<br />
overexpression <strong>of</strong> PDE9A wherein cAMP is hydrolyzed. Alternatively, since G-protein<br />
signaling has been shown to activate autophagy (Ogier-Denis et al., 2000), it is<br />
conceivable that overexpression <strong>of</strong> GIPC may also stimulate this pathway. If VPS41,<br />
ATG7, ULK2, and GIPC functionally converge as the components <strong>of</strong> endocytic and<br />
autophagic pathways, then it will be intriguing to examine if these proteins enhance<br />
degradation <strong>of</strong> α-syn found in the synaptic termini and/or degradation <strong>of</strong> presumably<br />
neurodegenerative receptors (e.g., D1-like receptor that stimulates cAMP synthesis).<br />
173
Defining Networks <strong>of</strong> <strong>neuroprotective</strong> <strong>genes</strong> by miRNAs<br />
miRNAs are 21-23 bp long, single-stranded RNAs that regulate gene expression<br />
(Ambros, 2001). Initially transcribed as approximately 1000 bp primary miRNAs, these<br />
molecules are further processed by the RISC complex to form mature miRNAs (Moss,<br />
2002). In mammalian neurons, miRNAs have been shown to regulate neuronal<br />
differentiation, neurite outgrowth, survival, and synaptic formation, thus dysregulation <strong>of</strong><br />
miRNAs may enhance susceptibility to neurodegenerative diseases (Bushati and Cohen,<br />
2008; Hebert and De Strooper, 2009). In PD, Kim et al. (2007) reported that mir-133b is<br />
deficient in the midbrain <strong>of</strong> PD patients. Further, Junn et al. (2009) reported that mir-7<br />
suppresses α-syn expression by directly binding to 3’UTR <strong>of</strong> α-syn mRNA.<br />
Collectively, the study <strong>of</strong> miRNAs represents an exciting field that is yet fully explored,<br />
especially with respect to its significance for disease etiology.<br />
As described in Chapter 3, in collaboration with Susan Lindquist at Whitehead<br />
Institute/MIT, we have identified potential therapeutic targets that suppress α-syn toxicity<br />
in multiple model organisms. <strong>The</strong>se candidates include the gene products that function in<br />
ER-Golgi trafficking, protein phosphorylation, ubiquitination, etc. Based on these yeast<br />
α-syn toxicity modifiers, as well as our list <strong>of</strong> <strong>neuroprotective</strong> <strong>genes</strong>, we utilized<br />
bioinformatic databases (miRBase and TargetScan) to mine worm miRNAs that are<br />
predicted to target worm putative <strong>neuroprotective</strong> <strong>genes</strong>. Despite the significant number<br />
<strong>of</strong> targets affected by a single miRNA, only a few miRNAs are predicted to target more<br />
than five <strong>genes</strong> implicated in neuroprotection. Most notably, mir-797, which is part <strong>of</strong><br />
174
mir-2/mir-43/mir-250/mir-797 superfamily may regulate as many as six <strong>genes</strong> that are<br />
predicted or shown to protect DA neurons (Fig. 5.1). For example, according to<br />
miRBase, mir-797 may regulate ykt-6, cmk-1, csnk-1, tag-278, hrd-1, and obr-1. Among<br />
them, we have shown that csnk-1 and hrd-1 rescue DA neurons from α-syn toxicity.<br />
Based on the bioinformatic associations, we examined the effect <strong>of</strong> mir-2 or mir-<br />
797 knockout on α-syn-induced neurodegeneration, and determined that the knockout <strong>of</strong><br />
these miRNAs enhanced neuroprotection (unpublished data). Given the role <strong>of</strong> miRNAs<br />
in suppressing the translation <strong>of</strong> their targets, these data suggest that, under mir-2 and<br />
mir-797 knockout backgrounds, <strong>neuroprotective</strong> <strong>genes</strong> may be up-regulated. To confirm<br />
this, we are currently performing quantitative real-time PCR <strong>of</strong> these targets in miRNA<br />
knockout backgrounds. While genetic analysis and quantitative real-time PCR provide<br />
evidence for the correlation between miRNAs and α-syn-induced neurodegeneration,<br />
these results do not indicate that the observed neuroprotection is specific to the cellular<br />
changes in DA neurons. To further validate the interaction between miRNAs and α-syn<br />
toxicity in DA neurons, we generated transgenic strains that overexpress these miRNAs<br />
selectively in DA neurons. Thus far, we have analyzed the worms that overexpress a<br />
transgene encoding mir-2 primary miRNA (encompassing approximately 500 bp both up-<br />
stream and down-stream <strong>of</strong> the miRNA) under the control <strong>of</strong> dat-1 (dopamine<br />
transporter) promoter. Interestingly, overexpression <strong>of</strong> mir-2 enhanced neurodegeneration<br />
