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idiopathic PD post-mortem midbrains, suggesting the potential neuroprotective function of this gene. As discussed in Chapter 3, we have shown that while knockdown of worm ATP13A2 enhances α-syn misfolding, overexpression of this gene rescues worm DA neurons from α-syn toxicity, demonstrating a novel genetic interaction between α-syn and ATP13A2 (Gitler et al., 2009). PD pathogenesis: mitochondrial and oxidative stress While cellular stress induced by misfolded or aggregated proteins may shed light on the neurodegenerative mechanisms leading to PD, defects in protein degradation machinery alone cannot explain the selective loss of DA neurons. PD pathogenesis consists of both genetic and environmental causes, which only 5-10% of all PD cases have been linked to genetic components. Studies on environmental PD factors have provided insights on the pathways involved in the selective DA neurodegeneration. Langston et al. (1986) studied four patients who exhibited features of clinical Parkinsonism after using a new “synthetic heroin.” Subsequent analysis of the drug components revealed 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) as a primary compound, and they proposed that MPTP might induce the selective loss of DA neurons. While MPTP was later found harmless, after crossing the blood-brain barrier, the compound is readily metabolized into toxic 1-methyl-4-phenylpyridinium (MPP+), which disrupts complex I of mitochondrial respiratory chain. Inhibition of complex I generates 10
ROS, which may oxidize DA (a neurotransmitter known to be readily oxidized) to produce highly toxic DA that results in DA neurodegeneration. Two PD genes, PINK1 and HTRA2 are also linked to mitochondria. Valente et al. (2004) mapped PINK1 from an Italian family with autosomal recessive PD, and determined that PINK1 when expressed in COS-7 and SH-SY5Y cells localized to mitochondria. Furthermore, using SH-SY5Y cells overexpressing wildtype or mutant PINK1, they demonstrated that, after MG-132 (a proteasome inhibitor) treatment, wildtype PINK1 enhanced cell survival without modifying mitochondrial membrane potential whereas mutant PINK1 displayed no protection with decreased membrane potential. Taken together, these findings provided the first evidence linking a genetic cause of PD to mitochondria, further confirming the relationship between mitochondrial malfunction and PD pathogenesis, and suggesting a potential neuroprotective function of PINK1. Strauss et al. (2005) identified HTRA2 in German PD patients. To further validate their findings, they overexpressed wildtype and mutant HTRA2 in cell culture, and found that mutant form failed to interact with XIAP (an inhibitor of apoptosis), suggesting that misregulation of HTRA2 might be linked to PD pathogenesis. Moreover, they determined that HTRA2 is predominantly localized to mitochondria by immunohistochemistry, and observed distinct mitochondrial morphological changes by electron microscopy. Focusing on mitochondrial function, they examined mitochondrial membrane potential and cell viability, and showed that mutant HTRA2 decreased the 11
Page 1 and 2: IDENTIFICATION OF NEUROPROTECTIVE G
Page 3 and 4: ABSTRACT Recent functional analyses
Page 5 and 6: E3 Ubiquitin ligase ER Endoplasmic
Page 7 and 8: RT-PCR Reverse transcriptase polyme
Page 9 and 10: TAG-278 Temporary assigned gene 278
Page 11 and 12: p38 Mitogen-activated protein kinas
Page 13 and 14: ACKNOWLEDGMENTS First and foremost,
Page 15 and 16: CONTENTS ABSTRACT .................
Page 17 and 18: a. Abstract .......................
Page 19 and 20: LIST OF FIGURES 1.1. Schematic repr
Page 21 and 22: Parkinson disease CHAPTER ONE INTRO
Page 23 and 24: al., 2006) and torsinA (Cao et al.,
Page 25 and 26: endoplasmic reticulum (ER) to Golgi
Page 27 and 28: c-Jun N-terminal kinase and caspase
Page 29: lysosomal protease promotes α-syn
Page 33 and 34: together, although anti-oxidant act
Page 35 and 36: multicellular eukaryotic model orga
Page 37 and 38: C. elegans PD models C. elegans off
Page 39 and 40: Current studies To investigate gene
Page 41 and 42: Specific outcomes and future direct
Page 43 and 44: Corti, O., Hampe, C., Koutnikova, H
Page 45 and 46: Lautier, C., Goldwurm, S., Dürr, A
Page 47 and 48: Scherzer, C.R., Jensen, R.V., Gulla
Page 49 and 50: Willcox, B.J., Donlon, T.A., He, Q.