(unpublished data), suggesting that mir-2 modifies gene expression and subsequently<br />
impacts a cellular threshold <strong>against</strong> α-syn toxicity in DA neurons.<br />
175
While speculative, it is interesting to note that the worm F16A11.2 gene and mir-2<br />
are co-expressed in a C. elegans operon unit. F16A11.2/HSPC117 encodes an<br />
uncharacterized conserved protein that was identified in mRNA granules in humans, and<br />
might be involved in mRNA transport in neurons (Kanai et al., 2004). Since<br />
overexpression <strong>of</strong> F16A11.2 and knockout <strong>of</strong> mir-2 enhance neuroprotection, this RNA-<br />
binding protein may physically interact with mir-2 to regulate the miRNA activity.<br />
Additional PD-related studies using C. elegans<br />
As described in Chapters 2-4, our worm α-syn neurodegeneration model has been<br />
utilized to verify putative <strong>neuroprotective</strong> <strong>genes</strong>, but it has also been extensively used to<br />
identify novel compounds that protect DA neurons. For example, we have explored<br />
chemical space by studying two cyclic peptides that rescue DA neurons from α-syn<br />
toxicity (Kritzer et al., 2009). Interestingly, these cyclic peptides resemble a common<br />
sequence found in thioredoxin, which suggests that these compounds may be<br />
<strong>neuroprotective</strong> via reduction <strong>of</strong> oxidative stress. Furthermore, we have identified<br />
several chemicals that suppress α-syn-induced neurodegeneration and this effect is<br />
conserved across species from yeast, to worms, and to rat neurons (Su et al., unpublished<br />
data).<br />
Focusing on genetic interactions among different PD <strong>genes</strong>, we have generated<br />
djr-1.1/DJ-1, djr-1.2/DJ-1, pdr-1/parkin, pink-1/PINK1, and ubh-1/UCHL1 worm mutant<br />
strains overexpressing α-syn and GFP in DA neurons, and determined that the depletion<br />
176
<strong>of</strong> djr-1.1 and djr-1.2 enhance α-syn toxicity (unpublished data). <strong>The</strong>se results suggest<br />
that in our model, protection <strong>against</strong> α-syn-induced oxidative stress may be a key feature<br />
for rescuing DA neurons. Further supporting this view is the result demonstrating that α-<br />
syn-induced neurodegeneration is enhanced in trx-1 (a C. elegans ortholog <strong>of</strong><br />
thioredoxin) knockout background (unpublished data). It will be interesting to further<br />
explore the association between α-syn-induced neurodegeneration and oxidative stress by<br />
using neurotoxins (e.g., 6-OHDA, MPTP/MPP+) or overexpressing proteins with anti-<br />
oxidant properties.<br />
We have also generated transgenic worms overexpressing wildtype or mutant<br />
LRRK2 and GFP in DA neurons, and determined that the overexpression <strong>of</strong> LRRK2<br />
G2019S enhanced DA neurodegeneration (unpublished data). Both C. elegans (Saha et<br />
al., 2009) and D. melanogaster (Liu et al., 2008; Venderova et al., 2009) models <strong>of</strong><br />
LRRK2 G2019S-induced neurodegeneration have been created showing LRRK2 G2019S<br />
more neurodegenerative than wildtype LRRK2. Using our LRRK2 G2019S<br />
neurodegeneration model, we have also identified kinase inhibitors that rescue DA<br />
neurons in worms, mammalian cell culture, and mouse primary neurons (unpublished<br />
data). Since increased kinase activity <strong>of</strong> LRRK2 is linked to enhanced toxicity (West et<br />
al., 2005), it will be intriguing to examine downstream components <strong>of</strong> LRRK2 (e.g.,<br />
MAPKK, MAPK, etc). In C. elegans, the MAPK signaling has been shown to regulate<br />
cell death, therefore, pmk-1/p38 mutant strain may suppress LRRK2 G2019S-induced<br />
neurodegeneration.<br />
177
Lastly, one <strong>of</strong> the 20 positive <strong>genes</strong> from the hypothesis-based RNAi screen<br />
(Chapter 2) is smf-1/SLC11A2. smf-1 encodes a divalent metal transporter that is<br />
predicted to transfer iron, manganese, and other divalent metals. In C. elegans, smf-1 is<br />
predicted to be co-expressed with djr-1.1 and pink-1. Consistent with the RNAi data<br />
indicating that knockdown <strong>of</strong> smf-1 enhanced α-syn misfolding, we have also<br />
demonstrated that smf-1 knockout enhances α-syn toxicity in worm DA neurons<br />
(unpublished data). Since manganese exposure has been linked to PD pathological<br />
features, and more recently through ATP13A2 protein (as mentioned in Chapter 3), it will<br />
be interesting to examine how decreased manganese uptake in smf-1 background<br />
enhances α-syn toxicity. For example, expression pattern or subcellular localization <strong>of</strong><br />