Page 51 and 52: Table 1.2. Summary of selected inve
Page 53 and 54: FIGURE LEGENDS Fig. 1.1. Schematic
Page 55 and 56: ABSTRACT Genomic multiplication of
Page 57 and 58: et al., 2003). Maintenance of DA ne
Page 59 and 60: Pdat-1::FLAG-R05D11.6, Punc-54::DsR
Page 61 and 62: Biochemicals) and 1:10000 horseradi
Page 63 and 64: was washed with 100 µl RNase-free
Page 65 and 66: and effectively discerned via RNAi
Page 67 and 68: protein. The primary RNAi screen of
Page 69 and 70: expressing a polyglutamine::GFP fus
Page 71 and 72: stage (Fig. 2.2B-D). Two genes exhi
Page 73 and 74: with strong expression localized to
Page 75 and 76: Our data demonstrating that the C.
Page 77 and 78: disparate datasets, such as those o
Page 79 and 80: Driscoll, M., Gerstbrein, B. (2003)
Page 81 and 82:
Singleton, A.B., Farrer, M., Johnso
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Table 2.2. Bioinformatic associatio
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Table 2.4. Results of all genes kno
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C09G12.9 Tumor susceptibility gene
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C32B5.13 Cathepsin H precursor C32B
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D1014.3 Alpha-soluble NSF attachmen
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F22B7.5 DNAJA3, mitochondrial precu
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F40F4.5 Tubulin alpha-ubiquitous ch
Page 97 and 98:
F54F7.5 Serine/threonine kinase 24
Page 99 and 100:
K07A9.2 CAMK1 K07C11.9 Hypothetical
Page 101 and 102:
R74.4 DNAJB1 precursor T01B7.4 Pept
Page 103 and 104:
T22D1.9 PSMD2 T22F3.2 Ubiquitin spe
Page 105 and 106:
Y38F2AL.3 Vacuolar ATP synthase sub
Page 107 and 108:
Y73C8C.8 CENPE variant protein (Fra
Page 109 and 110:
Table 2.5. Summary of RNAi knockdow
Page 111 and 112:
Figure 2.2 91
Page 113 and 114:
Figure 2.4 93
Page 115 and 116:
Figure 2.6 95
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FIGURE LEGENDS Figure 2.1. RNAi kno
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came from genes that were co-expres
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ABSTRACT Multiple Parkinson disease
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vacuoles (mammalian equivalent of l
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neuroprotection when the animals we
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To investigate the genetic interact
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illustrated that α-syn overexpress
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main contributor of α-syn phosphor
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Hoozemans, J.J., van Haastert, E.S.
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Figure 3.2 115
Page 137 and 138:
FIGURE LEGENDS Figure 3.1. RAB3A, R
Page 139 and 140:
CHAPTER FOUR RNA INTERFERENCE SCREE
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INTRODUCTION Central to Parkinson d
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thermotolerance (Lithgow et al., 19
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mCherry were transferred onto NGM p
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20°C for 72 hrs. The gravid adults
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pathways downstream of DAF-2 exclud
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were assayed by growing the RNAi-tr
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while DOG treatment enhanced α-syn
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enhance α-syn misfolding in a daf-
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REFERENCES Bar-On, P., Crews, L., K
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Lesage, S., Brice, A. (2009) Hum Mo
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Table 4.1. Summary of genes analyze
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C12D12.2 Excitatory amino acid tran
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C47B2.2 n/a C47C12.6 Amiloride-sens
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F18A1.5 Replication protein A F18E3
Page 169 and 170:
F46E10.10 Malate dehydrogenase F46E
Page 171 and 172:
K08H10.1 Dentin sialophosphoprotein
Page 173 and 174:
T05A10.5 Cysteine-rich secretory pr
Page 175 and 176:
W03G9.6 Platelet-activating factor
Page 177 and 178:
Y58G8A.1 Acetylcholine receptor sub
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Table 4.2. Summary of positive gene
Page 181 and 182:
Figure 4.1 161
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Figure 4.3 163
Page 185 and 186:
Figure 4.5 165
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Figure 4.7 167
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Figure 4.4. Graph illustrating the
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in yeast but not in humans. In yeas
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kinase that has also been shown to
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mir-2/mir-43/mir-250/mir-797 superf
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of djr-1.1 and djr-1.2 enhance α-s
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expensive, since they require high
Page 201 and 202:
Ambros, V. (2001) Cell 107, 823-826
Page 203 and 204:
Yi, J., Tang, X.M. (1999) Cell Res
Page 205 and 206:
Figure 5.2 185
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