smf-1 will significantly facilitate our understanding <strong>of</strong> PD patho<strong>genes</strong>is and manganese<br />
toxicity.<br />
Conclusion and future directions<br />
<strong>The</strong> experimental strategy we have employed using transgenic C. elegans to<br />
model primary pathological features <strong>of</strong> PD in vivo has enabled us to identify various and<br />
novel factors that modify α-syn misfolding and neurodegeneration. While<br />
vertebrate/mammalian models are the most suitable research tools for studying human<br />
diseases, generation <strong>of</strong> a degenerative model using α-syn in mammalian cell cultures has<br />
been difficult because overexpression <strong>of</strong> α-syn kills these cells, and rodent models are<br />
178
expensive, since they require high maintenance. <strong>The</strong>refore, invertebrate PD models,<br />
admittedly imperfect, remain both valuable and informative tools for discerning genetic,<br />
chemical, and environmental modifiers <strong>of</strong> PD patho<strong>genes</strong>is. Moreover, as described in<br />
Chapter 4, these invertebrate animals with short lifespans readily allow us to examine the<br />
effect <strong>of</strong> longevity on aging-related diseases, including PD.<br />
While C. elegans will continue to be a model organism <strong>of</strong> choice for genetic and<br />
chemical screens, another distinct advantage <strong>of</strong> this organism is a well-defined neuronal<br />
circuitry (White et al., 1986). As we begin to address PD patho<strong>genes</strong>is beyond DA<br />
neurons, C. elegans <strong>of</strong>fers accuracy and precision in studying neuronal connection<br />
unmatched in other model organisms. For instance, we know that CEP neurons (worm<br />
DA neurons) receive signals from ADE (another set <strong>of</strong> worm DA neurons), ALM, OLL,<br />
RIH, RIS, and URB (Fig. 5.2). Furthermore, CEP neurons send signals to AVE, IL1,<br />
OLL, OLQ, RIC, RMD, RMG, RMH, URA, and URB while forming gap junctions with<br />
OLQ and RIH. It will be interesting to examine how neurodegenerative <strong>genes</strong> affect<br />
synaptic plasticity and how DA neurodegeneration may influence synaptic transmission<br />
to or from other surrounding neurons (Desplats et al., 2009).<br />
In addition, since neurodegeneration is also linked to inflammation, it will be<br />
interesting to determine which neuronal types respond to DA neurodegeneration via<br />
inflammatory signals. For example, Styer et al. (2008) reported that npr-1 suppresses<br />
innate immune response in C. elegans. Interestingly, npr-1 is expressed in OLQ neurons,<br />
which form synapses as well as gap functions with CEP neurons. If DA neuron death is<br />
179
enhanced by inflammatory signals, then npr-1 knockout may further promote<br />
neurodegeneration. Conversely, if NPR-1 is overexpressed in the neurons surrounding<br />
CEP and ADE, then this may lead to neuroprotection.<br />
Taken together, the experimental outcomes, current and future directions<br />
collectively described herein provide a substantial scaffold on which further advances<br />
pertaining to therapeutic strategies and cellular mechanisms associated with PD can be<br />
built. Moreover, the capacity to more rapidly ascertain factors influencing<br />
neurodegeneration using the nematode system accelerates the pace toward defining novel<br />
<strong>neuroprotective</strong> strategies that may translate to the clinic. With available tools,<br />
techniques, and databases, C. elegans is deemed to <strong>of</strong>fer immeasurable opportunities for<br />
studying PD and other neurodegenerative diseases.<br />
180
Ambros, V. (2001) Cell 107, 823-826.<br />
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Figure 5.1<br />
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Figure 5.2<br />
185
FIGURE LEGENDS<br />
Figure 5.1. Schematic diagram illustrating predicted targets <strong>of</strong> mir-2/mir-43/mir-<br />
250/mir-797 superfamily by miRBase (top) and TargetScan (bottom). According to<br />
miRBase, mir-797 regulates 6 <strong>genes</strong> including ykt-6, cmk-1, csnk-1, tag-278, hrd-1, and<br />
obr-1. Among them, csnk-1 and hrd-1 have been shown to display <strong>neuroprotective</strong><br />
capacities <strong>against</strong> α-syn-induced neurodegeneration.<br />
Figure 5.2. Schematic diagram <strong>of</strong> CEP neuronal circuitry. CEP receives signal<br />
from 6 neurons, sends signal to 10 neurons, and forms gap junctions with 2 neurons. A<br />
marker for DA neurons, dat-1 (dopamine transporter) is expressed in both CEP and ADE<br />
neurons. Notice npr-1, which suppresses innate immune response in C. elegans is<br />
expressed in OLQ.<br />
186